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Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: Analysis of their mechanism of action
The FASEB Journal
Abstract
Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.
... AnxA1 and its N-terminal peptides have both been shown to be cardioprotective in various I/RI models [58,59]. D'Amico et al. demonstrated a decrease in infarct size (50%) upon infusion of recombinant AnxA1 and the protective effects of AnxA1 were further confirmed by La et al. as evidenced by the administration of the AnxA1 N-terminal peptide, Ac2-26, to decrease infarct size and reduce MPO and IL-1β content in infarcted hearts [58]. ...
... AnxA1 and its N-terminal peptides have both been shown to be cardioprotective in various I/RI models [58,59]. D'Amico et al. demonstrated a decrease in infarct size (50%) upon infusion of recombinant AnxA1 and the protective effects of AnxA1 were further confirmed by La et al. as evidenced by the administration of the AnxA1 N-terminal peptide, Ac2-26, to decrease infarct size and reduce MPO and IL-1β content in infarcted hearts [58]. Treatment with AnxA1 also attenuated loss of fiber organization, decreased MPO activity, reduced TNF-α and macrophage inflammatory protein (MIP-1α) levels and leukocyte extravagation in cardiac tissues [60]. ...
... In addition, platelet aggregates are diminished [7,31,47] and pro-inflammatory mediators (e.g., myeloperoxidase (MPO), reactive oxygen species (ROS) and cytokines) are moderated [88,90]. (B) AnxA1 promotes the balance between proinflammatory [7,31,47,58,60,88,90] and anti-inflammatory cytokines, thereby fostering homeostasis in the host. (C) The disruption of barrier function, due to the loss of junctional molecules and apoptosis/necrosis of endothelial/epithelial cells, is one of the main characteristics of I/RI. ...
Cardiovascular disease (CVD) continues to be the leading cause of death in the world. Increased inflammation and an enhanced thrombotic milieu represent two major complications of CVD, which can culminate into an ischemic event. Treatment for these life-threatening complications remains reperfusion and restoration of blood flow. However, reperfusion strategies may result in ischemia–reperfusion injury (I/RI) secondary to various cardiovascular pathologies, including myocardial infarction and stroke, by furthering the inflammatory and thrombotic responses and delivering inflammatory mediators to the affected tissue. Annexin A1 (AnxA1) and its mimetic peptides are endogenous anti-inflammatory and pro-resolving mediators, known to have significant effects in resolving inflammation in a variety of disease models. Mounting evidence suggests that AnxA1, which interacts with the formyl peptide receptor (FPR) family, may have a significant role in mitigating I/RI associated complications. In this review article, we focus on how AnxA1 plays a protective role in the I/R based vascular pathologies.
... AnxA1 has emerged as a potent anti-inflammatory therapeutic agent in a variety of diseases based on its ability to decrease inflammation both in vitro and in vivo, as well as to provide tissue protection [2]. Following IR injury, there was a significant increase in AnxA1 in the lung tissue, similar to that shown in previous studies [6,[10][11][12][13]. The increased expression of AnxA1 was accompanied by leukocytes infiltrating the damaged tissues of the myocardium, brain, lung, and kidney [6,[10][11][12][13]. ...
... Following IR injury, there was a significant increase in AnxA1 in the lung tissue, similar to that shown in previous studies [6,[10][11][12][13]. The increased expression of AnxA1 was accompanied by leukocytes infiltrating the damaged tissues of the myocardium, brain, lung, and kidney [6,[10][11][12][13]. As previously reported, the increased AnxA1 expression was at least partly due to transmigrating neutrophils [6,10,12,13]. ...
... The increased expression of AnxA1 was accompanied by leukocytes infiltrating the damaged tissues of the myocardium, brain, lung, and kidney [6,[10][11][12][13]. As previously reported, the increased AnxA1 expression was at least partly due to transmigrating neutrophils [6,10,12,13]. The increase was attenuated by the administration of the AnxA1 peptide Ac2-26 [2,6]. ...
Annexin A1 (AnxA1) is an endogenous protein that modulates anti-inflammatory processes, and its therapeutic potential has been reported in a range of inflammatory diseases. The effect of AnxA1 on ischemia-reperfusion (IR)-induced lung injury has not been examined. In this study, isolated, perfused rat lungs were subjected to IR lung injury induced by ischemia for 40 min, followed by reperfusion for 60 min. The rat lungs were randomly treated with vehicle (phosphate-buffered saline), and Ac2-26 (an active N-terminal peptide of AnxA1) with or without an N-formyl peptide receptor (FPR) antagonist N-Boc-Phe-Leu-Phe-Leu-Phe (Boc2). An in vitro study of the effects of Ac2-26 on human alveolar epithelial cells subjected to hypoxia-reoxygenation was also investigated. Administration of Ac2-26 in IR lung injury produced a significant attenuation of lung edema, pro-inflammatory cytokine production recovered in bronchoalveolar lavage fluid, oxidative stress, apoptosis, neutrophil infiltration, and lung tissue injury. Ac2-26 also decreased AnxA1 protein expression, inhibited the activation of nuclear factor-κB and mitogen-activated protein kinase pathways in the injured lung tissue. Finally, treatment with Boc2 abolished the protective action of Ac2-26. The results indicated that Ac2-26 had a protective effect against acute lung injury induced by IR, which may be via the activation of the FPR.
... The main mechanism of ANXA1 action is by the inhibition of phospholipase A2 (PLA2), an enzyme involved in the adhesive properties of neutrophils to endothelial cells, preventing the neutrophil transmigration through the endothelium [20,22]. This effect may also be beneficial in ischemia-reperfusion situations [23][24][25]. Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. ...
... Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. In some chronic inflammatory conditions, as Crohn's disease or sepsis, ANXA1 levels are usually reduced, supporting the progression and exacerbation of the inflammatory response [26,27]. ...
... Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. In some chronic inflammatory conditions, as Crohn's disease or sepsis, ANXA1 levels are usually reduced, supporting the progression and exacerbation of the inflammatory response [26,27]. ...
[This corrects the article DOI: 10.1371/journal.pone.0165833.].
... The ANXA1 derived peptide Ac2-26 has been described to be protective in mouse and rat models of myocardial infarction, however solely in models of acute damage after reperfusion. [111][112][113] Studies with a larger time frame looking at the effects of ANXA1 in the myocardial repair have not been reported. After an ischemia period of 25 min followed by a reperfusion period of 1-2 hours ANXA1 mimetic peptide Ac2-26 reduced the infarct area. ...
... Furthermore, inflammatory cytokine (e.g., TNFa and IL-1ß) and myeloperoxidase (as a marker of neutrophil recruitment) expression was reduced after Ac2-26 treatment. [111][112][113] Myocardial infarction is an acute life-threatening disorder and therefore timing of treatment is indispensable. Treatment potential was indicated by injecting Ac2-26 0, 30 and 60 min after the start of the reperfusion period to mimic the clinical situation of patients which likely cannot be treated directly upon restoration of the blood flow. ...
... Treatment potential was indicated by injecting Ac2-26 0, 30 and 60 min after the start of the reperfusion period to mimic the clinical situation of patients which likely cannot be treated directly upon restoration of the blood flow. 111 The most prominent effect of Ac2-26 was found when the protein was administered 30 min after the start of the reperfusion. One potential problem in using ANXA1 or Ac2-26 in clinics might be the cleavage and inactivation of those proteins by proteases since externalized and exogenous ANXA1 is cleaved by human proteinase and neutrophil elastase. ...
The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.
... The main mechanism of ANXA1 action is by the inhibition of phospholipase A2 (PLA2), an enzyme involved in the adhesive properties of neutrophils to endothelial cells, preventing the neutrophil transmigration through the endothelium [20,22]. This effect may also be beneficial in ischemia-reperfusion situations [23][24][25]. Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. ...
... Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. In some chronic inflammatory conditions, as Crohn's disease or sepsis, ANXA1 levels are usually reduced, supporting the progression and exacerbation of the inflammatory response [26,27]. ...
... Facio et al. (2010) demonstrated that ANXA1 has an important role in renal defense against ischemia-reperfusion injury, aborting neutrophil extravasation after reperfusion [24]. La et al. (2001) and Qin et al. (2014) showed that ANXA1 reduces the tissue damage in the myocardium caused by ischemia-reperfusion events [23,25]. In some chronic inflammatory conditions, as Crohn's disease or sepsis, ANXA1 levels are usually reduced, supporting the progression and exacerbation of the inflammatory response [26,27]. ...
Sickle cell disease (SCD) is an inherited hemolytic anemia whose pathophysiology is driven by polymerization of the hemoglobin S (Hb S), leading to hemolysis and vaso-occlusive events. Inflammation is a fundamental component in these processes and a continuous inflammatory stimulus can lead to tissue damages. Thus, pro-resolving pathways emerge in order to restore the homeostasis. For example there is the annexin A1 (ANXA1), an endogenous anti-inflammatory protein involved in reducing neutrophil-endothelial interactions , accelerating neutrophil apoptosis and stimulating macrophage efferocytosis. We investigated the expression of ANXA1 in plasma of SCD patients and its relation with anemic , hemolytic and inflammatory parameters of the disease. Three SCD genotypes were considered: the homozygous inheritance for Hb S (Hb SS) and the association between Hb S and the hemoglobin variants D-Punjab (Hb SD) and C (Hb SC). ANXA1 and proinflamma-tory cytokines were quantified by ELISA in plasma of SCD patients and control individuals without hemoglobinopathies. Hematological and biochemical parameters were analyzed by flow cytometry and spectrophotometer. The plasma levels of ANXA1 were about threefold lesser in SCD patients compared to the control group, and within the SCD genotypes the most elevated levels were found in Hb SS individuals (approximately threefold higher). Proinflammatory cytokines were higher in SCD groups than in the control individuals. Anemic and hemolytic markers were higher in Hb SS and Hb SD genotypes compared to Hb SC patients. White blood cells and platelets count were higher in Hb SS genotype and were positively correlated to ANXA1 levels. We found that ANXA1 is down-regulated and differentially expressed within the SCD genotypes. Its expression seems to depend on the inflammatory, hemolytic and vaso-occlusive characteristics of the diseased. These data may lead to new biological targets for therapeutic intervention in SCD.
... A top hit in this screen was Annexin A4 (ANXA4), which was significantly increased in response to prolonged strain. This result is consistent with other work describing annexin upregulation in glaucoma, ischemic injury, Alzheimer's disease, and related neurodegenerative processes (Eberhard et al., 1994;Facio et al., 2011;Johnson et al., 2007;Kulik et al., 2009;La et al., 2001;Ries et al., 2021;Ruan et al., 2013). Similar to other Annexin family members, ANXA4 translocates to cell membranes upon elevation of intracellular calcium and forms lateral assemblies of two-dimensional (2D) trimer structures (Newman et al., 1991). ...
... This model suggests a novel mechanism that requires no additional specialized membrane mechanosensor to initiate Ca 2+driven cellular responses to IOP-induced biomechanical strain. A variety of annexins are commonly reported to be upregulated in models of disease that experience chronic biomechanical insult, such as glaucoma, muscular dystrophy, and cardiovascular disease (Cagliani et al., 2005;Holland et al., 2013;Johnson et al., 2007;La et al., 2001;Monastyrskaya et al., 2009;Waddell et al., 2011). Therefore, the link between moderate biomechanical strain and PMR mechanisms may be relevant to a variety of chronic injury and disease mechanisms. ...
Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.
... Several recent reviews have described the role of Annexin A1 in the cardiovascular system, including its pro-resolving role in inflammation associated with atherosclerosis and myocardial infarction [20][21][22][23]. Results of some of these studies are summarized in Tables 2 and 3 [58][59][60][61][62][63][64][65][66][67]. Thus, the following section provides a review of the more recent studies in this field (Table 4). ...
... Reduced myeloperoxidase activity and interleukin-1β in the infarcted heart; protective effects were abrogated with an antagonist of Fpr receptors [61] Ac2-26 treatment of wild-type and Fpr null mice subjected to cardiac IRI Cardioprotection in both wild-type and Fpr null mice [62] Effects of of Annexin A1(2-50) peptide in wild-type, Fpr1 −/− , and Fpr2 −/− /ALX −/− mice. Effects of the metabolically stable form of this peptide (i.e., CR-Annexin A1 in the murine model of cardiac IRI Reduced leukocyte adhesion in wild-type and Fpr1 −/− , but not Fpr2 −/− /ALX −/− , mice Reduced infarct size and incidence of 24-h death [63] Effects of hrANXA1 in ANXA1 deficient STZ mice Ameliorated cardiac injury [46] ANXA1 overexpression in the rat model of myocardial infarction ...
