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Smad2-Dependent Protease Nexin-1 Overexpression Differentiates Chronic Aneurysms From Acute Dissections of Human Ascending Aorta
Abstract
Tissue activation of proteolysis is involved in acute intramural rupture (dissections, acute ascending aortic dissection) and in progressive dilation (aneurysms, thoracic aneurysm of the ascending aorta) of human ascending aorta. The translational aim of this study was to characterize the regulation of antiproteolytic serpin expression in normal, aneurysmal, and dissecting aorta.
We explored expression of protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 and their regulation by the Smad2 signaling pathway in human tissue and cultured vascular smooth muscle cells (VSMCs) of aneurysms (thoracic aneurysm of the ascending aorta; n=46) and acute dissections (acute ascending aortic dissection; n=10) of the ascending aorta compared with healthy aortas (n=10). Both PN-1 and plasminogen activator inhibitor-1 mRNA and proteins were overexpressed in medial tissue extracts and primary VSMC cultures from thoracic aneurysm of the ascending aorta compared with acute ascending aortic dissection and controls. Transforming growth factor-β induced increased PN-1 expression in control but not in aneurysmal VSMCs. PN-1 and plasminogen activator inhibitor-1 overexpression by aneurysmal VSMCs was associated with increased Smad2 binding on their promoters and, functionally, resulted in VSMC self-protection from plasmin-induced detachment and death. This phenomenon was restricted to aneurysms and not observed in acute dissections.
These results demonstrate that epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic VSMC phenotype and might favor progressive aneurysmal dilation, whereas absence of this counter-regulation in dissections would lead to acute wall rupture.
... In human biopsies, PN-1 expression was found to be increased in the medial layer of TAA compared with the aortic medial layer from healthy donors and the protein colocalized with vSMCs. Interestingly, cultured vSMCs from TAA continued to display an increased level of PN-1 mRNA expression compared with control vSMCs (36). This was found to be due to the permanent epigenetic activation of the smad2 pathway in vivo in the arterial wall of TAA, an activation which persisted in cultures of vSMCs of TAA origin. ...
... This was found to be due to the permanent epigenetic activation of the smad2 pathway in vivo in the arterial wall of TAA, an activation which persisted in cultures of vSMCs of TAA origin. Hence, human cultured vSMCs from TAA had a limited capacity to convert plasminogen into plasmin, and were therefore protected against apoptosis-induced detachment after plasminogen or plasmin treatment (36). Indeed, PN-1 overexpression was shown to be associated with aneurysmal dilatation, whereas the absence of PN-1 overexpression was associated with aortic dissections (36). ...
... Hence, human cultured vSMCs from TAA had a limited capacity to convert plasminogen into plasmin, and were therefore protected against apoptosis-induced detachment after plasminogen or plasmin treatment (36). Indeed, PN-1 overexpression was shown to be associated with aneurysmal dilatation, whereas the absence of PN-1 overexpression was associated with aortic dissections (36). Together, these data show that overproduction of PN-1 by vSMCs in vivo during TAA development may participate in the increased ability of the cells to resist the proteolytic environment. ...
The balance between proteases and protease inhibitors plays a critical role in tissue remodeling during cardiovascular diseases. Different serine protease inhibitors termed serpins, which are expressed in the cardiovascular system, can exert a fine-tuned regulation of protease activities. Among them, protease nexin-1 (PN-1, encoded by SERPINE2 ) is a very powerful thrombin inhibitor and can also inactivate plasminogen activators and plasmin. Studies have shown that this serpin is expressed by all cell subpopulations in the vascular wall and by circulating cells but is barely detectable in plasma in the free form. PN-1 present in platelet granules and released upon activation has been shown to present strong antithrombotic and antifibrinolytic properties. PN-1 has a broad spectrum of action related to both hemostatic and blood vessel wall protease activities. Different studies showed that PN-1 is not only an important protector of vascular cells against protease activities but also a significant actor in the clearance of the complexes it forms with its targets. In this context, PN-1 overexpression has been observed in the pathophysiology of thoracic aortic aneurysms (TAA) and during the development of atherosclerosis in humans. Similarly, in the heart, PN-1 has been shown to be overexpressed in a mouse model of heart failure and to be involved in cardiac fibrosis. Overall, PN-1 appears to serve as a “hand brake” for protease activities during cardiovascular remodeling. This review will thus highlight the role of PN-1 in the cardiovascular system and deliver a comprehensive assessment of its position among serpins.
... In parallel, it was observed that p-SMAD translocation to the nuclei of vSMCs is a common feature to all forms of TAA, whatever the etiology [35]. Since this phenomenon persisted when vSMCs from TAA were cultured and was conserved after several passages of the cells, it was identified as an epigenetic phenomenon, related to histone acetylation and HAT/transcription factor activation on the promoter of the SMAD2 gene [36]. The consequences of p-SMAD translocation to vSMC nuclei are an increase in the expression of genes controled by the SMAD2 pathway, including the synthesis of CTGF, adhesive glycoproteins (fibronectin), collagens, anti-proteases (serpins, TIMPs), anti-inflammatory mediators, and inhibition of lymphocyte proliferation. ...
... Tobacco, hypertension, intensive and diffuse muscle tonic contraction, creating high resistances to flow (weightlifting), are risk factors for acute dissections. As compared to chronic dilation of TAA, acute dissections of the ascending aorta are characterized by similar inwall activation of plasminogen, but in the absence of epigenetic modification of the SMAD2 pathway [36]. Since chromatin remodeling prevents vSMC anoïkis by promoting antiprotease expression and CTGF synthesis in response to plasminogen [39], in the absence of it, acute dissection (rupture) is facilited rather than chronic dilation [36]. ...
... As compared to chronic dilation of TAA, acute dissections of the ascending aorta are characterized by similar inwall activation of plasminogen, but in the absence of epigenetic modification of the SMAD2 pathway [36]. Since chromatin remodeling prevents vSMC anoïkis by promoting antiprotease expression and CTGF synthesis in response to plasminogen [39], in the absence of it, acute dissection (rupture) is facilited rather than chronic dilation [36]. ...
The study of the pathology of the aorta must necessarily take into account the role of biomechanical stress that continuously impact on the biology of the cellular and molecular components of its wall. In mammals, the circulation requires a highly organized system, in which organ-regulated directional blood flow is propelled through the conductance arterial tree with a defined wall structure, by the pumping action of the mammalian heart.
... Plasminogen can be activated on the vSMC membrane platform by t-PA (tissue plasminogen activator) and u-PA (urokinase) release (Borges et al., 2010). vSMCs also produce antiproteases to protect the vascular wall from proteolytic injury (Gomez et al., 2013). ...
... PN-1, a tissue serpin, is poorly diffusible due to its retention by glycosaminoglycans (GAGs) at the cell surface, thereby being barely detectable in the plasma (Bouton et al., 2012). PN-1 is synthesized and secreted under basal conditions by vSMCs, where its expression is up-regulated by mechanical load (Bouton et al., 2003) and transforming growth factor β (TGFβ) (Gomez et al., 2013) or down-regulated by thrombin (Richard et al., 2004). Overexpression of PN-1 limits spreading and migration of vSMCs (Richard et al., 2006). ...
... Our results also showed an increase in both mRNA and protein levels of PN-1 associated with the intima in early human atheroma. Plasmin is one of the main proteases responsible for the release of active TGF-β (Jenkins, 2008) which is a powerful inducer of PN-1 expression (Gomez et al., 2013). Therefore, TGFβ could be a link between plasmin and PN-1 in early atheroma. ...
Zymogens are delivered to the arterial wall by radial transmural convection. Plasminogen can be activated within the arterial wall to produce plasmin, which is involved in evolution of the atherosclerotic plaque. Vascular smooth muscle cells (vSMCs) protect the vessels from proteolytic injury due to atherosclerosis development by highly expressing endocytic LDL receptor-related protein-1 (LRP-1), and by producing anti-proteases, such as Protease Nexin-1 (PN-1). PN-1 is able to form covalent complexes with plasmin. We hypothesized that plasmin-PN-1 complexes could be internalized via LRP-1 by vSMCs during the early stages of human atheroma. LRP-1 is also responsible for the capture of aggregated LDL in human atheroma. Plasmin activity and immunohistochemical analyses of early human atheroma showed that the plasminergic system is activated within the arterial wall, where intimal foam cells, including vSMCs and platelets, are the major sites of PN-1 accumulation. Both PN-1 and LRP-1 are overexpressed in early atheroma at both messenger and protein levels. Cell biology studies demonstrated an increased expression of PN-1 and tissue plasminogen activator by vSMCs in response to LDL. Plasmin-PN-1 complexes are internalized via LRP-1 in vSMCs, whereas plasmin alone is not. Tissue PN-1 interacts with plasmin in early human atheroma via two complementary mechanisms: plasmin inhibition and tissue uptake of plasmin-PN-1 complexes via LRP-1 in vSMCs. Despite this potential protective effect, plasminogen activation by vSMCs remains abnormally elevated in the intima in early stages of human atheroma.
... Third, large amounts of t-PA are associated with enlarged arteries (66). Fourth, there is overexpression of the plasminogen activators t-PA and u-PA in TAA (67). These data suggest that plasmin generation is a causative factor for TAA development. ...
... Plasminogen activator inhibitor-1 (PAI-1) mRNA and protein are increased in the media of TAA (67). In vascular smooth muscle cells isolated from human TAA, there is an increased amount of PAI-1, which increases further when exposed to TGF-β 1 (67). ...
... Plasminogen activator inhibitor-1 (PAI-1) mRNA and protein are increased in the media of TAA (67). In vascular smooth muscle cells isolated from human TAA, there is an increased amount of PAI-1, which increases further when exposed to TGF-β 1 (67). PAI-1 is subject to regulatory control by the SMAD2 signaling pathway (67). ...
Understanding and unraveling the pathophysiology of thoracic aortic aneurysm (TAA), a vascular disease with a potentially high mortality rate, is one of the next frontiers in vascular biology. The processes leading to the formation of TAA that are not of known or suspected genetic origin, so-called degenerative TAA, are complex, interwoven and involve promoters and inhibitors at many potential sites. Some of these processes are discussed within this framework. Promoters of TAA development include age, blood pressure elevation, increased pulse pressure, neurohumeral factors increasing blood pressure, inflammation specifically IFN-γ, IL-1 β, IL-6, TNF-α and S100 A12; the coagulation system specifically plasmin, platelets and thrombin as well as matrix metalloproteinases (MMPs). Aortic calcification mechanisms and SMAD-2 signaling also promotes TAA development. The major factors inhibiting or opposing TAA development are the constituents of the aortic wall (elastic lamellae, collagen, fibulins, fibronectin, proteoglycans and vascular smooth muscle cells) which maintain normal aortic dimensions in the face of aortic wall stress. Inhibitors of MMP, specific tissue inhibitors of metalloproteinases (TIMPs), plasminogen activator inhibitor-1, protease nexin-1 and Syndecans act to oppose TAA formation. Increases in promoters and reductions in inhibitors expand the thoracic aorta leading to TAA formation.
... Immunostaining experiments previously demonstrated that PN-1 was overexpressed in the arterial wall of patients with a TAA compared with control aortas. 17 We confirmed this data by ELISA quantification of PN-1 released into conditioned medium from aorta incubated in RPMI ( Figure 5). Indeed, no PN-1 was detected in conditioned medium from healthy aortas, whereas the aorta from patients with TAA released measurable PN-1 ranging from 3.0 to 23.5 ng/mL (n = 6). ...
