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.

Calcium Signaling and Tissue Calcification.

Calcification is a regulated physiological process occurring in bones and teeth. However, calcification is commonly found in soft tissues in association with aging and in a variety of diseases. Over the last two decades, it has emerged that calcification occurring in diseased arteries is not simply an inevitable build-up of insoluble precipitates of calcium phosphate. In some cases, it is an active process in which transcription factors drive conversion of vascular cells to an osteoblast or chondrocyte-like phenotype, with the subsequent production of mineralizing "matrix vesicles." Early studies of bone and cartilage calcification suggested roles for cellular calcium signaling in several of the processes involved in the regulation of bone calcification. Similarly, calcium signaling has recently been highlighted as an important component in the mechanisms regulating pathological calcification. The emerging hypothesis is that ectopic/pathological calcification occurs in tissues in which there is an imbalance in the regulatory mechanisms that actively prevent calcification. This review highlights the various ways that calcium signaling regulates tissue calcification, with a particular focus on pathological vascular calcification.

Calcium phosphate particles stimulate interleukin-1ß release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.

AIMS: Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1ß (IL-1ß). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. METHODS AND RESULTS: Using ELISA to measure IL-1ß release from VSMCs, we demonstrated that CaP particles stimulated IL-1ß release from proliferating and senescent human VSMCs, but with substantially greater IL-1ß release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1ß release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1ß release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1ß and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1ß in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1ß release. CONCLUSIONS: CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1ß, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.

Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells.

Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥ 1 µM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms.

Human vascular smooth muscle cell culture.

Human vascular smooth muscle cells (VSMCs) in culture are an important tool in understanding how VSMCs function and contribute to vessel wall contraction as well as disease. In this chapter, we describe methodologies that enable the investigator to culture large numbers of proliferative VSMCs. These VSMCs are heterogeneous and vary in size, shape, and proliferative capacity depending on the disease state and location of the vessel of origin. Therefore, we also describe techniques to validate their identity as bone fide VSMCs. Briefly, the methods include information on how to dissect the blood vessel to remove the medial layer containing VSMCs, as well as methods on how to propagate these cells, by either allowing VSMCs to migrate from the explanted medial tissue or by enzymatically dispersing the cells from the tissue. Both methods are suitable for culturing VSMCs derived from most vessel types with modifications of the enzyme dispersal method suitable for the isolation of microvessel VSMCs. An important feature of VSMCs in culture is that they lose many of their in vivo contractile properties and so model disease-associated VSMCs in the vessel wall rather than a non-proliferative contractile cell. To overcome this limitation, we also describe alternate methods that enable the study of cultured VSMCs in their contractile state by allowing the VSMCs to remain within an intact vessel ring. Overall, these procedures enable the investigator to undertake a diverse array of experimental assays on cultured VSMCs.

Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization.

RATIONALE: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs. OBJECTIVES: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. METHODS AND RESULTS: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. CONCLUSIONS: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Nanocrystals seed calcification in more ways than one.

Although much progress has been made in the past five years in understanding the mechanisms leading to accelerated vascular calcification in patients with chronic kidney disease, it remains unclear how an environment high in phosphate can impinge so significantly on the calcification process. The study by Sage et al. highlights an important and novel role for calcium phosphate nanocrystals, produced in a high-phosphate environment, in rapidly driving calcification of vascular smooth muscle cells via enhanced production of bone morphogenetic protein-2.

Mineral surface in calcified plaque is like that of bone: further evidence for regulated mineralization.

OBJECTIVE: Cell biological studies demonstrate remarkable similarities between mineralization processes in bone and vasculature, but knowledge of the components acting to initiate mineralization in atherosclerosis is limited. The molecular level microenvironment at the organic-inorganic interface holds a record of the mechanisms controlling mineral nucleation. This study was undertaken to compare the poorly understood interface in mineralized plaque with that of bone, which is considerably better characterized. METHODS AND RESULTS: Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides powerful tools for studying the organic-inorganic interface in calcium phosphate biominerals. The rotational echo double resonance (REDOR) technique, applied to calcified human plaque, shows that this interface predominantly comprises sugars, most likely glycosaminoglycans (GAGs). In this respect, and in the pattern of secondary effects seen to protein (mainly collagen), calcified plaque strongly resembles bone. CONCLUSIONS: The similarity between biomineral formed under highly controlled (bone) and pathological (plaque) conditions suggests that the control mechanisms are more similar than previously thought, and may be adaptive. It is strong further evidence for regulation of plaque mineralization by osteo/chondrocytic vascular smooth muscle cells.

Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization.

Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 microm or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death.

Molecular mechanisms mediating vascular calcification: role of matrix Gla protein.

Patients with chronic kidney disease (CKD) have a higher incidence of vascular calcification and a greatly increased risk of cardiovascular death. The mechanisms involved in the accelerated vascular calcification observed in CKD have recently become clearer, leading to the hypothesis that a lack of natural inhibitors of calcification may trigger calcium deposition. One of these inhibitory factors, matrix Gla protein (MGP), is the focus of the present review. MGP, originally isolated from bone, is a vitamin K-dependent protein that is also highly expressed by vascular smooth muscle cells. MGP has been confirmed as a calcification-inhibitor in numerous studies; however, its mechanism of action is not completely understood. It potentially acts in several ways to regulate calcium deposition including: (i) binding calcium ions and crystals; (ii) antagonizing bone morphogenetic protein and altering cell differentiation; (iii) binding to extracellular matrix components; and (iv) regulating apoptosis. Its expression is regulated by several factors including retinoic acid, vitamin D and extracellular calcium ions, and a reduced form of vitamin K (KH2) is important in maintaining MGP in an active form. Therefore, strategies aimed at increasing its expression and activity may be beneficial in tipping the balance in favour of inhibition of calcification in CKD.

Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth muscle cells.

Lipid accumulation by vascular smooth muscle cells (VSMC) is a feature of atherosclerotic plaques. In this study we describe two mechanisms whereby human VSMC foam cell formation is driven by de novo synthesis of fatty acids leading to triacylglycerol accumulation in intracellular vacuoles, a process distinct from serum lipoprotein uptake. VSMC cultured in adipogenic differentiation medium accumulated lipids and were induced to express the adipocyte marker genes adipsin, adipocyte fatty acid-binding protein, C/EBPalpha, PPARgamma, and leptin. However, complete adipocyte differentiation was not observed as numerous genes present in mature adipocytes were not detected, and the phenotype was reversible. The rate of lipid accumulation was not affected by PPARgamma agonists, but screening for the effects of other nuclear receptor agonists showed that activation of the liver X receptors (LXR) dramatically promoted lipid accumulation in VSMC. Both LXRalpha and LXRbeta were present in VSMC, and their activation with TO901317 resulted in induction of the lipogenic genes fatty acid synthetase, sterol regulatory element binding protein (SREBP1c), and stearoyl-CoA desaturase. 27-Hydroxycholesterol, an abundant oxysterol synthesized by VSMC acted as an LXR antagonist and, therefore, may have a protective role in preventing foam cell formation. Immunohistochemistry showed that VSMC within atherosclerotic plaques express adipogenic and lipogenic markers, suggesting these pathways are present in vivo. Moreover, the development of an adipogenic phenotype in VSMC is consistent with their known phenotypic plasticity and may contribute to their dysfunction in atherosclerotic plaques and, thus, impinge on plaque growth and stability.

Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.