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Increased Endothelial Mitogen-Activated Protein Kinase Phosphatase-1 Expression Suppresses Proinflammatory Activation at Sites That Are Resistant to Atherosclerosis
Abstract
Atherosclerosis is a chronic inflammatory disease of arteries. It is triggered by proinflammatory mediators which induce adhesion molecules (eg, vascular cell adhesion molecule [VCAM]-1) in endothelial cells (ECs) by activating p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases by phosphorylation. Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on the inner surface of arteries, a force that alters endothelial physiology. Regions of the arterial tree exposed to high shear are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear are susceptible. We examined whether MAP kinase phosphatase (MKP)-1, a negative regulator of p38 and JNK, mediates the antiinflammatory effects of shear stress. We observed that expression of MKP-1 in cultured ECs was elevated by shear stress, whereas the expression of VCAM-1 was reduced. MKP-1 induction was shown to be necessary for the antiinflammatory effects of shear stress because gene silencing of MKP-1 restored VCAM-1 expression in sheared ECs. Immunostaining revealed that MKP-1 is preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and is necessary for suppression of EC activation at this site, because p38 activation and VCAM-1 expression was enhanced by genetic deletion of MKP-1. We conclude that MKP-1 induction is required for the antiinflammatory effects of shear stress. Thus, our findings reveal a novel molecular mechanism contributing to the spatial distribution of vascular inflammation and atherosclerosis.
... The activation of these protein kinases plays a critical role in inducing and stabilizing pro-inflammatory transcripts in vascular ECs. Studies have shown that in vitro and ex vivo exposure of ECs to high shear stress levels leads to activation of p38 mitogen-activated protein kinase (MAPK) that, in turn, is associated with increased monocyte chemotactic protein 1 (MCP1) expression (Zakkar et al., 2011(Zakkar et al., , 2008. MCP1 is a potent chemotactic agent to monocytes and has a crucial role in vein graft thickening (Xu et al., 2003). ...
... The underlying mechanism revealed that shear stress can lead to the upregulation of MKP-1 which is known to be a negative regulator of p38 in arterial but not venous ECs. This presents potential therapeutic benefits as the activation of MKP-1 in vein grafts can prevent them from mounting a pro-inflammatory response when exposed to acute high shear (12 dynes/cm 2 , consistent with arterial levels) and thus facilitates arterialisation (Zakkar et al., 2008(Zakkar et al., , 2011. ...
... This pathway is well established as having a role in regulating the leukocyte adhesion cascade, leukocyte transmigration and the release of pro-inflammatory mediators. Despite the recognised contribution of shear stress to EC dysfunction in vitro (Zakkar et al., 2011(Zakkar et al., , 2008, previous studies have shown that the local NF-κB inhibition in vivo is associated with the suppression of IH development in different vein graft models (Shintani et al., 2002;Wang et al., 2007). However, these studies focused on the impact of NF-κB inhibition on the alteration of SMC proliferation and migration, but not the role of NF-κB in the acute adaptive response of the vein graft ECs to its new haemodynamic environment. ...
The long saphenous vein is commonly used in cardiac surgery to bypass occluded coronary arteries. Its use is complicated by late stenosis and occlusion due to the development of intimal hyperplasia. It is accepted that intimal hyperplasia is a multifactorial inflammatory process that starts immediately after surgery. The role of acute changes in haemodynamic conditions when the vein is implanted into arterial circulation, especially shear stress, is not fully appreciated. This review provides an overview of intimal hyperplasia and the effect of acute shear stress changes on the activation of pro-inflammatory mediators.
... A reservoir containing 30 mL RPMI 1640 culture medium (ThermoFisher Scientific, 11875093), supplemented with 2% (v/v) FCS, 100 µg/mL penicillin, and 100 U/mL streptomycin attached to a closed-circuit loop of silicon tubing (VWR, Radnor, PA, USA and Elkay, Basingstoke, UK) was connected to the chambers. HUVECs were then cultured at 37 • C and 5% CO 2, and shear stress applied using a multi-channel peristaltic pump (Watson-Marlow, Marlow, UK) [19,20,26]. ...
... HUVECs were fixed and stained as previously reported [19,20,26] using pre-chilled 10% neutral buffered formalin (Sigma Aldrich, HT5014-1CS) for 15 min at 4 • C. Thereafter, slides were dehydrated in ethanol (50-100%) and allowed to dry. Cells were incubated in immunofluorescence blocking buffer (Cell Signaling Technology, 12411) for 30 min at room temperature followed by overnight primary antibody incubation at 4 • C and AlexaFluor fluorophore-conjugated secondary antibody for 1 h at room temperature. ...
Background:
Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin.
Methods:
We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography.
Results:
The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake.
Conclusions:
Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.
... MKP-1 is highly expressed in oxPAPC-treated HEAC, which may control MCP-1 expression via acting on MAPK pathway such as p38 (Reddy et al., 2001). Interestingly, MKP-1 also acts as a negative regulator of p38 and JNK to take anti-inflammatory effects under shear stress, p38 and JNK signaling pathways are related to the expression of IL-6, IL-8, and VCAM-1 ( Fig. 1a) (Cheng et al., 2019b;Zakkar et al., 2008). Therefore, the role of MKP-1 in atherosclerosis is controversial, and experiments demonstrate that partial loss of MKP-1 activity in monocytes can lead to monocyte recruitment and dysfunction, however, complete loss attenuates this effect (Kim et al., 2012). ...
... Anti (Reddy et al., 2001;Zakkar et al., 2008) Upregulate TF to promote coagulation, Pro (Berliner and Gharavi, 2008;Lee et al., 2012) Inhibit TFPI by linking the C-terminal basic region of TFPI Pro (Ohkura et al., 2004) Protect against LPS-induced endothelial dysfunction ...
Vascular ageing is an important factor in the morbidity and mortality of the elderly. Atherosclerosis is a characteristic disease of vascular ageing, which is closely related to the enhancement of vascular inflammation. Phospholipid oxidation products are important factors in inducing cellular inflammation. Through interactions with vascular cells and immune cells, they regulate intracellular signaling pathways, activate the expression of various cytokines, and affect cell behavior, such as metabolic level, proliferation, apoptosis, etc. Intervention in lipid metabolism and anti-inflammation are the two key pathways of drugs for the treatment of atherosclerosis. This review aims to sort out the signaling pathway of oxidized phospholipids-induced inflammatory factors in vascular cells and immune cells and the mechanism leading to changes in cell behavior, and summarize the therapeutic targets in the inflammatory signaling pathway for the development of atherosclerosis drugs.
... ECs respond to shear stress via mechanoreceptors that translate mechanical distortions into various molecular signals, including GR translocation (81,82). Regions of the arterial tree exposed to high shear stress are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear stress, are susceptible (83,84). The expression of DUSP1 in cultured ECs was elevated by shear stress, whereas vascular cell adhesion protein (VCAM)-1 levels were reduced; silencing of DUSP1 restored VCAM-1 expression. ...
... The expression of DUSP1 in cultured ECs was elevated by shear stress, whereas vascular cell adhesion protein (VCAM)-1 levels were reduced; silencing of DUSP1 restored VCAM-1 expression. In vivo, DUSP1 was preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and was necessary for the suppression of EC activation (84). ...
Glucocorticoids (GCs) potently inhibit pro-inflammatory responses and are widely used for the treatment of inflammatory diseases, such as allergies, autoimmune disorders, and asthma. Dual-specificity phosphatase 1 (DUSP1), also known as mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), exerts its effects by dephosphorylation of MAPKs, i.e., extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Endogenous DUSP1 expression is tightly regulated at multiple levels, involving both transcriptional and post-transcriptional mechanisms. DUSP1 has emerged as a central mediator in the resolution of inflammation, and upregulation of DUSP1 by GCs has been suggested to be a key mechanism of GC actions. In this review, we discuss the impact of DUSP1 on the efficacy of GC-mediated suppression of inflammation and address the underlying mechanisms.
... All rights reserved. The role of EC as inflammatory contributors to atherosclerosis is well established (44,67), and the role of myeloid trained immunity in the disease is also gaining attention (reviewed in (68,69). These publications and others (34,37,40,48,70) acknowledge the dangers of innate memory facilitating over-aggressive inflammatory responses based on priming by endogenous mediators. ...
... Shear stress (pressure exerted by blood flow on the vessel walls) is higher in straight vessel sections than arches or junctions. Work from the Evans lab has shown that EC under low shear stress have constitutively active c-Jun N-Terminal kinase (JNK) (43), which is suppressed by Dual Specificity Phosphatase 1 (DUSP1) in areas of high shear stress(44). This high level of JNK activity correlates with VCAM-1 expression and thus ability to facilitate leukocyte adhesion. ...
All organisms are constantly exposed to a variety of infectious and injurious stimuli. These induce inflammatory responses tailored to the threat posed. Whilst the innate immune system is the front line of response to each stimulant, it has been traditionally considered to lack memory, acting in a generic fashion until the adaptive immune arm can take over.
This outmoded simplification of the roles of innate and acquired arms of the immune system has been challenged by evidence of myeloid cells altering their response to subsequent encounters based on earlier exposure. This concept of “innate immune memory” has been known for nearly a century, and is accepted amongst myeloid biologists. In recent years, other innate immune cells, such as natural killer cells, have been shown to display memory, suggesting innate immune memory is a trait common to several cell types.
Over the last thirty years, evidence has slowly accumulated in favour of not only haematopoietic cells, but also stromal cells, being imbued with memory following inflammatory episodes. A recent publication showing this also to be true in epithelial cells suggests innate immune memory to be widespread, if underappreciated, in non‐haematopoietic cells.
In this review, we will examine the evidence supporting the existence of innate immune memory in stromal cells. We will also discuss the ramifications of memory in long‐lived tissue‐resident cells. Finally, we will pose questions we feel to be important in the understanding of these forgotten cells in the field of innate memory. This article is protected by copyright. All rights reserved.
... In einer weiteren Studie, in der mit Titanoxid-und Quartz-Nanopartikeln gearbeitet wurde, konnte wiederum keine Abhängigkeit zwischen der Aufnahmemenge und der potentiellen inflammatorischen Aktivität festgestellt werden (Höhr et al., 2002). Für oberflächenmodifizierte magnetische Nanopartikel wurden sogar eher gegenteilige Effekte beobachtet, was die Korrelation zwischen Partikelaufnahme und Zellaktivität angeht (Gupta and Gupta, 2005 (Huotari et al., 2012;Imaizumi et al., 2010;Kiemer et al., 2002a;Kiemer et al., 2002b;Kim et al., 2015;Prabhala et al., 2015;Shen et al., 2010;Zakkar et al., 2008). MKP-1 ist eine nukleäre Phosphatase, deren Expression durch verschiedene Stimuli, wie zum Beispiel Zytokine, LPS oder Glukokortikoide induziert wird und generell die Synthese von proinflammatorischen Zytokinen abschwächt (Huotari et al., 2012). ...
... Welche Rolle MKP-1 in der Arteriosklerose spielt, wird allerdings noch diskutiert. Es gibt Hinweise darauf, dass MKP-1 atheroprotektiv wirkt (Kim, Tavakoli et al., 2015, Zakkar, Chaudhury et al., 2008. Allerdings wurden auch Daten veröffentlicht, die das Gegenteil postulieren (Imaizumi, Grijalva et al., 2010, Shen, Chandrasekharan et al., 2010. ...
... By contract, irregular flow promotes endothelial expression of adhesion molecules and monocyte attachment to ECs (Morigi et al. 1995, Chappell et al. 1998, Sheikh et al. 2003. In arteries, endothelial expression of adhesion molecules is site specific and is upregulated in atherosusceptible regions challenged by low shear stress (Iiyama et al. 1999, Partridge et al. 2007, Zakkar et al. 2008. ...
... Nrf2 suppresses p38 MAPK, which is primarily involved in AP-1 activation by down-regulating the upstream activator MKK3/6 and by stimulating the negative regulator MKP-1 ). Expression of MKP-1 that negatively targets c-Jun and p38 MAPK by dephosphorylation is found to be increased at atheroprone vascular sites and is modulated by shear stress (Zakkar et al. 2008). ...
Hemodynamic forces influence functional properties of vascular endothelium. Endothelial cells have a variety of receptors, which sense flow and transmit mechanical signals through mechanosensitive signaling pathways to recipient molecules, that lead to phenotypic and functional changes. Arterial architecture varies greatly exhibiting bifurcations, branch points, and curved regions, which are exposed to various flow patterns. Clinical studies showed that atherosclerotic plaques develop preferentially at arterial branches and curvatures, i.e. in the regions exposed to disturbed flow and shear stress. In the atheroprone regions, the endothelium has a proinflammatory phenotype associated with low nitric oxide production, reduced barrier function, and increased proadhesive, procoagulant, and proproliferative properties. Atheroresistant regions are exposed to laminar flow and high shear stress that induce prosurvival antioxidant signals and maintain the quiescent phenotype in endothelial cells. Indeed, various flow patterns contribute to phenotypic and functional heterogeneity of arterial endothelium whose response to proatherogenic stimuli is differentiated. This may explain the preferential development of endothelial dysfunction in arterial sites with disturbed flow. This article is protected by copyright. All rights reserved.
