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[Figure: see text].

Objective: Vascular calcification is a pathology characterized by arterial mineralization, which is a common late-term complication of atherosclerosis that independently increases the risk of adverse cardiovascular events by fourfold. A major source of calcifying cells is transdifferentiating vascular smooth muscle cells (VSMCs). Previous studies showed that deletion of the collagen-binding receptor, DDR1 (discoidin domain receptor-1), attenuated VSMC calcification. Increased matrix stiffness drives osteogenesis, and DDR1 has been implicated in stiffness sensing in other cell types; however, the role of DDR1 as a mechanosensor in VSMCs has not been investigated. Here, we test the hypothesis that DDR1 senses increased matrix stiffness and promotes VSMC transdifferentiation and calcification. Approach and Results: Primary VSMCs isolated from Ddr1 +/+ (wild-type) and Ddr1 -/- (knockout) mice were studied on collagen-I-coated silicon substrates of varying stiffness, culturing in normal or calcifying medium. DDR1 expression and phosphorylation increased with increasing stiffness, as did in vitro calcification, nuclear localization of Runx2 (Runt-related transcription factor 2), and expression of other osteochondrocytic markers. By contrast, DDR1 deficient VSMCs were not responsive to stiffness and did not undergo transdifferentiation. DDR1 regulated stress fiber formation and RhoA (ras homolog family member A) activation through the RhoGEF (rho guanine nucleotide exchange factor), Vav2. Inhibition of actomyosin contractility reduced Runx2 activation and attenuated in vitro calcification in wild-type VSMCs. Finally, a novel positive feedforward loop was uncovered between DDR1 and actomyosin contractility, important in regulating DDR1 expression, clustering, and activation., Conclusions: This study provides mechanistic insights into DDR1 mechanosignaling and shows that DDR1 activity and actomyosin contractility are interdependent in mediating stiffness-dependent increases in VSMC calcification.

Objective: Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development., Approach and Results: A model of aortic dilatation by AngII (angiotensin II) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5 Δcat ). Adamts5 Δcat mice showed an attenuated rise in blood pressure while displaying increased dilatation of the ascending aorta (AsAo). Interestingly, a proteomic comparison of the aortic ECM from AngII-treated wild-type and Adamts5 Δcat mice revealed versican as the most upregulated ECM protein in Adamts5 Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine) and a decrease of LRP1 (low-density lipoprotein-related protein 1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5 , attenuated the generation of versikine, but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5 Δcat mice but was not sufficient to maintain versican processing and prevent aortic dilatation., Conclusions: Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation., (© 2018 The Authors.)

Objective: Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury., Approach and Results: Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). mmp13 and mmp2 were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE -/- (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition., Conclusions: FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease., (© 2018 The Authors.)

Matrix metalloproteinases (MMPs) can degrade strength-giving collagens and other structural proteins of the arterial extracellular matrix. Overproduction of MMPs by monocyte/macrophages could therefore promote atherosclerotic plaque rupture and myocardial infarction. Freshly-recruited monocyte macrophages appear to use a prostaglandin (PG)-dependent pathway to coordinately upregulate a broad and potentially highly-destructive spectrum of MMPs. Differentiated macrophages rely on a series of distinct pathways to selectively upregulate groups of MMPs. Moreover, recent evidence suggests that different macrophage phenotypes express characteristically different spectra of MMPs and their inhibitors. New therapies may result from targeting matrix MMP overproduction.

Objective: The cysteine proteases, cathepsins, have been implicated in vascular remodeling and atherosclerosis, processes known to be regulated by shear stress. It is not known, however, whether shear regulates cathepsins. We examined the hypothesis that shear stress regulates cathepsin activity in endothelial cells., Methods and Results: Mouse aortic endothelial cells (MAECs) exposed to atheroprotective, unidirectional laminar shear (LS) degraded significantly less BODIPY-labeled elastin and gelatin in comparison to static and proatherogenic oscillatory shear (OS). The cathepsin inhibitor E64 also reduced this activity. Gelatin zymography showed that cathepsin activity of MAECs was blunted by LS exposure and by a cathepsin L inhibitor but not by cathepsin B and S inhibitors, whereas a cathepsin K inhibitor had a minor effect. Cathepsin L siRNA knocked down cathepsin L expression, gelatinase, and elastase activity in OS and static MAECs. A partial reduction of cathepsin B protein raised the possibility that the siRNA effect on the matrix protease activity could have been attributable to cathepsin L or B. Cathepsin B activity study using the synthetic peptide showed it was not regulated by shear., Conclusions: These results suggest that cathepsin L is a shear-sensitive matrix protease and that it may play an important role in flow-mediated vascular remodeling and atherogenic responses.