... 16 In the present study, we also confirmed the overexpression of PN-1 observed previously by immunohistochemistry or western blot in aortic tissues from patients with a TAA. 17 By ELISA, we could indeed easily detect PN-1 released in the conditioned media of TAA samples, whereas it was barely detectable in that of control aorta. ...
Introduction:
Serpin E2 or protease nexin-1 (PN-1) is a glycoprotein belonging to the serpin superfamily, whose function is closely linked to its ability to inhibit thrombin and proteases of the plasminergic system.
Objectives:
In the absence of specific quantitative methods, an ELISA for the quantification of human PN-1 was characterized and used in biological fluids.
Methods:
The ELISA for human PN-1 was developed using two monoclonal antibodies raised against human recombinant PN-1. PN-1 was quantified in plasma, serum, platelet secretion from controls and patients with hemophilia A and in conditioned medium of aortic tissue.
Results:
A linear dose-response curve was observed between 2 and 35 ng/mL human PN-1. Intra- and interassay coefficients of variation were 6.2% and 11.1%, respectively. Assay recoveries of PN-1 added to biological samples were ≈95% in plasma, ≈97% in platelet reaction buffer, and ≈93% in RPMI cell culture medium. Levels of PN-1 secreted from activated human platelets from controls was similar to that of patients with hemophilia A. PN-1 could be detected in conditioned media of aneurysmal aorta but not in that of control aorta.
Conclusion:
This is the first fully characterized ELISA for human serpin E2 level in biological fluids. It may constitute a relevant novel tool for further investigations on the pathophysiological role of serpin E2 in a variety of clinical studies.
... In a fourth study, protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1), both inhibitors of serine-protease were overexpressed in medial tissue extracts and primary aSMCs from TAA compared with acute dissections of ascend-ing aortic and healthy aorta. 49 Furthermore, TGF-β increased PN-1 expression in control but not in aneurysmal aSMCs. PN-1 and PAI-1 overexpression by aneurysmal aSMCs was associated with increased Smad2 binding on their respective gene promoters. ...
... This phenomenon was restricted to aneurysms and not observed in acute dissections. 49 Thus, epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic aSMC phenotype which might favor progressive aneurysmal dilation. Absence of this counter regulation may lead to acute wall rupture (dissection, ►Fig. ...
Thoracic aortic aneurysm (TAA) is an asymptomatic and progressive dilatation of the thoracic aorta. Ascending aortic dissection (AAD) is an acute intraparietal tear, occurring or not on a pre-existing dilatation. AAD is a condition associated with a poor prognosis and a high mortality rate. TAA and AAD share common etiology as monogenic diseases linked to transforming growth factor β signaling pathway, extracellular matrix defect, or smooth muscle cell protein mutations. They feature a complex pathogenesis including loss of smooth muscle cells, altered phenotype, and extracellular matrix degradation in aortic media layer. A better knowledge of the mechanisms responsible for TAA progression and AAD occurrence is needed to improve healthcare, nowadays mainly consisting of aortic open surgery or endovascular replacement. Recent breakthrough discoveries allowed a deeper characterization of the mechanisms of gene regulation. Since alteration in gene expression has been linked to TAA and AAD, it is conceivable that a better knowledge of the causes of this alteration may lead to novel theranostic approaches. In this review article, the authors will focus on epigenetic regulation of gene expression, including the role of histone methylation and acetylation, deoxyribonucleic acid methylation, and noncoding ribonucleic acids in the pathogenesis of TAA and AAD. They will provide a translational perspective, presenting recent data that motivate the evaluation of the potential of epigenetics to diagnose TAA and prevent AAD.
... In addition, a series of studies from Gomez et al. suggests that the expression of SMAD2 is upregulated in medial smooth muscle cells (SMCs) due to epigenetic modification. The elevated production of total SMAD contributes to the phenomenon of "TGFβ paradox" in a mechanism independent of the local concentration of TGFβ proteins [15][16][17][18] . These lines of evidence have raised a critical question about the exact role of TGFβ in the development of aortic pathology that is linked to genetic TGFβ signaling defects. ...
... Further investigations have unraveled the epigenetic upregulation of the total SMAD2 production as a major contributor to the enhanced pSMAD2 accumulation in SMCs 15 . It is intriguing that the SMC-biased pSMAD2 confers a protective effect against aortic dissection by suppressing proteolytic 15,16 . This notion is further supported by experimental studies. ...
Transforming growth factor (TGF)-β signaling disorder has emerged as a common molecular signature for aortic aneurysm development. The timing of postnatal maturation plays a key role in dictating the biological outcome of TGF-β signaling disorders in the aortic wall. In this study, we investigated the impact of deficiency of TGFβ receptors on the structural homeostasis of mature aortas. We used an inducible Cre-loxP system driven by a Myh11 promoter to delete Tgfbr1, Tgfbr2, or both in smooth muscle cells (SMCs) of adult mice. TGFBR1 deficiency resulted in rapid and severe aneurysmal degeneration, with 100% penetrance of ascending thoracic aortas, whereas TGFBR2 deletion only caused mild aortic pathology with low (26%) lesion prevalence. Removal of TGFBR2 attenuated the aortic pathology caused by TGFBR1 deletion and correlated with a reduction of early ERK phosphorylation. In addition, the production of angiotensin (Ang)-converting enzyme was upregulated in TGFBR1 deficient aortas at the early stage of aneurysmal degeneration. Inhibition of ERK phosphorylation or blockade of AngII type I receptor AT1R prevented aneurysmal degeneration of TGFBR1 deficient aortas. In conclusion, loss of SMC-Tgfbr1 triggers multiple deleterious pathways, including abnormal TGFBR2, ERK, and AngII/AT1R signals that disrupt aortic wall homeostasis to cause aortic aneurysm formation.
... Vascular Smooth Muscle Cells (VSMCs) phenotype switching has been demonstrated to be the major contributor of many cardiovascular diseases including atherosclerosis, aneurysm and aortic dissection [1,2]. Changing of VSMCs from a contractile phenotype to a synthetic phenotype triggers their migration to the intima, promotes their proliferation and induce synthesis of extracellular matrix protein, which ultimately results in impaired contractility of vascular function [1]. ...
... Together, these findings suggest that AngII play an important role for VSMC phenotypic switch. [1,2]. The present research demonstrates a unique role of angiotensin II in manipulating VSMC phenotype through inducement of autophagy. ...
Background: Angiotension II (AngII) has important roles on Vascular Smooth Muscle Cell VSMC) biological functions. It has been indicated to promote the synthetic phenotype of VSMC, but the underlying mechanism is still needed to be elucidated.
Methods and Results: In this study, we found AngII infusion promotes a increased form of autophagy characterized by increased expression of Beclin1 and LC3-II in the cultured human aortic vascular smooth muscle cells. The growth of autophagy stimulated by AngII could enhance implication of synthetic proteins in VSMCs, while inhibition of autophagy with spautin-1or 3-MA can encourage the stability of the contractile phenotype in AngIItreated VSMCs.
Conclusion: These results suggest that up regulation of autophagy by AngII contributes to the increase of synthetic phenotype, which triggered the functional change in VSMCs.
... Such upregulation of PN-1 expression induced by TGF-b has been shown in many different cultured cells, including neuroblastoma, 26 myotubes, 27 renal epithelial cells or cortical collecting duct cells, 28 and vascular smooth muscle cells. 29 PAI-1 is also overexpressed by pulmonary fibroblasts stimulated by TGF-b, 30 and functional analysis of both PAI-1 and PN-1 promoters have identified a regulatory region for TGF-b induction, involving Smad complex binding. 29 The possibility that upregulation of PN-1 expression is a common process in fibrosis whatever the tissue affected remains to be determined. ...
... 29 PAI-1 is also overexpressed by pulmonary fibroblasts stimulated by TGF-b, 30 and functional analysis of both PAI-1 and PN-1 promoters have identified a regulatory region for TGF-b induction, involving Smad complex binding. 29 The possibility that upregulation of PN-1 expression is a common process in fibrosis whatever the tissue affected remains to be determined. ...
Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-beta, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.
... It is known that several proteases are increased in TAA: MMPs, but also proteases of the plasminergic system: t-PA and u-PA, which are produced by the SMCs. 4,12,22 Indeed, our results showed an increase in proteases: plasminogen, MMP-9, and MMP-19 in TAA media as well as u-PA in TAA adventitia ( Figure 6G and H and see Supplementary material online, Figure S2). These proteases might increase neovessel permeability by weakening the neovessel ultrastructural components such as pericytes and adherent junctions, especially since tissue metalloproteinase inhibitors, TIMP-1 and TIMP-4, were decreased in TAA. ...
... Our previous work showed also that overexpression of the serine protease inhibitors, protease nexin-1 and plasminogen activator inhibitor-1, in TAA SMCs was protective, in favour of a progressive aneurysmal dilation, avoiding acute wall dissection. 4 We have observed that neovessels were mostly located in the mucoid degeneration areas, suggesting that ECM degradation facilitates neovessel invasion. We postulate that the imbalance of expression between proteases and TIMPs induces ECM degradation leading to the release and activation of factors promoting angiogenesis such as MMPs. ...
Aims:
Human thoracic aneurysm of the ascending aorta (TAA) is a chronic disease characterized by dilatation of the aortic wall, which can progress to vessel dissection and rupture. TAA has several aetiologies, but all forms present common features, including tissue remodelling. Here, we determined and characterized the angiogenic process associated with TAA and its relation with wall remodelling.
Methods and results:
Immunostaining for blood vessels showed an increased density of microvessels originating from the adventitia in the external medial layer of TAA compared with healthy aortas. Proteomic array analysis of 55 angiogenic factors in medial and adventitial layers showed different expression profiles in both tissue compartments between aneurysmal and healthy aortas. Quantification by ELISA confirmed that all forms of TAA contained higher levels of several pro- and anti-angiogenic factors, including angiopoietin-1 and -2, fibroblast growth factor-acidic, and thrombospondin-1, than that of healthy aortas. However, all groups showed comparable levels of vascular endothelial growth factor-A. Quantitative RT-PCR demonstrated that angiopoietins were overexpressed in TAA media. Immunostaining and electron microscopy revealed that neovessels had defective endothelial junctions and poor mural cell coverage. This incomplete structure was associated with the accumulation of plasminogen and albumin in the media of TAA.
Conclusion:
We describe, for the first time, leaky neovessel formation in TAA media in association with an imbalance of angiogenic factor levels. Although the initiating mechanisms of neo-angiogenesis in TAA and the potential aetiology-related differences remain to be determined, our results suggest that neo-angiogenesis could participate in TAA wall remodelling and weakening through deposition of blood-borne zymogens.
... The authors further report a fine-tuning mechanism in which Myc-mediated transcriptional repression, present in healthy vessels, is replaced by p53, resulting in sustained transcriptional activity during TAA development [81]. Moreover, increased protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1) expression in TAA patients was reported to be induced by SMAD2 binding and affect anti-proteolytic aortic SMC phenotype, thus leading to progressive aneurysmal dilation [82]. ...
Background:
Thoracic aortic aneurysm (TAA) is a serious condition that affects the aorta, characterized by the dilation of its first segment. The causes of TAA (e.g., age, hypertension, genetic syndromes) are heterogeneous and contribute to the weakening of the aortic wall. This complexity makes treating this life-threatening aortopathy challenging, as there are currently no etiological therapy available, and pharmacological strategies, aimed at avoiding surgical aortic replacement, are merely palliative. Recent studies on novel therapies for TAA have focused on identifying biological targets and etiological mechanisms of the disease by using advanced -omics techniques, including epigenomics, transcriptomics, proteomics, and metabolomics approaches.