... Natural inhibitors of inflammation and stress signaling include glucocorticoid hormones which can decrease signaling through stress kinase pathways by inducing the synthesis of DUSP1 (25,26). JNK and p38 as targets of DUSP1 have been well-characterized in vascular cell types, and our data agree with previous reports showing that after p38 and JNK activation, DUSP1 is effective in decreasing activation and protecting cells from actin stress fiber formation that often leads to EC dysfunction (39,48,50). In this regard, our finding that heparin treatment of ECs after DUSP1 knockdown resulted in increased TNFα-induced pJNK and stress fibers indicates that DUSP1 may be critical for routine modulation of stress fiber levels and their remodeling in ECs. ...
... In this regard, our finding that heparin treatment of ECs after DUSP1 knockdown resulted in increased TNFα-induced pJNK and stress fibers indicates that DUSP1 may be critical for routine modulation of stress fiber levels and their remodeling in ECs. ECs express higher levels of DUSP1 in athero-protective regions of the vasculature resulting in the lower levels of active JNK and p38 found in those regions (50). The lower levels of DUSP1 in the areas of disturbed flow thus contribute to increased inflammation in these regions. ...
Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well-understood. Studies with vascular smooth muscle cells (VSMCs)5 indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends upon specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα;-induced JNK and p38 activity and downstream target phosphorylation as identified through western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα;-induced increases in stress fibers. MAbs that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as is seen in VSMCs. Thus, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.
... This indicated several entries related to glutathione S-transferase, whereby there seemed to be an isoform switch (isoforms omega1, omega 2, mu5 repressed in DMSO treated cells and isoforms alpha1, mu2, mu6-like and microsomal glutathione S-transferase 1 repressed in Yoda1 treated cells), potentially related to an adaptation to oxidative stress. [38] Additionally we found peaks in Yoda1 treated cells for arhgef2 (also known as GEF-H1, an inhibitor of podosome formation [39,40] ), calm1 (calmodulin, downregulation consistent with ABCA1 loss/foam cell formation), [41] acvr1 (activin A receptor, consistent with a loss of contractile phenotype), [42] dusp1 (also known as MKP1; expressed at atheroprotected regions [43] and reduced levels associated with increased VSMC proliferation [39] ). On the other hand, key pro-atherosclerotic genes including klf2 (Kruppel-like factor 2), sele (E-selectin), or fos (which is critical for foam cell formation and atherosclerosis [44] ) all had identifiable H3K9me3 peaks and thus were repressed in DMSO treated cells only. ...
Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy.
... DUSP1 is involved in the synthesis and metabolism of fatty acids in the brain 32 . In atherosclerosis, DUSP1 plays a promoter and suppressor role 33,34 . It is found that DUSP1 is up-regulated in IS and may act as a new biomarker for the diagnosis and treatment of IS 35,36 . ...
Molecule alterations are important to explore the pathological mechanism of ischemic stroke (IS). Ferroptosis, a newly recognized type of regulated cell death, is related to IS. Identification of the interactions between circular RNA (circRNA), microRNA (miRNA) and ferroptosis related mRNA may be useful to understand the molecular mechanism of IS. The circRNA, miRNA and mRNA transcriptome data in IS, downloaded from the Gene Expression Omnibus (GEO) database, was used for differential expression analysis. Ferroptosis related mRNAs were identified from the FerrDb database, followed by construction of circRNA-miRNA-ferroptosis related mRNA network. Enrichment and protein–protein interaction analysis of mRNAs in circRNA-miRNA-mRNA network was performed, followed by expression validation by reverse transcriptase polymerase chain reaction and online dataset. A total of 694, 41 and 104 differentially expressed circRNAs, miRNAs and mRNAs were respectively identified in IS. Among which, dual specificity phosphatase 1 (DUSP1), nuclear receptor coactivator 4 (NCOA4) and solute carrier family 2 member 3 (SLC2A3) were the only three up-regulated ferroptosis related mRNAs. Moreover, DUSP1, NCOA4 and SLC2A3 were significantly up-regulated in IS after 3, 5 and 24 h of the attack. Based on these three ferroptosis related mRNAs, 4 circRNA-miRNA-ferroptosis related mRNA regulatory relationship pairs were identified in IS, including hsa_circ_0071036/hsa_circ_0039365/hsa_circ_0079347/hsa_circ_0008857-hsa-miR-122-5p-DUSP1, hsa_circ_0067717/hsa_circ_0003956/hsa_circ_0013729-hsa-miR-4446-3p-SLC2A3, hsa_circ_0059347/hsa_circ_0001414/hsa_circ_0049637-hsa-miR-885-3p-SLC2A3, and hsa_circ_0005633/hsa_circ_0004479-hsa-miR-4435-NCOA4. In addition, DUSP1 is involved in the signaling pathway of fluid shear stress and atherosclerosis. Relationship of regulatory action between circRNAs, miRNAs and ferroptosis related mRNAs may be associated with the development of IS.
... Activation of MAPK or its upstream kinases in cells has been reported to induce apoptosis, while blocking MAPK activation can prevent the onset of apoptosis in many cells 32,33 . DUSP1 has been demonstrated to exert anti-in ammatory and anti-apoptotic vasoprotective effects by inhibiting JNK and p38 protein phosphorylation in atherosclerosis and reducing the expression of in ammatory adhesion molecules such as vascular cell adhesion molecule-1 34 . Another study demonstrated that DUSP1 expression inhibited p38 protein phosphorylation and down-regulated caspase-3 protein levels, which in turn inhibited cardiomyocyte apoptosis 35 . ...
Staphylococcus aureus ( S. aureus ) is a major human pathogen that causes apoptosis of immune cells during infections. The rate of apoptosis influences the severity and outcome of the disease, which can be fatal for infections including sepsis and septicemia. Dual specificity phosphatase-1 (DUSP1) is a negative regulator of MAPK signaling pathways in the host innate immune response, but its role in S. aureus -induced apoptosis remains unexplored. We used western blotting and immunofluorescence assays to show that S. aureus infection induced DUSP1 expression and promoted apoptosis in THP-1 cells. Knockdown of DUSP1 using an siRNA construct promoted the expression of key pro-apoptotic proteins, including cleaved-caspase3, cleaved-PARP1, cleaved-caspase9, cytochrome c and bax, whereas it inhibited the expression of key apoptosis inhibitory proteins bcl-2 and bcl-XL. These results were validated by flow cytometry. In addition, knockdown of DUSP1 promoted the accumulation of reactive oxygen species in S. aureus -induced macrophages, and mechanistically, knockdown of DUSP1 promoted the phosphorylation of target molecules in the MAPK signaling pathway, thereby promoting apoptosis in S. aureus -infected THP-1 macrophages. These data support a regulatory role for DUSP1 in S. aureus- mediated apoptosis and we suggest that DUSP1 be investigated as an anti-apoptotic therapeutic target.
... Mitogen-activated protein kinase phosphatases (MKPs) are dual-specificity protein phosphatases that directly inactivate their mitogen-activated protein kinase (MAPK) substrates, primarily extracellular signal-regulated protein kinase (ERK) 1/2, P38, and Jun N-terminal kinase, by dephosphorylating threonine and tyrosine residues (Farooq and Zhou, 2004;Murphy and Blenis, 2006). MKP-1 was the first MKP to be discovered and is widely expressed in multiple cell types within the central nervous system, e.g., neurons, microglia, and ECs (Zakkar et al., 2008;Horita et al., 2010;Liu et al., 2014). MKP-1 has been implicated in the pathogenesis and development of various neurological diseases (Collins et al., 2015), including cerebral stroke. ...
Ischemic stroke can cause blood-brain barrier (BBB) injury, which worsens brain damage induced by stroke. Abnormal expression of tight junction proteins in endothelial cells (ECs) can increase intracellular space and BBB leakage. Selective inhibition of mitogen-activated protein kinase, the negative regulatory substrate of mitogen-activated protein kinase phosphatase (MKP)-1, improves tight junction protein function in ECs, and genetic deletion of MKP-1 aggravates ischemic brain injury. However, whether the latter affects BBB integrity, and the cell type-specific mechanism underlying this process, remain unclear. In this study, we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke. We found that overexpression of MKP-1 in ECs reduced infarct volume, reduced the level of inflammatory factors interleukin-1β, interleukin-6, and chemokine C-C motif ligand-2, inhibited vascular injury, and promoted the recovery of sensorimotor and memory/cognitive function. Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase (ERK) 1/2 and the downregulation of occludin expression. Finally, to investigate the mechanism by which MKP-1 exerted these functions in ECs, we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose, and pharmacologically inhibited the activity of MKP-1 and ERK1/2. Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death, cell monolayer leakage, and downregulation of occludin expression, and that inhibiting ERK1/2 can reverse these effects. In addition, co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2. These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2, thereby protecting the integrity of BBB, alleviating brain injury, and improving post-stroke prognosis.
... Indeed, VCAM-1, an endothelial adhesion molecule, is recognized the early marker of vascular inflammation. Previous studies have reported that VCAM-1 plays a crucial role in contributing the inflammatory response by mediating the leukocyte adhesion to endothelial cells (Zakkar et al. 2008;Heo et al. 2011Heo et al. , 2015. We consistently observed that VCAM-1 expression was higher in the atheroprone areas in the aortic arch and descending area, compared with the s-flow area, where is the atheroprotective area (Fig. 7). ...
Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6′-sialylactose (6′-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6′-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500 ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6′-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6′-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6′-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6′-SL treatment. Altogether, our findings suggest that 6′-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.
... There is less known, however, on the role of JNK in endothelial cells. Zakkar et al (2008) found that JNK was activated constitutively in endothelial cells at atherosusceptible sites but expressed in its an inactive form at A miR-17-92 promoter region (positions 5786-8494 in accession# NG-032702) was inserted into pEZX-LvPG04 dual-reporter vector (WT promoter). This vector uses GLuc (Gaussia Luciferase) as the promoter reporter and SEAP (secreted alkaline phosphatase) as the internal control for signal normalization (top panel). ...
Background:
Our previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction.
Method:
Human lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock.
Result:
Hypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls.
Conclusion:
Transcription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.
... In vein ECs, mechanical stretching induces ROS production to a greater extent than arterial ECs as the former is more sensitive to ROS effects [64]. Furthermore, data from several studies suggest the significance of oxidative stress modulation in neointima formation in vein grafts [65][66][67]. During normal conditions, ROS production can occur because of aerobic metabolism in the mitochondria. ...
Endothelial cells comprise the intimal layer of the vasculature, playing a crucial role in facilitating and regulating aspects such nutrient transport, vascular homeostasis, and inflammatory response. Given the importance of these cells in maintaining a healthy haemodynamic environment, dysfunction of the endothelium is central to a host of vascular diseases and is a key predictor of cardiovascular risk. Of note, endothelial dysfunction is believed to be a key driver for vein graft disease—a pathology in which vein grafts utilised in coronary artery bypass graft surgery develop intimal hyperplasia and accelerated atherosclerosis, resulting in poor long-term patency rates. Activation and denudation of the endothelium following surgical trauma and implantation of the graft encourage a host of immune, inflammatory, and cellular differentiation responses that risk driving the graft to failure. This review aims to provide an overview of the current working knowledge regarding the role of endothelial cells in the onset, development, and modulation of vein graft disease, as well as addressing current surgical and medical management approaches which aim to beneficially modulate endothelial function and improve patient outcomes.
... Of note, the impact of DUSP1 on the inflammatory response was first observed in atherosclerosis. Reduced DUSP1 expression was detected in endothelial cells exposed to shear stress, and this promoted the expression of adhesion factors such as vascular cell adhesion molecule 1 [33]. DUSP1 deficiency was found to induce hypotension by augmenting inducible nitric oxide synthase expression [34]. ...
Objectives
Dual specificity phosphatase 1 (DUSP1) is regarded as an anti-inflammatory factor in cardiovascular disorders. Mitophagy removes damaged mitochondria and thus promotes mitochondrial regeneration. We investigated whether DUSP1 could attenuate inflammation-induced cardiomyopathy by improving mitophagy.
Methods
Lipopolysaccharide was used to induce septic cardiomyopathy in wild-type (WT) and DUSP1 transgenic (DUSP1TG) mice.
Results
Echocardiography revealed that lipopolysaccharide impaired heart function by reducing the cardiac systolic and diastolic capacities of WT mice. Freshly isolated single cardiomyocytes from lipopolysaccharide-treated WT mice also exhibited reduced contractile/relaxation parameters. However, DUSP1 overexpression not only maintained the mechanical properties of cardiomyocytes, but also improved heart performance. Lipopolysaccharide upregulated myocardial inflammatory gene transcription and adhesive factor expression, which increased myocardial neutrophil accumulation and cardiomyocyte apoptosis in WT mice. DUSP1 overexpression inhibited the inflammatory response and therefore promoted cardiomyocyte survival. Lipopolysaccharide disrupted mitochondrial respiration and metabolism in WT cardiomyocytes, but DUSP1 overexpression restored mitochondrial metabolism, maintained the mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species production, possibly by increasing FUN14 domain-containing 1 (FUNDC1)-dependent mitophagy. Silencing of FUNDC1 abolished the protective effects of DUSP1 overexpression on cardiomyocytes and their mitochondria following lipopolysaccharide treatment.
