Smooth Muscle Cells In Atherosclerotic Lesions Research Articles

Abstract 17038: Twist1 Modulates Smooth Muscle Cell Phenotypes in Atherosclerosis

Introduction: Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and Twist1 genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases Twist1 expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types, Twist1 is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis. Hypothesis: Twist1 modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability in vivo . Methods: Twist1 expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red. In vivo studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications. Results: Ectopic overexpression of Twist1 in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014). Twist 1 overexpression also decreased the total area of calcification (33% reduction, p=0.007). In vivo , both control and Twist 1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the Twist1 KO. Additionally, Twist1 KO mice have more prominent and larger focal calcifications. Conclusions: Twist1 promotes SMC proliferation and decreases calcification in vitro , and may affect the presence of subendothelial SMCs and calcification in vivo . This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing Twist1 to modulate SMC phenotypes.

Soluble LR11 is a novel biomarker for vascular lesions late after Kawasaki disease

ObjectiveCoronary artery lesions (CALs) and a risk for early onset of atherosclerosis are major concerns following Kawasaki disease (KD). Intimal smooth muscle cells (SMCs) have an important role in vascular lesions in KD. It is known that soluble LR11 (sLR11) is a novel biomarker for vascular lesions and LR11 is markedly expressed in intimal SMCs in atherosclerotic lesions. In this study, we hypothesized that sLR11 reflects the presence of vascular lesions late after KD. MethodsTwenty-three age-matched controls (group 1) and 59 patients with a history of KD were enrolled; 36 with KD had normal coronary arteries or regressed aneurysms (group 2), and 23 had CALs (group 3). ResultsSerum sLR11 levels in group 3 (median, interquartile range (IQR): 11.1 ng/mL, 9.3–13.9 ng/mL) were significantly higher than those in groups 1 (8.4 ng/mL, 7.1–10.2 ng/mL, p < 0.001) and 2 (9.0 ng/mL, 7.7–10.1 ng/mL, p < 0.01). Levels of sLR11 were positively correlated with levels of high-sensitivity C-reactive protein (r = 0.480, p < 0.01) and lipoprotein (a) (r = 0.486, p < 0.01). ConclusionThese findings suggest that sLR11 reflects the development of vascular lesions in patients with serious CALs.

Elevated mature macrophage expression of human ABHD2 gene in vulnerable plaque

We previously reported that the mouse α/β hydrolase domain containing 2 (Abhd2) was expressed in smooth muscle cells (SMCs) which suppressed their migration and inhibited the development of intimal hyperplasia by cuff placement; however, the role of ABHD2 in human remains to be elucidated. In this study, we examined ABHD2 expression in the human coronary atherosclerotic lesions of the patients with unstable angina (UA) and stable angina (SA). Our results showed that the ABHD2 was expressed in atherosclerotic lesions, and that the ABHD2 expression was significantly higher in the patients with UA than with SA. Immunohistochemistry analysis revealed abundant expression of ABHD2 in macrophages, but low expression in SMCs of atherosclerotic lesions. Using human vascular primary culture cell lines, we also demonstrated that the expression of ABHD2 was significantly higher in macrophages than in SMCs, and that the expression of ABHD2 significantly increased proportionally with differentiation from monocyte into macrophage.

Different Effects of Homocysteine and Oxidized Low Density Lipoprotein on Methylation Status in the Promoter Region of the Estrogen Receptor &amp;alpha; Gene

We investigated the effects of homocysteine (Hcy) and oxidized low density lipoprotein (ox-LDL) on DNA methylation in the promoter region of the estrogen receptor alpha (ERalpha) gene, and its potential mechanism in the pathogenesis of atherosclerosis. Cultured smooth muscle cells (SMCs) of humans were treated by Hcy and ox-LDL with different concentrations for different periods of time. The DNA methylation status was assayed by nested methylation-specific polymerase chain reaction, the lipids that accumulated in the SMCs and foam cell formations were examined with Oil red O staining. The proliferation of SMCs was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The results showed that ox-LDL in moderate concentrations (10-40 mg/L) induced de novo methylation in the promoter region of the ERalpha gene of SMCs. However, high concentrations (50 mg/L) of ox-LDL, resulted in demethylation of ERalpha. The Hcy treatment resulted in de novo methylation in the promoter region of the ERalpha gene with a concentration- and treating time-dependent manner, and a dose-dependent promoting effect on SMC proliferation. These data indicated that the two risk factors for atherosclerosis had the function of inducing de novo methylation in the promoter region of the ERalpha gene of SMCs. However, high concentrations (50 mg/L) of ox-LDL induced demethylation, indicating that different risk factors of atherosclerosis with different potency might cause different aberrant methylation patterns in the promoter region of the ERalpha gene. The atherogenic mechanism of Hcy might involve the hypermethylation of the ERalpha gene, leading to the proliferation of SMCs in atherosclerotic lesions.