Objective: T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death., Methods and Results: The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation., Conclusions: These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin.

Objective: The proteolytic activity of metalloproteinases, proinflammatory enzymes that degrade extracellular matrix, is elevated in lipid-rich atherosclerotic plaques, thereby contributing to plaque fragility and rupture. Pregnancy-associated plasma protein (PAPP-A) is a metalloproteinase, expressed in unstable atherosclerotic plaques, whose circulating levels are elevated in acute coronary syndromes. We evaluated serum PAPP-A levels as a marker of the premature development of atherosclerosis in hyperlipidemic subjects at elevated cardiovascular risk., Methods and Results: Serum PAPP-A levels were determined in asymptomatic hyperlipidemic male subjects (n=64; mean+/-SD age, 51+/-7 years) in whom intima-media thickness (IMT) and lesion status in the carotid artery were evaluated by noninvasive ultrasonography and compared with those of a normolipidemic control group (n=25). No difference was observed in circulating PAPP-A levels between hyperlipidemic subjects and controls (8.99+/-2.93 and 8.03+/-2.75 mIU/L, respectively; mean+/-SD) nor between hyperlipidemic subjects who presented with a luminal obstruction of the carotid artery (9.26+/-2.53 mIU/L) and those who did not (8.85+/-3.29 mIU/L). By contrast, in patients with atheromatous carotid plaques, a positive association between serum levels of PAPP-A and C-reactive protein was observed (P<0.05); moreover, subjects exhibiting hyperechoic or isoechoic, echogenic lesions had significantly higher PAPP-A levels compared with those with hypoechoic lesions (10.32+/-2.72 vs 8.27+/-2.18 mIU/L, P<0.05) and with normolipidemic controls (P<0.05)., Conclusions: Elevated serum PAPP-A levels represent a potential marker of the degree of echogenicity of carotid atherosclerotic plaques in asymptomatic hyperlipidemic patients at high cardiovascular risk and equally of an enhanced local inflammatory state involving remodeling of subendothelial extracellular matrix.

Objective: In human atherosclerotic lesions, degranulated mast cells are found in the vicinity of macrophage foam cells. Mast cell granules contain tryptase, a tetrameric serine protease requiring glycosaminoglycans for stabilization. No endogenous inhibitors have been described for tryptase, and the physiological functions of the enzyme are poorly understood. Here, we investigated the effects of human tryptase on the integrity of high density lipoprotein (HDL)3 and on its ability to release cholesterol from cultured mouse macrophage foam cells., Methods and Results: Incubation of HDL3 with tryptase led to degradation of its apolipoproteins. Tryptase predominantly degraded a quantitatively minor subfraction of HDL3 that is lipid poor, exhibits electrophoretic pre-beta mobility, and contains either apolipoprotein A-I or apolipoprotein A-IV as its sole apolipoprotein. Moreover, tryptase caused functional changes in HDL3 by destroying its ability to promote high-affinity efflux of cholesterol from macrophage foam cells, ie, the pre-beta-HDL-dependent component of the process. Human aortic proteoglycans increased the ability of tryptase to proteolyze HDL3, suggesting that the proteoglycan-rich extracellular matrix of the arterial intima provides an appropriate environment for the extracellular actions of tryptase., Conclusions: By depleting pre-beta-HDL, mast cell tryptase may impair the initial step of reverse cholesterol transport and will then favor cellular accumulation of cholesterol during atherogenesis.

Tumor necrosis factor (TNF) receptor superfamily 14 (TNFRSF14) is the cellular receptor for TNF superfamily 14 (LIGHT). Immunohistochemical staining of human carotid atherosclerotic plaques revealed a high level of expression of the TNFRSF14 in regions rich in macrophages/foam cells. To investigate the role of TNFRSF14 in the functioning of monocytes in relation to atherogenesis, we have analyzed TNFRSF14 expression levels and cellular events after stimulation of TNFRSF14 in peripheral blood monocytes or the human macrophage-like cell line, THP-1. A high level of expression of TNFRSF14 was detected in activated monocytes, in macrophages derived from monocytes, and in THP-1 cells. Concomitant activation of THP-1 cells with interferon-gamma and immobilized anti-TNFRSF14 monoclonal antibody resulted in synergistic induction of proatherogenic cytokines, such as TNF-alpha and interleukin-8. Activation of THP-1 cells with immobilized anti-TNFRSF14 monoclonal antibody induced expression of matrix metalloproteinase (MMP)-1, MMP-9, MMP-13, and tissue inhibitors of metalloproteinase-1 and -2. Furthermore, immunohistochemical staining of atherosclerotic plaques with severe infiltration of foam cells revealed that the expression patterns of TNFRSF14 and MMP-1, -9, and -13 overlapped. Treatment of THP-1 cells with soluble LIGHT also caused induction of MMP-9 and interleukin-8. These data suggest that TNFRSF14 is involved in atherosclerosis via the induction of proatherogenic cytokines and decreasing plaque stability by inducing extracellular matrix-degrading enzymes.