Conclusion
These results demonstrated that DUSP1 is a novel anti-inflammatory factor that protects against septic cardiomyopathy by improving FUNDC1-induced mitophagy.
... Wang et al. [34] confirmed that MKP1 served to limit the inflammatory responses through inactivating p38 and JNK. Additionally, DUSP1 was also been reported to play promoter and suppressor roles in atherosclerosis [35,36]. Compared with the above studies, the advantages of our study lie in not only verifying DUSP1 as a robust signature for identifying IS but also exploring the ceRNA regulatory mechanism and immune infiltration microenvironment of IS. ...
Background:
Oxidative stress (OS) and immune inflammation play complex intersections in the pathophysiology of ischemic stroke (IS). However, a competing endogenous RNA- (ceRNA-) based mechanism linked to the intersections in IS has not been explored. We aimed to identify potential OS-related signatures and analyze immune infiltration characteristics in IS.
Methods:
Three datasets (GSE22255, GSE110993, and GSE140275) from the GEO database were extracted. Differentially expressed long noncoding RNAs, microRNAs, and messenger RNAs (DElncRNAs, DEmiRNAs, and DEmRNAs) between IS patients and controls were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were explored. Moreover, a triple ceRNA network was constructed to reveal transcriptional regulation mechanisms. A comprehensive strategy among least absolute shrinkage and selection operator (LASSO) regression, DEmRNAs, uprelated DEmRNAs, and OS-related genes was adopted to select the best signature. Then, we evaluated and verified the discriminant ability of the signature via receiver operating characteristic (ROC) analysis. Immune infiltration characteristics were explored via the CIBERSORT algorithm. Moreover, the best signature was verified via qPCR and western blot methods in rat brain tissues and PC12 cells.
Results:
11 DEmRNAs were identified totally. Enrichment analysis showed that the DEmRNAs were primarily concentrated in MAPK-associated biological processes and immune or inflammation-involved pathways. DUSP1 was identified as the best signature with an area under the ROC curve of 73.5% (95%CI = 57.02-89.98, sensitivity = 95%, and specificity = 60%) in GSE22255 and 100.0% (95%CI = 100.00-100.00, sensitivity = 100%, and specificity = 100%) in GSE140275. Importantly, we also identified the AC079305/DUSP1 axis in the ceRNA network. Immune infiltration showed that resting mast cells infiltrate less in IS patients compared with controls. And DUSP1 was negatively correlated with resting mast cells (r = -0.703, P < 0.01), whereas it was positively correlated with neutrophils (r = 0.339, P < 0.05). Both in vivo and in vitro models confirmed the upregulated expression of DUSP1 and the downregulated expression of miR-429.
Conclusion:
This study identified the ceRNA-based AC079305/DUSP1 axis as a promising OS-related signature for IS. Immune infiltrating cells, especially mast cells, may exert a pivotal role in IS progression. Pharmacological agents targeting signatures, their receptors, or mast cells may shed a novel light on therapeutic targets for IS.
... The internal bend of the aortic arch and the areas of arterial bifurcation, where low and low/oscillatory WSS are realized, are associated with the localisation of atheroma. In the same areas, increased ex pression of active cJun Nterminal kinase 1 (JNK1), which belongs to mitogenactivated protein kinases (MAPK), was revealed (Zakkar et al., 2008), as well as increased expres sion and nuclear localisation of the RelA subunit of NF-κB (Cuhlmann et al., 2011). The hypothesis that physiologically low WSS regulates the expression of RelA and NF-κB target genes (VCAM-1 and others) through the JNK1dependent pathway was proved by modelling the haemodynamics of the carotid artery in mice. ...
A terrible disease of the cardiovascular system, atherosclerosis, develops in the areas of bends and branches of arteries, where the direction and modulus of the blood flow velocity vector change, and consequently so does the mechanical effect on endothelial cells in contact with the blood flow. The review focuses on topical research studies on the development of atherosclerosis – mechanobiochemical events that transform the proatherogenic mechanical stimulus of blood flow – low and low/oscillatory arterial wall shear stress in the chains of biochemical reactions in endothelial cells, leading to the expression of specific proteins that cause the progression of the pathological process. The stages of atherogenesis, systemic risk factors for atherogenesis and its important hemodynamic factor, low and low/oscillatory wall shear stress exerted by blood flow on the endothelial cells lining the arterial walls, have been described. The interactions of cell adhesion molecules responsible for the development of atherosclerosis under low and low/oscillating shear stress conditions have been demonstrated. The activation of the regulator of the expression of cell adhesion molecules, the transcription factor NFκB, and the factors regulating its activation under these conditions have been described. Mechanosensitive signaling pathways leading to the expression of NFκB in endothelial cells have been described. Studies of the mechanobiochemical signaling pathways and interactions involved in the progression of atherosclerosis provide valuable information for the development of approaches that delay or block the development of this disease.
... DUSP1 is a dual-specific phosphatase that regulates the MAPK pathway which controlled a vast of cellular processes. The endothelial exposure of high shear stress induces the persistent expression of DUSP1 and mitigates the activities of p38 MAPK and JNK pathway (Zakkar et al., 2008). Recent studies have revealed that the attenuation of ERK induced by DUSP1 inhibits intimal hyperplasia by reducing proliferation and inducing apoptosis of SMCs (Kim et al., 2011). ...
Objective: The aim was to study the preliminary screening of the crucial genes in intimal hyperplasia in the venous segment of arteriovenous (AV) fistula and the underlying potential molecular mechanisms of intimal hyperplasia with bioinformatics analysis.
Methods: The gene expression profile data (GSE39488) was analyzed to identify differentially expressed genes (DEGs). We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of DEGs. Gene set enrichment analysis (GSEA) was used to understand the potential activated signaling pathway. The protein–protein interaction (PPI) network was constructed with the STRING database and Cytoscape software. The Venn diagram between 10 hub genes and gene sets of 4 crucial signaling pathways was used to obtain core genes and relevant potential pathways. Furthermore, GSEAs were performed to understand their biological functions.
Results: A total of 185 DEGs were screened in this study. The main biological function of the 111 upregulated genes in AV fistula primarily concentrated on cell proliferation and vascular remodeling, and the 74 downregulated genes in AV fistula were enriched in the biological function mainly relevant to inflammation. GSEA found four signaling pathways crucial for intimal hyperplasia, namely, MAPK, NOD-like, Cell Cycle, and TGF-beta signaling pathway. A total of 10 hub genes were identified, namely, EGR1, EGR2, EGR3, NR4A1, NR4A2, DUSP1, CXCR4, ATF3, CCL4, and CYR61. Particularly, DUSP1 and NR4A1 were identified as core genes that potentially participate in the MAPK signaling pathway. In AV fistula, the biological processes and pathways were primarily involved with MAPK signaling pathway and MAPK-mediated pathway with the high expression of DUSP1 and were highly relevant to cell proliferation and inflammation with the low expression of DUSP1. Besides, the biological processes and pathways in AV fistula with the high expression of NR4A1 similarly included the MAPK signaling pathway and the pathway mediated by MAPK signaling, and it was mainly involved with inflammation in AV fistula with the low expression of NR4A1.
Conclusion: We screened four potential signaling pathways relevant to intimal hyperplasia and identified 10 hub genes, including two core genes (i.e., DUSP1 and NR4A1). Two core genes potentially participate in the MAPK signaling pathway and might serve as the therapeutic targets of intimal hyperplasia to prevent stenosis after AV fistula creation.
... ECs are sensors of blood fluid SS and transduce the frictional force from blood flow into biochemical signals that regulate gene expression and cell behavior through specialized pathways, such as caveolae or caveolin-1 (8). Endothelial dysfunction in atherosclerosis impairs endothelium-dependent vasodilation and may lead to other pathophysiological consequences, such as increased chemotactic and adhesion molecule expression (12), increased monocyte/macrophage recruitment and accumulation, decreased EC regeneration, and increased vascular smooth muscle cells (VSMCs) proliferation and migration. However, it is unknown whether VSMC phenotype affects EC properties during vascular remodeling and endothelium recovery. ...
Low blood fluid shear stress (SS) promotes vascular remodeling and atherosclerosis; however, the effects of high (H)SS on vascular remodeling and atherogenesis is not fully clarified. The major goal of this study was to investigate the role of HSS in atherosclerotic plaque formation. A perivascular SS modifier was implanted in the right carotid artery of apolipoprotein E (ApoE)‑/‑ mice to induce HSS, whereas the left carotid artery represented undisturbed (U)SS as a control in vivo. In vitro modeling used human umbilical vein endothelial cells and vascular smooth muscle cells exposed to HSS (2.5 Pa) using a parallel‑plate flow system. The results demonstrated that there were no plaque formations or endothelial cells in the HSS regions of the carotid artery in ApoE‑/‑ mice. The number of umbilical vein endothelial cells was markedly decreased in a time‑dependent manner in HSS. HSS significantly decreased α‑smooth muscle actin and increased osteopontin protein expression levels compared with USS in vascular smooth muscle cells (P<0.05). In addition, HSS significantly increased the protein expression levels of collagen α1(XVIII) chain/endostatin and matrix metalloproteinase‑8 in vascular smooth muscle cells. These data indicated that HSS may prevent atherosclerotic plaque formation through endothelium denudation and contractile‑to‑synthetic phenotypic conversion of smooth muscle cells.
... The expression levels of VCAM-1 were assessed in ECs at regions of the lesser curvature [low shear (LS) site], greater curvature [high shear (HS) site], and descending mouse aortae by en face staining as described previously. 17 Briefly, mice were treated with AngII (1.1 mg/kg/day) or saline for 48 h before killing by CO 2 inhalation. Mice were slowly perfused with cold phosphate-buffered saline and then perfusion-fixed with 2% formalin before harvesting. ...
Aims:
Endothelial activation and inflammatory cell infiltration have important roles in the development of cardiac fibrosis induced by renin-angiotensin system activation. NADPH oxidases (Nox proteins) are expressed in endothelial cells (ECs) and alter their function. Previous studies indicated that Nox2 in ECs contributes to angiotensin II (AngII)-induced cardiac fibrosis. However, the effects of EC Nox4 on cardiac fibrosis are unknown.
Methods and results:
Transgenic (TG) mice overexpressing endothelial-restricted Nox4 were studied alongside wild-type (WT) littermates as controls. At baseline, Nox4 TG mice had significantly enlarged hearts compared with WT, with elongated cardiomyocytes (increased by 18.5%, P < 0.01) and eccentric hypertrophy but well-preserved cardiac function by echocardiography and in vivo pressure-volume analysis. Animals were subjected to a chronic AngII infusion (AngII, 1.1 mg/kg/day) for 14 days. Whereas WT/AngII developed a 2.1-fold increase in interstitial cardiac fibrosis as compared with WT/saline controls (P < 0.01), TG/AngII mice developed significant less fibrosis (1.4-fold increase, P > 0.05), but there were no differences in cardiac hypertrophy or contractile function between the two groups. TG hearts displayed significantly decreased inflammatory cell infiltration with reduced levels of vascular cell adhesion molecule 1 in both the vasculature and myocardium compared with WT after AngII treatment. TG microvascular ECs stimulated with AngII in vitro supported significantly less leukocyte adhesion than WT ECs.
Conclusions:
A chronic increase in endothelial Nox4 stimulates physiological cardiac hypertrophy and protects against AngII-induced cardiac fibrosis by inhibiting EC activation and the recruitment of inflammatory cells.
... As TLRs and some adaptors are involved in many human disorders such as atherosclerosis [6], phosphatases regulating TLR signaling may also be implicated. For example, it has been suggested that DUSP1 is athero-protective, as DUSP1 induction is necessary for the anti-inflammatory effects of shear stress in endothelial cells [148]. In addition, Khadir et al. identified circulating DUSP1 as a potential biomarker for chronic inflammation in patients with cardiovascular diseases associated with atherosclerosis [149]. ...
Over the past 2 decades, pattern recognition receptors (PRRs) have been shown to be on the front line of many illnesses such as autoimmune, inflammatory, and neurodegenerative diseases as well as allergies and cancer. Among PRRs, toll-like receptors (TLRs) are the most studied family. Dissecting TLRs signaling turned out to be advantageous to elaborate efficient treatments to cure autoimmune and chronic inflammatory disorders. However, a broad understanding of TLR effectors is required to propose a better range of cures. In addition to kinases and E3 ubiquitin ligases, phosphatases emerge as important regulators of TLRs signaling mediated by NF-κB, type I interferons (IFN I) and Mitogen-Activated Protein Kinases signaling pathways. Here, we review recent knowledge on TLRs signaling modulation by different classes and subclasses of phosphatases. Thus, it becomes more and more evident that phosphatases could represent novel therapeutic targets to control pathogenic TLRs signaling.
