Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification.

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 reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5). METHODS AND RESULTS: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca2+. Phenotypic switching was accompanied by increased levels of ROS and Ca2+-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca2+-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca2+. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca2+ 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 Ca2+ uptake via EVs and show that Ca2+ 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 remodeling and calcification in the context of mineral imbalance. Graphic Abstract: A graphic abstract is available for this article.

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles.

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.

Calcium Signalling in Heart and Vessels: Role of Calmodulin and Downstream Calmodulin-Dependent Protein Kinases.

Cardiovascular disease is the major cause of death worldwide. The success of medication and other preventive measures introduced in the last century have not yet halted the epidemic of cardiovascular disease. Although the molecular mechanisms of the pathophysiology of the heart and vessels have been extensively studied, the burden of ischemic cardiovascular conditions has risen to become a top cause of morbidity and mortality. Calcium has important functions in the cardiovascular system. Calcium is involved in the mechanism of excitation-contraction coupling that regulates numerous events, ranging from the production of action potentials to the contraction of cardiomyocytes and vascular smooth muscle cells. Both in the heart and vessels, the rise of intracellular calcium is sensed by calmodulin, a protein that regulates and activates downstream kinases involved in regulating calcium signalling. Among them is the calcium calmodulin kinase family, which is involved in the regulation of cardiac functions. In this review, we present the current literature regarding the role of calcium/calmodulin pathways in the heart and vessels with the aim to summarize our mechanistic understanding of this process and to open novel avenues for research.

Role of Vascular Smooth Muscle Cell Phenotypic Switching and Calcification in Aortic Aneurysm Formation.

Aortic aneurysm is a vascular disease whereby the ECM (extracellular matrix) of a blood vessel degenerates, leading to dilation and eventually vessel wall rupture. Recently, it was shown that calcification of the vessel wall is involved in both the initiation and progression of aneurysms. Changes in aortic wall structure that lead to aneurysm formation and vascular calcification are actively mediated by vascular smooth muscle cells. Vascular smooth muscle cells in a healthy vessel wall are termed contractile as they maintain vascular tone and remain quiescent. However, in pathological conditions they can dedifferentiate into a synthetic phenotype, whereby they secrete extracellular vesicles, proliferate, and migrate to repair injury. This process is called phenotypic switching and is often the first step in vascular pathology. Additionally, healthy vascular smooth muscle cells synthesize VKDPs (vitamin K-dependent proteins), which are involved in inhibition of vascular calcification. The metabolism of these proteins is known to be disrupted in vascular pathologies. In this review, we summarize the current literature on vascular smooth muscle cell phenotypic switching and vascular calcification in relation to aneurysm. Moreover, we address the role of vitamin K and VKDPs that are involved in vascular calcification and aneurysm. Visual Overview- An online visual overview is available for this article.

Arterial "inflammaging" drives vascular calcification in children on dialysis.

Children on dialysis have a cardiovascular mortality risk equivalent to older adults in the general population, and rapidly develop medial vascular calcification, an age-associated pathology. We hypothesized that premature vascular ageing contributes to calcification in children with advanced chronic kidney disease (CKD). Vessels from children with Stage 5 CKD with and without dialysis had evidence of increased oxidative DNA damage. The senescence markers p16 and p21 were also increased in vessels from children on dialysis. Treatment of vessel rings ex vivo with calcifying media increased oxidative DNA damage in vessels from children with Stage 5 CKD, but not in those from healthy controls. Vascular smooth muscle cells cultured from children on dialysis exhibited persistent DNA damage, impaired DNA damage repair, and accelerated senescence. Under calcifying conditions vascular smooth muscle cells from children on dialysis showed increased osteogenic differentiation and calcification. These changes correlated with activation of the senescence-associated secretory phenotype (SASP), an inflammatory phenotype characterized by the secretion of proinflammatory cytokines and growth factors. Blockade of ataxia-telangiectasia mutated (ATM)-mediated DNA damage signaling reduced both inflammation and calcification. Clinically, children on dialysis had elevated circulating levels of osteogenic SASP factors that correlated with increased vascular stiffness and coronary artery calcification. These data imply that dysregulated mineral metabolism drives vascular "inflammaging" by promoting oxidative DNA damage, premature senescence, and activation of a pro-inflammatory SASP. Drugs that target DNA damage signaling or eliminate senescent cells may have the potential to prevent vascular calcification in patients with advanced CKD.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress, and extracellular vesicle release in vascular smooth muscle cells.

AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms. METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine-induced human primary VSMC calcification, and increased osteogenic gene expression (Runx2, Osx, BSP, and OPN) and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide, we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels. CONCLUSION: In this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

ER stress regulates alkaline phosphatase gene expression in vascular smooth muscle cells via an ATF4-dependent mechanism.

OBJECTIVE: Vascular calcification is the deposition of hydroxyapatite crystals in the blood vessel wall. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a key role in this process. Increased expression of alkaline phosphatase (ALP) occurs in some in vitro models of VSMC calcification and is thought to be crucial for mineralization, however, little is known about the transcriptional regulation of ALP in VSMCs. Recently, ALP upregulation was shown to coincide with endoplasmic reticulum (ER) stress-mediated vascular calcification, specifically with expression of the transcription factor ATF4. As no direct links between ALP expression and ER stress have previously been demonstrated in VSMCs, the aim of this study was to investigate whether ATF4 interacts directly with the ALP promoter. RESULTS: The present study shows that ALP mRNA and activity were significantly increased by ER stress treatment of human primary VSMCs in vitro and that this was ATF4-dependent. Bioinformatics analysis predicted two ATF4 binding sites in ER-stress responsive regions of the ALP promoter (- 3631 to - 2048 bp from the first intron). However, we found that ATF4 does not bind within this fragment of the ALP promoter region.

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling.

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP-/- mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, ß-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.

Endoplasmic Reticulum Stress in Arterial Smooth Muscle Cells: A Novel Regulator of Vascular Disease.

Cardiovascular disease continues to be the leading cause of death in industrialised societies. The idea that the arterial smooth muscle cell (ASMC) plays a key role in regulating many vascular pathologies has been gaining importance, as has the realisation that not enough is known about the pathological cellular mechanisms regulating ASMC function in vascular remodelling. In the past decade endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been recognised as a stress response underlying many physiological and pathological processes in various vascular cell types. Here we summarise what is known about how ER stress signalling regulates phenotypic switching, trans/dedifferentiation and apoptosis of ASMCs and contributes to atherosclerosis, hypertension, aneurysms and vascular calcification.