Renal and cardiovascular disorders are very prevalent and associated with significant morbidity and mortality. Among diverse pathogenic mechanisms, the dysregulation of immune and inflammatory responses plays an essential role in such disorders. Consequently, the discovery of Annexin A1, as a glucocorticoid-inducible anti-inflammatory protein, has fueled investigation of its role in renal and cardiovascular pathologies. Indeed, with respect to the kidney, its role has been examined in diverse renal pathologies, including acute kidney injury, diabetic nephropathy, immune-mediated nephropathy, drug-induced kidney injury, kidney stone formation, and renal cancer. Regarding the cardiovascular system, major areas of investigation include the role of Annexin A1 in vascular abnormalities, atherosclerosis, and myocardial infarction. Thus, this review briefly describes major structural and functional features of Annexin A1 followed by a review of its role in pathologies of the kidney and the cardiovascular system, as well as the therapeutic potential of its modulation for such disorders.
... Moreover, an FPR1/FPR2 dual agonist, compound 17b, preserves cardiac function in models of acute myocardial infarction (42), while another dual agonist, compound 43, affords cardio protection in experimental heart failure induced by permanent coronary artery ligation through its ability to modify macrophage phenotype (43). We have a long-dated interest in FPRs and their agonist AnxA1; the heart can be targeted by AnxA1 and AnxA1 peptide Ac2-26 protecting against myocardial ischemia reperfusion injury (15,44). These cardio-protective effects are mediated by FPR2 as demonstrated by loss of efficacy after administration of receptor antagonists (44) and persistence of efficacy in FPR1 null mice (45). ...
... We have a long-dated interest in FPRs and their agonist AnxA1; the heart can be targeted by AnxA1 and AnxA1 peptide Ac2-26 protecting against myocardial ischemia reperfusion injury (15,44). These cardio-protective effects are mediated by FPR2 as demonstrated by loss of efficacy after administration of receptor antagonists (44) and persistence of efficacy in FPR1 null mice (45). More recently, hrAnxA1 was showed to protect against myocardial inflammation in type I diabetes-induced cardiomyopathy, a result obtained through dampening cardiac MAPK signaling and activating the prosurvival Akt pathway (24). ...
Significance
Patients with rheumatoid arthritis (RA) are susceptible to heart failure accompanied by diastolic dysfunction. It is unknown what causes diastolic dysfunction in RA, and current therapies do not reduce the risk of heart problems. Here, we characterize a murine model of arthritis, which mirrors the diastolic dysfunction observed in RA patients; this dysfunction is associated with fibrous tissue formation, enlargement of the heart, and changes in the number/type of inflammatory cells within the heart. Importantly, treatment with the protein Annexin A1 not only halts the progression of diastolic dysfunction, but, when given at a later stage, it reverses established diastolic dysfunction and attenuates cardiac remodeling in arthritic mice. This model can help develop treatments for heart failure in RA.
... Annexin A1 and its derivative peptides have been shown to have therapeutic effect in various disease states, including sepsis Zhang et al., 2018), lung inflammation (da Cunha et al., 2012) and chronic obstructive pulmonary disease (COPD) (Possebon et al., 2018), myocardial infarction (D' Amico et al., 2000;La et al., 2001;Qin et al., 2015), and intestinal wound repair (Babbin et al., 2008). Studies confirmed that endogenous annexin A1 plays a role in promoting phagocytosis (Yona et al., 2006;Scannell et al., 2007), and its derived peptide Ac2-26 increases macrophage phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) (Maderna et al., 2005). ...
... In ischemic insult, Ac2-26 and annexin A1 demonstrate cardioprotective effects in rats and mice, reducing infarct size and myeloperoxidase activity (D' Amico et al., 2000;La et al., 2001;Qin et al., 2015), although not as completely as corticosteroid treatment (Ritchie et al., 2003). In mice models of ischemia and reperfusion, annexin A1 mimetics prevent white blood cell adhesion and markers of inflammation in cerebral infarct (Gavins et al., 2007) and inhibit macrophage infiltration, maintaining glomerular filtration rate and urine osmolality while preventing acute tubular necrosis in renal ischemia (Facio et al., 2011). ...
Sepsis is a continuing problem in modern healthcare, with a relatively high prevalence, and a significant mortality rate worldwide. Currently, no specific anti-sepsis treatment exists despite decades of research on developing potential therapies. Annexins are molecules that show efficacy in preclinical models of sepsis but have not been investigated as a potential therapy in patients with sepsis. Human annexins play important roles in cell membrane dynamics, as well as mediation of systemic effects. Most notably, annexins are highly involved in anti-inflammatory processes, adaptive immunity, modulation of coagulation and fibrinolysis, as well as protective shielding of cells from phagocytosis. These discoveries led to the development of analogous peptides which mimic their physiological function, and investigation into the potential of using the annexins and their analogous peptides as therapeutic agents in conditions where inflammation and coagulation play a large role in the pathophysiology. In numerous studies, treatment with recombinant human annexins and annexin analogue peptides have consistently found positive outcomes in animal models of sepsis, myocardial infarction, and ischemia reperfusion injury. Annexins A1 and A5 improve organ function and reduce mortality in animal sepsis models, inhibit inflammatory processes, reduce inflammatory mediator release, and protect against ischemic injury. The mechanisms of action and demonstrated efficacy of annexins in animal models support development of annexins and their analogues for the treatment of sepsis. The effects of annexin A5 on inflammation and platelet activation may be particularly beneficial in disease caused by SARS-CoV-2 infection. Safety and efficacy of recombinant human annexin A5 are currently being studied in clinical trials in sepsis and severe COVID-19 patients.
... protein in vivo (La et al., 2001a;Gavins et al., 2005;D'Amico et al., 2000;Dalli et al., 2012), at least over the short-term. The duration of reperfusion following ischemia employed in these earlier I-R studies in vivo has ranged from 45 min to 2 h (La et al., 2001a;Gavins et al., 2003aGavins et al., , 2005. ...
... protein in vivo (La et al., 2001a;Gavins et al., 2005;D'Amico et al., 2000;Dalli et al., 2012), at least over the short-term. The duration of reperfusion following ischemia employed in these earlier I-R studies in vivo has ranged from 45 min to 2 h (La et al., 2001a;Gavins et al., 2003aGavins et al., , 2005. A small number of studies have shown that Ac 2-26 reduces infarct size and leukocyte recruitment when administrated at reperfusion (D'Amico et al., 2000;La et al., 2001b;Gavins et al., 2005). ...
The anti-inflammatory, pro-resolving annexin-A1 protein acts as an endogenous brake against exaggerated cardiac necrosis, inflammation, and fibrosis following myocardial infarction (MI) in vivo. Little is known, however, regarding the cardioprotective actions of the N-terminal-derived peptide of annexin A1, Ac2-26, particularly beyond its anti-necrotic actions in the first few hours after an ischemic insult. In this study, we tested the hypothesis that exogenous Ac2-26 limits cardiac injury in vitro and in vivo. Firstly, we demonstrated that Ac2-26 limits cardiomyocyte death both in vitro and in mice subjected to ischemia-reperfusion (I-R) injury in vivo (Ac2-26, 1 mg/kg, i.v. just prior to post-ischemic reperfusion). Further, Ac2-26 (1 mg/kg i.v.) reduced cardiac inflammation (after 48 h reperfusion), as well as both cardiac fibrosis and apoptosis (after 7-days reperfusion). Lastly, we investigated whether Ac2-26 preserved cardiac function after MI. Ac2-26 (1 mg/kg/day s.c., osmotic pump) delayed early cardiac dysfunction 1 week post MI, but elicited no further improvement 4 weeks after MI. Taken together, our data demonstrate the first evidence that Ac2-26 not only preserves cardiomyocyte survival in vitro, but also offers cardioprotection beyond the first few hours after an ischemic insult in vivo. Annexin-A1 mimetics thus represent a potential new therapy to improve cardiac outcomes after MI.
... Animals subjected to myocardial I/R injury had higher expression of AnxA1 in their hearts compared to the control groups [166], which seems to be the result of de novo synth- esis of the protein in infiltrating neutrophils. It is also possible that AnxA1 might be upregulated aiming to attenuate inflam- mation in this condition. ...
... Indeed, AnxA1 attenuated the progres- sion of existing plaques of aortic arch and subclavian artery in vivo [78]. Furthermore, the expression of AnxA1 in neutrophils isolated from blood was significantly increased in patients with coronary artery disease compared to controls [32], simi- larly to the results found in the animal model of myocardial I/R injury [166], possibly in an attempt to attenuate disease- related immune activation, as suggested by the authors. ...
Introduction:
The timely resolution of inflammation is essential to restore tissue homeostasis and to avoid chronic inflammatory diseases. Resolution of inflammation is an active process modulated by various proresolving mediators, including annexin A1 (AnxA1) and specialized proresolving lipid mediators (SPMs), which counteract excessive inflammatory responses and stimulate proresolving mechanisms. Areas covered: The protective effects of AnxA1 and SPMs have been extensively explored in pre-clinical animal models. However, studies investigating the function of these molecules in human diseases are just emerging. This review highlights recent advances on the role of proresolving mediators, and pharmacological opportunities of promoting resolution pathways in preclinical models and patients with various human diseases. Expert opinion: Dysregulation or 'failure' in proresolving mechanisms might be involved the pathogenesis of chronic inflammatory diseases. Altered levels of proresolving mediators were found in a wide range of human diseases. In some cases, AnxA1 and SPMs are up-regulated in human blood and tissues but fail to engage in proresolving signaling and, hence, to regulate excessive inflammation. Thus, the new concept of 'resolution pharmacology' could be applied to compensate deficiency of endogenous proresolving mediators' generation and/or possible failures in the engagement of resolution pathways observed in many chronic inflammatory diseases.
... In this context, the therapeutic potential of glucocorticoid-regulated anti-inflammatory mediator annexin-A1 has been demonstrated in different systemic inflammatory disorders. Annexin-A1 is a glucocorticoidinducible 37 kDa protein, highly expressed by macrophages, that activates the family of formyl peptide receptors and inhibits different processes related to myocardial reperfusion injury such as polymorphonuclear leukocyte activation, migration, and infiltration (Ambrose et al., 1992;De Caterina et al., 1993;La et al., 2001;Perretti and Gavins, 2003;Qin et al., 2015). Due to the potent anti-inflammatory and cardioprotective properties of endogenous annexin-A1, several studies utilized experimental models to examine the role of the exogenous protein and its derived peptides (Perretti and Gavins, 2003). ...
... The main benefits attributed to annexin-A1 peptide mimetics include cardioprotection based on their anti-inflammatory effect to preserve myocardial viability after MI but also other inflammation-independent properties that directly protect cardiomyocytes viability and contractile function (Qin et al., 2015). The subcutaneous administration of annexin-A1 N-terminal derived peptide Ac2-26 has been shown to confer protection against ischemia-reperfusion injury by reducing myeloperoxidase activity and IL-1β levels in the infarcted heart, as well as down-regulate monocyte accumulation and inhibit phagocytic activity of macrophages in different rodent experimental models (Getting et al., 1997;La et al., 2001). Another annexin-A1 mimetic is CGEN-855A, a 21 amino acid peptide displays anti-inflammatory effects by inhibition of polymorphonuclear neutrophils recruitment and also provides protection against ischemia-reperfusion-mediated injury to the myocardium after being injected intravenously in mice (Hecht et al., 2009). ...
Cardiovascular disease (CVD) remains a leading cause of mortality and morbidity worldwide. Numerous therapies are currently under investigation to improve pathological cardiovascular complications, but yet, there have been very few new medications approved for intervention/treatment. Therefore, new approaches to treat CVD are urgently required. Attempts to prevent vascular complications usually involve amelioration of contributing risk factors and underlying processes such as inflammation, obesity, hyperglycaemia, or hypercholesterolemia. Historically, the development of peptides as therapeutic agents has been avoided by the Pharmaceutical industry due to their low stability, size, rate of degradation, and poor delivery. However, more recently, resurgence has taken place in developing peptides and their mimetics for therapeutic intervention. As a result, increased attention has been placed upon using peptides that mimic the function of mediators involved in pathologic processes during vascular damage. This review will provide an overview on novel targets and experimental therapeutic approaches based on peptidomimetics for modulation in CVD. We aim to specifically examine apolipoprotein A-I (apoA-I) and apoE mimetic peptides and their role in cholesterol transport during atherosclerosis, suppressors of cytokine signaling (SOCS)1-derived peptides and annexin-A1 as potent inhibitors of inflammation, incretin mimetics and their function in glucose-insulin tolerance, among others. With improvements in technology and synthesis platforms the future looks promising for the development of novel peptides and mimetics for therapeutic use. However, within the area of CVD much more work is required to identify and improve our understanding of peptide structure, interaction, and function in order to select the best targets to take forward for treatment.
