Mineralization Of Vascular Smooth Muscle Cells Research Articles

Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD.

IKK2/NF-κB pathway-mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.

Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using invitro and exvivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination

Introduction: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). Methods: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. Results: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. Conclusions: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.

Icariin attenuates the calcification of vascular smooth muscle cells through ERα - p38MAPK pathway.

To investigate the relationship between icariin and the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and the signal pathway involved. We applied a universally accepted calcification model of VSMCs induced by β glycerophosphate. Then the VSMCs calcification was observed by treatment with icariin and/or inhibitors of estrogen receptors (ERs) and p38-mitogen-activated protein kinase (MAPK) signaling. Icariin inhibited osteoblastic differentiation and mineralization of VSMCs due to decreased ALP activity and Runx2 expression. Further study demonstrated that icariin exerted this suppression effect through activating p38-MAPK but not extracellular-regulated kinase, JNK or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of icariin on osteoblastic differentiation. Interestingly, treatment of VSMCs with an ER antagonist ICI182780 and a selective ERα receptor antagonist PPT attenuated icariin-mediated inhibition effect of VSMCs calcification, associated with suppression of p38-MAPK phosphorylation. Icariin inhibited the osteoblastic differentiation of VSMCs, and that the inhibitory effects were mediated by p38-MAPK pathways through ERα.

Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways.

Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runt-related transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification.

Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear. VSMCs was treated with high-phosphate to study VSMC calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl2) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling, VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms. Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. Nidogen-2-/- mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl2 administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with KD value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization. Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.

BGP-15 Inhibits Hyperglycemia-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification associated with high plasma phosphate (Pi) level is a frequent complication of hyperglycemia, diabetes mellitus, and chronic kidney disease. BGP-15 is an emerging anti-diabetic drug candidate. This study was aimed to explore whether BGP-15 inhibits high Pi-induced calcification of human vascular smooth muscle cells (VSMCs) under normal glucose (NG) and high glucose (HG) conditions. Exposure of VSMCs to Pi resulted in accumulation of extracellular calcium, elevated cellular Pi uptake and intracellular pyruvate dehydrogenase kinase-4 (PDK-4) level, loss of smooth muscle cell markers (ACTA, TAGLN), and enhanced osteochondrogenic gene expression (KLF-5, Msx-2, Sp7, BMP-2). Increased Annexin A2 and decreased matrix Gla protein (MGP) content were found in extracellular vesicles (EVs). The HG condition markedly aggravated Pi-induced VSMC calcification. BGP-15 inhibited Pi uptake and PDK-4 expression that was accompanied by the decreased nuclear translocation of KLF-5, Msx-2, Sp7, retained VSMC markers (ACTA, TAGLN), and decreased BMP-2 in both NG and HG conditions. EVs exhibited increased MGP content and decreased Annexin A2. Importantly, BGP-15 prevented the deposition of calcium in the extracellular matrix. In conclusion, BGP-15 inhibits Pi-induced osteochondrogenic phenotypic switch and mineralization of VSMCs in vitro that make BGP-15 an ideal candidate to attenuate both diabetic and non-diabetic vascular calcification.

Nano-hydroxyapatite accelerates vascular calcification via lysosome impairment and autophagy dysfunction in smooth muscle cells

Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified to play an essential role in the development of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear. We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp stimulated osteogenic differentiation and accelerated mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H+-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. Lysosomal re-acidification by cyclic-3′,5′-adenosine monophosphate (cAMP) significantly enhanced autophagic degradation and attenuated nHAp-induced calcification. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition. Altogether, our results demonstrated a repressive effect of nHAp on lysosomal acidification, which inhibited autophagic degradation and promoted a conversion of the accumulated autophagic vacuoles into exosomes that were loaded with undissolved nHAp, Ca2+, Pi and ALP. These exosomes bud off the plasma membrane, deposit within ECM, and form calcium nodules. Vascular calcification was thus accelerated by nHAP through blockage of autophagic flux in VSMCs.

Role of the Scavenger Receptor CD36 in Accelerated Diabetic Atherosclerosis.