Graphic abstract
... Here, Nrf2 inactivated p38 by inhibiting MKK3/6 signaling pathway and by inducing the activity of a negative regulator MKP-1 [162]. Moreover, MKP-1 is preferentially expressed by endothelial cells in aorta region where it inhibits p38-VCAM-1 signaling [163]. In this regard, some studies demonstrated the importance of the antioxidant enzyme HO-1, suggesting that the anti-inflammatory action of Nrf2 might in part depend on the modulation of the redox homeostasis. ...
Inflammation is a key driver in many pathological conditions such as allergy, cancer, Alzheimer’s disease, and many others, and the current state of available drugs prompted researchers to explore new therapeutic targets. In this context, accumulating evidence indicates that the transcription factor Nrf2 plays a pivotal role controlling the expression of antioxidant genes that ultimately exert anti-inflammatory functions. Nrf2 and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH- associated protein 1 (Keap1), play a central role in the maintenance of intracellular redox homeostasis and regulation of inflammation. Interestingly, Nrf2 is proved to contribute to the regulation of the heme oxygenase-1 (HO-1) axis, which is a potent anti-inflammatory target. Recent studies showed a connection between the Nrf2/antioxidant response element (ARE) system and the expression of inflammatory mediators, NF-κB pathway and macrophage metabolism. This suggests a new strategy for designing chemical agents as modulators of Nrf2 dependent pathways to target the immune response. Therefore, the present review will examine the relationship between Nrf2 signaling and the inflammation as well as possible approaches for the therapeutic modulation of this pathway.
... Furthermore, the molecular cascades that occur after acute increases in WSS rates in LSV graft ECs remain poorly understood. We, and others, have previously shown that arterial and venous EC respond differently to acute high shear stress, in both in vivo and ex vivo graft models 13,[22][23][24] . Here, we show that the NF-κB classical pathway is activated in the endothelium of LSV in response to acute exposure to arterial rates of shear stress and is a critical regulator of vascular inflammation in veins. ...
The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.
... Yamawaki et al. (24) demonstrated that blocking p38 in a rabbit artery perfusion model inhibited TNF-ainduced VCAM-1 expression. Zakkar et al. (29) demonstrated that the dephosphorylation of p-p38 via the phosphatase MKP-1 was associated with atheroprotective regions of the mouse aorta. Upon deleting MKP-1 with RNA interference, they demonstrated a significant increase in VCAM-1. ...
Endothelial up‐regulation of VCAM‐1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x‐box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF‐α‐stimulated VCAM‐1 expression. High‐resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM‐1)‐dependent, outside‐in signaling as a function of SS magnitude. Differential expression of VCAM‐1 and intercellular adhesion molecule 1 (ICAM‐1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM‐1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM‐1 at low SS (2 dyn/cm²), whereas inhibition of ERK1/2 attenuated peak ICAM‐1 at high SS (12 dyn/cm²). A shear stress‐regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM‐1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine‐induced ER stress response to spatially regulate inflammation promoting atherosclerosis.—Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress‐mediated inflammation by arterial endothelium. FASEB J. 33, 12888–12899 (2019). www.fasebj.org
... One of the negative regulator of JNK, MAP kinase phosphatase-1 (MKP-1), has been investigated in several different models of atherosclerosis with mixed results. MKP-1 has been identified to prevent endothelial activation in sites resistant to atherosclerosis [94] suggesting that deletion would augment disease. However, two different studies demonstrated that MKP 1 deletion in ApoE mice protected mice from atherosclerotic lesions [95,96]. ...
Cardiometabolic syndrome (CMS) describes the cluster of metabolic and cardiovascular diseases that are generally characterized by impaired glucose tolerance, intra-abdominal adiposity, dyslipidemia, and hypertension. CMS currently affects more than 25% of the world's population and the rates of diseases are rapidly rising. These CMS conditions represent critical risk factors for cardiovascular diseases including atherosclerosis, heart failure, myocardial infarction, and peripheral artery disease (PAD). Therefore, it is imperative to elucidate the underlying signaling involved in disease onset and progression. The c-Jun N-terminal Kinases (JNKs) are a family of stress signaling kinases that have been recently indicated in CMS. The purpose of this review is to examine the in vivo implications of JNK as a potential therapeutic target for CMS. As the constellation of diseases associated with CMS are complex and involve multiple tissues and environmental triggers, carefully examining what is known about the JNK pathway will be important for specificity in treatment strategies.
... In addition, the CFD simulations and subsequent statistical post-processing indicted that several OCT-defined erosions occurred in regions with modest or no stenosis, where the predominant flow feature was oscillatory shear stress (defined through OSI) [48,49]. Low time averaged wall shear stress or elevated OSI values strongly correlate with the focal predilection sites for atherosclerosis [32,[50][51][52] and tend to activate endothelial cells, priming them for inflammatory activation and apoptosis [28,34,53]. Inducing endothelial apoptosis experimentally can initiate thrombosis [54] supporting a role for apoptosis triggering endothelial erosion. ...
Endothelial erosion of atherosclerotic plaques and resulting thrombosis causes approximately 30% of acute coronary syndromes (ACS). As changes in the haemodynamic environment strongly influence endothelial function and contribute to plaque development, we reconstructed the coronary artery geometries of plaques with thrombi overlying intact fibrous caps from 17 ACS patients and performed computational fluid dynamic analysis. The results demonstrated that erosions frequently occur within areas of stenosis exposed to elevated flow. We recapitulated this flow environment in vitro, exposing human coronary artery endothelial cells to elevated flow and modelled smoking (a risk factor for erosion) by exposure to a combination of aqueous cigarette smoke extract and TNFα. This treatment induced endothelial detachment, which increased with pharmacological activation of the antioxidant system controlled by transcription factor Nrf2 (encoded by NFE2L2). The expression of Oxidative Stress Growth INhibitor genes OSGIN1 and OSGIN2 increased under these conditions and also in the aortas of mice exposed to cigarette smoke. Sustained high level expression of OSGIN1+2 resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and dysregulation of autophagy. Overexpression of either Nrf2 or OSGIN1+2 induced cell detachment, which did not depend on apoptosis, and could be partially rescued by inhibition of HSP70 using VER-155008, or AMP kinase activation using metformin. These findings demonstrate that under elevated flow, smoking-induced hyperactivation of Nrf2 can trigger endothelial cell detachment, highlighting a novel mechanism that could contribute to ACS involving endothelial erosion overlying stenotic plaques.
... Received 7 October 2018; Received in revised form 21 December 2018; Accepted 21 December 2018 suppresses inflammation by removing phosphate groups and inhibiting the production of pro-inflammatory cytokines (Zhao et al., 2005). MKP-1 protects arteries from inflammatory injury by inactivating p38 MAPK in the vascular endothelium (Zakkar et al., 2008). The anti-atherogenic effects of MKP-1 are related to the inhibition of vascular smooth muscle cell growth and vasoconstriction, as well as the promotion of endothelial cell migration (Lai et al., 1996). ...
Atherosclerosis (AS) is the common pathological basis of chronic cardiovascular diseases and is associated with inflammation and lipid metabolism dysfunction. Geniposide, the main active ingredient of Gardenia jasminoides Ellis fruit, exhibits a variety of anti-inflammatory and anti-oxidative functions; however, its role in AS remains unclear. The aim of this study was to investigate the mechanisms of geniposide in alleviating inflammation and thereby attenuating the development of AS. ApoE−/− mice were fed a high fat diet to induce AS and were treated with geniposide (50 mg/kg) for 12 weeks. Blood glucose and lipid levels were measured by biochemical analysis. H&E, Masson and Oil red O staining were performed to observe morphological changes and lipid deposition in the aorta and liver. Serum inflammatory cytokines were detected by ELISA. Dual-luciferase reporter gene assay was used to verify the target relationship between microRNA-101 (miR-101) and mitogen-activated protein kinase phosphatase-1 (MKP-1). The levels of miR-101, p-p38, and MKP-1 in the aorta were detected by qPCR and western blotting. The anti-inflammatory effect of geniposide in vitro was investigated in the RAW264.7 macrophage cell line. A miR-101 mimic and an inhibitor were used to study the effect of miR-101 on regulating the expression of the target MKP-1 and the downstream inflammatory cytokines. Geniposide treatment reduced lipid levels and plaque size in the mouse model of AS. Geniposide downregulated miR-101 to upregulate MKP-1 and suppress the production of inflammatory factors in vitro and in vivo. Geniposide suppressed the levels of inflammatory factors in the presence of the miR-101 mimic, whereas no obvious effect was observed in the miR-101 inhibitor group. We concluded that geniposide reduced the plaque size and alleviated inflammatory injury in ApoE−/− mice and RAW264.7 cells. The specific anti-inflammatory mechanism was related to the miR-101/ MKP-1/p38 signaling pathway.
... Inactivation of MAPKs by dual-specificity phosphatase 1 (DUSP1), also called mitogen-activated protein kinase phosphatase (MKP-1), was recently shown to be increased in circulating blood cells of CVD patients even after treatment with conventional drugs, such as statins [6]. However, the question as to whether DUSP1 influences atherogenesis and thereby CVDs is still controversial and remains to be clarified [7]; indeed, DUSP1 has been reported to be both antiatherogenic [8] and proatherogenic [9]. Likewise, DUSP1 has been linked to the atherosclerosis process in various animal and cell models of CVD [7]. ...
Background:
Cardiovascular disease (CVD) risks persist in patients despite the use of conventional treatments. This might be due to chronic inflammation as reflected in epidemiological studies associating circulating low-grade inflammatory markers with CVD recurrent events. Here, we explored this potential link by assessing plasma dual-specificity phosphatase 1 (DUSP1) levels and comparing them to high-sensitivity CRP (hsCRP) and oxidized low-density lipoprotein (oxLDL) levels and their associations to conventional CVD risk factors in confirmed CVD patients.
Methods:
Human adults with reported CVD (n = 207) and controls (n = 70) living in Kuwait were used in this study. Anthropometric and classical biochemical parameters were determined. Plasma levels of DUSP1, oxLDL, and hsCRP were measured using human enzyme-linked immunosorbent assay kits.
Results:
DUSP1 and hsCRP plasma levels and their least square means were higher in CVD cases, while oxLDL plasma levels were lower (p < 0.05). Multivariate logistic regression analysis showed that DUSP1 and hsCRP are independently associated with CVD in the studied population, as reflected by 2-fold and 1.5-fold increased risks with increased levels of DUSP1 and hsCRP, respectively. In our study, DUSP1 levels were found to be associated with CVD despite statin treatment and diabetes status (p < 0.05), whereas hsCRP mainly correlated with obesity markers.
Conclusions:
Circulating DUSP1 might be a predictor of chronic subclinical inflammation and residual risk in CVD patients, whereas our data suggest that the association between hsCRP and CVD is largely accounted for adiposity risk factors.
... Although P2X7 has not been linked previously to p38 activation in atheroprone endothelium, P2X7 activation is known to promote p38 phosphorylation in other cell types. 3,43,44 Our observations also support considerable evidence demonstrating a role for p38 activity in vascular inflammation, 45,46 atherosclerosis, 25,[47][48][49] and specifically in IL-8 and E-selectin activation. [50][51][52] Our findings align with previous studies linking P2X7 with atherosclerosis. ...
Objective:
Atherosclerosis is a focal disease occurring at arterial sites of disturbed blood flow that generates low oscillating shear stress. Endothelial inflammatory signalling is enhanced at sites of disturbed flow via mechanisms that are incompletely understood. The influence of disturbed flow on endothelial ATP receptors and downstream signalling was assessed.
Methods and results:
Cultured human endothelial cells were exposed to atheroprotective (high uniform) or atheroprone (low oscillatory) shear stress for 72 hours prior to assessment of ATP responses. Imaging of cells loaded with a calcium-sensitive fluorescent dye revealed that atheroprone flow enhanced extracellular calcium influx in response to 300µM BzATP. Pretreatment with pharmacological inhibitors demonstrated that this process required P2X7 receptors. The mechanism involved altered expression of P2X7, which was induced by atheroprone flow conditions in cultured cells. Similarly, en face staining of the murine aorta revealed enriched P2X7 expression at an atheroprone site. Functional studies in cultured endothelial cells showed that atheroprone flow induced p38 phosphorylation and up-regulation of E-selectin and IL-8 secretion via a P2X7-dependent mechanism. Moreover, genetic deletion of P2X7 significantly reduced E-selectin at atheroprone regions of the murine aorta.
Conclusions:
These findings reveal that P2X7 is regulated by shear forces leading to its accumulation at atheroprone sites that are exposed to disturbed patterns of blood flow. P2X7 promotes endothelial inflammation at atheroprone sites by transducing ATP signals into p38 activation. Thus P2X7 integrates vascular mechanical responses with purinergic signalling to promote endothelial dysfunction and may provide an attractive potential therapeutic target to prevent or reduce atherosclerosis.
... This endothelial dysfunction results in impairment of nitric oxide production, re-distribution of inter-EC junctions, altered communication, and loss of barrier function [39] (Fig. 1). Thus, disturbed flow provides predisposition for atherogenic tendencies by inducing EC dysfunction whereas uniform flow shields against atherosclerosis by enhancing endothelium integrity [40][41][42]. ...