... The effect of AnxA1 was related to a reduction in tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α expression, as well as myeloperoxidase (MPO) activity, leading to reduced leukocyte extravasation into cardiac tissues (D'Amico et al., 2000). In addition, Ac2-26 administration decreased infarct size and diminished MPO activity and IL-1β levels in an experimental model of rat myocardial ischemia/reperfusion injury (La et al., 2001). Ac2-26 treatment preserved cardiomyocyte contractile function and decreased cardiac myocyte injury through activation of p38-MAPK, PKC, and ATP-sensitive potassium channels (Ritchie et al., 2005). ...
Cardiovascular diseases (CVD) remain the leading cause of mortality worldwide. The main cause underlying CVD is associated with the pathological remodeling of the vascular wall, involving several cell types, including endothelial cells, vascular smooth muscle cells, and leukocytes. Vascular remodeling is often related with the development of atherosclerotic plaques leading to narrowing of the arteries and reduced blood flow. Atherosclerosis is known to be triggered by high blood cholesterol levels, which in the presence of a dysfunctional endothelium, results in the retention of lipoproteins in the artery wall, leading to an immune-inflammatory response. Continued hypercholesterolemia and inflammation aggravate the progression of atherosclerotic plaque over time, which is often complicated by thrombus development, leading to the possibility of CV events such as myocardial infarction or stroke. Annexins are a family of proteins with high structural homology that bind phospholipids in a calcium-dependent manner. These proteins are involved in several biological functions, from cell structural organization to growth regulation and vesicle trafficking. In vitro gain- or loss-of-function experiments have demonstrated the implication of annexins with a wide variety of cellular processes independent of calcium signaling such as immune-inflammatory response, cell proliferation, migration, differentiation, apoptosis, and membrane repair. In the last years, the use of mice deficient for different annexins has provided insight into additional functions of these proteins in vivo , and their involvement in different pathologies. This review will focus in the role of annexins in CVD, highlighting the mechanisms involved and the potential therapeutic effects of these proteins.
... As AnxA1 is easily inactivated in vitro, Ac2-26 is often used as its substitute and is widely employed in experimental models of inflammation. Increasing evidence (10)(11)(12)(13) suggests that Ac2-26 exhibits protective effects against the I/R-induced inflammatory response and ameliorates organ dysfunctions. However, whether Ac2-26 can alleviate HIRI by inhibiting neutrophil infiltration through regulation of the HMGB1/TLR4/NF-κB signaling pathway remains to be elucidated. ...
Inflammation is one of the most crucial mechanism underlying hepatic ischemia-reperfusion injury (HIRI). Several studies have shown that Ac2-26, the active N-terminal peptide of Annexin A1, could modulate anti-inflammatory processes and protect the organs from ischemia-reperfusion injury (IRI). However the effects of Ac2-26 on an HIRI model have not been reported to date. The purpose of the present study was to determine whether Ac2-26 pretreatment could protect hepatocytes against acute HIRI by inhibiting neutrophil infiltration through regulation of the high mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. To this end, a total of 72 adult C57BL/6 mice were randomly divided into sham operation (sham), ischemia-reperfusion (I/R), I/R + Ac2-26 and Ac2-26 groups. The HIRI model was established by occluding the branch of the hepatic pedicle to the left and median liver lobes with an atraumatic vascular clamp for 45 min, followed by reperfusion for 24 h. The expression of HMGB1, TLR4, NF-κB, IκBα and lymphocyte antigen 6 complex locus G6D (Ly6G) was detected using reverse transcription-quantitative PCR, western blotting and immunohistochemical staining; serum levels of HMGB1 were evaluated using an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the proportion of neutrophil. The results indicated that Ac2-26 preconditioning rescued hepatocyte dysfunctions induced by HIRI. In addition, HIRI was associated with a significant increase in HMGB1 expression and release, accompanied by increased expression of TLR4, which was significantly inhibited by Ac2-26. Furthermore, the expression of phosphorylated (p)-NF-κB and the ratio of p-NF-κB to NF-κB were markedly increased, while the expression of IκBα was decreased in the I/R group compared with those in the sham group; however, these effects were reversed by Ac2-26 administration. Additionally, Ac2-26 administration significantly inhibited neutrophil infiltration and resulted in low levels of neutrophils and Ly6G as well as reduced myeloperoxidase activity. Taken together, these results indicated that Ac2-26 pretreatment serves a protective role against HIRI by regulating the HMGB1/TLR4/NF-κB signaling pathway and inhibiting neutrophil infiltration.
... Its N-terminal peptide Ac2-26 was shown to bind the formyl peptide receptor (FPR) family which inhibits neutrophil adhesion, migration, and infiltration (81,143,144). In 2001, the group of Perretti has reported that Ac2-26 (1 mg/kg, i.v.) administrated at the onset of reperfusion or during 60 min, revealed significant cardioprotection associated with lower myeloperoxidase activity and IL-1β levels a rat IR model (79). More recently, Qin and coworkers have confirmed a significant reduction in inflammation (48 h post R) associated with decreased IS, fibrosis, and apoptosis (7-days post R) in a murine IR model (80). ...
Cardiovascular diseases (CVD) including acute myocardial infarction (AMI) rank first in worldwide mortality and according to the World Health Organization (WHO), they will stay at this rank until 2030. Prompt revascularization of the occluded artery to reperfuse the myocardium is the only recommended treatment (by angioplasty or thrombolysis) to decrease infarct size (IS). However, despite beneficial effects on ischemic lesions, reperfusion leads to ischemia-reperfusion (IR) injury related mainly to apoptosis. Improvement of revascularization techniques and patient care has decreased myocardial infarction (MI) mortality however heart failure (HF) morbidity is increasing, contributing to the cost-intense worldwide HF epidemic. Currently, there is no treatment for reperfusion injury despite promising results in animal models. There is now an obvious need to develop new cardioprotective strategies to decrease morbidity/mortality of CVD, which is increasing due to the aging of the population and the rising prevalence rates of diabetes and obesity. In this review, we will summarize the different therapeutic peptides developed or used focused on the treatment of myocardial IR injury (MIRI). Therapeutic peptides will be presented depending on their interacting mechanisms (apoptosis, necroptosis, and inflammation) reported as playing an important role in reperfusion injury following myocardial ischemia. The search and development of therapeutic peptides have become very active, with increasing numbers of candidates entering clinical trials. Their optimization and their potential application in the treatment of patients with AMI will be discussed.
... Adapted from D'Acquisto et al [48] Image created with Biorender.com benefits of ANXA1 and its synthetic peptide in atherosclerosis [64], stroke [65] and MI [66]. ...
Annexin‐A1 has a well‐defined anti‐inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid‐induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin‐A1 primarily signals, have also been implicated in these diseases. In contrast, treatment with recombinant annexin‐A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin‐A1 in heath and disease and discuss the potential of this protein as a biomarker and therapeutic target.
... FPR is an important protein that regulates inflammation and host defense [22]. The protection and anti-inflammation of AnxA1 in organ injury has been exhibited through FPR [7,36]. The protection of AnxA1 and peptide Ac2-26 was attenuated in Fpr -/mice [37]. ...
Background
Acute respiratory distress syndrome (ARDS) is characterized by severe local and systemic inflammation. Ac2-26, an Annexin A1 Peptide, can reduce the lung injury induced by reperfusion via the inhibition of inflammation. The present study aims to evaluate the effect and mechanism of Ac2-26 in ARDS.
Methods
Thirty-two rats were anaesthetized and randomized into four groups: sham (S), ARDS (A), ARDS/Ac2-26 (AA), and ARDS/Ac2-26/BOC-2 (AAB) groups. Rats in the S group received saline for intratracheal instillation, while rats in the other three groups received endotoxin for intratracheal instillation, in order to prepare the ARDS and inject the saline, Ac2-26, and Ac2-26 combined with BOC-2. After 24 h, the PaO2/FiO2 ratio was calculated. The lung tissue wet-to-dry weight ratio and the protein level in bronchoalveolar lavage fluid (BALF) were tested. Then, the cytokines in BALF and serum, and the inflammatory cells in BALF were investigated. Afterwards, the oxidative stress response and histological injury was evaluated. Subsequently, the epithelium was cultured and analyzed to estimate the effect of Ac2-26 on apoptosis.
Results
Compared to the S group, all indexes worsened in the A, AA, and AAB groups. Furthermore, compared to the S group, Ac2-26 significantly improved the lung injury and alveolar-capillary permeability, and inhibited the oxidative stress response. In addition, Ac2-26 reduced the local and systemic inflammation through the regulation of pro- and anti-inflammatory cytokines, and the decrease in inflammatory cells in BALF. Moreover, Ac2-26 inhibited the epithelium apoptosis induced by LPS through the modulation of apoptosis-regulated proteins. The protective effect of Ac2-26 on ARDS was partially reversed by the FPR inhibitor, BOC-2.
Conclusion
Ac2-26 reduced the lung injury induced by LPS, promoted alveolar-capillary permeability, ameliorated the local and systemic inflammation, and inhibited the oxidative stress response and apoptosis. The protection of Ac2-26 on ARDS was mainly dependent on the FPR pathway.
... The ability of different ligands to induce specific conformational changes in FPR2 and the associated downstream signalling pathways elicited likely accounts for the divergent effects mediated via FPR2 [140]. Of relevance here, FPR2 agonists are also cardioprotective in experimental models of myocardial infarction [141][142][143]. Interestingly, the endogenous FPR2 agonist AnxA1 was first discovered as a downstream effector of the inhibitory effects of glucocorticoids on phospholipase A2 activity [144]. ...
Rheumatoid arthritis is a chronic, systemic inflammatory disease that carries an increased risk of mortality due to cardiovascular disease. The link between inflammation and atherosclerotic disease is clear; however, recent evidence suggests that inflammation may also play a role in the development of nonischemic heart disease in rheumatoid arthritis (RA) patients. We consider here the link between inflammation and cardiovascular disease in the RA community with a focus on heart failure with preserved ejection fraction. The effect of current anti-inflammatory therapeutics, used to treat RA patients, on cardiovascular disease are discussed as well as whether targeting resolution of inflammation might offer an alternative strategy for tempering inflammation and subsequent inflammation-driven comorbidities in RA.
... ANXA1 and its N-terminal peptides are protective in several models of IRI [33]. In fact, infusion of recombinant ANXA1 decreases myocardial infarct size [120] and the treatment with Ac2-26 reduces both infarct size, myeloperoxidase (MPO) and IL-1β levels [121]. Intra-cerebroventricular infusion of Ac2-26 decreases stroke volume and cerebral edema in rats [122], by reducing leukocyte-endothelial interactions [123]. ...
G protein-coupled receptors (GPCRs) are the most important regulators of cardiac function and are commonly targeted for medical therapeutics. Formyl-Peptide Receptors (FPRs) are members of the GPCR superfamily and play an emerging role in cardiovascular pathologies. FPRs can modulate oxidative stress through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) production whose dysregulation has been observed in different cardiovascular diseases. Therefore, many studies are focused on identifying molecular mechanisms of the regulation of ROS production. FPR1, FPR2 and FPR3 belong to the FPRs family and their stimulation triggers phosphorylation of intracellular signaling molecules and nonsignaling proteins that are required for NADPH oxidase activation. Some FPR agonists trigger inflammatory processes, while other ligands activate proresolving or anti-inflammatory pathways, depending on the nature of the ligands. In general, bacterial and mitochondrial formylated peptides activate a proinflammatory cell response through FPR1, while Annexin A1 and Lipoxin A4 are anti-inflammatory FPR2 ligands. FPR2 can also trigger a proinflammatory pathway and the switch between FPR2-mediated pro- and anti-inflammatory cell responses depends on conformational changes of the receptor upon ligand binding. Here we describe the detrimental or beneficial effects of the main FPR agonists and their potential role as new therapeutic and diagnostic targets in the progression of cardiovascular diseases.
... AnxA1 may be able to contribute here through actions on Dectin-1 (145). However, it is advisable that we also harness the wealth of past evidence that AnxA1 or its derivatives are useful when used to target FPR2/ALX to limit MImediated pathology and acute kidney injury (97)(98)(99). These data would support an investigation into using these reagents to limit early graft failure or IRI under cover of immunosuppression. ...