Diabetes mellitus entails increased atherosclerotic burden and medial arterial calcification, but the precise mechanisms are not fully elucidated. We aimed to investigate the implication of CD36 in inflammation and calcification processes orchestrated by vascular smooth muscle cells (VSMCs) under hyperglycemic and atherogenic conditions. We examined the expression of CD36, pro-inflammatory cytokines, endoplasmic reticulum (ER) stress markers, and mineralization-regulating enzymes by RT-PCR in human VSMCs, cultured in a medium containing normal (5 mM) or high glucose (22 mM) for 72 h with or without oxidized low-density lipoprotein (oxLDL) (24 h). The uptake of 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate-fluorescently (DiI) labeled oxLDL was quantified by flow cytometry and fluorimetry and calcification assays were performed in VSMC cultured in osteogenic medium and stained by alizarin red. We observed induction in the expression of CD36, cytokines, calcification markers, and ER stress markers under high glucose that was exacerbated by oxLDL. These results were confirmed in carotid plaques from subjects with diabetes versus non-diabetic subjects. Accordingly, the uptake of DiI-labeled oxLDL was increased after exposure to high glucose. The silencing of CD36 reduced the induction of CD36 and the expression of calcification enzymes and mineralization of VSMC. Our results indicate that CD36 signaling is partially involved in hyperglycemia and oxLDL-induced vascular calcification in diabetes.

Role of SGK1 in the Osteogenic Transdifferentiation and Calcification of Vascular Smooth Muscle Cells Promoted by Hyperglycemic Conditions.

In diabetes mellitus, hyperglycemia promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to enhance medial vascular calcification, a common complication strongly associated with cardiovascular disease and mortality. The mechanisms involved are, however, still poorly understood. Therefore, the present study explored the potential role of serum- and glucocorticoid-inducible kinase 1 (SGK1) during vascular calcification promoted by hyperglycemic conditions. Exposure to high-glucose conditions up-regulated the SGK1 expression in primary human aortic VSMCs. High glucose increased osteogenic marker expression and activity and, thus, promoted the osteogenic transdifferentiation of VSMCs, effects significantly suppressed by additional treatment with the SGK1 inhibitor EMD638683. Moreover, high glucose augmented the mineralization of VSMCs in the presence of calcification medium, effects again significantly reduced by SGK1 inhibition. Similarly, SGK1 knockdown blunted the high glucose-induced osteogenic transdifferentiation of VSMCs. The osteoinductive signaling promoted by high glucose required SGK1-dependent NF-κB activation. In addition, advanced glycation end products (AGEs) increased the SGK1 expression in VSMCs, and SGK1 inhibition was able to interfere with AGEs-induced osteogenic signaling. In conclusion, SGK1 is up-regulated and mediates, at least partly, the osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic conditions. Thus, SGK1 inhibition may reduce the development of vascular calcification promoted by hyperglycemia in diabetes.

Impact of \u03b2-glycerophosphate on the bioenergetic profile of vascular smooth muscle cells

In chronic kidney disease, hyperphosphatemia is a key pathological factor promoting medial vascular calcification, a common complication associated with cardiovascular events and mortality. This active pathophysiological process involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) via complex intracellular mechanisms that are still incompletely understood. Little is known about the effects of phosphate on the bioenergetic profile of VSMCs during the onset of this process. Therefore, the present study explored the effects of the phosphate donor β-glycerophosphate on cellular bioenergetics of VSMCs. Mitochondrial and glycolytic functions were determined utilizing extracellular flux analysis in primary human aortic VSMCs following exposure to β-glycerophosphate. In VSMCs, β-glycerophosphate increased basal respiration, mitochondrial ATP production as well as proton leak and decreased spare respiratory capacity and coupling efficiency, but did not modify non-mitochondrial or maximal respiration. β-Glycerophosphate-treated VSMCs had higher ability to increase mitochondrial glutamine and long-chain fatty acid usage as oxidation substrates to meet their energy demand. β-Glycerophosphate did not modify glycolytic function or basal and glycolytic proton efflux rate. In contrast, β-glycerophosphate increased non-glycolytic acidification. β-Glycerophosphate-treated VSMCs had a more oxidative and less glycolytic phenotype, but a reduced ability to respond to stressed conditions via mitochondrial respiration. Moreover, compounds targeting components of mitochondrial respiration modulated β-glycerophosphate-induced oxidative stress, osteo-/chondrogenic signalling and mineralization of VSMCs. In conclusion, β-glycerophosphate modifies key parameters of mitochondrial function and cellular bioenergetics in VSMCs that may contribute to the onset of phenotypical transdifferentiation and calcification. These observations advance the understanding of the role of energy metabolism in VSMC physiology and pathophysiology of vascular calcification during hyperphosphatemia.Key messagesβ-Glycerophosphate modifies key parameters of mitochondrial respiration in VSMCs.β-Glycerophosphate induces changes in mitochondrial fuel choice in VSMCs.β-Glycerophosphate promotes a more oxidative and less glycolytic phenotype of VSMCs.β-Glycerophosphate triggers mitochondrial-dependent oxidative stress in VSMCs.Bioenergetics impact β-glycerophosphate-induced VSMC calcification.

GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes.

SummaryExcessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.

The protective effects of long non-coding RNA-ANCR on arterial calcification

Arterial calcification is a major factor for cardiovascular events and is characterized by vascular smooth muscle cells (VSMCs) transformed into osteoblast-like cells. Long non-coding RNAs (lncRNA) were recognized as important regulators of diverse biological processes. Previous studies have demonstrated that lncRNAs could regulate the proliferation and apoptosis of VSMCs. LncRNA-ANCR (Anti-differentiation ncRNA) is an essential mediator governing the differentiation of human osteoblast. However, it is unclear whether ANCR could regulate the osteoblastic differentiation of VSMCs. In this study, we determined the effect of ANCR on VSMCs differentiation and arterial calcification. Both cellular and mouse model of arterial calcification were, respectively, established to investigate the role of ANCR in the mechanism of arterial calcification. ANCR overexpressing lentivirus were used to investigate the effects of ANCR on the expression of bone proteins and autophagy-related molecules. ANCR could inhibit β-glycerophosphate (β-GP)-induced VSMCs osteoblastic differentiation and mineralization due to decreased expressions of Runt-related transcription factor 2, bone morphogenetic protein-2, and formation of mineralized nodule, and attenuate high calcitriol-induced mice model of arterial calcification. Furthermore, ANCR could significantly increase LC3 and autophagy protein 5 expression in β-GP-stimulated VSMCs, and the effect could be inhibited by 3-methyladenine, a pharmacological inhibitor of autophagy. ANCR may inhibit the osteoblastic differentiation of VSMCs and attenuate mice arterial calcification through activating autophagy.

Zinc Inhibits HIF-Prolyl Hydroxylase Inhibitor-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22α) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the pro-calcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cell-specific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion - PHI promoted the loss of smooth muscle markers and augmented Pi-induced osteochondrogenic phenotypic switch leading to VSMCs calcification. This mineralization process was attenuated by zinc. Enhanced vascular calcification is a potential risk factor during PHI therapy in CKD which necessitates the strict follow up of vascular calcification and zinc supplementation.