Origin of Neointimal Smooth Muscle

The origin of smooth muscle cells (SMCs) in atherosclerotic lesions has been a hotly debated topic for more than four decades. Until recently, it was widely accepted that the majority of smooth muscle cells in atherosclerotic lesions originate from the medial layer of the vessel wall, with local SMC proliferation and migration leading to remodeling of the vessel wall and vessel lumen.1,2 Recent reports in the literature, however, claimed that SMCs in atherosclerotic lesions originate primarily from circulating bone marrow progenitor cells.3,4 Where these cells originate from is not merely academic, because the cells that give rise to SMCs within atherosclerotic lesions would likely serve as targets for current and future therapeutic interventions. The bone marrow–derived origin suggested by these articles would indicate that attempts to control SMC phenotype in in-stent restenosis, transplant atherosclerosis, or fibrous cap rupture should be targeted toward these cells from the marrow. However, we cautioned in 2004 that if anything, the bone marrow–derived intimal SMC paradigm was “Lost in Transdifferentiation”,5 and further studies were needed to clearly resolve this critically important issue. See page 2696 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Bentzon and colleagues provide excellent evidence that bone marrow–derived cells do not give rise to vascular SMCs in hyperlipidemia-induced atherosclerotic plaques in the apoE knockout mouse.6 These investigators studied plaques in apoE−/− mice that had their bone marrow reconstituted from sex-mismatched apoE−/−/eGFP+ animals, and then performed meticulous high-resolution z -axis sectioning of atherosclerotic lesions induced by Western diet to try to identify transdifferentiated cells staining for both the eGFP/Y chromosome lineage tags and the SMC marker smooth muscle α-actin (SMαA). Of critical importance, and in contrast to many previous studies in this area where investigators examined relatively small numbers of intimal SMCs …

Pravastatin Inhibits Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 (LOX-1) in Watanabe Heritable Hyperlipidemic Rabbits

LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mphis) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. Pravastatin downregulated LOX-1 expression in cultured human Mphis and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg(-1) d(-1) pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mphis and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.

Local generation of C-reactive protein in diseased coronary artery venous bypass grafts and normal vascular tissue.

Venous coronary artery bypass grafts (CABGs) are prone to accelerated atherosclerosis. In atherosclerotic diseases, serum C-reactive protein (CRP) levels have become an important diagnostic and prognostic marker. The origin of CRP in this setting remains to be elucidated. Monoclonal anti-CRP identified CRP expression in medial and intimal alpha-actin-positive smooth muscle cells (SMCs) of diseased CABGs with type V and VI lesions and also of native saphenous veins of atherosclerotic individuals. In addition, patent coronary arteries with type IV and V but not with type I through III lesions exhibited intense SMC staining for CRP. Calcified desobliterates of occluded coronary arteries with end-stage disease did not show SMC staining for CRP and were consistently negative for CRP mRNA, as detected by means of real-time polymerase chain reaction. However, CRP mRNA was expressed in 11 of 15 diseased CABGs and also in 10 of 15 native veins. By contrast, only 3 of 18 internal mammary and 4 of 12 radial arteries with virtually no atherosclerosis were positive for CRP mRNA. CRP is produced by SMCs of atherosclerotic lesions with active disease but not in end-stage plaques. The role of CRP constitutively expressed by normal vascular tissue in vein graft disease has yet to be elucidated.

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis.

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.

Heterogeneity of smooth muscle cells in advanced human atherosclerotic plaques: intimal smooth muscle cells expressing a fibroblast surface protein are highly activated by platelet-released products.

In vascular disease, smooth muscle cells (SMC) undergo phenotypic modulation and may acquire properties resembling those of fibroblasts in tissue wound healing. We aimed to show the differential expression of a fibroblast surface protein (FSP) by SMC in atherosclerotic lesions. In early human coronary atherosclerotic lesions the expression of FSP in the intima was absent. In contrast, 29 of 29 middle/advanced lesions contained intimal SMC expressing high levels of FSP. Fibroblast surface protein positive SMC were negative for desmin but expressed variable levels of alpha-SM actin, SM caldesmon, SM myosin heavy chain and vimentin. Explants from advanced atherosclerotic lesions yielded two main SMC subpopulations. SMC over-expressing FSP exhibited higher in vitro mitogenic response (premitotic DNA synthesis) to sera (2- to 8-fold) and platelet-released products (8- to 26-fold), especially from thrombin-activated platelets, than FSP-negative SMC. Our results suggest that the expression of FSP in SMC could indicate an activated phenotype, and the presence of highly positive FSP cells in the atherosclerotic lesions might be indicative of an increased SMC responsiveness to processes that locally generate thrombin and activate platelets.