Purpose of Review
The cell surface-attached extracellular glycocalyx (GCX) layer is a major contributor to endothelial cell (EC) function and EC-dependent vascular health and is a first line of defense against vascular diseases including atherosclerosis. Here, we highlight our findings regarding three GCX-dependent EC functions, which are altered when GCX is shed and in atherosclerosis. We discuss why the GCX is a viable option for the prevention and treatment of atherosclerosis.
Recent Findings
GCX regulated EC activities such as barrier and filtration function, active cell-to-cell communication, and vascular tone mediation contribute to function of the entire vascular wall. Atheroprone vessel regions, including bifurcation sites, exhibit breakdown in GCX. This GCX degradation allows increased lipid flux and thereby promotes lipid deposition in the vessel walls, a hallmark of atherosclerosis. GCX degradation also alters EC-to-EC communication while increasing EC-to-inflammatory cell interactions that enable inflammatory cells to migrate into the vessel wall. Inflammatory macrophages and foam cells, to be specific, appear in early stages of atherosclerosis. Furthermore, GCX degradation deregulates vascular tone, by causing ECs to reduce their expression of endothelial nitric oxide synthase (eNOS) which produces the vasodilator, nitric oxide. Loss of vasodilation supports vasoconstriction, which promotes the progression of atherosclerosis.
Summary
Common medicinal atherosclerosis therapies include lipid lowering and anti-platelet therapies. None of these treatments specifically target the endothelial GCX, although the GCX is at the front-line in atherosclerosis combat. This review demonstrates the viability of targeting the GCX therapeutically, to support proper EC functionality and prevent and/or treat atherosclerosis.
... When comparing the impact of altered haemodynamics (mainly shear stress), we noted that vECs respond to acute changes in shear stress by inducing a pro-inflammatory profile mediated by the activation of MAPK signalling pathway, while aECs remained resistant to such stimuli [67]. This differential response is related to the fact that aECs react to shear stress by upregulating antiinflammatory mediators such as MKP-1 and NRF-2, and this response appears to be innately reduced in vEC [68,69]. ...
The long saphenous vein is the most commonly used conduit in coronary artery bypass graft (CABG) surgery when bypassing multiple diseased arteries; however, its use is complicated by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis leading to compromised graft efficacy. Despite refinement of surgical techniques to improve graft patency, late vein graft failure remains a significant problem. Moreover, there is a lack of pharmacological interventions proven to be effective in the treatment of late vein graft failure. A greater understanding of the molecular nature of the disease and the interactions between endothelial and smooth muscle cells as a result of alterations in local haemodynamics may assist with designing future beneficial pharmacological interventions. Venous endothelial cells (ECs) are physiologically adapted to chronic low shear stress; however, once the graft is implanted into the arterial circulation, they become suddenly exposed to acute high levels of shear stress. A small number of in vitro and ex vivo studies have demonstrated that acute high shear stress is associated with the activation of a pro-inflammatory profile in saphenous vein ECs, which may be mediated by mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signalling pathways. The impact of acute changes in shear stress on venous ECs and the role of ECs in the development of intimal hyperplasia remains incomplete and is the subject of this review.
... Indeed, inhibition of PP2A in endothelial cells is associated with increased phosphorylation of p38 in unstimulated cells but does not affect oxidative stressinduced p38 activation [56]. Interestingly, in endothelial cells, MAP kinase phosphatase-1 (MKP-1) is also a negative regulator of p38 activity and p38-dependant VCAM expression in response to shear stress [57]. Recently, R. Singh proposed a model that predicts that MKP1 and TAB1 regulate p38 initial transient activation and its basal activity through positive and negative feedback loops in response to IL-1 [58]. ...
By gating the traffic of molecules and cells across the vessel wall, endothelial cells play a central role in regulating cardiovascular functions and systemic homeostasis and in modulating pathophysiological processes such as inflammation and immunity. Accordingly, the loss of endothelial cell integrity is associated with pathological disorders that include atherosclerosis and cancer. The p38 mitogen-activated protein kinase (MAPK) cascades are major signaling pathways that regulate several functions of endothelial cells in response to exogenous and endogenous stimuli including growth factors, stress and cytokines. The p38 MAPK family contains four isoforms p38α, p38β, p38γ and p38δ that are encoded by four different genes. They are all widely expressed although to different levels in almost all human tissues. p38α/MAPK14, that is ubiquitously expressed is the prototype member of the family and is referred here as p38. It regulates the production of inflammatory mediators, and controls cell proliferation, differentiation, migration and survival. Its activation in endothelial cells leads to actin remodeling, angiogenesis, DNA damage response and thereby has major impact on cardiovascular homeostasis, and on cancer progression. In this manuscript, we review the biology of p38 in regulating endothelial functions especially in response to oxidative stress and during the metastatic process.
... JNK can phosphorylate several transcription factors, including c-Jun, ATF2, Elk-2, RXRa, NFAT4, HSF-1, and p53, which can contribute to both endothelial inflammation and apoptosis [9][10][11][12][13]. It has been well established that the induction of VCAM-1, ICAM-1, and E-selectin by TNF-a is regulated by JNK [14][15][16]. ...
Atherosclerosis rarely develops in the region of arteries exposed to undisturbed flow (u-flow, unidirectional flow). Instead, atherogenesis occurs in the area exposed to disturbed flow (d-flow, multidirectional flow). Based on these general pathohistological observations, u-flow is considered to be athero-protective, while d-flow is atherogenic. The fact that u-flow and d-flow induce such clearly different biological responses in the wall of large arteries indicates that these two types of flow activate each distinct intracellular signaling cascade in vascular endothelial cells (ECs), which are directly exposed to blood flow. The ability of ECs to differentially respond to the two types of flow provides an opportunity to identify molecular events that lead to endothelial dysfunction and atherosclerosis. In this review, we will focus on various molecular events, which are differentially regulated by these two flow types. We will discuss how various kinases, ER stress, inflammasome, SUMOylation, and DNA methylation play roles in the differential flow response, endothelial dysfunction, and atherosclerosis. We will also discuss the interplay among the molecular events and how they coordinately regulate flow-dependent signaling and cellular responses. It is hoped that clear understanding of the way how the two flow types beget each unique phenotype in ECs will lead us to possible points of intervention against endothelial dysfunction and cardiovascular diseases.
... Laminar shear stress promotes glycocalyx formation in ECs [75][76][77] and thick and negatively charged endothelial glycocalyx improves the barrier function [78,79]. In contrast, the exposure to disturbed, non-uniform shear stress renders endothelial cells prone to inflammatory activation, and increases the permeability of monolayer to immune cells and blood components [74,[80][81][82]. Additionally, upon exposure to non-uniform shear stress, the glycocalyx becomes stiffer and loses its buffering function [75][76][77], thus further increasing the endothelial permeability [83]. ...
Abstract. Recent years have brought about substantial advances in nanomedical approaches to human diseases. Novel therapeutic and diagnostic nanoparticulate agents can be delivered via several routes, including enteral, transdermal, inhalational, as well as parenteral application. For nanoparticles administered via intravascular route, endothelial cells represent the firstcontact cells and their responses to the candidate nanosystems should be considered before clinical applications. Additionally, a number of drug-delivery nanosystems that target endothelium are currently proposed. It is increasingly evident that the future
progress in the treatment of many human maladies, including cardiovascular diseases and cancer, will be closely bound with the development of endothelium-targeting nanosystems. As endothelial cells in circulation are constantly exposed to shear stress, investigating nanoparticle-endothelial interactions under flow conditions is necessary to estimate the cell Responses in physiological-like settings. This mini-review focuses on the recently reported studies assessing the uptake of circulating nanoparticles and their biological effects on endothelial cells, and summarizes the targeting approaches to enhance endothelial internalization of nanoparticles under flow conditions.
... Thus we hypothesise that ivabradine suppresses EC expression of VCAM-1 via WSS-dependent induction of eNOS. However, it is also plausible that the enhancement of WSS and/or modulation of vascular mechanical stress in response to ivabradine reduces inflammation via other mechanosensitive anti-inflammatory molecules including KLF2, Nrf2 and MKP-1 (38,39). ...
Blood flow generates wall shear stress (WSS) which alters endothelial cell (EC) function. Low WSS promotes vascular inflammation and atherosclerosis whereas high uniform WSS is protective. Ivabradine decreases heart rate leading to altered haemodynamics. Besides its cardio-protective effects, ivabradine protects arteries from inflammation and atherosclerosis via unknown mechanisms. We hypothesised that ivabradine protects arteries by increasing WSS to reduce vascular inflammation. Hypercholesterolaemic mice were treated with ivabradine for seven weeks in drinking water or remained untreated as a control. En face immunostaining demonstrated that treatment with ivabradine reduced the expression of pro-inflammatory VCAM-1 (p<0.01) and enhanced the expression of anti-inflammatory eNOS (p<0.01) at the inner curvature of the aorta. We concluded that ivabradine alters EC physiology indirectly via modulation of flow because treatment with ivabradine had no effect in ligated carotid arteries in vivo, and did not influence the basal or TNFα-induced expression of inflammatory (VCAM-1, MCP-1) or protective (eNOS, HMOX1, KLF2, KLF4) genes in cultured EC. We therefore considered whether ivabradine can alter WSS which is a regulator of EC inflammatory activation. Computational fluid dynamics demonstrated that ivabradine treatment reduced heart rate by 20 % and enhanced WSS in the aorta. In conclusion, ivabradine treatment altered haemodynamics in the murine aorta by increasing the magnitude of shear stress. This was accompanied by induction of eNOS and suppression of VCAM-1, whereas ivabradine did not alter EC that could not respond to flow. Thus ivabradine protects arteries by altering local mechanical conditions to trigger an anti-inflammatory response.
... The mechanisms that link WSS with MAP kinases involve KLF2, which is induced by high WSS and subsequently inhibits phosphorylation and nuclear localization of AP-1 family members c-Jun and ATF2 [64][65][66] . High WSS also activates the transcription factor Nrf2 67-69 which function together with MAP kinase phosphatase-1 to reduce inflammation by dephosphorylating p38 and JNK 69,70 . The inhibitory effect of high WSS on inflammatory MAP kinases is also mediated via downregulation of thioredoxin interacting protein (TXNIP) leading to inhibition of apoptosis signal-regulating kinase 1, a kinase that acts upstream of p38 and JNK 71 . ...
Atherosclerosis remains a major cause of morbidity and mortality worldwide, and a thorough understanding of the underlying pathophysiological mechanisms is crucial for the development of new therapeutic strategies. Although atherosclerosis is a systemic inflammatory disease, coronary atherosclerotic plaques are not uniformly distributed in the vascular tree. Experimental and clinical data highlight that biomechanical forces, including wall shear stress (WSS) and plaque structural stress (PSS), have an important role in the natural history of coronary atherosclerosis. Endothelial cell function is heavily influenced by changes in WSS, and longitudinal animal and human studies have shown that coronary regions with low WSS undergo increased plaque growth compared with high WSS regions. Local alterations in WSS might also promote transformation of stable to unstable plaque subtypes. Plaque rupture is determined by the balance between PSS and material strength, with plaque composition having a profound effect on PSS. Prospective clinical studies are required to ascertain whether integrating mechanical parameters with medical imaging can improve our ability to identify patients at highest risk of rapid disease progression or sudden cardiac events.
Endothelial erosion of atherosclerotic plaques is the underlying cause of approximately 30% of acute coronary syndromes (ACS). As the vascular endothelium is profoundly affected by the haemodynamic environment to which it is exposed, we employed computational fluid dynamic (CFD) analysis of the luminal geometry from 17 patients with optical coherence tomography (OCT)-defined plaque erosion, to determine the flow environment permissive for plaque erosion. Our results demonstrate that 15 of the 17 cases analysed occurred on stenotic plaques with median 31% diameter stenosis (interquartile range 28–52%), where all but one of the adherent thrombi located proximal to, or within the region of maximum stenosis. Consequently, all flow metrics related to elevated flow were significantly increased (time averaged wall shear stress, maximum wall shear stress, time averaged wall shear stress gradient) with a reduction in relative residence time, compared to a non-diseased reference segment. We also identified two cases that did not exhibit an elevation of flow, but occurred in a region exposed to elevated oscillatory flow. Our study demonstrates that the majority of OCT-defined erosions occur where the endothelium is exposed to elevated flow, a haemodynamic environment known to evoke a distinctive phenotypic response in endothelial cells.
Introduction:
Ischemic stroke is the third leading cause of death. There is no known treatment or cure for the disease. Moreover, the pathological mechanism of ischemic stroke remains unclear.
Objective:
We aimed to identify potential microRNAs (miRNAs) and mRNAs, contributing to understanding the pathology of ischemic stroke.
Methods:
First, the data of miRNA and mRNA were downloaded for differential expression analysis. Then, the regulatory network between miRNA and mRNAs was constructed. Third, top 100 differentially expressed mRNAs were used to construct a protein-protein interaction network followed by the function annotation of mRNAs. In addition, in vitro experiment was used to validate the expression of mRNAs. Last, receiver operating characteristic diagnostic analysis of differentially methylated genes was performed.