Detrimental inflammatory responses after solid organ transplantation are initiated when immune cells sense pathogen-associated molecular patterns (PAMPs) and certain damage-associated molecular patterns (DAMPs) released or exposed during transplant-associated processes, such as ischemia/reperfusion injury (IRI), surgical trauma, and recipient conditioning. These inflammatory responses initiate and propagate anti-alloantigen (AlloAg) responses and targeting DAMPs and PAMPs, or the signaling cascades they activate, reduce alloimmunity, and contribute to improved outcomes after allogeneic solid organ transplantation in experimental studies. However, DAMPs have also been implicated in initiating essential anti-inflammatory and reparative functions of specific immune cells, particularly Treg and macrophages. Interestingly, DAMP signaling is also involved in local and systemic homeostasis. Herein, we describe the emerging literature defining how poor outcomes after transplantation may result, not from just an over-abundance of DAMP-driven inflammation, but instead an inadequate presence of a subset of DAMPs or related molecules needed to repair tissue successfully or re-establish tissue homeostasis. Adverse outcomes may also arise when these homeostatic or reparative signals become dysregulated or hijacked by alloreactive immune cells in transplant niches. A complete understanding of the critical pathways controlling tissue repair and homeostasis, and how alloimmune responses or transplant-related processes disrupt these will lead to new immunotherapeutics that can prevent or reverse the tissue pathology leading to lost grafts due to chronic rejection.
... The reason may be that (a) the inflammation caused by lung IR was inhibited after adding Ac2-26 33 ; (b) in the COPD model, 35 Lucas et al proposed that the reduction in endogenous ANX-A1 after Ac2-26 injection was associated with negative feedback regulation. (c) There are also studies 36 indicating that Ac2-26 may regulate the conversion of endogenous ANX-A1 between 37 and 34 kDa. In lung injury caused by distal intestinal ischaemia, 37 ANX-A1 expression at 37 kDa was decreased in the lung after Ac2-26 treatment, and ANX-A1 expression at 33 kDa was significantly increased, which was consistent with the results of this experiment and further research. ...
The main causes of lung injury after cardiopulmonary bypass (CPB) are systemic inflammatory response syndrome (SIRS) and pulmonary ischaemia‐reperfusion injury (IR‐I). SIRS and IR‐I are often initiated by a systemic inflammatory response. The present study investigated whether the annexin A1 (ANX‐A1) peptidomimetic Ac2‐26 by binding to formyl peptide receptors (FPRs) inhibit inflammatory cytokines and reduce lung injury after CPB. Male rats were randomized to the following five groups (n=6, each): sham, exposed to pulmonary ischaemic‐reperfusion (IR‐I), IR‐I plus Ac2‐26, IR‐I plus the FPR antagonist, BoC2 (N‐tert‐butyloxycarbonyl‐Phe‐Leu‐Phe‐Leu‐Phe), and IR‐I plus Ac2‐26 and BoC2. Treatment with Ac2‐26 improved the oxygenation index, an effect blocked by BoC2. Histopathological analysis of the lung tissue revealed that the degree of lung injury was significantly less (p<0.05) in the Ac2‐26‐treated rats compared to the other experimental groups exposed to IR‐I. Ac2‐26 treatment reduced the levels of the inflammatory cytokines TNF‐α, IL‐1β, ICAM‐1, and NF‐κB‐p65 (p<0.05) compared to the vehicle‐treated group exposed to IR‐I. In conclusion, the annexin A1 (ANX‐A1) peptidomimetic Ac2‐26 by binding to formyl peptide receptors inhibit inflammatory cytokines and reduce ischaemic‐reperfusion lung injury after cardiopulmonary bypass.
... Annexin A1 (AnxA1) is a 37-kDa phospholipid-binding, calcium-dependent pro-resolving protein that is present intracellularly 1 and extracellularly. 2 AnxA1 inhibits leukocyte recruitment into inflammation sites and reduces the expression of inflammatory cytokines after ischemia-reperfusion (I-R) injury, 3 including renal 4 and cardiovascular tissues. 5 After renal I-R injury, AnxA1 is upregulated in epithelial cells, ameliorating acute tubular necrosis. ...
Objectives: The study sought to identify the role of Annexin A1 as a congestion marker in acute heart failure, and to identify its putative role in predicting clinical outcomes.
Background: Annexin A1 (AnxA1) is a protein that inhibits inflammation following ischemia-reperfusion (I-R) injury in cardiorenal tissues. Since acute heart failure (AHF) is a state of tissue hypoperfusion, we hypothesized that plasma AnxA1 levels are altered in AHF.
Methods: In The Renal Optimization Strategies Evaluation (ROSE) trial, patients hospitalized for AHF with kidney injury were randomized to receive dopamine, nesiritide, or placebo for 72 hours in addition to diuresis. In a sub-analysis, plasma AnxA1 levels were measured at baseline and at 72 hours in 275 patients. Participants were divided into three tertiles based on their baseline AnxA1 levels.
Results: The prevalence of peripheral edema 2+ increased with increasing AnxA1 levels (p < 0.007). Cystatin C, blood urea nitrogen, and kidney injury molecule-1 plasma levels were higher among participants in tertile 3 vs tertiles 1 or 2 (p < 0.05). Patients with a congestion score of 4 had a mean baseline AnxA1 level 8.63 units higher than those with a congestion score of 0 (p= 0.03). Patients in tertiles 2 and 3 were twice as likely to experience creatinine elevation as patients in tertile 1 (p= 0.03). Patients in tertiles 2 and 3 were at a higher risk of 60-day all-cause mortality or heart failure hospitalization and 180-day all-cause mortality (p <0.05).
Conclusions: Among patients hospitalized for AHF with impaired kidney function, elevated AnxA1 levels are associated with worse congestion, higher risk for further creatinine elevation, and higher rates of 60-day morbidity or all-cause mortality & 180-day all-cause mortality.
Clinical Trial Registration: clinicaltrials.gov Identifier: NCT01132846
... Additional studies of cardiac muscle injury further suggest a role for annexin proteins in modulating the repair response. Administration of recombinant annexin A1 or the N-terminal annexin A1 peptide (AC2-26) elicited a cardioprotective response in a rat model of myocardial ischemia-reperfusion-induced injury (51). Meng et al. demonstrated that downregulation of annexin A3 resulted in cardioprotection, decreasing rat myocardial infarct size through activation of AKT signaling (52). ...
Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.
... Systemically administered ANXA1sp was also associated with anti-inflammatory effects in the heart (reduced levels of IL-6 and TNFα) following HS-EPR. Consistent with previous reports in experimental models of regional myocardial I/RI (La et al., 2001;Qin et al., 2015), these results support the broader therapeutic roles attributed to ANXA1 and its peptide mimetics in reducing systemic inflammation and conferring organ protection from a variety of insults involving IR/I. ...
Neuroinflammation initiated by damage-associated molecular patterns, including high mobility group box 1 protein (HMGB1), has been implicated in adverse neurological outcomes following lethal hemorrhagic shock and polytrauma. Emergency preservation and resuscitation (EPR) is a novel method of resuscitation for victims of exsanguinating cardiac arrest, shown in preclinical studies to improve survival with acceptable neurological recovery. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has emerged as a key regulator of metabolic and energy stress response pathways in the brain and a pharmacological target to induce a neuronal pro-survival phenotype. This study aims to examine whether systemic administration of an Annexin-A1 bioactive peptide (ANXA1sp) could resolve neuroinflammation and induce sirtuin-3 regulated cytoprotective pathways in a novel rat model of exsanguinating cardiac arrest and EPR. Adult male rats underwent hemorrhagic shock and ventricular fibrillation, induction of profound hypothermia, followed by resuscitation and rewarming using cardiopulmonary bypass (EPR). Animals randomly received ANXA1sp (3 mg/kg, in divided doses) or vehicle. Neuroinflammation (HMGB1, TNFα, IL-6, and IL-10 levels), cerebral cell death (TUNEL, caspase-3, pro and antiapoptotic protein levels), and neurologic scores were assessed to evaluate the inflammation resolving effects of ANXA1sp following EPR. Furthermore, western blot analysis and immunohistochemistry were used to interrogate the mechanisms involved. Compared to vehicle controls, ANXA1sp effectively reduced expression of cerebral HMGB1, IL-6, and TNFα and increased IL-10 expression, which were associated with improved neurological scores. ANXA1sp reversed EPR-induced increases in expression of proapoptotic protein Bax and reduction in antiapoptotic protein Bcl-2, with a corresponding decrease in cerebral levels of cleaved caspase-3. Furthermore, ANXA1sp induced autophagic flux (increased LC3II and reduced p62 expression) in the brain. Mechanistically, these findings were accompanied by upregulation of the mitochondrial protein deacetylase Sirtuin-3, and its downstream targets FOXO3a and MnSOD in ANXA1sp-treated animals. Our data provide new evidence that engaging pro-resolving pharmacological strategies such as Annexin-A1 biomimetic peptides can effectively attenuate neuroinflammation and enhance the neuroprotective effects of EPR after exsanguinating cardiac arrest.
... D'Amico et al. demonstrated a decrease in myocardial infarct size upon infusion of recombinant ANXA1 (57). Treatment with Ac2-26 decreased infarct size and reduced MPO and IL-1β content in infarcted hearts (58). The peptide Ac2-26 preserved cardiomyocyte contractility by activating PKC, p38, and K ATP channels (59). ...
Annexin-A1 (ANXA1) was first discovered in the early 1980's as a protein, which mediates (some of the) anti-inflammatory effects of glucocorticoids. Subsequently, the role of ANXA1 in inflammation has been extensively studied. The biology of ANXA1 is complex and it has many different roles in both health and disease. Its effects as a potent endogenous anti-inflammatory mediator are well-described in both acute and chronic inflammation and its role in activating the pro-resolution phase receptor, FPR2, has been described and is now being exploited for therapeutic benefit. In the present mini review, we will endeavor to give an overview of ANXA1 biology in relation to inflammation and functions that mediate pro-resolution that are independent of glucocorticoid induction. We will focus on the role of ANXA1 in diseases with a large inflammatory component focusing on diabetes and microvascular disease. Finally, we will explore the possibility of exploiting ANXA1 as a novel therapeutic target in diabetes and the treatment of microvascular disease.
... Anxa1 overexpression inhibits ET-1-induced inflammatory cytokine secretion and PASMC proliferation (IL-6; IL-1β; TNFα); however, Anxa1 is stimulated by IL-6, suggesting feedback between IL-6 and Anxa1 [44]. Upregulation of Anxa1 in vivo, improves atherosclerosis (decreased lesion size, inflammation, improved plaque stability) [45][46][47], myocardial infarction (decreased infarct size, inflammation and increased survival) [48][49][50][51] and stroke (decreased infarct size, inflammation) [52][53][54][55]. In addition to increased inflammation, Anxa1-knockout mice exhibited significant increases of fibrosis eight weeks post-myocardial infarction [56]. ...
Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (−0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (−3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (−2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (−2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation.
... Since its discovery, nearly four decades ago, ANXA1 has been shown to be capable of modulating the immune system to affect a number of anti-and proinflammatory events. including inhibition of leukocyte activation and migration [19], apoptosis and tissue growth [20,21] and ischemia-reperfusion injury [22]. ANXA1 is also involved in an inflammation-pro-resolving process, which is associated with a wide range of cellular and molecular steps of the inflammatory response and is deeply involved in the endogenous mechanisms that are activated to bring about the resolution of inflammation. ...
Targeting inflammation in cancer has shown promise to improve and complement current therapies. The tumor microenvironment plays an important role in cancer growth and metastasis and -tumor associated macrophages possess pro-tumoral and pro-metastatic properties. Annexin A1 (ANXA1) is an immune-modulating protein with diverse functions in the immune system and in cancer. In breast cancer, high ANXA1 expression leads to poor prognosis and increased metastasis. Here, we will review ANXA1 as a modulator of inflammation, and discuss its importance in breast cancer and highlight its new role in alternative macrophage activation in the tumor microenvironment. This review may provide an updated understanding into the various roles of ANXA1 which may enable future therapeutic developments for the treatment of breast cancer.
... Annexin A1 (ANXA1), which originally believed to inhibit phospholipase activity (22), has been reported to be able to modulate various cellular functions in multiple cell types (23,24). Protective roles of ANXA1 have been observed (25,26). A recent study demonstrated that ANXA1 is able to accelerate the healing of gastric ulcers that caused by indomethacin (27). ...