Mast cell chymase induces apoptosis of vascular smooth muscle cells.

In human coronary atheromas, the numbers of degranulated mast cells and of apoptotic smooth muscle cells (SMCs) are increased. Accordingly, the possibility exists that mast cells participate in the regulation of SMC apoptosis in the lesions. Mast cells isolated from the serosal cavities of rats were stimulated to release their secretory granules. The neutral protease chymase, present in the exocytosed granules, was found to induce apoptosis when added to rat aortic SMCs in culture. The chymase-induced apoptosis of SMCs was detected by flow cytometry, microscopic analysis of cellular morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and electrophoretic demonstration of DNA laddering. Chymase induced SMC apoptosis in a dose- and time- dependent manner, and its proteolytic activity was essential for the proapoptotic effect. In addition to rat chymase, recombinant human chymase was also found to induce apoptosis of human coronary artery SMCs in culture. These results suggest that mast cells may participate in the apoptotic regulation of SMCs in atherosclerotic lesions.

Mast cell chymase induces apoptosis of vascular smooth muscle cells

In human coronary atheromas, the numbers of degranulated mast cells and of apoptotic smooth muscle cells (SMCs) are increased. Accordingly, the possibility exists that mast cells participate in the regulation of SMC apoptosis in the lesions. Mast cells isolated from the serosal cavities of rats were stimulated to release their secretory granules. The neutral protease chymase, present in the exocytosed granules, was found to induce apoptosis when added to rat aortic SMCs in culture. The chymase-induced apoptosis of SMCs was detected by flow cytometry, microscopic analysis of cellular morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and electrophoretic demonstration of DNA laddering. Chymase induced SMC apoptosis in a dose- and time- dependent manner, and its proteolytic activity was essential for the proapoptotic effect. In addition to rat chymase, recombinant human chymase was also found to induce apoptosis of human coronary artery SMCs in culture. These results suggest that mast cells may participate in the apoptotic regulation of SMCs in atherosclerotic lesions.

Cardiac alpha-actin in smooth muscle cells: detection in umbilical cord vessels and in atherosclerotic lesions.

Phenotypic modulation of smooth muscle cells (SMC) is a key event during the development of atherosclerotic and restenotic lesions. During this process, the composition of the cytoskeleton is substantially altered, with changes predominantly in actin expression reflecting a shift from smooth muscle alpha-actin to the non-muscle beta-isoform. We now demonstrate that yet another actin isoform, cardiac alpha-actin, is synthesized, de novo, in SMC of various atherosclerotic lesions. Using a highly specific monoclonal antibody against cardiac alpha-actin, we analyzed and compared the accumulation of this actin isoform in diverse SMC by immunofluorescence microscopy and immunoblotting. As expected, cardiac alpha-actin was present in human myocardium but not in healthy SMC of adult aorta, coronary arteries, trabeculae of the spleen, colon, stomach or skeletal muscle. Interestingly, the presence of cardiac alpha-actin was detected in umbilical cord vessels, human myometrium, in atherosclerotic coronary lesions and atherosclerotic lesions from peripheral vascular disease. The distribution of cardiac alpha-actin often paralleled that of cytokeratins 8 and 18, intermediate filament proteins typically found in dedifferentiated SMC. Taken together, the data presented here illustrate the expression of cardiac alpha-actin to be limited to either fetal vessels or those vessels or tissue having suffered damage or atrophy, outside its 'native' environment in the heart. The demonstration of cardiac alpha-actin in SMC of umbilical cord vessels and in atherosclerotic lesions but not in apparently healthy vessels supports the notion that SMC in atherosclerotic lesions exhibit a dedifferentiated phenotype.

Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions.

Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease.

Ciliated smooth muscle cells in aortic atherosclerotic lesions of rabbit

Single rudimentary cilia were observed by electron microscopy in smooth muscle cells (SMCs) of aortic fatty streaks in hypercholesterolemic rabbits, but not in aortae of controls. Similar cilia are known to occur in several tissues and various species, but it is believed that they have not so far been identified in the SMCs of atherosclerotic lesions. These cilia differ structurally from the classical type characterising ciliated epithelium. It is currently thought that a sudden transformation from mitotic replicative to nonmitotic structuring tissue may be correlated with the disappearance of centrioles and formation of cilia. The possible implications of the above concept in the overall process of atherosclerosis in the context of our present-day knowledge is briefly discussed.