Results:
Totally, up to 26 differentially expressed miRNAs and 1,345 differentially expressed mRNAs were identified. Several regulatory interaction pairs between miRNA and mRNAs were identified, such as hsa-miR-206-HMGCR/PICALM, hsa-miR-4491-TMEM97, hsa-miR-3622b-5p/hsa-miR-548k-KLF12, and hsa-miR-302a-3p/hsa-miR-3145-3p-CTSS. MAPK signaling pathway (involved DUSP1) and the Notch signaling pathway (involved NUMB and CREBBP) were identified. The expression validation of KLF12, ARG1, ITGAM, SIRT4, SERPINH1, and DUSP1 was consistent with the bioinformatics analysis. Interestingly, hsa-miR-206, hsa-miR-4491, hsa-miR-3622b-5p, hsa-miR-548k, hsa-miR-302a-3p, hsa-miR-3145-3p, KLF12, and ID3 had the potential diagnostic value of ischemic stroke.
Conclusions:
The identified differentially expressed miRNAs and mRNAs may be associated with the development of ischemic stroke.
Background and objectives:
Wall shear stress plays a critical role in neointimal hyperplasia after stent implantation. It has been found that there is an inverse relation between wall shear stress and neointimal hyperplasia. This study hypothesized that the increase of arterial wall shear stress caused by arteriovenous fistula could reduce neointimal hyperplasia after stents implantation.
Methods and results:
Thirty-six male rabbits were randomly divided into three groups: STENT, rabbits received stent implantation into right common carotid artery; STENT/arteriovenous fistula, rabbits received stent implantation into right common carotid artery and carotid-jugular arteriovenous fistula; Control, rabbits received no treatment. After 21 days, stented common carotid artery specimens were harvested for histological staining and protein expression analysis. In STENT group, wall shear stress maintained at a low level from 43.2 to 48.9% of baseline. In STENT/arteriovenous fistula group, wall shear stress gradually increased to 86% over baseline. There was a more significant neointimal hyperplasia in group STENT compared with the STENT/arteriovenous fistula group (neointima area: 0.87 mm2 versus 0.19 mm2; neointima-to-media area ratio: 1.13 versus 0.18). Western blot analysis demonstrated that the protein level of endothelial nitric oxide synthase in STENT group was significantly lower than that in STENT/arteriovenous fistula group, but the protein levels of proliferating cell nuclear antigen, vascular cell adhesion molecule 1, phospho-p38 mitogen-activated protein kinase (Pp38), and phospho-c-Jun N-terminal kinase in STENT group were significantly higher than that in the STENT group.
Conclusion:
High wall shear stress caused by arteriovenous fistula as associated with the induction in neointimal hyperplasia after stent implantation. The underlying mechanisms may be related to modulating the expression and activation of endothelial nitric oxide synthase, vascular cell adhesion molecule 1, p38, and c-Jun N-terminal kinase.
Background:
Inflammatory process is one of the mechanisms by which our body upholds us from pathogens such as parasites, bacteria, viruses, and other harmful microorganisms. Inflammatory stimuli activate many intracellular signaling pathways such as nuclear factor-kB (NF-kB) pathway and three mitogen-activated protein kinase (MAPK) pathways which are mediated through extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. The p38 has evolved as an enticing target in treating many persistent inflammatory diseases. Hence, designing novel p38 inhibitors targeting MAPK pathways has acquired significance.
Objective:
Peruse to identify the lead target to discover novel p38MAPK inhibitors with different scaffolds having improved selectivity over the prototype drugs.
Methods:
Structure and the binding sites of p38MAPK were focused. Various scaffolds designed for inhibition and the molecules which have entered the clinical trials are discussed.
Results:
This review aspires to present the available information on the structure and the 3D binding sites of p38MAPK, various scaffolds designed for imidazole, urea, benzamide, azoles, quinoxaline, chromone, ketone as a potent p38MAPK inhibitors and their SAR studies and the molecules which have entered the clinical trials.
Conclusion:
Development of successful selective p38MAPK inhibitors in inflammatory diseases are in progress despite of all challenges. It was speculated that p38MAPK also plays an important role in treating diseases such as neuroinflammation, arterial inflammation, vascular inflammation, cancer and so on which are posing the world with treatment challenges. In this review, clinical trials of drugs are discussed related to inflammatory and its related diseases. Research is in progress to design and develop novel p38MAPK inhibitors with minimal side effects.
Flowing blood generates a frictional force called shear stress that has major effects on vascular function. Branches and bends of arteries are exposed to complex blood flow patterns that exert low or low oscillatory shear stress, a mechanical environment that promotes vascular dysfunction and atherosclerosis. Conversely, physiologically high shear stress is protective. Endothelial cells are critical sensors of shear stress but the mechanisms by which they decode complex shear stress environments to regulate physiological and pathophysiological responses remain incompletely understood. Several laboratories have advanced this field by integrating specialized shear-stress models with systems biology approaches, including transcriptome, methylome and proteome profiling and functional screening platforms, for unbiased identification of novel mechanosensitive signalling pathways in arteries. In this Review, we describe these studies, which reveal that shear stress regulates diverse processes and demonstrate that multiple pathways classically known to be involved in embryonic development, such as BMP–TGFβ, WNT, Notch, HIF1α, TWIST1 and HOX family genes, are regulated by shear stress in arteries in adults. We propose that mechanical activation of these pathways evolved to orchestrate vascular development but also drives atherosclerosis in low shear stress regions of adult arteries.
The vascular endothelium is a critical interface, which separates the organs from the blood and its contents. The endothelium has a wide variety of functions and maintenance of endothelial homeostasis is a multi-dimensional active process, disruption of which has potentially deleterious consequences if not reversed. Vascular injury predisposes to endothelial apoptosis, dysfunction and development of atherosclerosis. Endothelial dysfunction is an end-point, a central feature of which is increased ROS generation, a reduction in endothelial nitric oxide synthase and increased nitric oxide consumption. A dysfunctional endothelium is a common feature of diseases including rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus and chronic renal impairment. The endothelium is endowed with a variety of constitutive and inducible mechanisms that act to minimise injury and facilitate repair. Endothelial cytoprotection can be enhanced by exogenous factors such as vascular endothelial growth factor, prostacyclin and laminar shear stress. Target genes include endothelial nitric oxide synthase, heme oxygenase-1, A20 and anti-apoptotic members of the B cell lymphoma protein-2 family. In light of the importance of endothelial function, and the link between its disruption and the risk of atherothrombosis, interest has focused on therapeutic conditioning and reversal of endothelial dysfunction. A detailed understanding of cytoprotective signalling pathways, their regulation and target genes is now required to identify novel therapeutic targets. The ultimate aim is to add vasculoprotection to current therapeutic strategies for systemic inflammatory diseases, in an attempt to reduce vascular injury and prevent or retard atherogenesis.
This chapter deals with the understanding of protein phosphorylation events regulated by protein kinases and phosphatases and the mechanisms potentially relevant for atherosclerosis. The mitogen-activated protein kinase (MAPK) signaling cascades are a universal signaling system used by eukaryotic cells. The MAPK cascade consists of a set of kinases that are activated in series to transduce an external signal into the nucleus. Regarding atherosclerosis, MAPK pathways have been implicated in foam cell formation, endothelial cell activation, and vascular smooth muscle migration and proliferation. Several receptor tyrosine kinases (RTKs), in a variety of cells, have been shown to be important for the development of atherosclerosis. Finally, the chapter provides much information about knockout mouse experiments involving protein kinases implicated in pathophysiological signal transduction and demonstrates critical roles for these kinases and the downstream protein phosphorylation events which are likely to be involved in atherosclerosis in humans.
The vascular endothelium is continually exposed to hemodynamic stress induced by the frictional force of blood flow across its surface and pressure changes throughout each cardiac cycle. The interaction between fluid shear stress (FSS) and the endothelium is critical in maintaining vascular homeostasis via the integration of biomechanical forces with signal transduction to maintain redox balance.1 High unidirectional laminar shear (US) forces characteristic of straight regions of the vasculature have been demonstrated to be protected from atherosclerosis, whereas areas of the vasculature exposed to oscillatory disturbed shear (OS) forces, prevalent in curvatures and branches in blood vessels with complex geometry where blood flow forms localized flow-separation zones that include regions of low shear and flow reversal, are prone to atherogenesis.2 Endothelial cells (ECs) respond to the changes in shear stress to modulate redox signaling,3 which leads to alterations of pro- and antioxidant gene expression, inflammatory phenotype, cell alignment, and structural remodelling of vessels.4 The transcription factor nuclear factor (erythroid-derived 2)–like 2 (Nrf2) has been well characterized to play an important role in the antioxidant response element (ARE)–mediated expression of a group of genes encoding phase II detoxification enzymes and antioxidant proteins, such as glutathione-S-transferase, heme oxygenase-1 (HO-1), peroxiredoxin 1 (Prx-1), and nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase-1 (NQO1).5–7 The transcriptional regulation of these genes by Nrf2 can be enhanced by phytonutrients, and it plays a key role for the protection of vascular EC from oxidative stress during the pathogenesis of vascular diseases.8–10 It has been reported that EC exposed to OS but not to US flow patterns in both in vivo and in vitro models exhibits greater nuclear factor-κB (NF-κB) activity and deficiencies in the Nrf2/ARE redox signaling pathway and …
Endothelial cell surface expression of VCAM-1 is one of the initial steps in the pathogenesis of atherosclerosis. The inflammatory
response transcription factor nuclear factor (NF)-κB plays an important role in the regulation ofVCAM-1 expression by various stimuli including tumor necrosis factor (TNF)-α. Other transcription factors may modulate this response
through interaction with NF-κB factors. Since c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular endothelium
during proinflammatory conditions, we investigated the role of AP-1 proteins in the expression of VCAM-1 by TNF-α in SV40 immortalized human microvascular endothelial cells (HMEC). TNF-α induced expression of both early protooncogenes,
c-fos and c-jun. The ability of TNF-α to activate the κB-motif (κL-κR)-dependent VCAM-1promoter-chloramphenicol acetyltransferase (CAT) reporter gene lacking a consensus AP-1 element was markedly inhibited by
co-transfection of the expression vector encoding c-fosribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or c-jun oligonucleotides. Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-κB-mediated
transactivation of the VCAM-1promoter-CAT reporter gene. Furthermore the c-Fos encoding expression vector potentiated by 2-fold the transactivation activity
of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain of p65 and the DNA binding domain of the
yeast transcriptional factor Gal-4). Consistent with the promoter studies, curcumin and NDGA, inhibitors of AP-1 activation,
markedly inhibited the ability of TNF-α to activate the expression of VCAM-1 mRNA levels at concentrations that did not inhibit the activation of NF-κB. In gel mobility supershift assays, the antibodies
to c-Fos or c-Jun inhibited the binding of TNF-α-activated nuclear NF-κB to the κL-κR, suggesting that both c-Fos and c-Jun
interacted with NF-κB. These results suggest that AP-1 proteins may mediate the effect of TNF-α in the regulation of VCAM-1 expression through interaction with NF-κB factors in endothelial cells.
The inflammatory cytokine TNF-alpha stimulates several presumed pro-atherogenic signaling events in endothelial cells (ECs), including activation of c-Jun NH(2)-terminal kinase (JNK) and induction of E-selectin. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, is required for TNF-mediated JNK activation. TNF activates ASK1 in part by dissociating ASK1 from its inhibitor 14-3-3. Because the risk of atherosclerosis is decreased in regions of steady laminar flow, we hypothesized that laminar flow inhibits proinflammatory cytokine-mediated activation of JNK. Steady laminar flow inhibited both TNF activation of ASK1 and JNK. Inhibition of ASK1 by flow correlated with increased association of ASK1 with 14-3-3. A constitutively active form of ASK1 lacking the 14-3-3-binding site (ASK1-Delta NS967A) was not inhibited by flow. These data establish ASK1 as a target for flow-mediated inhibition of cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator in ECs.
VCAM-1 and ICAM-1 are endothelial adhesion molecules of the Ig gene superfamily that may participate in atherogenesis by promoting monocyte accumulation in the arterial intima. Both are expressed in regions predisposed to atherosclerosis and at the periphery of established lesions, while ICAM-1 is also expressed more broadly. To evaluate functions of VCAM-1 in chronic disease, we disrupted its fourth Ig domain, producing the murine Vcam1(D4D) allele. VCAM-1(D4D) mRNA and protein were reduced to 2-8% of wild-type allele (Vcam1(+)) levels but were sufficient to partially rescue the lethal phenotype of VCAM-1-null embryos. After crossing into the LDL receptor-null background, Vcam1(+/+) and Vcam1(D4D/D4D) paired littermates were generated from heterozygous intercrosses and fed a cholesterol-enriched diet for 8 weeks. The area of early atherosclerotic lesions in the aorta, quantified by en face oil red O staining, was reduced significantly in Vcam1(D4D/D4D) mice, although cholesterol levels, lipoprotein profiles, and numbers of circulating leukocytes were comparable to wild-type. In contrast, deficiency of ICAM-1 either alone or in combination with VCAM-1 deficiency did not alter nascent lesion formation. Therefore, although expression of both VCAM-1 and ICAM-1 is upregulated in atherosclerotic lesions, our data indicate that VCAM-1 plays a dominant role in the initiation of atherosclerosis.