The incidence of asthma is increasing worldwide. Bronchial epithelium injury is common in asthma. The regulatory role of Annexin A1 (ANXA1) in bronchial epithelium injury is currently not well understood. The aim of the present study was to evaluate the role of ANXA1 on bronchial epithelium injury. The cell viability and levels of apoptosis were respectively tested by Cell Counting Kit‑8 and flow cytometry. Reactive oxygen species (ROS) content and the activity of oxidative indicators were assessed by commercial kits. Enzyme linked immunosorbent assay was performed to detect the activity of active caspase‑3. Reverse transcription‑quantitative polymerase chain reaction and western blot assays were used to determine the expression levels of the target factors. The results demonstrated that ANXA1 improved the viability of benzo[a]pyrene (Bap)‑treated bronchial epithelial cells. The Bap‑induced oxidative stress was mitigated by the reduction in ROS generation, and the regulation of the activity of superoxide dismutase, glutathione peroxidases, malondialdehyde and lactic dehydrogenase. In addition, apoptosis was decreased by ANXA1 via the reduction of the expression of B‑cell lymphoma 2 (Bcl‑2), and the increase in the expression of Bcl‑2‑associated X protein and cyclin D1. Furthermore, the expression of phosphatase and tensin homolog (PTEN) and focal adhesion kinase (FAK) was rescued and the phosphorylation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) was depressed by ANXA1, when compared with the Bap group. SF1670 treatment reversed the anti‑apoptotic effect of ANXA1. In conclusion, the results highlighted the protective effects of ANXA1 on bronchial epithelium injury, which most likely occurred via the PTEN/FAK/PI3K/Akt signaling pathway. Thus, the present study contributes to a potential therapeutic strategy for asthma patients.
... It has been reported that administration of exogenous human recombinant ANX-A1, and its N-terminal derived peptide Ac2-26, limits infarct size after 25 min ischaemia and 2 h reperfusion 11 . In this study, we demonstrated that both the protein and gene expression of endogenous ANX-A1 increase after a myocardial ischaemic insult, and that deficiency in ANX-A1 resulted in exaggerated inflammatory responses. ...
Endogenous anti-inflammatory annexin-A1 (ANX-A1) plays an important role in preserving left ventricular (LV) viability and function after ischaemic insults in vitro, but its long-term cardioprotective actions in vivo are largely unknown. We tested the hypothesis that ANX-A1-deficiency exaggerates inflammation, haematopoietic stem progenitor cell (HSPC) activity and LV remodelling in response to myocardial ischaemia in vivo. Adult ANX-A1−/− mice subjected to coronary artery occlusion exhibited increased infarct size and LV macrophage content after 24–48 h reperfusion compared with wildtype (WT) counterparts. In addition, ANX-A1−/− mice exhibited greater expansion of HSPCs and altered pattern of HSPC mobilisation 8 days post-myocardial infarction, with increased circulating neutrophils and platelets, consistent with increased cardiac inflammation as a result of increased myeloid invading injured myocardium in response to MI. Furthermore, ANX-A1−/− mice exhibited significantly increased expression of LV pro-inflammatory and pro-fibrotic genes and collagen deposition after MI compared to WT counterparts. ANX-A1-deficiency increased cardiac necrosis, inflammation, hypertrophy and fibrosis following MI, accompanied by exaggerated HSPC activity and impaired macrophage phenotype. These findings suggest that endogenous ANX-A1 regulates mobilisation and differentiation of HSPCs. Limiting excessive monocyte/neutrophil production may limit LV damage in vivo. Our findings support further development of novel ANX-A1-based therapies to improve cardiac outcomes after MI.
... FPRs thus represent a novel therapeutic target in MI, where inflammation is a major contributing mechanism 3,[5][6][7][8] . Endogenous non-selective FPR agonists annexin-A1 and its N-terminal-peptide Ac2-26 confer significant cardioprotection early after myocardial I-R injury 7,[9][10][11][12] . Three FPR subtypes are expressed in humans; FPR1 and FPR2 (previously known as aspirin-triggered lipoxin receptor/FPR-like receptor-1 (FPRL1)) 13 are widely distributed. ...
Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.
... 18 There is evidence of ANX1 protection from ischemia and reperfusion in the mesentery, myocardium and brain. [19][20] In a previous study, we demonstrated that ANX1 contributes to an improvement EF particularly through the protective effect of a single dose of 100 μg kg − 1 intravenously, before CN injury in rats. 21 The present study extends knowledge in the field of ED associated with CN injury by further exploring the cellular mechanisms in the penis that are associated with CN injury and examining the consequence of a plausible treatment that targets these mechanisms. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function (EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and three groups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injection of either ANX1 (50 or 100 μg kg− 1) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury with histomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase in intracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservation of the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (real P-value vs injured control). ANX1 treatment prevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelial growth factor and decreased tumor necrosis factor-α in the lumen of the blood vessels of the organ. ANX1 proved effective in preserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery, enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of this compound merits application in penile rehabilitation studies following radical prostatectomy.
... 18 There is evidence of ANX1 protection from ischemia and reperfusion in the mesentery, myocardium and brain. [19][20] In a previous study, we demonstrated that ANX1 contributes to an improvement EF particularly through the protective effect of a single dose of 100 μg kg − 1 intravenously, before CN injury in rats. 21 The present study extends knowledge in the field of ED associated with CN injury by further exploring the cellular mechanisms in the penis that are associated with CN injury and examining the consequence of a plausible treatment that targets these mechanisms. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function(EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and threegroups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injectionof either ANX1 (50 or 100μgkg−1) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury withhistomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase inintracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservationof the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (realP-value vs injured control). ANX1 treatmentprevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelialgrowth factor and decreased tumor necrosis factor-αin the lumen of the blood vessels of the organ. ANX1 proved effective inpreserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery,enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of thiscompound merits application in penile rehabilitation studies following radical prostatectomy.
... 18 There is evidence of ANX1 protection from ischemia and reperfusion in the mesentery, myocardium and brain. [19][20] In a previous study, we demonstrated that ANX1 contributes to an improvement EF particularly through the protective effect of a single dose of 100 μg kg − 1 intravenously, before CN injury in rats. 21 The present study extends knowledge in the field of ED associated with CN injury by further exploring the cellular mechanisms in the penis that are associated with CN injury and examining the consequence of a plausible treatment that targets these mechanisms. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function(EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and three groups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injection
of either ANX1 (50 or 100 μg kg − 1
) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury with
histomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase in intracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservation of the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (real P-value vs injured control). ANX1 treatment prevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelial growth factor and decreased tumor necrosis factor-α in the lumen of the blood vessels of the organ. ANX1 proved effective in preserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery,
enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of this compound merits application in penile rehabilitation studies following radical prostatectomy
... 363 The N-terminal fragment of annexin-1, Anx (2-26) is an endogenous ligand agonist of FPR1 (Fig. 25). 364 Anx (2-26) promoted chemotactic migration and OB differentiation of MSCs via FPR1 in the presence of osteogenic medium. 361,362 ...
Loss of bone occurs in the age-related skeletal disorder, osteoporosis, leading to bone fragility and increased incidence of fractures, which are associated with enormous costs and substantial morbidity and mortality. Recent data indicate that osteoporotic fractures are more common than other diseases, which usually attract public attention (e.g., heart attack and breast cancer). The prevention and treatment of this skeletal disorder are therefore of paramount importance. Majority of osteoporosis medications restore skeletal balance by reducing osteoclastic activity, thereby reducing bone resorption. These agents, however, do not regenerate damaged bone tissue, leaving limited options for patients once bone loss has occurred. Recently, attention has turned to bone-anabolic agents. Such agents have the ability to increase bone mass and strength, potentially reversing structural damage. To date, only one bone-anabolic drug is available in the market. The discovery of more novel, cost-effective bone anabolic agents is therefore a priority to treat those suffering from this disabling condition. Short peptides offer an important alternative for the development of novel bone-anabolic agents given their high target binding specificity, which translates into potent activity with limited side effects. This review summarizes attempts in the identification of bone-anabolic peptides, and their development for promoting bone growth.
... Ac2-26 exerts antiinflammatory actions in vivo and was shown to be protective in several disease models, including myocardial ischemiareperfusion injury, allergic inflammation, and endotoxininduced cerebral inflammation, and the zymosan-induced peritonitic model. 48,66,67 Upon administration of collagen IV-Ac2-26 nanoparticles in an LDLr À/À mouse, targeted nanoparticles increased collagen I production and fibrous cap thickness, while decreasing metalloproteinase activity in lesions of the aortic root compared to free Ac2-26 or therapeutic nanoparticles with a scrambled collage IV peptide. Moreover, lesion area, necrotic core, and oxidative stress decreased in plaques within the brachiocephalic artery. ...
Peptides in atherosclerosis nanomedicine provide structural, targeting, and therapeutic functionality and can assist in overcoming delivery barriers of traditional pharmaceuticals. Moreover, their inherent biocompatibility and biodegradability make them especially attractive as materials intended for usein vivo In this review, an overview of nanoparticle-associated targeting and therapeutic peptides for atherosclerosis is provided, including peptides designed for cellular targets such as endothelial cells, monocytes, and macrophages as well as for plaque components such as collagen and fibrin. An emphasis is placed on recent advances in multimodal strategies and a discussion on current challenges and barriers for clinical applicability is presented.
Although medical treatment is sucessful in most cases in patients with inflammatory bowel diseases (IBD), a percentage of patients require surgical resection of diseased bowel segments at least once in their lifetime. Healing success of the intestinal anastomosis is at high risk, especially in presence of acute inflammation. Failure of anastomotic healing is a life-threatening complication and causes high socioeconomic costs. Common anti-inflammatory medications can have detrimental effects on wound healing. Thus, targeted perioperative therapeutics supporting anastomotic healing during colitis are an urgent medical need. Here, we develop a novel basal membrane targeted controlled release, pectin-coated polymeric nanoparticle (NP) encapsulating a highly potent inflammation resolving mediator, the peptide Ac2-26. These NPs can undergo gastric passage and facilitate localized release of the therapeutic peptide in the colon via degradation of their pectin-chitosan coating by microbial pectinases, which subsequently exposes a collagen IV targeted NP surface, allowing for further binding and retention of the NPs at the intestinal wound. To test these NPs, we used a murine surgical model combining the formation of an intestinal anastomosis with the induction of a preoperative colitis by dextran sodium sulfate. In this model, perioperative administration of pectin-chitosan coated NPs containing Ac2-26 (P-C-Col IV-Ac2-26-NP) led to the reduction of colitis activity in the postoperative phase. Macroscopic wound closure was improved by P-C-Col IV-Ac2-26-NP treatment as evaluated by endoscopy and intraabdominal adhesion scoring. Microscopic analysis of the healing process showed an improved semiquantitative healing score in the treatment group. In this proof-of-concept study we demonstrate that novel P-C-Col IV-Ac2-26-NP could be a promising and clinically feasible perioperative treatment strategy for IBD patients.
Significance:
Chronic inflammation has emerged as a major underlying cause of many prevalent conditions in the Western world, including cardiovascular diseases. While targeting inflammation has emerged as a promising avenue by which to treat cardiovascular disease, it is also associated with increased risk of infection.
Recent advances:
Resolution has been identified as an active process, mediated by unique immunoresolving mediators and mechanisms, that terminates acute inflammation and promotes tissue repair. Recent work has determined that failures of resolution contribute to chronic inflammation and disease progression.
Critical issues:
Pro-resolving therapies offer the promise of limiting chronic inflammation without impairing host defense. Therefore, it is imperative to better understand the mechanisms underlying resolution in order to identify therapeutic targets.
Future directions:
Here, we review what is known about the role of inflammation resolution in arterial hypertension, atherosclerosis, myocardial infarction, and ischemic heart disease.
Using a global formyl-peptide receptor (Fpr)2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (abbreviated to KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome and impaired bacteria clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers, but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 up-regulation was absent in myeloid cells and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist Annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice, but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages towards an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2 competent septic mice.
Formyl peptide receptor type 2 (FPR2) regulates the initiation and resolution phases of the inflammatory response. In the setting of heart injury and disease, dysregulated inflammation can potentiate maladaptive healing and pathological remodeling of the heart leading to cardiac dysfunction and failure. The potential to regulate and resolve adverse inflammation is postulated to improve outcome in the setting of heart disease. This review covers emerging concepts on the role of FPR2 in heart disease and strategies to activate pro-resolution processes to limit disease progression. We summarize key preclinical studies that support use of FPR2 agonists in heart disease. Finally, we briefly discuss the status of FPR2 agonists under evaluation in the clinic.
Myocardial infarction (MI) initiates an intense inflammatory response that induces neutrophil infiltration into the infarct region. Neutrophils commence the pro-inflammatory response that includes upregulation of cytokines and chemokines (e.g., interleukin-1 beta) and degranulation of pre-formed proteases (e.g., matrix metalloproteinases -8 and -9) that degrade existing extracellular matrix to clear necrotic tissue. An increase or complete depletion of neutrophils both paradoxically impair MI resolution, indicating a complex role of neutrophils in cardiac wound healing. Following pro-inflammation, the neutrophil shifts to a reparative phenotype that promotes inflammation resolution and aids in scar formation. Across the shifts in phenotype, the neutrophil communicates with other cells to coordinate repair and scar formation. This review summarizes our current understanding of neutrophil crosstalk with cardiomyocytes and macrophages during MI wound healing.
