PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling.

RATIONALE: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. OBJECTIVE: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. METHODS AND RESULTS: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6-/- versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6-/- mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. CONCLUSIONS: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions.

RATIONALE: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. OBJECTIVE: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. METHODS AND RESULTS: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction-based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. CONCLUSIONS: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

The role of the FPR2/ALX receptor in atherosclerosis development and plaque stability.

AIMS: The formyl peptide receptor (FPR) subtype FPR2/ALX transduces pro-inflammatory responses and participates in the resolution of inflammation depending on activation. The aim of the present study was to unravel the role of FPR2/ALX signalling in atherosclerosis. METHODS AND RESULTS: Expression of FPR2/ALX was analysed in 127 human carotid atherosclerotic lesions and revealed that this receptor was expressed on macrophages, smooth muscle cells (SMCs), and endothelial cells. Furthermore, FPR2/ALX mRNA levels were significantly up-regulated in atherosclerotic lesions compared with healthy vessels. In multiple regression, age, creatinine, and clinical signs of increased cerebral ischaemia were independent predictors of FPR2/ALX expression. To provide mechanistic insights into these observations, we generated Ldlr(-/-)xFpr2(-/-) mice, which exhibited delayed atherosclerosis development and less macrophage infiltration compared with Ldlr(-/-)xFpr2(+/+) mice. These findings were reproduced by transplantation of Fpr2(-/-) bone marrow into Ldlr(-/-) mice and further extended by in vitro experiments, demonstrating a lower inflammatory state in Fpr2(-/-) macrophages. FPR2/ALX expression correlated with chemo- and cytokines in human atherosclerotic lesions and leucocytes. Finally, atherosclerotic lesions in Ldlr(-/-)xFpr2(-/-) mice exhibited decreased collagen content, and Fpr2(-/-) SMCs exhibited a profile of increased collagenase and decreased collagen production pathways. CONCLUSION: FPR2/ALX is proatherogenic due to effects on bone marrow-derived cells, but promoted a more stable plaque phenotype through effects on SMCs. Taken together, these results suggest a dual role of FPR2/ALX signalling in atherosclerosis by way of promoting disease progression and but increasing plaque stability.

Sphingosine-1-phosphate: A bioactive lipid that confers high-density lipoprotein with vasculoprotection mediated by nitric oxide and prostacyclin.

Sphingosine-1-phosphate (S1P) is a bioactive lipid generated in the intracellular membranes from the metabolism of sphingomyelin. Once secreted/exported by cells of haematopoietic origin and vascular cells S1P interacts with plasma proteins and accumulates in high-density lipoprotein (HDL). Growing evidence indicates that HDL-associated S1P is responsible for the beneficial effects of these lipoproteins on vasorelaxation, cell survival, cell adhesiveness, angiogenesis and synthesis of two powerful endogenous anti-atherogenic and anti-thrombotic molecules such as nitric oxide (NO) and prostacyclin (PGI(2)). It is likely that vascular effects of HDL-S1P are regulated by the local expression of S1P receptors. Five G protein-coupled receptors (S1P(1) to S1P(5)), with differential expression patterns and dissimilar coupling mechanism to G protein subunits, have been identified in the vasculature. This review is focused on the central role of S1P as a bioactive component that confers vasculoprotective properties to HDL by eliciting a wide range of biological responses on endothelial and smooth muscle cells largely dependent on the up-regulation of NO and prostacyclin.

Prostacyclin induction by high-density lipoprotein (HDL) in vascular smooth muscle cells depends on sphingosine 1-phosphate receptors: effect of simvastatin.