Methods:
This review presents the latest findings from -omics approaches and underscores the importance of integrating multi-omics data to gain more comprehensive understanding of TAA.
Results:
Literature suggests that the alterations in TAA mediators frequently involve members of pro-fibrotic process (i.e., TGF-β signaling pathways) or proteins associated with cell/extracellular structures (e.g., aggrecans). Further analyses often reported the importance in TAA of processes as inflammation (PCR, CD3, leukotriene compounds), oxidative stress (chromatin OXPHOS, fatty acids), mitochondrial respiration and glycolysis/gluconeogenesis (e.g., PPARs and HIF1a). Of note, more recent metabolomics studies added novel molecular markers to the list of TAA-specific detrimental mediators (proteoglycans).
Conclusion:
It is increasingly clear that integrating data from different -omics branches, along with clinical data, is essential as well as complicated both to reveal hidden relevant information and to address complex diseases such as TAA. Importantly, recent progresses in metabolomics highlighted novel potential and unprecedented marks in TAA diagnosis and therapy.
... 165 SMC therefore trigger a defensive response to oxidation in abdominal AA 166 and respond to mechanical dilation in thoracic AA by producing antiproteases, 167 and subsequently performing clearance of protease/antiprotease complexes, 84 preventing acute dissection (acute intramural rupture). 168 Therefore, different types of overload of the arterial wall clearance functions take place in abdominal and thoracic aneurysms of the aorta and are involved in the progression of aneurysmal dilations. ...
Evolutionary organization of the arterial wall into layers occurred concomitantly with the emergence of a highly muscularized, pressurized arterial system that facilitates outward hydraulic conductance and mass transport of soluble substances across the arterial wall. Although colliding circulating cells disperse potential energy within the arterial wall, the different layers counteract this effect: (1) the endothelium ensures a partial barrier function; (2) the media comprises smooth muscle cells capable of endocytosis/phagocytosis; (3) the outer adventitia and perivascular adipocytic tissue are the final receptacles of convected substances. While the endothelium forms a physical and a biochemical barrier, the medial layer is avascular, relying on the specific permeability properties of the endothelium for metabolic support. Different components of the media interact with convected molecules: medial smooth muscle cells take up numerous molecules via scavenger receptors and are capable of phagocytosis of macro/micro particles. The outer layers—the highly microvascularized innervated adventitia and perivascular adipose tissue—are also involved in the clearance functions of the media: the adventitia is the seat of immune response development, inward angiogenesis, macromolecular lymphatic drainage, and neuronal stimulation. Consequently, the clearance functions of the arterial wall are physiologically essential, but also may favor the development of arterial wall pathologies. This review describes how the walls of large conductance arteries have acquired physiological clearance functions, how this is determined by the attributes of the endothelial barrier, governed by endocytic and phagocytic capacities of smooth muscle cells, impacting adventitial functions, and the role of these clearance functions in arterial wall diseases.
... 46 In TAA, alteration in histone modifications was also shown to lead to dysregulation of SMAD2 and overexpression of certain serine proteases that could affect aortic wall degeneration. [47][48][49] WHAT ABOUT HEMODYNAMICS? ...
Smooth muscle cells and endothelial cells have a remarkable level of plasticity in vascular pathologies. In thoracic and abdominal aortic aneurysms, smooth muscle cells have been suggested to undergo phenotypic switching and to contribute to degradation of the aortic wall structure in response to, for example, inflammatory mediators, dysregulation of growth factor signaling or oxidative stress. Recently, endothelial-to-mesenchymal transition, and a clonal expansion of degradative smooth muscle cells and immune cells, as well as mesenchymal stem–like cells have been suggested to contribute to the progression of aortic aneurysms. What are the factors driving the aortic cell phenotype changes and how vascular flow, known to affect aortic wall structure and to be altered in aortic aneurysms, could affect aortic cell remodeling? In this review, we summarize the current literature on aortic cell heterogeneity and phenotypic switching in relation to changes in vascular flow and aortic wall structure in aortic aneurysms in clinical samples with special focus on smooth muscle and endothelial cells. The differences between thoracic and abdominal aortic aneurysms are discussed.
... Serine proteinase inhibitor clade E member 2 (SERPINE2), also known as protease nexin-1, is a member of the serine protease inhibitor superfamily and was first identified as a neurite-promoting factor for the release of cultured glioma cells [6]; various other cells such as fibroblasts, vascular smooth muscle cells, endothelial cells, platelet particles, and chondrocytes have also been reported to secrete the same [7][8][9][10]. As a serine protease inhibitor, SERPINE2 has been known for its anti-serine protease activity against thrombin, urokinase, plasminogen, and other serine proteinases [11][12][13]. Tumor cells have also been found to secrete SERPINE2, with studies showing that abnormal expression of SERPINE2 contributes to tumorigenesis and tumor invasion in various cancers, including breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, testicular cancer, skin melanoma, osteosarcoma, thyroid cancer, oral squamous cell carcinoma, endometrial cancer, esophageal squamous cell carcinoma, lung adenocarcinoma, and hepatocellular cell carcinoma [8,[14][15][16][17][18][19][20][21][22][23][24][25][26]. A recent study on UBUC demonstrated that SERPINE2 might play important roles in activating and recruiting immune cells, which can significantly impact tumor behavior regulation and treatment response [27]. ...
Recent studies have reported that SERPINE2 contributes to the development of various cancers. However, its association with urothelial carcinoma (UC) remains unclear. In this study, data on urinary bladder UC (UBUC) cases from The Cancer Genome Atlas (TCGA) database were used to investigate the prognostic value of SERPINE2 mRNA expression. Then, SERPINE2 expression was analyzed with tissue microarrays constructed from 117 upper tract UC (UTUC) and 84 UBUC tissue specimens using immunohistochemical staining. Results were compared to clinicopathologic data by multivariate analysis. In the TCGA database, high SERPINE2 mRNA expression indicated a poor prognosis in patients with UBUC. Furthermore, Mann–Whitney U test showed that high SERPINE2 immunoexpression was significantly associated with adverse pathologic parameters including invasion, high grade, coexistence of UC in situ, and advanced pT stage (all p < 0.05, except for a marginal association with high-grade UBUC, p = 0.066). Kaplan–Meier analysis revealed that high SERPINE2 expression was associated with worse overall survival (OS; UTUC, p = 0.003; UBUC, p = 0.014) and disease-free survival (UTUC, p = 0.031; UBUC, p = 0.033). Moreover, multivariate analysis identified high SERPINE2 expression as an independent prognostic factor for OS (UTUC, p = 0.002; UBUC, p = 0.024). Taken together, our findings demonstrated that increased SERPINE2 expression is associated with adverse pathologic features and may serve as a prognostic biomarker for UC.
... SERPINE2 can regulate cellular differentiation (15)(16)(17) and can function as a regulator of angiogenesis (18), apoptosis (19,20), and endothelial barrier function (20). SERPINE2 overexpression promotes development of an antiproteolytic vascular smooth muscle cell phenotype and might favor progressive aneurysmal dilation (21). Injuryrelated cytokines can increase SERPINE2 expression in different cell types (22,23). ...
Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell-and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2 2/2) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2 2/2 mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2 2/2 mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-kB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo. These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation
... We performed elastase infusion in rat abdominal aortic arteries which often induces marked aneurysm 7 days after the infusion [25]. Immunohistochemistry on aorta cross sections indicated that Smad2 increased in aneurysmal aortic arteries versus non-aneurysmal arteries, in agreement with previous reports [11,15]. More interestingly, whereas Fas increased, Met decreased in elastase-treated arteries as compared to normal artery controls, in particular in the medial layer where SMCs reside (Fig. 7, Fig. S2). ...
Background
Vascular smooth muscle cell (SMC) apoptosis is involved in major cardiovascular diseases. Smad2 is a transcription factor implicated in aortic aneurysm. The molecular mediators of Smad2-driven SMC apoptosis are not well defined. Here we have identified a Smad2-directed mechanism involving MET and FAS, both encoding cell membrane signaling receptors.
Methods and results
Guided by microarray analysis in human primary aortic SMCs, loss/gain-of-function (siRNA/overexpression) indicated that Smad2 negatively and positively regulated, respectively, the gene expression of Met which was identified herein as anti-apoptotic and that of Fas, a known pro-apoptotic factor. While co-immunoprecipitation suggested a physical association of Smad2 with p53, chromatin immunoprecipitation followed by quantitative PCR revealed their co-occupancy in the same region of the MET promoter. Activating p53 with nutlin3a further potentiated the suppression of MET promoter-dependent luciferase activity and the exacerbation of SMC apoptosis that were caused by Smad2 overexpression. These results indicated that Smad2 in SMCs repressed the transcription of MET by cooperating with p53, and that Smad2 also activated FAS, a target gene of its transcription factor activity.
Conclusions
Our study suggests a pro-apoptotic mechanism in human SMCs, whereby Smad2 negatively and positively regulates MET and FAS, genes encoding anti-apoptotic and pro-apoptotic factors, respectively.
... Large vessels including arteries and veins are composed of three main microscopic wall layers: an outer layer made up of collagen fibers and elastic tissue, a middle layer encompassing collagen fibers, elastic tissue and smooth muscle, and an inner layer comprising endothelium [32][33][34]. Arteries contain more percentage of elastic tissue than veins that enables them to enhance their blood conduction capacity as the blood pressure increases [35]. Small vessels including venules arterioles and capillaries have much thinner and narrower walls in proportion to arteries and veins. ...
Efficient strategies to promote microvascularization in vascular tissue engineering, a central priority in regenerative medicine, are still scarce; nano- and micro-sized aggregates and spheres or beads harboring primitive microvascular beds are promising methods in vascular tissue engineering. Capillaries are the smallest type and in numerous blood vessels, which are distributed densely in cardiovascular system. To mimic this microvascular network, specific cell components and proangiogenic factors are required. Herein, advanced biofabrication methods in microvascular engineering, including extrusion-based and droplet-based bioprinting, Kenzan, and biogripper approaches, are deliberated with emphasis on the newest works in prevascular nano- and micro-sized aggregates and microspheres/microbeads.
... Beneficial roles of TGF-β in TAA may include the suppression of AT1R signaling, induction of protective factors such as nexin-1 and proteases inhibitors, and promotion of contractile proteins expression [317,[409][410][411][412][413]. In addition, TGF-β-dependent induction of collagen, lysyl oxidases, and other pro-fibrotic factors might contribute to thickening of the adventitial layer, which, as discussed, can be protective [71,269,364,[414][415][416][417]. ...
Thoracic aortic aneurysms (TAA) are permanent and localized dilations of the aorta that predispose patients to a life-threatening risk of aortic dissection or rupture. The identification of pathogenic variants that cause hereditary forms of TAA has delineated fundamental molecular processes required to maintain aortic homeostasis. Vascular smooth muscle cells (VSMCs) elaborate and remodel the extracellular matrix (ECM) in response to mechanical and biochemical cues from their environment. Causal variants for hereditary forms of aneurysm compromise the function of gene products involved in the transmission or interpretation of these signals, initiating processes that eventually lead to degeneration and mechanical failure of the vessel. These include mutations that interfere with transduction of stimuli from the matrix to the actin–myosin cytoskeleton through integrins, and those that impair signaling pathways activated by transforming growth factor-β (TGF-β). In this review, we summarize the features of the healthy aortic wall, the major pathways involved in the modulation of VSMC phenotypes, and the basic molecular functions impaired by TAA-associated mutations. We also discuss how the heterogeneity and balance of adaptive and maladaptive responses to the initial genetic insult might contribute to disease.