Introduction
Cardiopulmonary bypass (CPB) –induced lung ischemia-reperfusion (I/R) injury remains a large challenge in cardiac surgery; up to date, no effective treatment has been found. Annexin A1 (AnxA1) has an anti-inflammatory effect, and it has been proven to have a protective effect on CPB-induced lung injury. However, the specific mechanism of AnxA1 in CPB-induced lung injury is not well studied. Therefore, we established a CPB-induced lung injury model to explore the relevant mechanism of AnxA1 and try to find an effective treatment for lung protection.
Methods
Male rats were randomized into five groups ( n = 6, each): sham (S group), I/R exposure (I/R group), I/R + dimethyl sulfoxide (D group), I/R + Ac2-26 (AnxA1 peptide) (A group), and I/R + LY294002 (a PI3K specific inhibitor) (AL group). Arterial blood gas analysis and calculation of the oxygenation index, and respiratory index were performed. The morphological changes in lung tissues were observed under light and electron microscopes. TNF-α and IL-6 and total protein in lung bronchoalveolar lavage fluid were detected via enzyme-linked immunosorbent assay. The expressions of PI3K, Akt, and NF-κB (p65) as well as p-PI3K, p-Akt, p-NF-κB (p65), and AnxA1 were detected via western blotting.
Results
Compared with the I/R group, the A group showed the following: lower lung pathological damage score; decreased expression of IL-6 and total protein in the bronchoalveolar lavage fluid, and TNF-α in the lung; increased lung oxygenation index; and improved lung function. These imply the protective role of Ac2-26, and show that LY294002 inhibited the ameliorative preconditioning effect of Ac2-26.
Conclusion
This finding suggested that the AnxA1 peptide Ac2-26 decreased the inflammation reaction and CPB-induced lung injury in rats, the lung protective effects of AnxA1may be correlated with the activation of PI3K/Akt signaling pathway.
This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.
The Coronavirus Diseases-2019 (COVID-19) pandemic leads many researchers around the world to study the SARS-CoV-s2 infection and pathology to find a treatment for it. This generates a massive production of papers including pre-clinical, clinical and revisions but till now no specific treatment were identified. Meanwhile, like other coronavirus infections, COVID-19 leads to the cytokine storm syndrome resulting in hyperinflammation, exacerbated immune response and multiple organ dysfunctions indicating that drugs that modulate this response, as glucocorticoids could be a treatment option. However glucocorticoids have several side effects or usage limitations. In this sense a drug with anti-inflammatory effects and capable to reduce inflammation but with less after-effects could be a powerful tool to combat COVID-19. Thus the Ac2-26 Mimetic Peptide of Annexin A1 emerges as a possible therapy. The peptide has many anti-inflammatory effects described including the reduction of interleukin (IL)-6, one of the main mediators of cytokine storm syndrome. Therefore the hypothesis to use the Ac2-26 peptide to treat severe COVID-19 will be highlighted in this paper.
BACKGROUND:
Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration.
OBJECTIVES:
This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI.
METHODS:
AnxA1 knockout (AnxA1-/-) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1.
RESULTS:
AnxA1-/- mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1-/- mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx3cr1creERT2Vegfflox/flox or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus.
CONCLUSIONS:
AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair.
This chapter presents future nanotechnology approaches for the treatment of cardiovascular diseases (CVD) such as atherosclerosis and heart attack, responsible for about half of all CVD-related deaths. Nanotechnology-based approaches offer a route into providing stable formulations to deliver such therapeutic agents and bypass their detrimental systemic side effects. The chapter discusses a cardio-specific subspecialty for nanomedicine, that is, nanocardiology. In the field of invasive cardiology procedures, nanocardiology can help to better classify high-risk atherosclerotic lesions to present a more differentiated risk-benefit analysis. Perfluorocarbon nanoparticles are lipid-encapsulated nanoemulsions with per-fluorinated chemicals in their core. They show promising theranostic properties and great potential for characterizing high-risk atherosclerotic plaques. Furthermore, anionic nanoparticles (NPs) could lower the cellular uptake of highly oxidized low-density lipoprotein molecules. Nanoparticles capable of responding to physical changes present an exciting class of stimulus-responsive treatments for diseases such as atherosclerosis.
Increasing evidence suggests that circulating angiogenic cells (CACs) promote repair of ischemic tissues. Activation of formyl peptide receptor 2 (Fpr2) has been reported to stimulate repair of ischemic heart. This study was conducted to investigate the role of Fpr2 on CAC mobilization and cardiac protection in myocardial infarction (MI). WKYMVm, a strong agonist for Fpr2, was administered in a murine model of acute MI, and mobilization of CACs including endothelial progenitor cells (CD34(+) Flk1(+) or Sca1(+) Flk1(+) cells) in peripheral blood was monitored. CAC mobilization by daily injection of WKYMVm for the first 4 days after MI was as efficient as G-CSF and provided myocardial protection from apoptosis with increased vascular density and preservation of cardiac function. Bone marrow (BM) from GFP mice showed that BM-derived cells homed to ischemic heart after WKYMVm treatment and contributed to tissue protection. BM from Fpr2 knockout mice showed that Fpr2 in BM cells is critical in mediation of WKYMVm-stimulated myocardial protection and neovascularization after MI. These results suggest that activation of Fpr2 in BM after WKYMVm treatment provides cardiac protection through mobilization of CACs after MI, which may lead to the development of a new clinical protocol for treating patients with ischemic heart conditions. This article is protected by copyright. All rights reserved.
Inflammation is essential to protect the host from exogenous and endogenous dangers that ultimately lead to tissue injury. The consequent tissue repair is intimately associated with the fate of the inflammatory response. Restoration of tissue homeostasis is achieved through a balance between pro-inflammatory and anti-inflammatory/pro-resolving mediators. In chronic inflammatory diseases such balance is compromised resulting in persistent inflammation and impaired healing. During the last two decades the glucocorticoid-regulated protein Annexin A1 (AnxA1) has emerged as a potent pro-resolving mediator acting on several facets of the innate immune system. Here, we review the therapeutic effects of AnxA1 on tissue healing and repairing together with the molecular targets responsible for these complex biological properties.
Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintainance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis.
Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial
injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO−) and nitric oxide (NO⋅), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed
by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 μmol/kg S-nitrosoglutathione (GSNO) (an NO⋅ donor), or 3 μmol/kg Angeli’s salt (AS) (a source of NO−) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved
cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase
activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but,
rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine
kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron
oxidant that converts NO− to NO⋅, completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO.
These results demonstrate that, although NO⋅ is protective, NO− increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute
to postischemic myocardial injury.
Lipocortins are structurally related, glucocorticoid-inducible proteins that inhibit phospholipase A2 (PLA2), thereby reducing the liberation of arachidonic acid from phospholipids and so limiting the synthesis of eicosanoid inflammatory mediators. This study is the first demonstration of one lipocortin, lipocortin 1 (Lc 1; 37 kDa), in human lung lavage supernatants. In lavage fluid from healthy volunteers, a higher percentage (greater than 70%) of the detected Lc 1 was in its native form, compared to that from patients with abnormal lungs. In patients' lavage fluids, Lc 1 was more likely to be partially degraded (34 kDa). In abnormal bronchoalveolar lavage fluid (BALF), the more polymorphonuclear neutrophils (PMN)/lavage, the lower the proportion of Lc 1 in the native (37 kDa) form (n = 7 pairs, rs = -0.8214, p less than 0.05). Furthermore, when BALF cells were cultured and the harvested conditioned media incubated with pure human recombinant Lc 1, degradation of the 37 kDa form increased with the percentage of PMN (n = 10 pairs, s = -0.7200 after 1 hr; n = 6 pairs, rs = -0.9241 after 6 hr). These results suggest that factors released from the PMN are responsible for Lc 1 degradation in man. When recombinant human Lc 1 was incubated with human neutrophil elastase, the enzyme degraded Lc 1 in a dose-dependent way, suggesting that neutrophil elastase may be one such factor. Since PMNs are ubiquitous at sites of inflammation, it is possible that Lc 1 degradation is a permissive mechanism, which ensures that sufficient inflammation occurs to destroy the provocative stimulus. However, it is equally possible that, in some circumstances, the mechanism may be pathological and that the inactivation of Lc 1 leads to chronic, uncontrolled inflammation.
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FIGURE 3
FIGURE 4.
FIGURE 4.
alpha-Melanocyte stimulating hormone has been shown to prevent endotoxin shock. A heptapeptide analog (HP-228) has recently been synthesized and shown to be an even more potent protective agent. Because the hypotensive and toxic actions of lipopolysaccharide (LPS) appear to involve the induction of type II nitric oxide synthase (iNOS), we have examined the actions of HP-228 on nitric oxide production using an endotoxemia model in conscious rats given E. coli LPS (5 mg/kg i.v.) and monitored for 6 h. A group of rats received HP-228 (30 micrograms/kg) 30 min before LPS. Using nitro L-arginine methyl ester-sensitive cGMP production as an estimate of nitric oxide synthase activity in aortic segments, ex vivo, we determined that LPS increases iNOS activity and that HP-228 pretreatment markedly reduces this response. Additionally, the rate of conversion of 3[H]-arginine to 3[H]-citrulline was significantly reduced in lung homogenates from HP-228-treated rats. HP-228 did not alter the activity of the constitutive nitric oxide synthase in aortic rings or in cerebella. In isolated rat aortic smooth muscle cells, LPS or interleukin-1 beta caused prominent rises in nitric oxide generated by iNOS. HP-228 did not antagonize the effect of these inducing agents. However, in these cells, plasma obtained from rats 1 h after administration of HP-228 prevented the induction of iNOS by both LPS and interleukin-1 beta. In conclusion, HP-228 prevents the in vivo induction of nitric oxide synthase by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.
Reperfusion of the ischemic myocardium results in the generation of oxygen-derived free radicals, NO, and presumably peroxynitrite. These, in turn, may cause strand breaks in DNA, which activate the nuclear enzyme poly(ADP ribose) synthetase (PARS). This results in a rapid depletion of intracellular NAD and ATP. When this reaction is excessive, there is ultimately cell death. Here we demonstrate that 3-aminobenzamide (and several other, chemically distinct, inhibitors of PARS activity) reduces the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle of the rabbit. Inhibition of PARS activity also attenuates the myocardial dysfunction caused by global ischemia and reperfusion in the isolated, perfused heart of the rabbit. In skeletal muscle, inhibition of the activity of neuronal NO synthase reduces infarct size, indicating that the formation of NO contributes to the activation of PARS there. There is no significant neuronal NO synthase activity in the heart, and hence NO synthase inhibitors did not reduce myocardial infarct size. Thus, activation of PARS contributes to the cell death caused by ischemia-reperfusion, and PARS inhibitors may constitute a novel therapy for ischemia-reperfusion injury.
A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.
Splanchnic artery occlusion and reperfusion (SAO/R) shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (60-min reperfusion). Following this reperfusion period, rats developed a fall in mean arterial blood pressure, associated with a significant increase in tissue myeloperoxidase (MPO) activity in the intestine and a marked histologic injury to the distal ileum. Treatment of rats with a lipocortin-1 (LC1)-derived N-terminal peptide, peptide Ac(2-26), dose-dependently (0.125-0.5 mg/kg s.c.) reduced the progressive fall in blood pressure and prevented the infiltration of neutrophils into the reperfused intestine (reduced MPO activity). The LC1 peptide also reduced the degree of ischemia/reperfusion injury in the bowel as evaluated by histologic examination. The glucocorticoid dexamethasone (0.1 mg/kg s.c., -1 h) also produced a marked improvement in SAO/R shock (i.e., maintained mean arterial blood pressure and reduced tissue MPO activity), and this was reversed by pretreatment with two different antisera raised against the LC1 pharmacophore. Peptide Ac(2-26) (0.5 mg/kg s.c., -30 min) reduced (>60%) the extent of IL-1beta-induced cell emigration and significantly attenuated (approximately 45%) the number of adherent leukocytes in the rat mesenteric vascular bed, as assessed by video microscopy. These results suggest that LC1 inhibits neutrophil migration and accumulation into reperfused tissues, thereby ameliorating the outcome of SAO/R shock.
In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-micrometers of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2-26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (>/=50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg/kg) or recombinant human LC1 (0.7-2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 +/- 0.75 min and 2.36 +/- 0.31 min, respectively (n = 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.
Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.
Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.
We assessed here the effect of the glucocorticoid-regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25-min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with approximately 50% inhibition at the highest dose tested of 50 microg per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose-dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor a, and macrophage inflammatory protein-1a. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 microg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte-dependent myocardial damage associated with an ischemia-reperfusion procedure.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We determined the binding affinities of the MSH analogues MSH-B, HP-228 and 153N-6 and of the enkephalin analogue GHRP-6 on a single eukaryotic cell line transiently expressing the human MC1, MC3, MC4 and MC5 receptors. Moreover, we tested the binding and cAMP response of MSH-B in comparison with α-MSH on murine B16 melanoma cells. Our results indicate that MSH-B has a potency similar to that of α-MSH and that these two peptides induce similar cAMP responses in murine B16 melanoma cells. HP-228 has its highest affinity for the MC1 receptor. For the other receptors, it has slightly higher affinity for the MC5 receptor than for the MC3 and MC4 receptors. 153N-6 was found to be selective for the MC1 receptor. GHRP-6 was found to bind to the MC1 and the MC5 receptors despite its low structural homology with α-MSH. [D-Lys3]GHRP-6 bound to all the four MC receptors with similar affinities. The structurally related Met-enkephalin and the functionally related GHRH, as well as LHRH and somatostatin-14 did not bind to these MC receptors. The low affinity of the GH-releasing/enkephalin peptides may indicate that they do not interact with the MC receptors at pharmacologically relevant concentrations.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested.
Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15×106 cells per mouse).
Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.).
Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide.
Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle- and vinblastine-treated mice, respectively.
Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2-26 (200 μg s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle- and peptide Ac2-26-treated mice, respectively (P<0.05).
Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05).
In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.
British Journal of Pharmacology (1997) 120, 1075–1082; doi:10.1038/sj.bjp.0701029
Annexin 1 is a protein induced by glucocorticoids endowed with extracellular anti-inflammatory properties. In this study, the local expression and secretion of annexins 1–6, in rat proximal colon, were studied at different times after intracolonic administration of 30 mg trinltrobenzenesulfonic acid in 50% ethanol. Secretion was identified by incubating colonic tissues in a culture medium. The expression of annexins was detected by immunoblotting in tissue homogenates and incubation media. Inflammatory stages were evaluated by measuring myeloperoxidase activity. Annexin 1 expression in colons increased after trinitrobenzenesulfonic acid treatment and was maximal between days 1 to 9, during the cellular stage of the inflammation that corresponded to maximal myeloperoxidase activity. From 12 h to 9 days after trinitrobenzenesulfonic acid/ethanol treatment, annexin 1 was specifically secreted. Annexin 3 was also overexpressed during the cellular stage, but the expression of annexins 2, 4, 5, and 6 was unchanged; none of these annexins were secreted. Annexin 1 was shown to be physiologically secreted because its release was specific, abundant, and not correlated with cellular lysis. Annexin 1 may be considered as a putative candidate in the control of the gut inflammatory processes.
Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
N-formyl peptides, such as fMet-Leu-Phe, are one of the most potent chemoattractants for phagocytic leukocytes. The interaction of N-formyl peptides with their specific cell surface receptors has been studied extensively and used as a model system for the characterization of G-protein-coupled signal transduction in phagocytes. The cloning of the N-formyl peptide receptor cDNA from several species and the identification of homologous genes have allowed detailed studies of structural and functional aspects of the receptor. Recent findings that the receptor is expressed in nonhematopoietic cells and that nonformylated peptides can activate the receptor suggest potentially novel functions and the existence of additional ligands for this receptor.
Reperfusion of ischemic myocardium is necessary to salvage tissue from eventual death. However, reperfusion after even brief periods of ischemia is associated with pathologic changes that represent either an acceleration of processes initiated during ischemia per se, or new pathophysiological changes that were initiated after reperfusion. This 'reperfusion injury' shares many characteristics with inflammatory responses in the myocardium. Neutrophils feature prominently in this inflammatory component of postischemic injury. Ischemia-reperfusion prompts a release of oxygen free radicals, cytokines and other proinflammatory mediators that activate both the neutrophils and the coronary vascular endothelium. Activation of these cell types promotes the expression of adhesion molecules on both the neutrophils and endothelium, which recruits neutrophils to the surface of the endothelium and initiate a specific cascade of cell-cell interactions, leading first to adherence of neutrophils to the vascular endothelium, followed later by transendothelial migration and direct interaction with myocytes. This specific series of events is a prerequisite to the phenotypic expression of reperfusion injury, including endothelial dysfunction, microvascular collapse and blood flow defects, myocardial infarction and apoptosis. Pharmacologic therapy can target the various components in this critical series of events. Effective targets for these pharmacologic agents include: (a) inhibiting the release or accumulation of proinflammatory mediators, (b) altering neutrophil or endothelial cell activation and (c) attenuating adhesion molecule expression on endothelium, neutrophils and myocytes. Monoclonal antibodies to adhesion molecules (P-selectin, L-selectin, CD11, CD18), complement fragments and receptors attenuate neutrophil-mediated injury (vascular injury, infarction), but clinical application may encounter limitations due to antigen-antibody reactions with the peptides. Humanized antibodies and non-peptide agents, such as oligosaccharide analogs to sialyl Lewis, may prove effective in this regard. Both nitric oxide and adenosine exhibit broad spectrum effects against neutrophil-mediated events and, therefore, can intervene at several critical points in the ischemic-reperfusion response, and may offer greater benefit than agents that interdict at a single point in the cascade. The understanding of the molecular processes regulating actions of neutrophils in ischemic-reperfusion injury may be applicable to other clinical situations, such as trauma, shock and organ or tissue (i.e. vascular conduits) transplantation.
The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We have developed two monoclonal antibodies to human lipocortin-1 (103 and 105) as reagents for quantitating the protein in biological systems and neutralizing its activity. Lipo 105 is a high affinity antibody that is functional in ELISA and Western blot formats. The antibody recognizes a site between amino acids 30 and 55 in the lipocortin-1 sequence and can be used on native or denatured protein. Lipo 103 is an antibody that neutralizes the phospholipase A2 inhibitory activity of lipocortin-1 by blocking binding of the protein to phospholipid surfaces. The antibody is specific for native human lipocortin-1. Lipo 103 was recently shown to block lipocortin-1-dependent differentiation of a squamous carcinoma cell line, demonstrating its usefulness as a probe for function.
There are several clinical situations in which large epicardial coronary arteries are deprived of blood flow, such as occurs when an obstructing thrombus or embolus lodges within a vessel or during coronary dissection. There is little information concerning the effect of flow deprivation on large epicardial coronary arteries.
We studied a model in which a segment of a large epicardial coronary artery was deprived of blood flow using both proximal and distal clamps for 3 hours followed by reperfusion. On examination by light microscopy of cross sections of the arteries, 19 +/- 6 neutrophils were present in the intima of ischemic/reperfused vessels, whereas only a mean of 4 +/- 3 (SEM) were present in the intima of nonischemic vessels (p less than 0.02). On average, there were 17 +/- 9 neutrophils just under the elastic lamina in ischemic/reperfused vessels versus none in the nonischemic vessels (p less than 0.05); there were 16 +/- 10 neutrophils present within the media of ischemic/reperfused vessels, and none (p less than 0.05) in the nonischemic vessels. Electron microscopic analysis revealed that neutrophils in the ischemic/reperfused vessels were often "sandwiched" between the endothelial cells and the elastic lamina. Ultrastructural abnormalities within the myocardium also revealed damage to the microvasculature, including the presence of neutrophils within the vessels and erythrocyte stasis. To rule out the possibility that findings in the large epicardial arteries were due to toxic substances from static blood within the isolated arterial segment, a protocol was performed in which blood was removed from the isolated segment. Again, neutrophil infiltration into the vessel was observed. Resting mean epicardial coronary artery blood flow before coronary occlusion was 19 +/- 3 ml/min; mean coronary blood flow 2.5 hours after reperfusion was identical at 19 +/- 3 ml/min. Response to both endothelial-dependent vasodilation (acetylcholine) and endothelial-independent vasodilation (nitroglycerin) challenges was normal early after reperfusion but was depressed late after reperfusion, suggesting progressive vascular dysfunction and hence a form of vascular reperfusion injury in this model.
When large epicardial coronary arteries are deprived of blood flow, followed by reperfusion in this model, neutrophils migrate into the vessel wall as well as into the microvasculature. These abnormalities are associated with reduced endothelial-dependent and endothelial-independent coronary vasodilator reserve.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
Reperfusion of ischemic myocardium may accelerate necrosis of injured myocytes. To determine the role of neutrophil leukocytes in this process, we examined whether neutrophil depletion during reperfusion could modify infarct size in anesthetized dogs. The proximal circumflex coronary artery was occluded for 90 minutes and then reperfused for 2 hours via an extracorporeal circuit with either whole blood (n = 11) or with blood depleted of neutrophils by leukocyte filters (n = 11). The leukocyte filters caused near-total neutropenia in blood reperfusing the ischemic myocardium (7 +/- 7 neutrophils/microliters compared with 2,551 +/- 317/microliters in controls, mean +/- SEM; p less than 0.001. Infarct size was measured by planimetry of myocardial slices stained with triphenyltetrazolium chloride (TTC), and the accuracy of TTC for identifying necrotic myocardium was verified by electron microscopy. The size of the ischemic risk region was the same in the control (41.6 +/- 1.0%) and neutropenic (41.8 +/- 2.1%) groups. Collateral blood flow to the risk region was the same in control (0.15 +/- 0.03 ml/min/g) and neutropenic (0.13 +/- 0.03 ml/min/g) groups. Among dogs with collateral flow less than 0.2 ml/min/g, infarct size was reduced in the neutropenic group (27.7 +/- 6.7% of risk region, n = 8), compared with control dogs (52.5 +/- 5.7%; n = 7; p = 0.02). Multiple linear regression described the relation between infarct size, risk region size, and collateral flow in the control group, and the same regression relation was used to predict infarct size for the neutropenic group. Mean predicted infarct size in the neutropenic group (n = 11) was 16.8 +/- 3.4% of left ventricle, whereas mean observed infarct size was 9.6 +/- 3.1% (p less than 0.01). The extent of the no-reflow zone (absence of thioflavin-S-fluorescence) was also less in the neutropenic than the control group (2.2 +/- 0.8% vs. 8.1 +/- 2.7% of the risk region, p less than 0.05). Neutropenia limited to the reperfusion period is associated with significant reductions in the extent of the infarct and no-reflow zones after 90 minutes of ischemia. These findings support the hypothesis that reperfusion necrosis occurs after prolonged myocardial ischemia and indicate that neutrophil leukocytes are important mediators of such reperfusion injury.
Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.1.4), may provide such a dual action and recent research has concentrated on the role of PLA2-inhibitory proteins as possible anti-inflammatory agents. Blastokinin or uteroglobin is a steroid-induced rabbit secretory protein with PLA2-inhibitory activity. Its biochemical and biological properties have been extensively studied and its crystallographic structure has been resolved at 1.34 A (refs 15, 16). Lipocortins are a family of related proteins, which, it has been suggested, mediate the anti-inflammatory effects of glucocorticoids (for a review, see ref. 23). Some proteins of this group have been purified and the complementary DNA sequences of two human lipocortins are known. Lipocortins inhibit PLA2 in vitro, although their mechanism of action is still unclear. Recombinant lipocortin I inhibits eicosanoid synthesis in isolated perfused lungs from the guinea pig. Here, we report that synthetic oligopeptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity in vitro and striking anti-inflammatory effects in vivo.
The infiltration of neutrophils into ischemic myocardium exacerbates myocardial damage upon reperfusion, whereas drugs that inhibit neutrophil activity or function reduce infarct size. Consequently, it is important to accurately assess the myocardial neutrophil content. Histologic sections and radiolabeled cells have been used, but have major limitations. We have developed a method to measure the neutrophils present in cardiac tissue by utilizing a spectrophotometric assay for the neutrophil-specific myeloperoxidase enzyme (MPO) (Bradley et al., 1982a). Coronary artery occlusion and reperfusion in the anesthetized dog induces neutrophil accumulation into the ischemic heart, which shows a linear relationship with time. An increase in activity from 0.014 +/- 0.001 units (u) MPO/100 mg tissue to 0.091 +/- 0.02 u MPO/100 mg is already apparent at the end of the 90-min occlusion period. This activity increases over 5 hr reperfusion to 0.32 +/- 0.018 u MPO/100 mg tissue. Histologic analyses confirmed the temporal association of neutrophil accumulation. Moreover, there is a correlation between infarct size and tissue MPO activity. Measuring the MPO content in preparations of canine neutrophils, which is directly correlated with cell number, allows units of MPO activity to be converted into a tissue neutrophil content. This assay is simple, sensitive, and provides a quantitative index of myocardial neutrophil accumulation that can be used to study the relationship between leukocyte infiltration and myocardial injury.