Potential role of calcifying nanoparticles in the etiology of multiple sclerosis

Nanobacteria or calcifying nanoparticles are 80–500 nm sized nano-organisms that are physically associated with carbonate apatite mineral formations. They have been indicated in various diseases, including kidney stone formation, Alzheimer’s disease, and atherosclerosis. Nanoparticles contain calcium and apatite-binding protein fetuin-A, a calcification inhibitor. However, recent evidence indicates that fetuin-A can form nucleation seeds or nidi that grow in size through ion sedimentation to become larger amorphous nanoparticles in the presence of excess calcium and apatite ions.Fetuin-A also functions as an inhibitor of meprin, a metalloproteinase implicated in inflammation and neurodegenerative diseases. During inflammation, meprin functions to regulate chemokine activity of monocyte chemotactic protein 1, which is associated with chronic inflammatory diseases, including atherosclerosis, renal inflammatory diseases, and multiple sclerosis (MS). In addition, calcium phosphate nanocrystals that contain fetuin-A are pro-inflammatory to macrophages and promote vascular smooth muscle cell mineralization, potentiating a vicious cycle of inflammation and calcification. Thus, mineral stress and inflammation appear to be associated with each other. Furthermore, fetuin-A deficient mice exhibited reduced experimental autoimmune encephalomyelitis severity. Thus, fetuin-A plays a direct role in the neuroinflammatory response. Indeed, the level of fetuin-A in cerebrospinal fluid has been defined as a biomarker of disease activity in MS.MS is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) with an unknown etiology. The “inside-out” model of MS, supported by recent data, states that the initial axonal degeneration in the CNS occurs before demyelination, which then stimulates an auto-immune attack. It was shown very recently that influx of calcium from the extracellular space through nanoscale ruptures of the axonal plasma membrane predict axon degeneration in neuroinflammation. Calcium is an activator of calpains, proteases that function to break down the cytoskeleton, leading to neurodegeneration. Nanoruptures of the plasma membrane were suggested to occur at the early stages of axon damage, especially at nodes of Ranvier, which are devoid of myelin.Here, I propose that calcifying nanoparticles may have a role in the etiology and/or pathophysiology of MS. The initial event causing neurodegeneration may be due to the nanoparticles that have been suggested to easily cross the blood-brain barrier. Following this, the nanoparticles may create nanoruptures in the axonal membrane and also increase the calcium concentration around and within the neurons by forming nidi for calcification, eventually causing neurodegeneration. Nanoparticles can self-replicate; hence, they may represent an infectious causative agent for the development of MS.

Klotho/FGF23 axis mediates high phosphate-induced vascular calcification in vascular smooth muscle cells via Wnt7b/β-catenin pathway.

Vascular calcification (VC) plays as a critical role on cardiovascular disease (CVD) and acts as a notable risk factor in cardiovascular system. Vascular smooth muscle cells (VSMCs) calcification can be triggered by high phosphate treatment; however, the explicit mechanism remains unclear. In the present study, we isolated VSMCs from primary rat artery, applied β-GP (β-glycerophosphate) for inducing VSMCs calcification in vitro to explore the mechanism of phosphate-induced calcification in VSMCs. Alizarin red staining was performed to assess the mineralization in VSMCs. Calcium deposition experiment was taken to evaluate the calcium content. ALP staining was determined to assess the ALP activity. The recombinant adenoviruses were constructed for the overexpression of Klotho and FGF23, respectively. qRT-PCR and western blot analysis were subjected to measure the expression of Klotho/FGF23 and correlated genes among Wnt7b/β-catenin pathway. We found that the calcium content was obviously increased and Alizarin red staining was positive in calcification group exposure with high phosphate in a time-dependent manner. The expression of Klotho and FGF23 was significantly decreased in the calcification group. However, overexpression of Klotho and FGF23 markedly reversed VSMCs calcification stimulating with high phosphate treatment. Moreover, Wnt7b/β-catenin inhibitor DKK1 could partly attenuate the effect of high phosphate on calcified VSMCs. These findings demonstrated that Klotho/FGF23 axis could modulate high phosphate-induced VSMCs calcification via Wnt7b/β-catenin signaling pathway. Our findings unravel that Klotho/FGF23- Wnt7b/β-catenin axis functions as a crucial role in the VSMCs calcification.

Death-associated protein kinase 3 deficiency alleviates vascular calcification via AMPK-mediated inhibition of endoplasmic reticulum stress

Vascular calcification (VC) is a critical feature of chronic kidney disease (CKD), diabetes, hypertension, and atherosclerosis. Death-associated protein kinase 3 (DAPK3) is involved in vascular remodeling in hypertension. However, it remains to be clarified whether DAPK3 controls vascular smooth muscle cell (VSMC) phenotypic transition into an osteogenic cell phenotype, which is an important process for VC. In vivo VC was induced in rats by vitamin D3 and nicotine. VSMCs were incubated with calcifying media containing β-glycerophosphate and Ca2+ to induce VC in vitro. Herein, we demonstrated increased expression of DAPK3 in the aortas of VC rats and VSMCs cultured in calcifying media. Knockdown of DAPK3 significantly inhibited calcifying media-induced VSMC mineralization and retarded the phenotypic transformation of VSMCs into osteogenic cells. Silencing of DAPK3 suppressed endoplasmic reticulum stress (ERS) related protein expressions, but upregulated the phosphorylation level of AMP-activated protein kinase (AMPK) in calcified VSMCs. Moreover, pretreatment with AMPK inhibitor Compound C abolished DAPK3 shRNA-mediated inhibition of ERS in VSMCs. In vivo, DAPK inhibitor significantly prevented calcium deposition in the aortas of VC rats. The present results revealed that DAPK3 modulated VSMC calcification through AMPK-mediated ERS signaling.