... Although the investigation of these mechanisms is still at its beginning, this is an interesting and important field of research in BAV-associated aortopathy. Previous studies on altered DNA methylation and epigenetic regulation originate from aneurysms in general [136,137] and were not focused on BAV aortopathy. Pan et al., conducted the first explorative study on DNAmlandscape, and revealed that BAV patients are generally characterized by a non-CpG hypomethylation signature [138]. ...
Herein we summarize the current knowledge on the bicuspid aortic valve (BAV)-associated aortopathy regarding clinical presentation and disease sub-classification, genetic background, hemodynamics, histopathology, cells and signaling, animal models, and biomarkers. Despite enormous efforts in research in all of the above areas, important issues remain unknown: (i) what is the ontogenetic basis of BAV development? (ii) how can we explain the diversity of BAV and associated aortopathy phenotypes? (iii) what are the signaling processes in aortopathy pathogenesis and how can we interfere with these processes? Despite undoubtedly great progress that has been made in the understanding of BAV-associated aortopathy, so far researchers have put together a heap of Lego bricks, but at present it is unclear if the bricks are compatible, how they fit together, and which parts are missing to build the true model of the BAV aorta. A joint approach is needed to accelerate research progress.
... Interestingly, this chromatin remodelling is observed only during progressive chronic dilation, i.e. a progressive increase in wall tension, but not during acute intramural rupture. 88 Thereby chromatin remodelling in vSMCs may be a hallmark of TAAs as compared with aortic dissection. ...
Vascular Smooth Muscle Cells (vSMCs) play a crucial role in both the pathogenesis of Aneurysms and Dissections of the ascending thoracic aorta (TAAD) in humans and in the associated adaptive compensatory responses, since thrombosis and inflammatory processes are absent in the majority of cases. Aneurysms and dissections share numerous characteristics, including aetiologies and histopathological alterations: vSMC disappearance, medial areas of mucoid degeneration, and ExtraCellular Matrix (ECM) breakdown. Three aetiologies predominate in TAAD in humans: i) genetic causes in heritable familial forms, ii) an association with bicuspid aortic valves and iii) a sporadic degenerative form linked to the aortic aging process. Genetic forms include mutations in vSMC genes encoding for molecules of the ECM or the TGF-β pathways, or participating in vSMC tone. On the other hand, aneurysms and dissections, whatever their aetiologies, are characterized by an increase in wall permeability leading to transmural advection of plasma proteins which could interact with vSMCs and ECM components. In this context, blood-borne plasminogen appears to play an important role, because its outward convection through the wall is increased in TAAD, and it could be converted to active plasmin at the vSMC membrane. Active plasmin can induce vSMC disappearance, proteolysis of adhesive proteins, activation of MMPs and release of TGF-β from its ECM storage sites. Conversely, vSMCs could respond to aneurysmal biomechanical and proteolytic injury by an epigenetic phenotypic switch, including constitutional overexpression and nuclear translocation of Smad2 and an increase in antiprotease and ECM protein synthesis. In contrast, such an epigenetic phenomenon is not observed in dissections. In this context, dysfunction of proteins involved in vSMC tone are interesting to study, particularly in interaction with plasma protein transport through the wall and TGF-β activation, to establish the relationship between these dysfunctions and ECM proteolysis.
... 424 Furthermore, PAI-1 overexpression in SMCs protected SMCs from plasmin-induced detachment and death. 413 These findings suggest that PAI-1 has a protective role against aortic destruction and AAD development. ...
... SERPINE2 can regulate cellular differentiation (15)(16)(17) and can function as a regulator of angiogenesis (18), apoptosis (19,20), and endothelial barrier function (20). SERPINE2 overexpression promotes development of an antiproteolytic vascular smooth muscle cell phenotype and might favor progressive aneurysmal dilation (21). Injuryrelated cytokines can increase SERPINE2 expression in different cell types (22,23). ...
Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2(-/-)mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2(-/-)mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expressionin vivo These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.-Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel-Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.
... Although the best available option when studying human tissue, the authors hypothesize that apoptosis resistance in human aneurysm specimens likely represents a compensatory epigenetic reprogramming response to the disease process, instead of an initiating event. 21 Importantly, apoptosis of aortic root/ascending aortic SMCs cannot explain localized aneurysm growth alone because aneurysms still develop in Marfan mice despite caspase-inhibition, albeit at a slower growth rate. In addition, we cannot exclude the possibility that the caspase-inhibitor cross reacts and blocks other matrix-degrading enzymes. ...
Background
Aortic aneurysms (AA) are pathological dilations of the aorta, associated with an overall mortality rate up to 90% in case of rupture. In addition to dilation, the aortic layers can separate by a tear within the layers, defined as aortic dissections (AD). Vascular smooth muscle cells (vSMC) are the major cell type within the aortic wall and dysregulation of vSMC functions contributes to AA and AD development and progression. However, since the exact underlying mechanism is poorly understood, finding potential treatment targets for AA and AD is challenging and surgery remains the only treatment option.
Methods
In this review, we summarize current knowledge about vSMC functions within the aortic wall and give an overview of how vSMC functions are dysregulated in AA and AD pathogenesis, organized per anatomical location (abdominal or thoracic aorta).
Results
Important functions of vSMC are apoptosis, phenotypic switch, extracellular matrix regeneration and degradation, proliferation and contractility. Stressors within the aortic wall, including inflammatory cell infiltration and (epi)genetic changes, modulate vSMC functions and cause disturbance of processes within vSMC, such as changes in TGF-β signaling and regulatory RNA expression.
Conclusion
This review underscores a central role of vSMC dysfunction in both abdominal and thoracic AA and AD development and progression. Further research focused on vSMC dysfunction in the aortic wall is necessary to find potential targets for non-invasive AA and AD treatment options.
The evolution of the circulatory system from invertebrates to mammals has involved the passage from an open system to a closed in-parallel system via a closed in-series system, accompanying the increasing complexity and efficiency of life's biological functions. The archaic heart enables pulsatile motion waves of hemolymph in invertebrates, and the in-series circulation in fish occurs with only an endothelium, whereas mural smooth muscle cells appear later. The present review focuses on evolution of the circulatory system. In particular, we address how and why this evolution took place from a closed, flowing, longitudinal conductance at low pressure to a flowing, highly pressurized and bifurcating arterial compartment.The general teleonomy of the evolution of species is the differentiation of individual organ function, supported by specific fueling allowing and favoring partial metabolic autonomy. This was achieved via the establishment of an active contractile tone in resistance arteries, which permitted the regulation of blood supply to specific organ activities via its localized function-dependent inhibition. The resistance to viscous blood flow is the peripheral increase in frictional forces caused by the tonic change in arterial radius, which backscatter as systemic arterial blood pressure. Consequently, the arterial pressure gradient from blood to the adventitial interstitium generates the outward radial advective conductance of plasma solutes across the wall. This hemodynamic evolution was accompanied by important changes in arterial wall structure, supported by smooth muscle cell functional plasticity. These adaptive phenotypic shifts are due to epigenetic regulation, mainly related to mechanotransduction. These paradigms actively participate in cardio-arterial pathologies.
The spectrum of bicuspid aortic valve (BAV) disease encompasses not only maldevelopment of the aortic valve but also abnormalities of the thoracic aorta, including aortic root and ascending aortic dilatation. Insidious formation of thoracic aortic aneurysm (TAA) can lead to aortic dissection or rupture with necropsy series suggesting a 5–10 times higher risk of aortic dissection in BAV compared to patients with a trileaflet aortic valve.KeywordsBicuspid aortic valveElastinGlycoproteinFibrillinAortopathyAneurysmDissection
Plasminogen is a circulating zymogen which enters the arterial wall by radial, transmural hydraulic conductance, where it is converted to plasmin by tissue plasminogen activator t-PA on an activation platform involving S100A4 on the vascular smooth muscle cell (vSMC) membrane. Plasmin is involved in the progression of human thoracic aneurysm of the ascending aorta (TAA). vSMCs protect the TAA wall from plasmin-induced proteolytic injury by expressing high levels of antiproteases. Protease nexin-1 (PN-1) is a tissue antiprotease belonging to the serpin superfamily, expressed in the vascular wall, and is able to form a covalent complex with plasmin. LDL receptor-related protein-1 (LRP-1) is a scavenger receptor implicated in protease–antiprotease complex internalization. In this study, we investigated whether PN-1 and LRP-1 are involved in the inhibition and clearance of plasminogen by the SMCs of human TAA.
We demonstrated an overexpression of S100A4, PN-1, and LRP-1 in the medial layer of human TAA. Plasminogen activation taking place in the media of TAA was revealed by immunohistochemical staining and plasmin activity analyses. We showed by cell biology studies that plasmin–PN-1 complexes are internalized via LRP-1 in vSMCs from healthy and TAA media.
Thus, two complementary mechanisms are involved in the protective role of PN-1 in human TAA: one involving plasmin inhibition and the other involving tissue clearance of plasmin-PN1 complexes via the scavenger receptor LRP-1.
Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis.
Protease nexin-1 (PN-1), also known as SERPINE2, is the phylogenetically closest relative of plasminogen activator type 1 (PAI-1). Unlike PAI-1, antithrombin (AT), protein C inhibitor (PCI) or heparin cofactor II (HCII), other members of the serpin family that share its ability to inhibit thrombin, PN-1 is not detectable in the circulating blood. In contrast, PN-1 is found within diverse organs, including the brain, the blood vessel wall and the lungs, among many others. PN-1 expression is tightly and finely regulated in different organs at distinct stages of development, suggesting a dynamic role in vivo depending on the cell type, the tissue and the stage of development. PN-1 is now recognized as a central regulator of vascular thrombosis and appears to play a protective role in the different tissues where it is expressed, by preventing pathological states induced by excessive proteolytic activities. In this chapter, we examine current knowledge of PN-1 in the pathophysiology of the nervous, vascular, respiratory, renal and reproductive systems, as well as in the development of tumours.
The main goal of The International Meeting on Aortic Diseases (IMAD) is to gather all cardiovascular clinicians and scientists to share their experiences on basic research, genetic aspects of aortic aneurysms, aortic dissections, aortitis, and their treatment as well as on the new pathophysiological concepts in bicuspid aortic valve, TAVI and surgical treatment of aortic valve diseases and also to provide information about the latest innovations in perfusion. Moreover, it is a great pleasure to share reflections with all worldwide scientists and clinicians interested in aortic diseases thanks to AORTA.
Different forms of Ehlers-Danlos Syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by a fragile vasculature with a high risk of catastrophic vascular events at young age. Classic EDS, characterized by fragile, hyperextensible skin and joint laxity, is caused by heterozygous mutations in the COL5A1 and COL5A2 genes. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation was reported. We describe a familial case of clinically vascular EDS, diagnosed in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3 and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). This revealed a novel heterozygous mutation in COL5A1, resulting in an essential glycine substitution in the triple helix domain, nearby the C-terminus of the protein (c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. This is the first report of familial clinical diagnosed vascular EDS with a mutation in COL5A1 displaying a similar phenotype to that caused by a COL3A1 mutation.
