Rationale: Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive precluding mechanism-based therapies. Objective: Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent ROS-forming NADPH oxidase 5 (Nox5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN1, αSMA and SM22α and an increase in synthetic marker S100A4 compared to contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca 2+ . Phenotypic switching was accompanied by increased levels of reactive oxygen species (ROS) and Ca 2+ -dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knock-down of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca 2+ -dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca 2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca 2+ . Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca 2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. Conclusions: We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca 2+ uptake via EVs and show that Ca 2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodelling and calcification in the context of mineral imbalance.

Objective: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. Conclusions: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Recent investigations have suggested that inflammasome activation plays an important role during atherosclerosis. Upon activation, the inflammasome induces processing and release of pro‐inflammatory cytokines interleukin 1β (IL‐1β) and interleukin 18 (IL‐18) via activation of caspase‐1/11. Previously, it was shown that complete caspase‐1 deficiency is protective against atherosclerosis development. However, while macrophages are the main inflammatory cells involved in atherosclerosis, the exact role of macrophage‐specific caspase‐1/11 activation during development of cardiovascular disease has never been investigated. We hypothesized that hematopoietic caspase‐1/11 deficiency leads to reduced atherosclerosis development. To investigate the specific contribution of hematopoietic caspase‐1/11 activation to atherosclerosis development, Ldlr−/− mice received a transplant (tp) of wild‐type (WT) or caspase‐1/11−/− bone marrow, to create WT‐tp mice and caspase‐1/11−/−‐tp mice, and fed a high‐fat, high‐cholesterol diet for 12 weeks. Our results showed an increase in anti‐inflammatory blood leukocytes in caspase‐1/11−/−‐tp mice compared with WT‐tp mice, as indicated by a decreased level of Ly6Chigh monocytes and an increased level of Ly6Clow monocytes. In line with our hypothesis, hematopoietic deletion of caspase‐1/11 resulted in a strong reduction in atherosclerotic plaque size. Furthermore, necrotic core content was dramatically decreased in caspase‐1/11−/−‐tp mice. Our data indicate that hematopoietic caspase‐1/11 activation is involved in vascular inflammation and atherosclerosis, and plays an important role in cardiovascular disease progression.

Annexin A5 (AnxA5) exerts anti-inflammatory, anticoagulant and anti-apoptotic effects through its binding to cell surface expressed phosphatidylserine. We previously showed that AnxA5 can stabilize advanced atherosclerotic plaques by reducing macrophage infiltration. We now investigated the effects of AnxA5 administration on the onset of atherosclerosis development. Eight-week-old ApoE-/-mice were fed a western diet while being administered AnxA5 or control (M1234) for a total of 6 weeks. AnxA5 administration reduced plaque size in the aortic root as well as the aortic arch by 36% and 55% respectively. As determined by immunohistochemistry, administration of AnxA5 further stabilized plaque by reducing macrophage content and increasing smooth muscle cell content. Furthermore, the pre-treatment of HUVEC’s with AnxA5 reduced monocyte adhesion under flow-conditions. Finally, AnxA5 administration results in a trend to reduced cell death more pronounced in the aortic arch than the aortic root. In conclusion, treatment with AnxA5 before the onset of atherosclerosis reduces plaque formation in a murine model of atherosclerosis in part by reducing apoptotic rates further to its beneficial effect on macrophage infiltration and activation.

Background:Rapid and accurate new biomarkers to predict risk of cardiovascular disease (CVD) are essential. The utility of extracellular vesicles in predicting the CVD risk is postulated, yet it remains unknown whether their expression is altered in response to statin therapy.Methods:We performed in-vitro studies with human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (hVSMC), and conducted a nested case–control study (nCCS) in hypertensive patients (n = 40) randomized to either atorvastatin or placebo in the ASCOT-LLA. Cases had a major adverse cardiovascular event or death (MACE) during 3.5 years of follow-up (median) from the time of extracellular vesicle characterization while controls, matched for age and duration of treatment, remained event-free. Conditional logistic regression models determined the risk of MACE. Additionally, the relationship of extracellular vesicle levels with statin therapy was assessed.Results:Added to HUVEC, extracellular vesicles increased neutrophil recruitment, and to hVSMC, aggravated calcification and proliferation. In the nCCS, compared with controls, cases (i.e. with MACE) had preceding higher levels of CD14+ and CD14+/CD41+ extracellular vesicles (P = 0.009 and P = 0.012, respectively) and a significant reduction in the median size of the vesicles (P = 0.037). On matched analysis, higher CD14+ extracellular vesicles were associated with a 3.7-fold increased risk of MACE (P = 0.032). Patients treated with atorvastatin (vs. placebo) had both reduced size of extracellular vesicles and the proportion of CD146+ extracellular vesicles (P = 0.034 and P = 0.020, respectively).Conclusion and relevance:These pilot analyses suggest a mechanistic role for extracellular vesicles in the development of CVD, with significant and differential changes in extracellular vesicles amongst those at risk of MACE, and those on atorvastatin therapy.