Prostacyclin (PGI2) is an important regulator of vascular homeostasis. Our goal was to analyze the role of sphingosine 1-phosphate (S1P) and its receptors in the up-regulation of cyclooxygenase-2 (Cox-2) induced by HDL in human vascular smooth muscle cells (VSMC). S1P induces Cox-2 expression in a time-and dose-dependent manner at concentrations (0.02-1 microM) compatible with those present in physiological HDL levels. The effect was mimicked by dihydro-S1P (DhS1P), a S1P derivative that only acts through cell surface S1P receptors. Desensitization of S1P receptors with S1P (or DhS1P) abolished HDL-induced Cox-2 up-regulation and PGI2 release. Inhibition of S1P receptors by suramin (inhibitor of S1P3), JTE013 (inhibitor of S1P2) or VPC23019 (inhibitor of S1P1 and S1P3) reduced the up-regulation of Cox-2 induced by HDL and S1P. The combination of suramin and JTE013 increased the inhibitory effect compared to that observed in cells treated with each inhibitor alone. siRNA against S1P2 or S1P3 significantly reduced the ability of HDL and S1P to up-regulate Cox-2. Simvastatin induced over-expression of S1P3 and potentiated the induction of Cox-2 expression produced by HDL (or S1P). Finally, suramin, JTE013 and VPC23019 inhibited p38 MAPK and ERK1/2 signaling pathways activated by HDL (or S1P) and the downstream activation of CREB, a key transcription factor involved in Cox-2 transcriptional up-regulation. These results indicate that S1P receptors, in particular S1P2 and S1P3, are involved in the Cox-2-dependent effects of HDL on vascular cells. Strategies aimed to therapeutically modulate S1P or S1P receptors could be useful to improve cardiovascular protection.

Oleanolic acid induces prostacyclin release in human vascular smooth muscle cells through a cyclooxygenase-2-dependent mechanism.

Oleanolic acid is a triterpenoid that may contribute to the cardio-protective effects of olive oil. Our goal was to assess whether oleanolic acid could modulate eicosanoid biosynthesis and to determine the mechanism involved in this effect. Human coronary smooth muscle cells (SMC) were treated with oleanolic acid, erythrodiol, or hydroxytyrosol and eicosanoid release was measured by enzyme immunoassay. Cyclooxygenase (Cox)-1 and Cox-2 protein and messenger sRNA levels were analyzed by Western blot and real-time PCR, respectively. Mitogen-activated protein kinase (MAPK) pathways were assessed using specific antibodies. Oleanolic acid induced prostaglandin I2 (PGI2) release by human coronary SMC, an effect that was prevented by celecoxib (a specific inhibitor of Cox-2). The increased PGI2 was time-and dose-dependent and was associated to the up-regulation of Cox-2. No effects were observed on thromboxane A2. Erythrodiol but not hydroxytyrosol upregulated Cox-2 expression and induced PGI2 synthesis. Oleanolic acid induced an early phosphorylation of p38 MAPK and p42/44 MAPK but not c-Jun N-terminal kinase-1 (JNK-1). SB203580 (p38MAPK inhibitor) and U0126 (MAPK kinase1/2 inhibitor) abrogated the upregulation of Cox-2 and PGI2 release induced by oleanolic acid. A peptide inhibitor of JNK-1 (L-JNKI1) did not produce any effect. The induction of Cox-2 was preceded by an early activation of cAMP regulatory element-binding protein, a key transcription factor involved in Cox-2 transcriptional upregulation. Therefore, oleanolic acid contributes to vascular homeostasis by inducing PGI2 release in a Cox-2-dependent manner. Oleanolic acid could be regarded as a bioactive molecule that may contribute to the beneficial effects of the Mediterranean diet.

Lysyl oxidase and endothelial dysfunction: mechanisms of lysyl oxidase down-regulation by pro-inflammatory cytokines.

Lysyl oxidase (LOX) plays a pivotal role in extracellular matrix (ECM) maturation. Furthermore, novel biological functions has been ascribed to LOX, among them cell differentiation, migration, transformation and regulation of gene expression. In this context, it has been suggested that abnormalities of LOX expression could underlie the development of multiple pathological processes including cardiovascular diseases. LOX seems to be crucial in the preservation of endothelial barrier function. In fact, accumulating evidences suggest a role of this enzyme in atherogenesis and endothelial dysfunction triggered by atherosclerotic risk factors and pro-inflammatory cytokines. Indeed, cytokines such as tumour necrosis factor-alpha (TNF-alpha) modulate vascular LOX expression. This cytokine decreases LOX expression and activity in endothelial cells through a transcriptional mechanism that involves TNF receptor-2 and protein kinase C activation. Interestingly, in vivo studies reveal that TNF-alpha causes a down-regulation of vascular LOX expression. Thus, LOX down-regulation seems to be associated to the endothelial dysfunction elicited by multiple pathological factors. LOX rises as a promising target gene for the development of therapeutic strategies in the treatment of cardiovascular diseases.