The aim of this study was to evaluate the involvement of the mitogen-activated protein kinase (ERK1/2) pathway in response to stretch in a blood vessel developing myogenic tone on stretch. Indeed, in resistance arteries and veins, the main effect of pressure is to induce a maintained vasoconstrictor (myogenic) tone. Isolated segments of rabbit facial vein were mounted in organ baths and submitted to isometric stretch. In this experimental model, myogenic tone was absent when the bath temperature was 33 degrees C. ERK1/2 activity was determined in each isolated segment by an in-gel kinase assay. Wall tension and ERK1/2 activity were measured in the same samples in the presence (39 degrees C) or in the absence of myogenic tone (33 degrees C). At 39 degrees C, a 5-mN wall tension induced myogenic tone (5.7+/-1.8 mN) and an increase in ERK1/2 activity (282+/-52% versus unstretched vessels, P<0.05). At 33 degrees C, in the absence of myogenic tone, ERK1/2 activity was similarly increased by stretch (254+/-35% versus unstretched vessels). The calcium-dependent and -independent protein kinase C (PKC) blocker Ro-31-8220 (5 x 10(-7) mol/L), but not the calcium-dependent PKC blocker Go-6976 (10(-6) mol/L), inhibited myogenic tone. However, ERK1/2 activity was not affected by either PKC blocker. Genistein (10(-7) mol/L), a general tyrosine kinase inhibitor, but not herbimycin A (5 x 10(-7) mol/L), a cSrc-family tyrosine kinase inhibitor, suppressed stretch-induced ERK1/2 activation (P<0.05) without affecting myogenic tone. Nifedipine (10(-6) mol/L), a voltage-dependent calcium entry inhibitor, and ryanodine (10(-6) mol/L), which depletes calcium stores, both inhibited ERK1/2 activity (113+/-12% and 121+/-7%, respectively; P<0. 05) without affecting myogenic tone. The mitogen-activated protein kinase kinase inhibitor PD 98059 (5 x 10(-6) mol/L) also inhibited ERK1/2 activation without affecting myogenic tone. The present results suggest that stretching the rabbit facial vein induced 2 distinct pathways, one leading to myogenic tone (via a non-calcium-dependent PKC activation) and one leading to ERK1/2 activation through a calcium-dependent pathway involving tyrosine kinase.

We recently reported on the immunolocalization of type II secretory nonpancreatic phospholipase A2 (snpPLA2) in human atherosclerotic lesions. In the present study, we present data on the distribution and ultrastructural localization of snpPLA2 in adjacent nonatherosclerotic and atherosclerotic regions of human arteries. Electron microscopy (EM) of immunogold labeling techniques with a monoclonal antibody was used to analyze arterial tissue. The human specimens analyzed were obtained from autopsy and surgery cases. The results with EM showed a stronger snpPLA2 immunoreactivity in regions of arteries with atherosclerotic lesions than in regions without lesions from the same individual. snpPLA2 immunoreactivity was stronger in the arterial intima of atherosclerotic than of nonatherosclerotic tissue. EM-immunogold examination revealed that the majority of snpPLA2 was localized along the extracellular matrix, associated with collagen fibers and other extracellular matrix structures. Intracellular snpPLA2 was observed in electron-dense vesicles in intimal cells. snpPLA2 was also found in contact with large, extracellular lipid droplets. These results support the hypothesis that extracellular snpPLA2 is localized at sites where it may hydrolyze phospholipids from lipoproteins and lipid aggregates retained in the extracellular matrix of the arterial wall. This may be a mechanism for in situ release of proinflammatory lipids, free fatty acids, and lysophosphatidylcholine in regions of apolipoprotein B accumulation, which are abundant in atherosclerotic lesions.