Association of astragaloside IV-inhibited autophagy and mineralization in vascular smooth muscle cells with lncRNA H19 and DUSP5-mediated ERK signaling

Defective autophagy in vascular smooth muscle cells (VSMCs) is the principal cause of atherosclerosis. This study aimed to investigate the effect of astragaloside IV (AS-IV) on VSMCs autophagy. In vivo, ApoE−/− mice were fed with high-fat diet ad libitum for eight weeks, with or without AS-IV (25 mg/kg, daily). In vitro, human VSMCs were cultured and treated with β-Glycerophosphate (10 mmol/L) and AS-IV (50 μg/ml). VSMCs autophagy, mineralization, expression of p-ERK1/2, p-mTOR, and autophagy-related proteins (LC3 II/I, p62, and Beclin 1) were detected. Increased autophagy and mineralization was observed in VSMCs in thoracic aorta of mice and in in vitro VSMCs model of atherosclerosis. AS-IV administration attenuated the autophagy and mineralization in VSMCs. Reverse expression profiles of H19 and DUSP5 were observed. AS-IV inhibited DUSP5 and autophagy-related proteins and increased expression of H19, level of p-ERK1/2 and p-mTOR. Further, autophagy and mineralization level in VSMCs were in line with DUSP5 expression level, but in contrast to H19, p-ERK1/2, and p-mTOR profiles. We demonstrated that AS-IV could attenuate autophagy and mineralization of VSMCs in atherosclerosis, which may be associated with H19 overexpression and DUSP5 inhibition.

MiR-34a Promotes Vascular Smooth Muscle Cell Calcification by Downregulating SIRT1 (Sirtuin 1) and Axl (AXL Receptor Tyrosine Kinase).

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.

Conditioned medium from bone marrow-derived mesenchymal stem cells inhibits vascular calcification through blockade of the BMP2\u2013Smad1/5/8 signaling pathway

BackgroundArterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis and is a significant contributor to cardiovascular morbidity and mortality. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important role in arterial calcification and is characterized by cellular necrosis, inflammation, and lipoprotein and phospholipid complexes, especially in atherosclerotic calcification. The conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) is well known as a rich source of autologous cytokines and is universally used for tissue regeneration in current clinical medicine. Here, we demonstrate that MSC-CM inhibits beta-glycerophosphate (β-GP)-induced vascular calcification through blockade of the bone morphogenetic protein-2 (BMP2)–Smad1/5/8 signaling pathway.MethodsVSMC calcification was induced by β-GP followed by treatment with MSC-CM. Mineral deposition was assessed by Alizarin Red S staining. Intracellular calcium content was determined colorimetrically by the o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured by the para-nitrophenyl phosphate method. Expression of BMP2, BMPR1A, BMPR1B, BMPR2, msh homeobox 2 (Msx2), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC), representative osteoblastic markers, was assessed using real-time polymerase chain reaction analysis while the protein expression of BMP2, Runx2, and phosphorylated Smad1/5/8 was detected by western blot analysis.ResultsOur data demonstrated that MSC-CM inhibits osteoblastic differentiation and mineralization of VSMCs as evidenced by decreased calcium content, ALP activity, and decreased expression of BMP-2, Runx2, Msx2, and OC. MSC-CM suppressed the expression of phosphorylated Smad1/5/8 and the β-GP-induced translocation from the cytoplasm to the nucleus. Further study demonstrated that human recombinant BMP-2 overcame the suppression of VSMC calcification by MSC-CM.ConclusionMSC-CM may act as a novel therapy for VSMC calcification by mediating the BMP2–Smad1/5/8 signaling pathway