Accumulation of polymorphonuclear neutrophils during the acute inflammatory response may exacerbate tissue injury through the release of activated oxygen products or proteolytic enzymes or both. To assess the role of neutrophils in acute myocardial infarction, circulating neutrophil levels in dogs were reduced by 77 +/- 2% (mean +/- SEM) by administering rabbit antiserum to dog neutrophils. Acute myocardial infarction was induced in open-chest anesthetized dogs by 90 minutes of left circumflex coronary artery occlusion followed by 6 hours of reperfusion. Dogs treated with neutrophil antiserum (n = 8) developed myocardial infarcts that were an average of 43% smaller than infarcts in dogs treated with nonimmune rabbit serum (n = 7) (27.0 +/- 4.5% vs 47.1% +/- 7.5% of the area at risk, p less than 0.05). In a saline-treated control group (n = 8), infarct size was 48.0 +/- 4.7% of the area at risk, a value not significantly different from that of the nonimmune serum group but significantly greater than that in the neutrophil antiserum dogs (p less than 0.05). There were no major hemodynamic differences between groups. Histopathologic examination revealed that infarcted myocardium from dogs given saline or treated with nonimmune serum had a substantial neutrophilic infiltrate, which was virtually absent in infarcted tissue from dogs treated with neutrophil antiserum. These observations suggest that neutrophil accumulation in response to myocardial ischemia may be responsible for a substantial portion of the irreversible myocardial injury resulting from temporary coronary artery occlusion.
Summary Myocardial oxygen consumption indices that are frequently applied to man such as tension-time index (TTI), pressure-rate product (P·HR) and triple product (TP) have not been fully validated so far. These easily obtainable indices and a modified TTI
, therefore, were examined in 10 closed-chest dogs with very broad variations of hemodynamics and oxygen consumption (3–36 ml/min·100 g) analyzing 162 steady states. Myocardial blood flow was directly measured by a differential pressure coronary sinus catheter.
was varied by administration of catecholamines and other inotropic drugs, atropine, beta-blocking agents and hypo- and hypervolemia. Over a wide range of hemodynamic states, correlations with directly measured
of TTI (r=0.63), P·HR (r=0.87), TP (r=0.65) and
(r=0.80) are not satisfactory due to neglect of contractility and cardiac volumes by these terms. Better correlations are obtained when relating these indices to
under different inotropic states. At normal and moderately increased contractility, correlations with
rose as follows: TTI (r=0.96), P·HR (r=0.91), TP (r=0.96) and
(r=0.94). Significant rises in correlation are due to the close relationship between peak pressure and dP/dtmax at only moderately increased contraction velocity. Correlation differences within this inotropic range must be related to incorporation or neglect of ejection time as a partial determinant of
. At markedly increased contractility, results for these indices, however, are in part very poor: TTI (r=0.40), P·HR (r=0.81), TP (r=0.38) and
(r=0.76). Within this inotropic state neglect of dP/dtmax as a major determinant of
and the inverse relationship between ejection time and dP/dtmax mainly account for these correlation shifts. It is concluded that non-invasively obtainable indices, currently in use, are no reliable predictors of actual overall
of the left ventricle if the contractile state of the myocardium is not checked invasively before. The broad variability of the relation of the energy demand of velocity of tension development to maintenance of systolic wall tension is not sufficiently considered by these terms. Appropriate caution, therefore, is necessary when applying those indirect indices of
to humans.
Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules.
Peptide Ac2-26, drawn from the sequence of human lipocortin 1, inhibited the release of elastase activity from cytoplasmic granules of human neutrophils, and neutrophil adhesion to monolayers of endothelial cells, in a concentration-dependent manner (approximate IC50 of 100 micrograms/ml, 33 microM). The effect of peptide Ac2-26 was not restricted to a specific neutrophil activator, being effective against formyl-Met-Leu-Phe (FMLP), leukotriene B4 (LTB4) and platelet-activating factor (PAF). Peptide Ac2-26 did not alter FMLP binding to its receptor. These in vitro observations complement in vivo data obtained with this peptide and may enable a better understanding of its pharmacology and, perhaps, that of of lipocortin 1 too.
In the intact organism, ischemic myocardial injury initiates an acute inflammatory response in which polymorphonuclear leukocytes (PMNs) are major participants. Evidence indicates that the interplaying inflammatory reactions are augmented by reperfusion and that accumulating PMNs can contribute to myocardial damage, eg, by release of oxygen-derived free radicals, proteases, and leukotrienes. In experimental models, interventions aimed at PMN inhibition can exert cardioprotective effects, and some of these strategies raise hope for future clinical applications. A greater understanding of the mechanisms involved in PMN-mediated myocardial damage is necessary for designing a rational approach to reduce the putative detrimental effects of PMNs without antagonizing their favorable consequences in tissue healing.
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
Lipocortin 1, a member of the annexin superfamily of calcium and phospholipid binding proteins, mediates some of the anti-inflammatory actions of the glucocorticoid hormones. Lipocortin 1 binds to the surface of murine peripheral blood monocytes and polymorphonuclear leukocytes (Kd estimate 2 x 10(-8) M) but not lymphocytes. Resident peritoneal macrophages exhibit binding (Kd estimate 1.3 x 10(-8) M) but lymphocytes do not. A 95-98% reduction in lipocortin 1 binding was observed to leukocytes obtained from air pouch or peritonitis models of inflammation. When given intravenously, lipocortin 1 binds rapidly to murine leukocytes within 5 min but disappears before 10 min, leaving the binding capacity of the cells unaltered. Modulation of lipocortin 1 binding sites could be an important step in regulating the function of inflammatory cells.
Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.
Neutrophils are pivotal in the pathogenesis of reperfusion injury leading to myocardial infarction. Firm adhesion of PMN to endothelium may be initiated by the interaction between constitutively expressed intercellular adhesion molecule-1 (ICAM-1) on endothelium and beta2 integrin (CD11b/CD18) on neutrophils. We tested the hypothesis that a monoclonal antibody (mAb RR1/1) against ICAM-1 would preserve postischemic myocardial blood flow and attenuate myocardial injury in an anesthetized rabbit model of coronary occlusion and reperfusion. Either mAb RR1/1 or isotypematched control mAb (R3.1) was injected 10 min before reperfusion. Postischemic myocardial blood flow in the area at risk (Ar) and necrotic area was significantly improved with mAb RR1/1 treatment compared with vehicle and mAb R3.1 during the reperfusion period. RR1/1 had no effect on nonischemic zone blood flow. The Ar as a percent of left ventricle was comparable between groups. Infarct size (TTC) as a percent of Ar was significantly reduced by mAb RR1/1 compared with saline vehicle and mAb R3.1. Plasma creatine kinase activity confirmed the reduction of infarct size in mAb RR1/1 group. In in vitro studies, 40 microg/mL mAb RR1/l, which approximates the plasma concentration of 2 mg/kg mAb RR1/1, markedly inhibited platelet-activating factor-stimulated neutrophil adherence to rabbit aortic endothelium. We conclude that blockade of ICAM-1 during reperfusion reduces postischemic perfusion defects and attenuates the progression of myocardial injury leading to necrosis. This cardioprotection by mAb RR1/1 may be due to inhibition of neutrophil adhesion to the coronary endothelium.
Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.
Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid-induced anti-inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme-digested synovium and treated with lipopolysaccharide (LPS) 1 microg/ml, dexamethasone (DEX) 10(-7) M, and anti-lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 +/- 2.1 micromol/24 h). iNOS was detected in synovial macrophages (ED-1+) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS-induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti-lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin I in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.
We determined the binding affinities of the MSH analogues MSH-B, HP-228 and 153N-6 and of the enkephalin analogue GHRP-6 on a single eukaryotic cell line transiently expressing the human MC1, MC3, MC4 and MC5 receptors. Moreover, we tested the binding and cAMP response of MSH-B in comparison with alpha-MSH on murine B16 melanoma cells. Our results indicate that MSH-B has a potency similar to that of alpha-MSH and that these two peptides induce similar cAMP responses in murine B16 melanoma cells. HP-228 has its highest affinity for the MC1 receptor. For the other receptors, it has slightly higher affinity for the MC5 receptor than for the MC3 and MC4 receptors. 153N-6 was found to be selective for the MC1 receptor. GHRP-6 was found to bind to the MC1 and the MC5 receptors despite its low structural homology with alpha-MSH. [D-Lys3]GHRP-6 bound to all the four MC receptors with similar affinities. The structurally related Met-enkephalin and the functionally related GHRH, as well as LHRH and somatostatin-14 did not bind to these MC receptors. The low affinity of the GH-releasing/enkephalin peptides may indicate that they do not interact with the MC receptors at pharmacologically relevant concentrations.
Annexins constitute a family of Ca2+- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman's capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues.
Postischemic reperfusion may profoundly alter cardiac function. Principal mediators of this phenomenon are oxygen radicals and neutrophils. Upon reflow, oxygen radicals are generated in large amounts, overwhelming cellular defenses and inducing oxidative tissue damage; biochemical markers of oxygen radical formation and attack can be found in postischemic myocardium. Reintroduction of neutrophils in postischemic tissues is accompanied by their activation, with release of lytic enzymes that directly induce tissue damage and proinflammatory mediators that amplify the local inflammatory reaction. Neutrophils may also plug capillaries, mechanically blocking flow. Oxidants can also modulate various events, ultimately leading to tissue injury, such as nitric oxide formation, platelet-activating factor metabolism, tissue factor synthesis, and exposure of adhesion molecules. In the clinical setting, important consequences of postischemic reperfusion are reversible contractile dysfunction ("stunning"), which is mostly caused by oxygen radical attack, and impairment to flow at the microvascular level ("no-reflow") secondary to neutrophil plugging and vasoconstriction.
Antiflammin-1 and antiflammin-2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid-inducible proteins lipocortin-1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin-1 nor antiflammin-2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor or interleukin-8 with IC(50) values of 4-20 micromol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin-1 (100 microgram/ml). Unlike dexamethasone (100 nmol/l), the antiflammins had little effect on LPS-stimulated expression of E-selectin and ICAM-1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflammins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation-induced up-regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.
The only way to rescue ischaemic tissue is to re-instate the oxygen supply to the tissue. However reperfusion of the ischaemic area not only oxygenates the tissue but also initiates a cascade of processes, which may in some cases result in temporary dysfunction of the myocardium. In order to devise protective measures, it is essential to understand the mechanisms and the triggers of this reperfusion phenomenon. In this review we will mainly focus on the inflammatory response caused by reperfusion. We will cover the different steps of polymorphonuclear leukocyte activation and will briefly discuss the molecular biology of the receptors involved. The currently used pharmacological medications in acute cardiology will be reviewed and in particular their actions on polymorphonuclear leukocyte activation, adhesion and degranulation. This review is a compilation of the current knowledge in the field and the therapeutic progress in the prevention of reperfusion injury made today.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.
Leukocyte recruitment in cremaster microcirculation was visualized by intravital microscopy, either in ovalbumin sensitized and challenged animals, or in response to TNF-alpha. In antigen-challenged mice a significant increase in leukocyte rolling (approximately 50 to 200-300 cells/min) and adhesion (2 to 15-20 cells/100 microm), and a very dramatic increase in emigration ( approximately 1 to >40 cells/field) was observed over 24 h. Although rolling and adhesion was dramatically blunted in P-selectin- or P selectin/ICAM-1-deficient mice, emigrated cell number was similar to that observed in wild-type mice. Leukocyte rolling, adhesion and emigration was almost entirely abrogated over 24 h in E/P-selectin-deficient mice, demonstrating that antigen-induced leukocyte recruitment can be entirely disrupted in the absence of both endothelial selectins. However, E/P-selectin-deficient mice were able to recruit leukocytes at 24 h after TNF-alpha challenge. Rolling 24 h post-TNF-alpha in E/P-selectin-deficient mice was not inhibitable with anti-L-selectin antibody, suggesting an entirely selectin-independent pathway of rolling. We identified this pathway to be alpha (4)-integrin dependent and demonstrated that VCAM-1 expression was increased only in mice challenged with TNF-alpha. These data demonstrate that, in vivo, sufficient amounts of TNF-alpha can recruit leukocytes independently of selectins, whereas inhibition of endothelial selectins is the optimal intervention in reducing the immune response to antigen.
Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.