Perlecan Heparan Sulfate Is Required for the Inhibition of Smooth Muscle Cell Proliferation by All-trans-Retinoic Acid.

Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti-proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti-proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all-trans-retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild-type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate-deficient perlecan, indicating that the inhibition is perlecan heparan sulfate-dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild-type and mutant SMCs, consistent with previous studies of perlecan-dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate-chains.

High plasma adiponectin concentration is associated with all-cause mortality in patients with carotid atherosclerosis.

BACKGROUND: The role of adiponectin as a risk factor for mortality and recurrent ischemic cardiovascular events in patients with carotid artery disease is unknown. METHODS: Consecutive patients (n = 292) undergoing carotid endarterectomy for symptomatic and asymptomatic carotid stenosis were included in the study. Mortality and cardiovascular ischemic events were recorded during a median follow-up of 5.2 years. Baseline plasma concentrations of adiponectin were measured. Cox regression models stratified for gender were used for estimation of risk of events. RESULTS: Fifty-two patients died and 73 had an ischemic event (ischemic stroke, n = 52 and/or MI, n = 28) during follow-up. In univariate analyses, adiponectin was associated with mortality, hazard ratio (HR) per standard deviation (SD) increase of adiponectin, 1.46 (95% confidence interval [CI], 1.14-1.86). In multivariate analysis age, type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), plasma interleukin-6 (IL-6) and plasma adiponectin (HR per SD increase of adiponectin, 1.73 [95% CI, 1.29-2.32]) were independently associated with mortality. T2DM, CHD, fibrinogen, contralateral carotid artery stenosis, systolic blood pressure, symptomatic carotid artery stenosis were independently associated with ischemic events, whereas plasma adiponectin was not (HR per SD increase of adiponectin, 0.96 [95% CI, 0.75-1.23]). CONCLUSIONS: High plasma adiponectin concentration is associated with mortality in patients with established atherosclerosis undergoing surgery for carotid artery stenosis. Further studies to determine the role for adiponectin as a biomarker are warranted.

Prediction of ischemic events on the basis of transcriptomic and genomic profiling in patients undergoing carotid endarterectomy.

Classic risk factors, including age, smoking, serum cholesterol, diabetes and blood pressure, constitute the basis of present risk prediction models but fail to identify all individuals at risk. The objective of this study was to investigate if genomic and transcriptional patterns improve prediction of ischemic events in patients with established carotid artery disease. Genotype and gene expression profiles were obtained from carotid plaque tissue (n = 126) and peripheral blood mononuclear cells (n = 97) of patients undergoing carotid endarterectomy. Patients were followed for an average of 44 months, and 25 ischemic events occurred (18 ischemic strokes and 7 myocardial infarctions). Blinded leave-one-out cross-validation on Cox regression coefficients was used to assign gene expression-based risk scores to each patient. When compared with classic risk factors, addition of carotid plaque gene expression-based risk score improved the prediction of future ischemic events from an area under the curve (AUC) of 0.66 to an AUC of 0.79. The inclusion of gene expression risk score from peripheral blood mononuclear cells or from 25 established myocardial infarction risk single nucleotide polymorphisms only exhibited marginal effects on the prediction of ischemic events. Prediction of ischemic events is improved by inclusion of gene expression profiling from carotid endarterectomy tissue compared with prediction on the basis of classic risk markers alone in patients with atherosclerosis. The method may be developed to identify subjects at very high risk of ischemic events.

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: associations with cerebrovascular symptoms.

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.

Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival.

OBJECTIVE: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. METHODS: Laminar FSS of arterial level (14 dynes/cm(2)) was applied to SMC cultures for 24 hours in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology, and results further verified by real time polymerase chain reaction (RT-PCR) and immunoblotting. Tissue factor pathway inhibitor-2 (TFPI-2) and caspase-3 protein expression was studied in the rat carotid artery after balloon-injury, and the effect of TFPI-2 on SMC DNA synthesis and apoptosis was examined in vitro. RESULTS: Microarrays identified TFPI-2 as one of the most differentially expressed gene by FSS in cultured SMCs (P < .001). Gene set enrichment analysis revealed significant regulation of genes linked to proliferation, apoptosis, and cell cycle regulation. TFPI-2 induction was confirmed by RT-PCR and immunoblotting demonstrating a more than 400-fold (P < .001) increase in TFPI-2 mRNA in SMCs exposed to FSS compared with static controls, and a consistent protein upregulation. Functionally, SMC proliferation was decreased by FSS (P < .001), and recombinant TFPI-2 was found to inhibit SMC proliferation (P < .001) and induce SMC apoptosis as indicated by activation of caspase-3 (P < .01). In vivo, TFPI-2 expression was found to be upregulated 5, 10, and 20 hours (P < .01) after rat carotid balloon injury, and immunohistochemistry demonstrated TFPI-2 protein in FSS-exposed luminal SMCs, co-localized with caspase-3 in the rat carotid neointima. CONCLUSION: FSS influenced gene expression associated with cell growth and apoptosis in cultured SMCs and strongly induced expression of TFPI-2 mRNA and protein. TFPI-2 was expressed in luminal, FSS-exposed SMCs together with caspase-3 in the rat carotid neointima after balloon injury. Functionally, TFPI-2 may play a role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 controls SMC function.

Reduced perlecan expression and accumulation in human carotid atherosclerotic lesions.

Heparan sulfate in the extracellular matrix of the artery wall has been proposed to possess anti-atherogenic properties by interfering with lipoprotein retention, suppression of inflammation, and inhibition of smooth muscle cell growth. Previously, the amount of heparan sulfate in atherosclerotic lesions from humans and animals has been shown to be reduced but the identity or identities of the heparan sulfate molecules being down regulated in this disease are not known. In this study, atherosclerotic lesions were retrieved from 44 patients undergoing surgery for symptomatic carotid stenosis. Normal iliac arteries from organ donors were used as controls. Analysis of the specimens by gene microarray showed a selective reduction in perlecan gene expression, whereas, expression of the other heparan sulfate proteoglycans in the artery wall, agrin and collagen XVIII, remained unchanged. Expression of the large chondroitin sulfate proteoglycan, versican, also remained unchanged. Real-time PCR confirmed the decrease in perlecan gene expression and the unchanged expression of versican. The findings were supported by immunohistochemical analysis demonstrating a reduced accumulation of both perlecan core protein and heparan sulfate in carotid lesions. The study demonstrates a reduction of perlecan mRNA-expression and protein deposition in human atherosclerosis, which in part explains the low levels of heparan sulfate in this disease.