The TicAAA-trial (Does Ticagrelor inhibit growth of small AAA? A randomised controlled trial). A key limitation of contemporary treatment strategies of AAA is the lack of therapy directed at reducing expansion. Platelet activation and intraluminal thrombus renewal are key events in AAA progression, and clinical and experimental data suggests that antiplatelet treatment with aspirin may be associated with reduced growth rates for AAAs. The TicAAA-trial is an ongoing investigator sponsored multi-centre randomized controlled trial investigating whether treatment with Ticagrelor (Brilinta, Brilique) inhibits growth of small AAA. A total of 140 ASA-naïve patients with an AAA of 35–49 mm are randomized to Ticagrelor or placebo (1:1). Ticagrelor is a potent antiplatelet drug that acts by a selectively and reversibly binding to the platelet P2Y12 receptor, blocking the actions of the platelet agonist adenosine diphosphate (ADP). The primary efficacy variable is difference in log-transformed AAA-volume determined by MRI at 12 months vs. log-transformed volume at baseline. The study is powered to detect a 20% reduction in growth rate.
It is well established that blocking the renin-angiotensin-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of the RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here, we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone.
TGFbeta1 suppresses the adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps, from 10 to 300% of normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10% hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes.
These results identify TGFbeta signalling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism, as well as assist in the development of novel therapeutic strategies for hypertension.
Objective:
Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome.
Approach and results:
Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs.
Conclusions:
Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.
Purpose of review:
A lot of new data have been obtained in familial thoracic aortic aneurysms, including description of new entities and better understanding of pathophysiology. The aim of this review is to put them in perspective.
Recent findings:
The new data have been collected, put together, and allowed a new classification scheme to be proposed by the Montalcino Aortic Consortium on the basis of the role of proteins coded by the culprit gene (either protein of the extracellular matrix or protein of the transforming growth factor-beta pathway, or protein of the contractile apparatus of the smooth muscle cell). These groups of diseases include aortic aneurysm, but the extent of extra-aortic vascular risk and the presence of extra-aortic (skeletal, ophthalmologic, neurological, or immunological) features vary according to the gene involved. This understanding also sheds light on the therapeutic benefits that can be foreseen for new molecules, or old molecules used in a newer way.
Summary:
Classification of familial forms of thoracic aortic aneurysm should allow a better understanding of these diseases and therefore standardization of initial evaluation of the patients (vascular evaluation limited or not to the aorta, and extravascular evaluation, including or not skeleton, eyes, neurology, digestive tract, and immunological diseases) and individualization of therapy (adapted to both the genotype and the phenotype).
Aortic diseases are common in many populations and are receiving increasing research focus. There are a broad spectrum of aortic diseases that occur in specific regions and appear to have different causes. For example, abdominal aortic aneurysms (AAAs) are most common in aged men.1 In contrast, many forms of thoracic aortic aneurysms (TAAs) occur early in life with a strong genetic basis and no sex discrimination.2 Both aortic aneurysms are amenable to surgical repair. Although surgical approaches have become increasingly sophisticated and less invasive,3 there remains an urgent need to determine factors that predispose to susceptibility and to divert treatment from surgical to medical approaches.4,5 This switch to medical treatment will require an increased knowledge of the mechanisms for several facets of aneurysms that cover the span of initiation, progression, and rupture. In this regard, many recent publications in ATVB have provided further insight into established pathways contributing to aneurysm development such as proteolysis, inflammation, and attenuation of the medial smooth muscle cell population, and a few publications have raised the possibility of new pathways such as adipokines and mineralocorticoid signaling. This article highlights these recent publications within a brief context of the literature.
Cigarette smoking remains the major risk factor for development and progression of AAAs.6,7 Several experimental studies have demonstrated that smoke exposure augments AAA induced in mice by either subcutaneous angiotensin II (AngII) infusion or intra-aortic elastase perfusion.8,9 However, it is unclear whether cessation of smoking impacts the development of AAAs. The study of Jin et al10 demonstrated that cessation of cigarette smoking exposure did not immediately decrease the augmentation of AAAs. This sustained effect was attributable to regulation of leukocytic metabolism. Also of note is that cigarette smoking–induced augmentation of AAAs was unaffected …
Objective— Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats.
Methods and Results— The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N G-nitro-l-arginine methyl ester (l-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional.
Conclusions— PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.
It is estimated worldwide that over 6 million people per annum experience a burn injury. Despite advances in management and improved survival rates, the incidence of hypertrophic scarring remains high. These scars are particularly common after burns and are often raised, red, hard and may cause abnormal sensations. Such pathological scarring can lead to severe functional impairment, psychological morbidity, and costly long term healthcare. Wound healing is an inherent process which restores the integrity of the skin after injury and although scarring is a frequent by-product, the scarless wound healing observed in early human gestational fetuses suggests that it is not an essential component of the response. This has lead to a large body of research attempting to understand the mechanisms behind scarring and in turn prevent it. One of the main focuses of recent research has been the role played by the growth factor TGF-β in the process of both wound healing and scar formation. The three isoforms (TGF-β1, TGF-β2 and TGF-β3) appear to have overlapping functions and predominantly mediate their effects through the intracellular SMAD pathway. Initial research suggested that TGF-β1 was responsible for the fibrotic scarring response whereas the scarless wound healing seen in fetal wounds was due to increased levels of TGF-β3. However, the reality appears to be far more complex and it is unlikely that simply altering the ratio of TGF-β isoforms will lead to scarless wound healing. Other aspects of the TGF-β system that appear promising include the downstream mediator CTGF, the proteoglycan decorin and the binding protein p311. Other putative mechanisms which may underlie the pathogenesis of hypertrophic scars include excessive inflammation, excessive angiogenesis, altered levels of matrix metalloproteinases, growth factors, and delayed apoptosis of fibrotic myofibroblasts either due to p53 genetic alterations or tensile forces across the wound. If an effective treatment for hypertrophic scars following burns injury is to be developed then further work must be carried out to understand the basic mechanisms of pathological scarring.
Vascular smooth muscle cells (VSMCs) are the stromal cells of the vascular wall, and, due to their myosin/actin interactions, they are also responsible for arterial contractile tonus and regulating blood pressure and flow in relation to specific metabolic demands. VSMCs show considerable differences depending on their position in the arterial tree (large conduit vs. small resistance vessels), their embryologic origin, and their organ-dependent microenvironment; the heart, brain, kidney, etc. As the stromal cells of the arterial wall, VSMCs synthesize and secrete insoluble extracellular matrix (ECM) molecules that assume the function of withstanding the high pressure of the circulating blood in the arterial system. For example, totally decellularized aortic grafts, created using sodium dodecyl sulphate detergent, are experimentally able to resist arterial blood pressure in the aorta in vivo , without dilating.1 This mechanical function predominates in large vessels, since pressure-dependent stress of the wall is also dependent on the diameter.2 Therefore, cell–ECM interactions are of particular importance in these large arteries. The contractile tonus of VSMCs, dependent on sympathetic innervation, is responsible for the peripheral resistance to blood flow generated by the beating heart, leading to the genesis of blood pressure. This function is mainly the characteristic of resistance arteries and arterioles and involves cell–cell interactions.
Due to pressure-dependent radial hydraulic conductance, the arterial wall is constantly submitted to outward convection of circulating plasma molecules.3 This physiological outward convection of plasma peptides or macromolecules is the main source of extrinsic stimuli, which, with time, cause damage to arterial VSMCs (pressure-dependent strain, lipid overload, hyperglycaemia, zymogens). In response to changes in their plasma-dependent microenvironment, VSMCs may either die or adapt, modifying their phenotype through acute, ligand/receptor-dependent signalling pathways or chronic nuclear signalling involving epigenetic molecular remodelling of their nuclear chromatin. VSMCs can also be the target of intrinsic …
Vascular smooth muscle cells (VSMCs) are the stromal cells of the vascular wall, continually exposed to mechanical signals and biochemical components generated in the blood compartment. They are involved in all the physiological functions and the pathological changes taking place in the vascular wall. Owing to their contractile tonus, VSMCs of resistance vessels participate in the regulation of blood pressure and also in hypertension. VSMCs of conduit arteries respond to hypertension-induced increases in wall stress by an increase in cell protein synthesis (hypertrophy) and extracellular matrix secretion. These responses are mediated by complex signalling pathways, mainly involving RhoA and extracellular signal-regulated kinase1/2. Serum response factor and miRNA expression represent main mechanisms controlling the pattern of gene expression. Ageing also induces VSMC phenotypic modulation that could have influence on cell senescence and loss of plasticity and reprogramming. In the early stages of human atheroma, VSMCs support the lipid overload. Endocytosis/phagocytosis of modified low-density lipoproteins, free cholesterol, microvesicles, and apoptotic cells by VSMCs plays a major role in the progression of atheroma. Migration and proliferation of VSMCs in the intima also participate in plaque progression. The medial VSMC is the organizer of the inwardly directed angiogenic response arising from the adventitia by overexpressing vascular endothelial growth factor in response to lipid-stimulated peroxisome proliferator-activated receptor-γ, and probably also the organizer of the adventitial immune response by secreting chemokines. VSMCs are also involved in the response to proteolytic injury via their ability to activate blood-borne proteases, to secrete antiproteases, and to clear protease/antiprotease complexes.
Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.
Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. Indeed, studies of PN-1-deficient mice have established important antithrombotic and antifibrinolytic properties of this serpin that have heretofore gone unrecognized. The roles of PN-1 in the areas of hemostasis and thrombosis summarized here provide insights that may allow the development of drugs and treatment strategies to prevent or limit thrombotic disorders.
Plasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-β1. Identification of molecular events underlying to PAI-1 induction in response to TGF-β1 may yield novel targets for the therapy of cardiovascular disease.
Reactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60(c-src) activation and PAI-1 expression. TGF-β1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-β1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-β1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-β1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-β1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-β1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1(Y14) phosphoryation and initiation of downstream signaling.
TGF-β1-initiated Src-dependent caveolin-1(Y14) phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene.
Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained.
This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner.
Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.
Aims Blood D-dimer testing has been proposed as diagnostic marker with high sensitivity for exclusion of acute aortic dissection (AAD). We performed a systematic review and validated the findings in a prospective patient cohort. Methods and results We searched MEDLINE, EMBASE, CINAHL, and BIOSIS from inception until January 2007 using a combination of search terms for aortic dissection and D-dimer. Study type, type of assay used, predefined cutoff level, result of D-dimer testing, sensitivity, and specificity were abstracted. In 16 identified studies (437 patients), the reported cutoff values ranged from 0.1 to 0.9 mg/mL. D-dimer testing provided high sensitivity (0.97 95% CI 0.94–0.98) and negative likelihood ratio (0.06 95% CI 0.02–0.13). In our cohort of 65 patients (36 male, 55%; median age 59 years, IQR 49–67) with proven AAD, D-dimer levels scattered from 0.24 to 137.88 mg/mL (median 3.47; IQR 1.55–14.49). Mean NPV for the different cutoff levels ranged from 92 % for a cutoff level of 0.9 mg/mL to 100% for a cutoff level of 0.1 mg/mL in our study population. Conclusion Current evidence supports a routine measurement of D-dimer in excluding AAD. A D-dimer ,0.1 mg/mL will exclude AAD in all cases.
Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in regulation of fibrinolytic system, cell-associated proteolysis and migration of smooth muscle cells (SMC). This study is focused on the types of PAI-1 expressing cells, quantification of PAI-1 expression in the walls of aneurysmatic abdominal aortas (AAA) and correlation between histological and clinical findings.