MAP kinases are among the most ancient signal transduction pathways and are widely used throughout evolution in many physiological processes. In mammalian species, MAP kinases are involved in all aspects of immune responses, from the initiation phase of innate immunity, to activation of adaptive immunity, and to cell death when immune function is complete. In this review, we summarize recent progress in understanding the function and regulation of MAP kinase pathways in these phases of immune responses.
The endothelium expresses a large repertoire of genes under apparent transcriptional control of biomechanical forces, many of which are neither cell-type nor flow specific. We set out to identify genes that are uniquely flow responsive in human vascular endothelial cells. Transcriptional profiling using commercial DNA microarrays identified 12 of 18 000 genes that were modulated at least 5-fold after 24 hours of steady laminar flow (25 dyne/cm(2)). After a 7-day exposure to unidirectional pulsatile flow (19 +/- 12 dyne/cm(2)), only 3 of 12 remained elevated at least 5-fold. A custom microarray of ~300 vascular cell-related gene fragments was constructed, and expression analysis revealed that many flow-induced genes are also induced by at least one of the following agents: tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), transforming growth factor-beta, vascular endothelial growth factor, or thrombin, indicating a more general role in adaptive or stress responses. Most flow-induced genes were also induced by TNF-alpha but not IL-1beta, suggesting the involvement of reactive oxygen species. A limited panel of genes that are unique for flow-exposed cultures was identified, including lung Krüppel-like factor (LKLF/KLF2) and cytochrome P450 1B1 (CYP1B1). In marked contrast, both these genes were substantially repressed by TNF-alpha. LKLF but not CYP1B1 mRNA was detected exclusively in the vascular endothelium of healthy human aorta by in situ hybridization and appeared to be flow regulated. To date LKLF is the first endothelial transcription factor that is uniquely induced by flow and might therefore be at the molecular basis of the physiological healthy, flow-exposed state of the endothelial cell.
The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1beta and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.
Atherosclerotic lesion localization to regions of disturbed flow within certain arterial geometries, in humans and experimental animals, suggests an important role for local hemodynamic forces in atherogenesis. To explore how endothelial cells (EC) acquire functional/dysfunctional phenotypes in response to vascular region-specific flow patterns, we have used an in vitro dynamic flow system to accurately reproduce arterial shear stress waveforms on cultured human EC and have examined the effects on EC gene expression by using a high-throughput transcriptional profiling approach. The flow patterns in the carotid artery bifurcations of several normal human subjects were characterized by using 3D flow analysis based on actual vascular geometries and blood flow profiles. Two prototypic arterial waveforms, "athero-prone" and "athero-protective," were defined as representative of the wall shear stresses in two distinct regions of the carotid artery (carotid sinus and distal internal carotid artery) that are typically "susceptible" or "resistant," respectively, to atherosclerotic lesion development. These two waveforms were applied to cultured EC, and cDNA microarrays were used to analyze the differential patterns of EC gene expression. In addition, the differential effects of athero-prone vs. athero-protective waveforms were further characterized on several parameters of EC structure and function, including actin cytoskeletal organization, expression and localization of junctional proteins, activation of the NF-kappaB transcriptional pathway, and expression of proinflammatory cytokines and adhesion molecules. These global gene expression patterns and functional data reveal a distinct phenotypic modulation in response to the wall shear stresses present in atherosclerosis-susceptible vs. atherosclerosis-resistant human arterial geometries.
The Nrf2-Keap1 system coordinately regulates cytoprotective gene expression via the antioxidant responsive element (ARE). The expression of several ARE-regulated genes was found to be up-regulated in endothelial cells by laminar shear stress, suggesting that Nrf2 contributes to the anti-atherosclerosis response via the ARE. To gain further insight into the roles that Nrf2 plays in the development of atherosclerosis, we examined how Nrf2 regulates gene expression in response to anti-atherogenic laminar flow (L-flow) or pro-atherogenic oscillatory flow (O-flow). Exposure of human aortic endothelial cells (HAECs) to L-flow, but not to O-flow, induced the expression of cytoprotective genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1) by 5-fold and heme oxygenase-1 by 8-fold. The critical contribution of Nrf2 to the expression induced by L-flow was ascertained in siRNA-mediated knock-down experiments. Two cyclooxygenase-2 (COX-2) specific inhibitors attenuated Nrf2 nuclear accumulation in the acute phase of L-flow exposure. A downstream product of COX-2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), activated the Nrf2 regulatory pathway in HAECs through binding to the cysteines of Keap1. These results demonstrate that 15d-PGJ2 is essential for L-flow to activate Nrf2 and induce anti-atherosclerotic gene expression. Whereas both L-flow and O-flow induced the nuclear accumulation of Nrf2 to comparable levels, chromatin immunoprecipitation analysis revealed that Nrf2 binding to the NQO1 ARE was significantly diminished in the case of O-flow compared with that of L-flow. These results suggest that O-flow inhibits Nrf2 activity at the DNA binding step, thereby suppressing athero-protective gene expression and hence predisposing the blood vessels to the formation of atherosclerosis.
Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodelling, cardiac development and atherogenesis, but the mechanisms of transduction are poorly understood. Previous work showed that the conversion of integrins to a high-affinity state mediates a subset of shear responses, including cell alignment and gene expression. Here we investigate the pathway upstream of integrin activation. PECAM-1 (which directly transmits mechanical force), vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol-3-OH kinase) comprise a mechanosensory complex. Together, these receptors are sufficient to confer responsiveness to flow in heterologous cells. In support of the relevance of this pathway in vivo, PECAM-1-knockout mice do not activate NF-kappaB and downstream inflammatory genes in regions of disturbed flow. Therefore, this mechanosensing pathway is required for the earliest-known events in atherogenesis.
Regions of the arterial tree exposed to laminar flow, which exerts high shear stress, are protected from inflammation, endothelial cell (EC) death and atherosclerosis. TNFalpha activates NF-kappaB transcription factors, which potentially exert dual functions by inducing both proinflammatory and cytoprotective transcripts. We assessed whether laminar shear stress protects EC by modulating NF-kappaB function. Human umbilical vein EC (HUVEC) were cultured under shear stress (12 dynes/cm2 for 16 h) using a parallel-plate flow chamber or were maintained in static conditions. Comparative real-time PCR revealed that preshearing significantly alters transcriptional responses to TNFalpha by enhancing the expression of cytoprotective molecules (Bcl-2, MnSOD, GADD45beta, A1) and suppressing proinflammatory transcripts (E-selectin, VCAM-1, IL-8). We demonstrated using assays of nuclear localization, NF-kappaB subunit phosphorylation, DNA-binding, and transcriptional activity that NF-kappaB is activated by TNFalpha in presheared HUVEC. Furthermore, a specific inhibitor revealed that NF-kappaB is essential for the induction of cytoprotective transcripts in presheared EC. Finally, we observed that NF-kappaB can be activated in vascular endothelium exposed to laminar shear stress in NF-kappaB-luciferase reporter mice, thus validating our cell culture experiments. We conclude that shear stress primes EC for enhanced NF-kappaB-dependent cytoprotective responsiveness while attenuating proinflammatory activation. Thus modulation of NF-kappaB function may underlie the atheroprotective effects of laminar shear stress.
Toll-like receptors (TLRs) are pattern recognition receptors of innate immunity. TLRs initiate inflammatory pathways that may exacerbate chronic inflammatory diseases like atherosclerosis. En face laser scanning confocal microscopy (LSCM) of isolated aortic segments revealed the distribution of intimal TLR2 expression and the atheroprotective outcomes resulting from a TLR2 deficiency. TLR2 expression was restricted to endothelial cells in regions of disturbed blood flow, such as the lesser curvature region, in atherosclerosis-prone, low-density lipoprotein receptor-deficient (LDLr(-/-)) mice. Diet-induced hyperlipidemia in LDLr(-/-) mice increased this regional endothelial TLR2 expression. Bone marrow (BM) reconstitution of LDLr(-/-) and LDLr(-/-)TLR2(-/-) mice created chimeric mice with green fluorescent protein (GFP) expression in BM-derived cells (BMGFP(+)). Lesser curvature BMGFP(+) leukocyte accumulation, lipid accumulation, foam cell generation and endothelial cell injury were all increased by hyperlipidemia, whereas hyperlipidemic double mutant BMGFP(+)LDLr(-/-)TLR2(-/-) mice had reduced BMGFP(+) leukocyte accumulation, lipid accumulation, foam cells, and endothelial cell injury. This is the first report of in vivo site-specific expression of endothelial cell TLR2. Expression of this receptor on endothelial cells contributed to early atherosclerotic processes in lesion-prone areas of the mouse aorta.
Atherosclerosis, formerly considered a bland lipid storage disease, actually involves an ongoing inflammatory response. Recent advances in basic science have established a fundamental role for inflammation in mediating all stages of this disease from initiation through progression and, ultimately, the thrombotic complications of atherosclerosis. These new findings provide important links between risk factors and the mechanisms of atherogenesis. Clinical studies have shown that this emerging biology of inflammation in atherosclerosis applies directly to human patients. Elevation in markers of inflammation predicts outcomes of patients with acute coronary syndromes, independently of myocardial damage. In addition, low-grade chronic inflammation, as indicated by levels of the inflammatory marker C-reactive protein, prospectively defines risk of atherosclerotic complications, thus adding to prognostic information provided by traditional risk factors. Moreover, certain treatments that reduce coronary risk also limit inflammation. In the case of lipid lowering with statins, this anti-inflammatory effect does not appear to correlate with reduction in low-density lipoprotein levels. These new insights into inflammation in atherosclerosis not only increase our understanding of this disease, but also have practical clinical applications in risk stratification and targeting of therapy for this scourge of growing worldwide importance.
c-Jun NH2-terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 μmol/L hydrogen peroxide (H2O2) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3±1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress=12 dyne/cm²) for 10 minutes significantly decreased H2O2-induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H2O2-induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells.
The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level.
Endothelial cell surface expression of VCAM-1 is one of the initial steps in the pathogenesis of atherosclerosis. The inflammatory response transcription factor nuclear factor (NF)-kappaB plays an important role in the regulation of VCAM-1 expression by various stimuli including tumor necrosis factor (TNF)-alpha. Other transcription factors may modulate this response through interaction with NF-kappaB factors. Since c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular endothelium during proinflammatory conditions, we investigated the role of AP-1 proteins in the expression of VCAM-1 by TNF-alpha in SV40 immortalized human microvascular endothelial cells (HMEC). TNF-alpha induced expression of both early protooncogenes, c-fos and c-jun. The ability of TNF-alpha to activate the kappaB-motif (kappaL-kappaR)-dependent VCAM-1 promoter-chloramphenicol acetyltransferase (CAT) reporter gene lacking a consensus AP-1 element was markedly inhibited by co-transfection of the expression vector encoding c-fos ribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or c-jun oligonucleotides. Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-kappaB-mediated transactivation of the VCAM-1 promoter-CAT reporter gene. Furthermore the c-Fos encoding expression vector potentiated by 2-fold the transactivation activity of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain of p65 and the DNA binding domain of the yeast transcriptional factor Gal-4). Consistent with the promoter studies, curcumin and NDGA, inhibitors of AP-1 activation, markedly inhibited the ability of TNF-alpha to activate the expression of VCAM-1 mRNA levels at concentrations that did not inhibit the activation of NF-kappaB. In gel mobility supershift assays, the antibodies to c-Fos or c-Jun inhibited the binding of TNF-alpha-activated nuclear NF-kappaB to the kappaL-kappaR, suggesting that both c-Fos and c-Jun interacted with NF-kappaB. These results suggest that AP-1 proteins may mediate the effect of TNF-alpha in the regulation of VCAM-1 expression through interaction with NF-kappaB factors in endothelial cells.
Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.
The recruitment of mononuclear leukocytes and formation of intimal macrophage-rich lesions at specific sites of the arterial tree are key events in atherogenesis. Inducible endothelial cell adhesion molecules may participate in this process. In aortas of normal chow-fed wild-type mice and rabbits, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), but not E-selectin, were expressed by endothelial cells in regions predisposed to atherosclerotic lesion formation. En face confocal microscopy of the mouse ascending aorta and proximal arch demonstrated that VCAM-1 expression was increased on the endothelial cell surface in lesion-prone areas. ICAM-1 expression extended into areas protected from lesion formation. Hypercholesterolemia induced atherosclerotic lesion formation in rabbits, LDL receptor and apolipoprotein E knockout mice, and Northern blot analysis demonstrated increased steady-state mRNA levels of VCAM-1 and ICAM-1, but not of E-selectin. Immunohistochemical staining revealed that VCAM-1 and ICAM-1 were expressed predominantly by endothelium in early lesions and by intimal cells in more advanced lesions. In early and advanced lesions, staining was most intense in endothelial cells at and adjacent to lesion borders. ICAM-1 staining extended into the uninvolved aorta. These expression patterns were highly reproducible in both species. The only difference was that VCAM-1 expression in endothelium over the central portions of lesions was found frequently in rabbits and rarely in mice. The expression of VCAM-1 by arterial endothelium in normal animals may represent a pathogenic mechanism or a phenotypic marker of predisposition to atherogenesis.
