Fibromodulin and Dystrophin in Atherosclerosis: Novel roles for extracellular matrix in plaque development

Cardiovascular disease represents nearly half the cases of noncommunicable diseases worldwide and is the leading global cause of death. The main underlying cause is atherosclerosis, and in atherosclerotic plaque progression the structure, composition and integrity of a dynamic extracellular matrix (ECM) is one very important factor. This thesis discusses the importance of the connection between the ECM and cells for atherosclerotic lesion development. It also tests the hypothesis that changes in this connection – whether through a modified collagenous ECM, or modification in a cellular protein directly linking cells to ECM components – have a significant impact on atherosclerotic plaque structure and stability. Fibromodulin is a small leucine-rich repeat proteoglycan of the ECM involved in the regulation of collagen fiber synthesis. In the fibrous murine atherosclerotic plaque, we found collagen fibrils synthesized in the absence of fibromodulin to be thicker and more heterogeneous, compared to fibrils generated in the control mouse. Murine plaques with an inflammatory phenotype, with a fibromodulin-deficient ECM, were also smaller with decreased lipid accumulation, whereas cell proliferation was increased. In addition, we show, for the first time, that high fibromodulin expression in the most stenotic region of a human carotid artery plaque is found in plaques from symptomatic patients, and in patients with diabetes. Fibromodulin expressed in human plaques co-localize with Oil Red O-staining and correlates with the area stained for lipids (quantified as percentage). Fibromodulin also correlates with the pro-inflammatory cytokines MIP-1β and sCD40L, as well as with VEGF and inversely with the anti-inflammatory cytokine IL-10. Dystrophin, and the dystrophin-glycoprotein complex (DGC), link the actin cytoskeleton to the basement membrane. This thesis provides novel data on dystrophin deficiency, as illustrated by the mdx mouse. The absence of dystrophin stimulates neointimal hyperplasia, but inhibits atherosclerotic lesion development driven by lipid-retention. In the mdx-mouse, laminin expression is decreased in the endothelium of atherosclerotic lesions with an inflammatory phenotype, suggesting that altered endothelial cell function and an abnormal basement membrane may be a possible explanation for the attenuated plaque development. In summary, this thesis shows that collagen structure and remodeling affects the growth, development and composition of atherosclerotic lesions, on the one hand promoting mechanical stability, and on the other hand affecting lipid accumulation and inflammation. In addition, growth of vascular lesions can be inhibited or promoted by a functional connection between cells and the ECM mediated by dystrophin and the DGC

Increased Neointimal Thickening in Dystrophin-Deficient mdx Mice

BACKGROUND: The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation. METHODOLOGY/PRINCIPAL FINDINGS: We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice. CONCLUSIONS/SIGNIFICANCE: These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening

Expression of fibromodulin in carotid atherosclerotic plaques is associated with diabetes and cerebrovascular events.

The small leucine-rich proteoglycans fibromodulin and lumican are functionally related extracellular matrix proteins involved in the regulation of collagen fiber formation. Fibromodulin-deficient apolipoprotein E-null mice have decreased vascular retention of lipids and reduced development of atherosclerosis suggesting that fibromodulin may influence the disease process. The aim of the present study was to investigate if fibromodulin and lumican are expressed in human carotid plaques and to determine if their expression is associated with the occurrence of preoperative symptoms and with risk for postoperative cardiovascular events

Soluble CD40 levels in plasma are associated with cardiovascular disease and in carotid plaques with a vulnerable phenotype

Background and Purpose CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability. Methods Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro-and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort. Results Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling. Conclusions Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.</p

Fibromodulin Deficiency Reduces Low-Density Lipoprotein Accumulation in Atherosclerotic Plaques in Apolipoprotein E-Null Mice.