A group of nine patients who underwent surgery for AAA: asymptomatic (aAAA), symptomatic (sAAA) and ruptured (rAAA) and one control specimen (CA) were included in the study. Samples underwent histological processing and immunohistochemistry in comparison with in situ hybridisation. In order to assess the PAI-1 area fraction in histological sections through the aortic wall the Line System module of Ellipse software was used. PAI-1 expressing cells were measured in CA and AAA: endothelium, SMC, and foam cells. Photomicrographs with a total area of 0.7 mm(2) for each specimen were analysed by two independent observers. Mean values of PAI-1 positive components per section area were calculated as average values.
The results of both observers are as follows: 28.6% in CA; 18.1% in aAAA; 10.9% in sAAA; 11.0% in rAAA. During the progression of AAA, the SMC (PAI-1 expression was found mainly in them) became less abundant in agreement with the values of PAI-1 area fraction. In rAAA immunohistochemistry detected PAI-1 in necrotic centres of atheromathous plaques.
AAA may be evaluated as the result of gradual changes in regulation of fibrinolysis that is observed as redistribution of cells expressing PAI-1. The area fraction of PAI-1 positive components correlates with clinical classification of AAA.
Overexpression of plasminogen activator inhibitor-1 (SERPINE1, PAI-1), the major physiological inhibitor of pericellular plasmin generation, is a significant causative factor in the progression of vascular disorders (e.g. arteriosclerosis, thrombosis, perivascular fibrosis) as well as a biomarker and a predictor of cardiovascular-disease associated mortality. PAI-1 is a temporal/spatial regulator of pericellular proteolysis and ECM accumulation impacting, thereby, vascular remodeling, smooth muscle cell migration, proliferation and apoptosis. Within the specific context of TGF-beta1-initiated vascular fibrosis and neointima formation, PAI-1 is a member of the most prominently expressed subset of TGF-beta1-induced transcripts. Recent findings implicate EGFR/pp60c-src-->MEK/ERK1/2 and Rho/ROCK-->SMAD2/3 signaling in TGF-beta1-stimulated PAI-1 expression in vascular smooth muscle cells. The EGFR is a direct upstream regulator of MEK/ERK1/2 while Rho/ROCK modulate both the duration of SMAD2/3 phosphorylation and nuclear accumulation. E-box motifs (CACGTG) in the PE1/PE2 promoter regions of the human PAI-1 gene, moreover, are platforms for a MAP kinase-directed USF subtype switch (USF-1-->USF-2) in response to growth factor addition suggesting that the EGFR-->MEK/ERK axis impacts PAI-1 expression, at least partly, through USF-dependent transcriptional controls. This paper reviews recent data suggesting the essential cooperativity among the EGFR-->MAP kinase cascade, the Rho/ROCK pathway and SMADs in TGF-beta1-initiated PAI-1 expression. The continued clarification of mechanistic controls on PAI-1 transcription may lead to new targeted therapies and clinically-relevant options for the treatment of vascular diseases in which PAI-1 dysregulation is a major underlying pathogenic feature.
The clotting protease thrombin might contribute to cell damage following brain injury by its ability to retract processes on neurons and astrocytes. Protease nexin-1 (PN-1), a potent inhibitor of thrombin, is localized around cerebral blood vessels where it may protect these cells from extravasated thrombin during injury or alteration of the blood-brain barrier. Here we examined the effects of several injury-related factors on the regulation of PN-1 in cultured brain cells. Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta stimulated the secretion of PN-1 by the neuroblastoma cell line SK-N-SH. This cell line comprises both neuronal and glial cells. Analyses using cloned derivatives of these two cell types showed that PN-1 was secreted by the glial cells; PN-1 secretion was stimulated 90-fold by interleukin-1, 15-fold by tumor necrosis factor-alpha, 10-fold by tumor growth factor-beta, and 4-fold by platelet-derived growth factor. Measurements of newly synthesised PN-1 demonstrated that these factors produced an equivalent stimulation of PN-1 synthesis. The neuronal cells secreted two thrombin-binding proteins distinct from PN-1. Interactions between these two cell types regulated the secretion of PN-1 and the two thrombin-binding proteins.
Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5- to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis.
To address a potential role for p53, Bcl2 associated protein X (Bax), and apoptosis in the processes associated with cell turnover during cystic medial degeneration (CMD) of the aorta.
Histochemical, immunohistochemical, biochemical, and morphometric methods were used to assess the presence and distribution of p53 immunoreactivity (p53-IR) and Bax immunoreactivity (Bax-IR), as well as the presence of apoptosis and tissue repair processes.
Immunohistochemical staining disclosed evidence for p53-IR in all specimens in 26.1 (11.5)% of vascular smooth muscle cells (VSMCs) (controls 0.8 (1.3)%; p < 0. 001). Bax-IR was present in all specimens in 10 (5.4)% of medial cells (controls 0.3 (0.5)%; p < 0.001). Medial VSMCs (alpha-actin positive) with cytoplasmic staining for an apoptosis specific protein (c-jun/ASP) were present in 20/20 specimens (0.7 (0.6)% of VSMCs, controls 0%, p < 0.001), whereas terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) positive VSMCs were present in 17/20 specimens (1 (1.5)% of VSMCs, controls 0%, p < 0.001). The presence of apoptosis was confirmed by electron microscopy and the demonstration of oligonucleosomal DNA fragments after agarose gel electrophoresis. As shown by double labeling and investigation of serial sections, p53-IR, Bax-IR, c-jun/ASP-IR, and positive TUNEL labeling localised to the same compartments of the aortic media, raising a possible role for p53 and Bax in the triggering of apoptosis of VSMC during CMD. MIB1/Ki-67 positive medial VSMCs (alpha-actin positive) and mesenchymal cells (vimentin positive) were present in all specimens (2.5 (2.8)% of medial cells; controls 0.3 (0.9)%, p < 0.001) mainly in the region around the vasa vasorum, indicating that cell regeneration during CMD may originate mainly from the mesenchyme surrounding the vasa vasorum.
This study shows that the formal pathogenesis of CMD is characterised by p53 accumulation, Bax upregulation, cell death by apoptosis, and cell regeneration. Nevertheless, the precise stimuli of p53 activation and Bax upregulation as well as the role of p53 and apoptosis in the dissection process itself remain elusive.
We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.
Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats.
The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N(G)-nitro-L-arginine methyl ester (L-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional.
PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.
Smooth muscle cell (SMC) rarefaction is involved in the development of several vascular pathologies. We suggest that the plasminogen activation system is a potential extracellular signal that can induce pericellular proteolysis and apoptosis of vascular SMCs. Using primary cultures of arterial SMCs, we show that plasmin generated from plasminogen on the cell surface induces cell retraction and fibronectin fragmentation, leading to detachment and morphological/biochemical changes characteristic of apoptosis (also called anoikis). The generation of cell-bound plasmin mediated by tissue-type plasminogen activator (t-PA), constitutively expressed by VSMCs, requires binding of plasminogen to the cell surface and is inhibited by epsilon-aminocaproic acid (IC50=0.9+/-0.2 mM), a competitor of plasminogen binding to membrane glycoproteins. Conversely, addition of alpha2-antiplasmin, which blocks free plasmin in the cell supernatant, could not fully prevent anoikis. Finally, an MMP inhibitor failed to prevent VSMC anoikis, arguing for a direct involvement of plasmin in this phenomenon. Indeed, similar changes are induced by plasmin directly added to VSMCs or to arterial rings, ex-vivo. We show for the first time that pathological anoikis can be triggered by a process that requires functional assembly of the plasminogen activation system on the surface of VSMCs.
Significant alterations of the vascular wall occurs in abdominal aortic aneurysm (AAA) and atherosclerotic occlusive disease (AOD) that ultimately may lead to either vascular rupture or obstruction. These modifications have been ascribed to one or a group of proteases, their inhibitors or to the matrix macromolecules involved in the repair process without considering the extent of the observed variations.
The mRNA steady-state level of a large spectrum of proteolytic enzymes (matrix metalloproteinases: MMP-1, -2, -3, -8, -9, -11, -12, -13, -14; urokinase plasminogen activator: u-PA), their physiological inhibitors (tissue inhibitors of MMPs: TIMP-1, -2, -3; plasminogen activator inhibitor: PAI-1) and that of structural matrix proteins (collagens type I and III, decorin, elastin, fibrillins 1 and 2) was determined by RT-PCR made quantitative by using a synthetic RNA as internal standard in each reaction mixture. The profile of expression was evaluated in AAA (n=7) and AOD (n=5) and compared to non-diseased abdominal (CAA, n=7) and thoracic aorta (CTA, n=5).
The MMPs -8, -9, -12 and -13 mostly associated with inflammatory cells were not or barely detected in CAA and CTA while they were largely and similarly expressed in AAA and AOD. Expression of protease inhibitors or structural proteins were only slightly increased in both pathological conditions with the exception of elastin which was reduced. The main significant difference between AAA and AOD was a lower expression of TIMP-2 and PAI-1 in the aneurysmal lesions.
The remodeling of the aortic wall in AAA and AOD involves gene activation of a large and similar spectrum of proteolytic enzymes while the expression of two physiological inhibitors, TIMP-2 and PAI-1, is significantly lower in AAA compared to AOD. The repair process in the aneurysmal disease seems similar to that of the occlusive disease.
Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin (FBN1) at 15q21.1 (ref. 1). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2, at 3p25-p24.2 in a large French family (family MS1). Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-beta receptor 2 (TGFBR2) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G-->A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-beta signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.
We report heterozygous mutations in the genes encoding either type I or type II transforming growth factor beta receptor in ten families with a newly described human phenotype that includes widespread perturbations in cardiovascular, craniofacial, neurocognitive and skeletal development. Despite evidence that receptors derived from selected mutated alleles cannot support TGFbeta signal propagation, cells derived from individuals heterozygous with respect to these mutations did not show altered kinetics of the acute phase response to administered ligand. Furthermore, tissues derived from affected individuals showed increased expression of both collagen and connective tissue growth factor, as well as nuclear enrichment of phosphorylated Smad2, indicative of increased TGFbeta signaling. These data definitively implicate perturbation of TGFbeta signaling in many common human phenotypes, including craniosynostosis, cleft palate, arterial aneurysms, congenital heart disease and mental retardation, and suggest that comprehensive mechanistic insight will require consideration of both primary and compensatory events.
Protease nexin-1 (PN-1) is a non-circulating pericellular serpin expressed by vascular cells. PN-1 inhibits different proteases but when associated with glycosaminoglycans, its activity is mainly directed towards thrombin. Fucoidans are sulphated polysaccharides which can interact with several serpins and have antithrombotic and anticoagulant properties in vivo with a lower hemorrhagic risk than heparin. The purpose of this study was to compare the effects of low (LMW) or high molecular weight (HMW) fucoidans to those of standard heparin and LMW heparin on PN-1 properties. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and affinity coelectrophoresis, we observed that polysaccharides bound to thrombin, PN-1 and the thrombin/PN-1 complex. Progress curve kinetics showed that LMW and HMW fucoidans accelerate thrombin inhibition by PN-1 (111 and 402 fold, respectively) whereas the acceleration by LMW heparin and standard heparin was only of 36- and of 307-fold, respectively. Moreover, the formation of PN-1/(125)I-thrombin complex was increased in the presence of heparin, HMW and LMW fucoidans, but barely by LMW heparin. The dose response followed a bell shape curve, again suggesting the formation of ternary complexes between thrombin, PN-1 and polysaccharides. We also investigated the ability of polysaccharides to remove PN-1 bound to the cell membrane of smooth muscle cells in culture. PN-1 was detached by fucoidans and heparins and was still able to inhibit thrombin. In conclusion, fucoidans reduce cell-associated PN-1 and thrombin/PN-1 complexes and increase the antithrombin activity of PN-1. The capacity of PN-1 to regulate the pericellular activity of thrombin amongst other proteases reinforces the therapeutical interest of fucoidans.
Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor-beta (TGF-beta) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-beta signaling and can be prevented by TGF-beta antagonists such as TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.
Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta1 (TGF-beta1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I(Na)) were measured. The effect of TGF-beta1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I(Na) by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-beta1 significantly increased protein expression of PN-1 by 3.8+/-0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7+/-6.7%. Basolateral, but not apical, application of TGF-beta1 significantly reduced I(eq). To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I(eq) by 1.6+/-0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.
Blood D-dimer testing has been proposed as diagnostic marker with high sensitivity for exclusion of acute aortic dissection (AAD). We performed a systematic review and validated the findings in a prospective patient cohort.
We searched MEDLINE, EMBASE, CINAHL, and BIOSIS from inception until January 2007 using a combination of search terms for aortic dissection and D-dimer. Study type, type of assay used, predefined cut-off level, result of D-dimer testing, sensitivity, and specificity were abstracted. In 16 identified studies (437 patients), the reported cut-off values ranged from 0.1 to 0.9 microg/mL. D-dimer testing provided high sensitivity (0.97 95% CI 0.94-0.98) and negative likelihood ratio (0.06 95% CI 0.02-0.13). In our cohort of 65 patients (36 male, 55%; median age 59 years, IQR 49-67) with proven AAD, D-dimer levels scattered from 0.24 to 137.88 microg/mL (median 3.47; IQR 1.55-14.49). Mean NPV for the different cut-off levels ranged from 92 % for a cut-off level of 0.9 microg/mL to 100% for a cut-off level of 0.1 microg/mL in our study population.
Current evidence supports a routine measurement of D-dimer in excluding AAD. A D-dimer <0.1 microg/mL will exclude AAD in all cases.
Objective: Significant alterations of the vascular wall occurs in abdominal aortic aneurysm (AAA) and atherosclerotic occlusive disease (AOD) that ultimately may lead to either vascular rupture or obstruction. These modifications have been ascribed to one or a group of proteases, their inhibitors or to the matrix macromolecules involved in the repair process without considering the extent of the observed variations. Methods: The mRNA steady-state level of a large spectrum of proteolytic enzymes (matrix metalloproteinases: MMP-1, -2, -3, -8, -9, -11, -12, -13, -14; urokinase plasminogen activator: u-PA), their physiological inhibitors (tissue inhibitors of MMPs: TIMP-1, -2, -3; plasminogen activator inhibitor: PAI-1) and that of structural matrix proteins (collagens type I and III, decorin, elastin, fibrillins 1 and 2) was determined by RT-PCR made quantitative by using a synthetic RNA as internal standard in each reaction mixture. The profile of expression was evaluated in AAA (n = 7) and AOD (n = 5) and compared to non-diseased abdominal (CAA, n = 7) and thoracic aorta (CTA, n = 5). Results: The MMPs -8, -9, -12 and -13 mostly associated with inflammatory cells were not or barely detected in CAA and CTA while they were largely and similarly expressed in AAA and AOD. Expression of protease inhibitors or structural proteins were only slightly increased in both pathological conditions with the exception of elastin which was reduced. The main significant difference between AAA and AOD was a lower expression of TIMP-2 and PAI-1 in the aneurysmal lesions. Conclusions: The remodeling of the aortic wall in AAA and AOD involves gene activation of a large and similar spectrum of proteolytic enzymes while the expression of two physiological inhibitors, TIMP-2 and PAI-1, is significantly lower in AAA compared to AOD. The repair process in the aneurysmal disease seems similar to that of the occlusive disease.
Astrocytes play a diverse role in central nervous system (CNS) injury. Production of the serine protease inhibitors (serpins) plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1) by astrocytes may counterbalance excessive serine protease activity associated with CNS pathologies such as ischemic stroke. Knowledge regarding the regulation of these genes in the brain is limited, so the objective of the present study was to characterize the effects of injury-related factors on serpin expression in human astrocytes. Native human astrocytes were exposed to hypoxia or cytokines, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), IL-10, transforming growth factor-α (TGF-α), and TGF-β for 0–20 hr. Serpin mRNA expression and protein secretion were determined by real-time RT-PCR and ELISA, respectively. Localization of PAI-1 and PN-1 in human brain tissue was examined by immunohistochemistry. Hypoxia and all assayed cytokines induced a significant increase in PAI-1 expression, whereas prolonged treatment with IL-1β or TNF-α resulted in a significant down-regulation. The most pronounced induction of both PAI-1 and PN-1 was observed following early treatment with TGF-α. In contrast to PAI-1, the PN-1 gene did not respond to hypoxia. Positive immunoreactivity for PAI-1 in human brain tissue was demonstrated in reactive astrocytes within gliotic areas of temporal cortex. We show here that human astrocytes express PAI-1 and PN-1 and demonstrate that this astrocytic expression is regulated in a dynamic manner by injury-related factors. © 2010 Wiley-Liss, Inc.
Post-translational modification of histones, ATP-dependent chromatin remodeling, and DNA methylation are interconnected nuclear mechanisms that ultimately lead to the changes in chromatin structure necessary to carry out epigenetic gene expression control. Tissue differentiation is characterized by a specific gene expression profile in association with the acquisition of a defined tissue architecture and function. Elements critical for tissue differentiation, like extracellular stimuli, adhesion and cell shape properties, and transcription factors all contribute to the modulation of gene expression and thus, are likely to impinge on the nuclear mechanisms of epigenetic gene expression control. In this review, we analyze how these elements modify chromatin structure in a hierarchical manner by acting on the nuclear machinery. We discuss how mechanotransduction via the structural continuum of the cell and biochemical signaling to the cell nucleus integrate to provide a comprehensive control of gene expression. The role of nuclear organization in this control is highlighted, with a presentation of differentiation-induced nuclear structure and the concept of nuclear organization as a modulator of the response to incoming signals.
Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues, including during development and progression of vascular diseases such as atherosclerosis. Although much is known regarding factors and mechanisms that control SMC phenotypic plasticity in cultured cells, our knowledge of the mechanisms controlling SMC phenotypic switching in vivo is far from complete. Indeed, the lack of definitive SMC lineage-tracing studies in the context of atherosclerosis, and difficulties in identifying phenotypically modulated SMCs within lesions that have down-regulated typical SMC marker genes, and/or activated expression of markers of alternative cell types including macrophages, raise major questions regarding the contributions of SMCs at all stages of atherogenesis. The goal of this review is to rigorously evaluate the current state of our knowledge regarding possible phenotypes exhibited by SMCs within atherosclerotic lesions and the factors and mechanisms that may control these phenotypic transitions.
Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (Km = 46 nM). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.
Marfan syndrome has changed over the last few years: new diagnostic criteria have been proposed, new clinical entities recognised and life expectancy increased. The role of fibrillin 1, which was initially thought to be mainly structural, has been shown to also be functional. The altered transforming growth factor β pathway is better understood, the importance of epigenetic factors has been demonstrated and recent data suggest that many of the observations made in Marfan syndrome can actually be made in thoracic aortic aneurysm from diverse aetiologies. Besides transforming growth factor β, the role of metalloproteinase, the fibrinolytic/coagulation system, is being suggested in the progression of the disease. A relationship between the type of fibrillin 1 (FBN1) gene mutation and the mechanism for the disease (haplo-insufficiency vs negative dominance), as well as some genotype/phenotype correlations, has been observed, although the main challenge of recognising gene modifiers has yet to explain tremendous variability despite similar mutation. This progress has led to new hopes for tomorrow's therapies, some of which are being tested in clinics, whereas others are still in the field of animal models. Here we review some of the new data obtained in the understanding of the pathophysiology and genetics of this disease.
Aortic aneurysm is common, accounting for 1-2% of all deaths in industrialized countries. Early theories of the causes of human aneurysm mostly focused on inherited or acquired defects in components of the extracellular matrix in the aorta. Although several mutations in the genes encoding extracellular matrix proteins have been recognized, more recent discoveries have shown important perturbations in cytokine signalling cascades and intracellular components of the smooth muscle contractile apparatus. The modelling of single-gene heritable aneurysm disorders in mice has shown unexpected involvement of the transforming growth factor-β cytokine pathway in aortic aneurysm, highlighting the potential for new therapeutic strategies.
Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.
Surgery for acute aortic dissection (AAD) is frequently complicated by excessive postoperative bleeding and blood product transfusion. Blood flow through the nonendothelialized false lumen is a potential trigger for the activation of the hemostatic system; however, the physiopathology of the aortic dissection induced coagulopathy has never been precisely studied. The aim of the present study is the evaluation of the coagulation and fibrinolytic systems and platelet activation in patients undergoing surgery for AAD.
Eighteen patients undergoing emergent surgery for Stanford type A AAD were enrolled in the study. The activation of the coagulation and fibrinolytic systems and platelet activation were evaluated at 6 different time points before, during, and after the operation, measuring prothrombin fragment 1.2 (F1.2), plasmin-antiplasmin complex, and platelet factor 4, respectively.
All measured biomarkers were increased before, during, and after the operations indicating a systemic activation of coagulation, fibrinolysis, and platelets. These changes were pronounced even preoperatively (T0), and soon after the beginning of cardiopulmonary bypass (T1) when the influence of hypothermia and prolonged cardiopulmonary bypass time were not yet involved. Time from symptom onset to intervention inversely correlated with preoperative F1.2 (r=-0.75; p=0.002) and plasmin-antiplasmin levels (r=-0.57; p=0.034).
Blood flow through the false lumen is a powerful activator of the hemostatic system even before the operation. This remarkable activation may influence postoperative outcome of AAD patients.
Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.
Thoracic aortic dissection (TAD) is estimated to occur at a rate of 3-4 cases per 100,000 persons per year and is associated with a high mortality. Reported rates are probably underestimates of the true incidence of TAD because of difficulties in diagnosis. The incidence of TAD appears to have been increasing over time. TAD is most common in men and older individuals. Aortic dilatation is a well-established risk factor for TAD but is not a prerequisite; most ascending aortic dissections occur when aortic diameter is <5.5 cm. Although atherosclerosis and typical cardiovascular risk factors, such as hypertension and smoking, are associated with TAD, evidence supporting their direct causal role is lacking. Notably, diabetes mellitus is remarkably uncommon in patients with TAD. Other risk factors for TAD include inflammatory diseases, iatrogenic aortic injury, and drug use. Congenital cardiovascular defects, such as bicuspid aortic valve, and certain genetic syndromes, such as Marfan syndrome, are the genetic factors most commonly associated with TAD. Specific nonsyndromic genetic mutations in families and single nucleotide polymorphisms have also been identified as possible risk factors for TAD.
Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-β/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-β, the aim was to investigate the plasminergic system in TAA.
Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochemically in dense clumps around SMCs and colocalized with latent TGF-β binding protein-1.
The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-β bioavailability.