Atherosclerotic lesions form at distinct sites in the arterial tree, suggesting that hemodynamic forces influence the initiation of atherogenesis. If NF-kappaB plays a role in atherogenesis, then the activation of this signal transduction pathway in arterial endothelium should show topographic variation. The expression of NF-kappaB/IkappaB components and NF-kappaB activation was evaluated by specific antibody staining, en face confocal microscopy, and image analysis of endothelium in regions of mouse proximal aorta with high and low probability (HP and LP) for atherosclerotic lesion development. In control C57BL/6 mice, expression levels of p65, IkappaBalpha, and IkappaBbeta were 5- to 18-fold higher in the HP region, yet NF-kappaB was activated in a minority of endothelial cells. This suggested that NF-kappaB signal transduction was primed for activation in HP regions on encountering an activation stimulus. Lipopolysaccharide treatment or feeding low-density lipoprotein receptor knockout mice an atherogenic diet resulted in NF-kappaB activation and up-regulated expression of NF-kappaB-inducible genes predominantly in HP region endothelium. Preferential regional activation of endothelial NF-kappaB by systemic stimuli, including hypercholesterolemia, may contribute to the localization of atherosclerotic lesions at sites with high steady-state expression levels of NF-kappaB/IkappaB components.
c-Jun NH2-terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 micro mol/L hydrogen peroxide (H2O2) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3+/-1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress=12 dyne/cm2) for 10 minutes significantly decreased H2O2-induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H2O2-induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells.
Abundant data link hypercholesterolaemia to atherogenesis. However, only recently have we appreciated that inflammatory mechanisms couple dyslipidaemia to atheroma formation. Leukocyte recruitment and expression of pro-inflammatory cytokines characterize early atherogenesis, and malfunction of inflammatory mediators mutes atheroma formation in mice. Moreover, inflammatory pathways promote thrombosis, a late and dreaded complication of atherosclerosis responsible for myocardial infarctions and most strokes. The new appreciation of the role of inflammation in atherosclerosis provides a mechanistic framework for understanding the clinical benefits of lipid-lowering therapies. Identifying the triggers for inflammation and unravelling the details of inflammatory pathways may eventually furnish new therapeutic targets.
Regions in the vasculature exposed to steady laminar flow have a lower likelihood for atherosclerosis than regions exposed to disturbed flow with low shear stress. We previously found that laminar flow of short duration inhibited tumor necrosis factor (TNF)-alpha-mediated proinflammatory signaling in cultured endothelial cells (ECs). However, mechanisms responsible for the atheroprotective effects of physiological shear stress remain undefined. Therefore, we examined the effects of chronic shear stress on TNF-alpha-induced inflammatory responses using an ex vivo perfusion organ culture system.
Rabbit aortas were exposed to low or normal shear stress (0.4 or 12 dyne/cm2) at a constant pressure for 24 to 26 hours. EC and vascular smooth muscle cell (VSMC) proteins were selectively purified. After exposure to low shear stress, TNF-alpha (50 ng/mL, 6 hours) specifically stimulated vascular cell adhesion molecule (VCAM)-1 expression in ECs but not VSMCs. TNF-alpha-stimulated VCAM expression was inhibited significantly by preexposure to normal shear stress. Normal shear stress inhibited TNF (15 minutes) activation of mitogen-activated protein (MAP) kinases (c-Jun NH2-terminal kinase [JNK], p38, extracellular signal-regulated kinase [ERK]) in ECs. Specific pharmacological inhibitors of JNK and p38 but not ERK significantly inhibited TNF-induced VCAM expression. Normal shear stress prevented the association of TNF receptor (TNFR)-1 with TNFR-associated factor (TRAF)-2. There was no effect of low or normal shear stress on TNF-alpha-induced nuclear factor-kappaB activation. A nitric oxide synthesis inhibitor, NG-nitro-l-arginine methyl ester, did not reverse the inhibitory effects of shear stress on VCAM expression.
These results suggest that physiological shear stress is antiinflammatory by specifically inhibiting MAP kinase signaling and inhibiting TRAF-2 interaction with TNFR-1.
Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes in ECs.
Shear stress (20 dynes/cm2) was applied to ECs prior to and/or following the addition of tumor necrosis factor (TNF)-alpha. Shear stress increased the TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) and E-selectin. Transfection studies using promoter reporter gene constructs of ICAM-1, VCAM-1, and E-selectin demonstrated that these shear stress modulations of gene expression occur at the transcriptional levels. After 24-hour preshearing followed by 1 hour of static incubation, the effect of preshearing on TNF-alpha-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-alpha-induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of >6 hours (complete recovery at 24 hours). Pre- and postshearing caused a reduction in the nuclear factor-kappaB-DNA binding activity induced by TNF-alpha in the EC nucleus.
Our findings suggest that shear stress plays differential roles in modulating the TNF-alpha-induced expressions of ICAM-1 versus VCAM-1 and E-selectin genes in ECs.
The rapid and transient induction of E-selectin gene expression by inflammatory tumor necrosis factor (TNF)-α in endothelial cells is mediated by signaling pathways which involve c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) kinase pathways. To explore this regulation, we first observed that in the continuous presence of cytokine TNF, activation of JNK-1 in both nuclear and cytoplasmic compartments peaked at 15-30 min, with activity returning to uninduced levels by 60 min. Phosphorylation of both the p38 kinase and its molecular target, the nuclear transcription factor, activating transcription factor-2, were transient after TNF-α or interleukin (IL)-1β induction. However, cycloheximide treatment prolonged the TNF-α-induced JNK-1 kinase activity beyond 60 min, suggesting that protein synthesis is required to limit this signaling cascade. We investigated the possible role of the dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-2 in limiting cytokine-induced MAPK signaling. Maximum induction of MKP-1 mRNA and nuclear protein levels by TNF-α or IL-1β were noted at 60 min and their expression correlated with the termination of JNK kinase activity, whereas nuclear levels of MKP-2 were not significantly affected by treatment with TNF-α or IL-1β. Transient overexpression of MKP-1 demonstrated significant specific inhibition of E-selectin promoter activity consistent with a regulatory role for dual-specificity phosphatases. Inhibition of MKP-1 expression through the use of small interfering RNAs prolonged the cytokine-induced p38 and JNK kinase phosphorylation. Our results suggest that endogenous inhibitors of the MAPK cascade, such as the dual-specificity phosphatases like MKP-1 may be important for the postinduction repression of MAPK activity and E-selectin transcription in endothelial cells. Thus, these inhibitors may play an important role in limiting the inflammatory effects of TNF-α and IL-1β.
Mitogen-activated protein kinase phosphatase-1 (MKP-1) is one of several oxidized-l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC)-induced genes identified in human aortic endothelial cells (HAEC). We previously reported that MKP-1 activity is required for Ox-PAPC-mediated endothelial/monocyte interactions; however, an in vivo role of MKP-1 in atherogenesis has not been investigated.
We now report that MKP-1 protein is expressed in the atherosclerotic lesions of mice. MKP-1 mRNA expression is highly induced in C57BL6/J mice on an atherogenic diet, low-density lipoprotein receptor (LDLR) (-/-) mice on a Western diet, and 10-week or older ApoE (-/-) mice on a chow diet. In ApoE (-/-) mice treated with 1 mg/mL of sodium orthovanadate (NaOV), a specific inhibitor of tyrosine phosphatases including MKP-1, total phosphatase activity and MKP-1 protein were decreased in both the aortic lesions and liver lysates. In 3 animal models of atherosclerosis [C57BL6/J mice on an atherogenic diet for 15 weeks, LDLR (-/-) mice on a Western diet for 10 weeks, and ApoE (-/-) mice on a chow diet for 8 weeks], mice treated with NaOV had significantly smaller atherosclerotic lesions when compared with the control group.
MKP-1 expression is associated with hypercholesterolemia and atherosclerosis, and inhibition of MKP-1 activity may prevent atherosclerotic lesion development in mice. MKP-1 is required for Ox-PAPC-mediated endothelial/monocyte interactions; however, an in vivo role of MKP-1 in atherogenesis has not been investigated. We now report that MKP-1 protein is expressed in the atherosclerotic lesions of mice and inhibition of tyrosine phosphatase activity and MKP-1 protein reduce atherosclerotic lesions in mouse models.
TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.
Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies.
We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38+/-0.68 versus 0.22+/-0.04); contained fewer smooth muscle cells (1.9+/-1.6% versus 26.3+/-9.7%), less collagen (15.3+/-1.0% versus 22.2+/-1.0%), and more lipids (15.8+/-0.9% versus 10.2+/-0.5%); and showed more outward vascular remodeling (214+/-19% versus 117+/-9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II-induced intraplaque hemorrhages occurred in the lowered shear stress regions only.
Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.
Endothelial cells regulate vascular tone and mural remodelling in a shear-dependent manner that is commonly assumed to keep wall shear stress constant across arteries and species. Allometric arguments show that aortic flow velocity is constant across species, a deduction that is consistent with much experimental data, but the same arguments also show that the shear stress experienced by aortic endothelium will depend inversely on body mass to the 3/8th power, and hence will be 20-fold higher in mice than in men. This conclusion is robust and has important implications for the study of shear-dependent vascular biology and pathology.
The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is known about local variations in wall shear stress (WSS) in the mouse and whether the levels of WSS are comparable to those in humans. The objective of this study was to determine WSS values in the mouse aorta and to relate these to expression of gene products associated with atherosclerosis.
Using micro-CT and ultrasound methodologies we developed a computational fluid dynamics model of the mouse aorta and found values of WSS to be much larger than those for humans. We also used a quantum dot-based approach to study vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression on the aortic intima and demonstrated that increased expression for these molecules occurs where WSS was relatively low for the mouse.
Despite large differences in WSS in the two species, the spatial distributions of atherogenic molecules in the mouse aorta are similar to atherosclerotic plaque localization found in human aortas. These results suggest that relative differences in WSS or in the direction of WSS, as opposed to the absolute magnitude, may be relevant determinants of flow-mediated inflammatory responses.
Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are protein phosphatases that dephosphorylate both the phosphothreonine and phosphotyrosine residues on activated MAPKs. Removal of the phosphates renders MAPKs inactive, effectively halting their cellular function. In recent years, evidence has emerged that, similar to MAPKs, MKPs are pivotal in the regulation of immune responses. By deactivating MAPKs, MKPs can modulate both innate and adaptive immunity. A number of immunomodulatory agents have been found to influence the expression of MKP1 in particular, highlighting the central role of this phosphatase in immune regulation. This Review discusses the properties, function and regulation of MKPs during immune responses.
Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-alpha signaling by preventing activation of JNK. Recently, PKCzeta was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKCzeta activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKCzeta is cleaved by caspases after TNF-alpha stimulation to generate a 50-kDa truncated form (CATzeta, catalytic domain of PKCzeta) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-alpha-mediated PKCzeta cleavage and thereby CATzeta formation. We found that PKCzeta activity was required for TNF-alpha-mediated JNK and caspase-3 activation in ECs. PKCzeta was rapidly cleaved to generate CATzeta in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-alpha. This truncated form of PKCzeta enhanced JNK and caspase-3 activation. Interestingly, PKCzeta cleavage was prevented by inhibitors of PKCzeta, JNK, and caspase activities, suggesting that these enzymes, via regulating CATzeta formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKCzeta and caspase-3 activation. These results define a novel role for PKCzeta as a shared signaling mediator for flow and TNF-alpha, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.
Exposure to disturbed flow, including oscillatory shear stress, stimulates endothelial cells (ECs) to produce bone morphogenic protein (BMP) 4, which in turn activates inflammation, a critical atherogenic step. BMP activity is regulated by the level of BMP antagonists. Until now it was not known whether shear also regulates the expression of BMP antagonists and whether they play a role in EC pathophysiology.
BMP antagonists follistatin, noggin, and matrix Gla protein were expressed in cultured bovine and human arterial ECs. Surprisingly, oscillatory shear stress increased expression of the BMP antagonists in ECs, whereas unidirectional laminar shear decreased such expression. Immunohistochemical studies with mouse aortas showed data consistent with in vitro findings: Only ECs in the lesser curvature exposed to disturbed flow, but not those in the greater curvature and straight arterial regions exposed to undisturbed flow, showed coexpression of BMP4 and the BMP antagonists. Similarly, in human coronary arteries, expression of BMP4 and BMP antagonists in ECs positively correlated with the severity of atherosclerosis. Monocyte adhesion induced by oscillatory shear stress was inhibited by knockdown of BMP4 or treatment with recombinant follistatin or noggin, whereas it was increased by knockdown of follistatin and/or noggin.
The present results suggest that ECs coexpress BMP antagonists along with BMP4 in an attempt to minimize the inflammatory response by oscillatory shear stress as part of a negative feedback mechanism. The balance between the agonist, BMP4, and its antagonists may play an important role in the overall control of inflammation and atherosclerosis.
We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.
Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity.
Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation.
These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.