OBJECTIVE: The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. METHODS AND RESULTS: Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. CONCLUSIONS: The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability

Plasma levels of PRO-C3, a type III collagen synthesis marker, are associated with arterial stiffness and increased risk of cardiovascular death

Background and aims: The N-terminal propeptide of type III collagen (PRO-C3) assay measures a pro-peptide released during type III collagen synthesis, an important feature of arterial stiffening and atherogenesis. There is a clinical need for improved non-invasive, cheap and easily accessible methods for evaluating individuals at risk of cardiovascular disease (CVD). In this study, we investigate the potential of using circulating levels of PRO-C3 to mark the degree of vascular stenosis and risk of cardiovascular events. Methods: Baseline plasma levels of PRO-C3 were measured by ELISA in subjects belonging to the SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort (N = 1354). Associations between PRO-C3 levels with vascular characteristics, namely stiffness and stenosis, and risk of future cardiovascular events were explored. Subjects were followed up after a median of 35 months (interquartile range 34–36 months), with recorded outcomes cardiovascular death and all-cause mortality. Results: We found a correlation between PRO-C3 levels and pulse wave velocity (rho 0.13, p = 0.000009), a measurement of arterial stiffness. Higher PRO-C3 levels were also associated with elevated blood pressure (rho 0.07, p = 0.014), as well as risk of cardiovascular mortality over a three-year follow-up period (OR 1.56, confidence interval 1.008–2.43, p = 0.046). Conclusions: Elevated circulating PRO-C3 levels are associated with arterial stiffness and future cardiovascular death, in the SUMMIT cohort, suggesting a potential value of PRO-C3 as a novel marker for declining vascular health.</p

Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review

Extracellular matrix: paving the way to the newest trends in atherosclerosis

PURPOSE OF REVIEW: The extracellular matrix (ECM) is critical for all aspects of vascular pathobiology. In vascular disease the balance of its structural components is shifted. In atherosclerotic plaques there is in fact a dynamic battle between stabilizing and proinflammatory responses. This review explores the most recent strides that have been made to detail the active role of the ECM - and its main binding partners - in driving atherosclerotic plaque development and destabilization. RECENT FINDINGS: Proteoglycans-glycosaminoglycans (PGs-GAGs) synthesis and remodelling, as well as elastin synthesis, cross-linking, degradation and its elastokines potentially affect disease progression, providing multiple steps for potential therapeutic intervention and diagnostic targeted imaging. Of note, GAGs biosynthetic enzymes modulate the phenotype of vascular resident and infiltrating cells. In addition, while plaque collagen structure exerts very palpable effects on its immediate surroundings, a new role for collagen is also emerging on a more systemic level as a biomarker for cardiovascular disease as well as a target for selective drug-delivery. SUMMARY: The importance of studying the ECM in atherosclerosis is more and more acknowledged and various systems are being developed to visualize, target and mimic it

Glucocorticoid induced TNF receptor family-related protein (GITR)-A novel driver of atherosclerosis

Atherosclerosis is a lipid-driven, chronic inflammatory disease. In spite of efficient lipid lowering treatments, such as statins and PCSK9 inhibitors, patients, especially those with elevated inflammatory biomarkers, still have a significant residual cardiovascular disease risk. Novel drugs targeting inflammatory mediators are needed to further reduce this residual risk. Agonistic immune checkpoint proteins, including CD86, CD40L and CD40, have been shown to be drivers of atherosclerosis. Recently, glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR), a co-stimulatory immune checkpoint protein, was identified to be pivotal in cardiovascular disease. Cardiovascular patients have elevated soluble GITR plasma levels compared to healthy controls. Furthermore, in human carotid endarterectomy plaques, GITR expression was higher in plaques from symptomatic compared to asymptomatic patients and correlated with features of plaque vulnerability. Moreover, depleting GITR reduced atherosclerotic plaque development in mice. GITR-deficient monocytes and macrophages exhibited less inflammatory potential and reduced migratory capacity. In this review, we discuss GITR's effects on various immune cells, mechanisms, signalling pathways and finally GITR's potential as a novel drug target in atherosclerosis