Bicuspid aortic valve (BAV) disease is the most common congenital cardiac defect. While the BAV can be found in isolation, it is often associated with other congenital cardiac lesions. The most frequent associated finding is dilation of the proximal ascending aorta secondary to abnormalities of the aortic media. Changes in the aortic media are present independent of whether the valve is functionally normal, stenotic, or incompetent. Although symptoms often manifest in adulthood, there is a wide spectrum of presentations ranging from severe disease detected in utero to asymptomatic disease in old age. Complications can include aortic valve stenosis or incompetence, endocarditis, aortic aneurysm formation, and aortic dissection. Despite the potential complications, 2 large contemporary series have demonstrated that life expectancy in adults with BAV disease is not shortened when compared with the general population. Because BAV is a disease of both the valve and the aorta, surgical decision making is more complicated, and many undergoing aortic valve replacement will also need aortic root surgery. With or without surgery, patients with BAV require continued surveillance. Recent studies have improved our understanding of the genetics, the pathobiology, and the clinical course of the disease, but questions are still unanswered. In the future, medical treatment strategies and timing of interventions will likely be refined. This review summarizes our current understanding of the pathology, genetics, and clinical aspects of BAV disease with a focus on BAV disease in adulthood.
Extracellular matrix degradation is a sentinel pathologic feature of abdominal aortic aneurysm (AAA) disease. Diabetes mellitus, a negative risk factor for AAA, may impair aneurysm progression through its influence on the fibrinolytic system. We hypothesize that hyperglycemia limits AAA progression through effects on endogenous plasminogen activator inhibitor-1 (PAI-1) levels and subsequent reductions in plasmin generation.
Experimental AAAs were induced in diabetic and control mice via the intra-aortic elastase infusion method. Serial transabdominal high-frequency ultrasound examinations were performed to monitor aortic diameter following elastase infusion. Circulating PAI-1 and plasmin alpha2-antiplasmin (PAP) complex concentrations were determined by ELISA and local expression of PAI-1 levels was examined by RT-PCR and immunohistochemistry.
Hyperglycemia was associated with reduced AAA diameter, increased plasma PAI-1 concentration and reduced plasmin generation. Aneurysmal aortic PAI-1 gene expression increased in parallel with plasma concentration, with peak expression occurring early after aneurysm initiation.
Hyperglycemia increases PAI-1 expression and attenuates AAA diameter in experimental AAA disease. These results emphasize the role of the fibrinolytic pathway in AAA pathophysiology, and suggest a candidate mechanism for hyperglycemic inhibition of AAA disease.
Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta1 mRNA level. The increase in stored TGF-beta1 was associated with enhanced LTBP-1 protein and mRNA levels. These dysregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta1 and LTBP-1 staining and between their respective mRNA expressions. In parallel, decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta.
Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases.
Arterial aneurysms exhibit a loss of elastin and an increase in the plasminogen activators urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). Because u-PA, t-PA, and plasmin have a limited proteolytic activity against elastin, the role of plasminogen activators in the aneurysmal disease is unclear. To investigate this question, we overexpressed plasminogen activator inhibitor-1 (PAI-1), an inhibitor of t-PA and u-PA, in a rat model of aortic aneurysm.
Guinea pig-to-rat aortic xenografts were seeded with syngeneic Fischer 344 rat smooth muscle cells retrovirally transduced with the rat PAI-1 gene (LPSN group) or the vector alone (LXSN group). Some grafts were not seeded with cells (NO group). Western blots showed increased PAI-1 in grafts from the LPSN group compared with LXSN and NO groups. All grafts in the NO group (n=8) and 40% in the LXSN group ruptured between days 4 and 14. At 4 weeks in the LXSN group, the remaining unruptured grafts (n=6) were aneurysmal (diameter increase > or =100%), whereas in the LPSN group (n=6) none of the grafts had ruptured or were aneurysmal. Elastin was preserved in the LPSN group. t-PA, the major PA expressed in the model, was decreased in the LPSN group compared with the other groups, as determined by zymography. Quantitative zymography showed decreased levels of two matrix metalloproteinases (MMPs), a 28-kD caseinase, and activated MMP-9 in the LPSN group.
The blockade of plasminogen activators prevents formation of aneurysms and arterial rupture by inhibiting MMP activation.
The etiopathogenesis of thoracic aortic aneurysms is currently an issue of debate. The present study investigated ultrastructural, morphometric, and immunohistochemical aspects of smooth muscle cells (SMCs) in chronic aneurysm of the thoracic aorta (aneurysm group), aortic dilatation associated with valvular disease (valvular group), and dissection of the thoracic aorta (dissection group). Fragments of the ascending aorta that had been taken from the patients during coronary bypass surgery were used as controls. No significant difference was observed in the density of SMCs between the 3 pathologic groups put together and the controls. Only separate analysis of SMC density in each of the pathologic groups showed that the valvular group samples had significantly smaller amounts of SMCs in the internal layer of the media than the dissection group samples and controls. Ultrastructural analysis, in situ end labeling, propidium iodide assay, and DNA laddering did not show apoptosis of SMCs in the samples investigated. Ultrastructure of SMCs characteristic of the synthetic phenotype, together with increased expression of osteopontin in the media of pathologic thoracic aortas indicated the transition of SMCs from the contractile to the synthetic phenotype. Immunohistochemical investigation showed that medial SMCs in the samples taken from aortas of all 3 pathologic groups expressed stronger immunoreactivity for matrix metalloproteinase 1, 2, and 9 and tissue inhibitor of metalloproteinase 1 and 2 than the controls. The present study shows that during the formation of aneurysms, dissection of the thoracic aorta, or aortic dilatation associated with valvular disease, loss of SMCs was not of great importance with respect to their transition from the contractile to the synthetic type in leading to increased production of matrix metalloproteinases.
Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of plasminogen activator in vivo. Increased PAI-1 expression is associated with arteriosclerosis. Transforming growth factor-beta (TGF-beta) induces PAI-1 production via Smads.
In vivo, TGF-beta receptors (TbetaRs), Smad2, Smad3, and Smad4, PAI-1, and Smad2 phosphorylation were examined by immunohistochemistry in 3 native aortas, 14 rat aortic syngrafts, and 19 allografts collected at 15, 30, and 45 days post-transplantation. In vitro, phosphorylation of Smad2 and induction of PAI-1 mRNA in human aortic smooth muscle cells (SMCs) in response to TGF-beta treatment were detected by Western blot and by TaqMan real-time RT-PCR, respectively.
Immunohistochemical staining revealed that vascular parenchymal cells contained TbetaRI, TbetaRII, Smad2, Smad3, and Smad4, known signaling transducers for TGF-beta/Smad pathway, in all samples. Intense staining for phospho-Smad2 was observed in 94% of endothelial cells (ECs), 86% of intimal cells, 27% of medial SMCs, and 38% of adventitial cells at all 3 time points in all aortic allografts, but only in 5% of ECs in syngrafts. PAI-1 immunoreactivity was detected in similar number of cells, and from consecutive sections, phospho-Smad2 colocalized with PAI-1, in the aortic allografts. Low basal level PAI-1 expression was observed in aortic syngrafts and native vessels. Smad2 phosphorylation and time-dependent PAI-1 induction were detected in cultured SMCs upon TGF-beta treatment.
Phospho-Smad2 staining in aortic allografts indicates the activation of TGF-beta signaling in allo-transplantation; and co-localization of PAI-1 and phospho-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF-beta/Smad pathway in aortic allografts.
Protease nexin-1 (PN-1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN-1 expression by rat aortic smooth muscle cells (RASMCs). PN-1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi-quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down-regulated PN-1 expression in a dose- and time-dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR-1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN-1 expression. PN-1 secreted by smooth muscle cells remained essentially associated to cell-surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN-1 was released by heparin from TRAP-stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri-cellular plasmin was increased in cells with a low PN-1 expression. Pre-treatment of smooth muscle cells with cycloheximide abolished the reduction of PN-1 expression by thrombin. Furthermore, conditioned media from thrombin-treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor-2 (FGF-2)-neutralizing antibodies abolished thrombin effect whereas FGF-2 reproduced it, indicating that FGF-2 is one of the involved mediator. Together, these results indicate that (i) PN-1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down-regulates PN-1 expression and thus may increase its own activity on cells.
Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P = .001). Metabolic radiolabeling with (35)S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). This functional assay showed that the majority of the new protein was in an active configuration and could complex-bind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots.
neurysms of the aorta are at times evaluated and treated by physicians from a number of specialties. Indeed, whereas cardiac surgeons operate on the ascending aorta and arch and vascular surgeons manage abdominal aortic aneu- rysms, at present the responsibility often falls to cardiologists to oversee the medical care of patients with aortic disease of all types. However, although formally trained in "cardiovas- cular medicine," most cardiologists devote their attention to the heart and its coronary arteries, and relatively few have experience in the management of diseases of the aorta. It is therefore important that cardiologists acquire a sufficient knowledge base so that they can confidently evaluate and manage patients with aortic disease and know when it is appropriate to refer them for surgery. Toward this end, the purpose of this review is to summarize the current under- standing of thoracic and abdominal aortic aneurysms.
The cell response to transforming growth factor-beta1 (TGF-beta1), a multipotent cytokine with healing potential, varies according to tissue context. We have evaluated the ability of TGF-beta1 overexpression by endovascular gene therapy to stabilize abdominal aortic aneurysms (AAAs) already injured by inflammation and proteolysis.
Active TGF-beta1 overexpression was obtained in already-developed experimental AAAs in rats after endovascular delivery of an adenoviral construct encoding for a mutated form of active simian TGF-beta1 and in an explant model using human atherosclerotic AAA fragments incubated with recombinant active TGF-beta1. Transient exogenous TGF-beta1 overexpression by endovascular gene delivery was followed by induction of endogenous rat TGF-beta1. Overexpression of active TGF-beta1 in experimental AAAs was associated with diameter stabilization, preservation of medial elastin, decreased infiltration of monocyte-macrophages and T lymphocytes, and a decrease in matrix metalloproteinase-2 and -9, which was also observed in the explant model, in both thrombus and wall. In parallel with downregulation of the destructive process, active TGF-beta1 overexpression triggered endoluminal reconstruction, replacing the thrombus by a vascular smooth muscle cell-, collagen-, and elastin-rich intima.
Local TGF-beta1 self-induction after transient exogenous overexpression reprograms dilated aortas altered by inflammation and proteolysis and restores their ability to withstand arterial pressure without further dilation. This first demonstration of stabilization of expanding AAAs by delivery of a single multipotent self-promoting gene supports the view that endovascular gene therapy should be considered for treatment of aneurysms.
Transforming growth factor-beta (TGF-beta) participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy and heart failure. TGF-beta exerts pleiotropic effects on cardiovascular cells, regulating cell growth, fibrosis and inflammation. TGF-beta has long been believed to be the most important extracellular matrix regulator. We review the complex mechanisms involved in TGF-beta-mediated vascular fibrosis that includes the Smad signaling pathway, activation of protein kinases and crosstalk between these pathways. TGF-beta blockade diminishes fibrosis in experimental models, however better antifibrotic targets are needed for an effective therapy in human fibrotic diseases. A good candidate is connective tissue growth factor (CTGF), a downstream mediator of TGF-beta-induced fibrosis. Among the different factors involved in vascular fibrosis, Angiotensin II (AngII) has special interest. AngII can activate the Smad pathway independent of TGF-beta and shares with TGF-beta many intracellular signals implicated in fibrosis. Blockers of AngII have demonstrated beneficial effects on many cardiovascular diseases and are now one of the best options to block TGF-beta fibrotic responses. A better knowledge of the intracellular signals of TGF-beta can provide novel therapeutic approaches for fibrotic diseases.