Vitamin D deficiency promotes intracranial aneurysm rupture.

Intracranial aneurysm rupture causes severe disability and high mortality. Epidemiological studies show a strong association between decreased vitamin D levels and an increase in aneurysm rupture. However, the causality and mechanism remain largely unknown. In this study, we tested whether vitamin D deficiency promotes aneurysm rupture and examined the underlying mechanism for the protective role of vitamin D against the development of aneurysm rupture utilizing a mouse model of intracranial aneurysm. Mice consuming a vitamin D-deficient diet had a higher rupture rate than mice with a regular diet. Vitamin D deficiency increased proinflammatory cytokines in the cerebral arteries. Concurrently, vitamin D receptor knockout mice had a higher rupture rate than the corresponding wild-type littermates. The vitamin D receptors on endothelial and vascular smooth muscle cells, but not on hematopoietic cells, mediated the effect of aneurysm rupture. Our results establish that vitamin D protects against the development of aneurysmal rupture through the vitamin D receptors on vascular endothelial and smooth muscle cells. Vitamin D supplementation may be a viable pharmacologic therapy for preventing aneurysm rupture.

Downregulation of HDAC9 by the ketone metabolite ß-hydroxybutyrate suppresses vascular calcification.

Vascular calcification is an actively regulated process resembling bone formation and contributes to the cardiovascular morbidity and mortality of chronic kidney disease (CKD). However, an effective therapy for vascular calcification is still lacking. The ketone body ß-hydroxybutyrate (BHB) has been demonstrated to have health-promoting effects including anti-inflammation and cardiovascular protective effects. However, whether BHB protects against vascular calcification in CKD remains unclear. In this study, Alizarin Red staining and calcium content assay showed that BHB reduced calcification of vascular smooth muscle cells (VSMCs) and arterial rings. Of note, compared with CKD patients without thoracic calcification, serum BHB levels were lower in CKD patients with thoracic calcification. Supplementation with 1,3-butanediol (1,3-B), the precursor of BHB, attenuated aortic calcification in CKD rats and VitD3-overloaded mice. Furthermore, RNA-seq analysis revealed that BHB downregulated HDAC9, which was further confirmed by RT-qPCR and western blot analysis. Both pharmacological inhibition and knockdown of HDAC9 attenuated calcification of human VSMCs, while overexpression of HDAC9 exacerbated calcification of VSMCs and aortic rings, indicating that HDAC9 promotes vascular calcification under CKD conditions. Of note, BHB treatment antagonized HDAC9-induced vascular calcification. In addition, HDAC9 overexpression activated the NF-κB signaling pathway and inhibition of NF-κB attenuated HDAC9-induced VSMC calcification, suggesting that HDAC9 promotes vascular calcification via activation of NF-κB. In conclusion, our study demonstrates that BHB supplementation inhibits vascular calcification in CKD via modulation of the HDAC9-dependent NF-κB signaling pathway. Moreover, we unveil a crucial mechanistic role of HDAC9 in vascular calcification under CKD conditions; thus, nutritional intervention or pharmacological approaches to enhance BHB levels could act as promising therapeutic strategies to target HDAC9 for the treatment of vascular calcification in CKD. © 2022 The Pathological Society of Great Britain and Ireland.

Zinc Ameliorates the Osteogenic Effects of High Glucose in Vascular Smooth Muscle Cells.

In diabetic patients, medial vascular calcification is common and associated with increased cardiovascular mortality. Excessive glucose concentrations can activate the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) and trigger pro-calcific effects in vascular smooth muscle cells (VSMCs), which may actively augment vascular calcification. Zinc is able to mitigate phosphate-induced VSMC calcification. Reduced serum zinc levels have been reported in diabetes mellitus. Therefore, in this study the effects of zinc supplementation were investigated in primary human aortic VSMCs exposed to excessive glucose concentrations. Zinc treatment was found to abrogate the stimulating effects of high glucose on VSMC calcification. Furthermore, zinc was found to blunt the increased expression of osteogenic and chondrogenic markers in high glucose-treated VSMCs. High glucose exposure was shown to activate NF-kB in VSMCs, an effect that was blunted by additional zinc treatment. Zinc was further found to increase the expression of TNFα-induced protein 3 (TNFAIP3) in high glucose-treated VSMCs. The silencing of TNFAIP3 was shown to abolish the protective effects of zinc on high glucose-induced NF-kB-dependent transcriptional activation, osteogenic marker expression, and the calcification of VSMCs. Silencing of the zinc-sensing receptor G protein-coupled receptor 39 (GPR39) was shown to abolish zinc-induced TNFAIP3 expression and the effects of zinc on high glucose-induced osteogenic marker expression. These observations indicate that zinc may be a protective factor during vascular calcification in hyperglycemic conditions.

Bioactive Compounds From Coptidis Rhizoma Alleviate Pulmonary Arterial Hypertension by Inhibiting Pulmonary Artery Smooth Muscle Cells' Proliferation and Migration.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.

Xinmai 'an extract enhances the efficacy of sildenafil in the treatment of pulmonary arterial hypertension via inhibiting MAPK signalling pathway.

CONTEXT: Xinmai 'an tablet has been used to improve myocardial blood supply. Recently, some compounds from its formula have shown that they can treat pulmonary arterial hypertension (PAH). OBJECTIVE: This study investigates the effects of Xinmai 'an extract (XMA) on PAH and further tests the co-therapeutic enhancement with sildenafil (SIL). MATERIALS AND METHODS: Pulmonary artery smooth muscle cells were subjected to stimulation with SIL (12.5 µM) and XMA (250 µg/mL) for 48 h. Sprague-Dawley rats were randomly grouped into eight groups (n = 8 per group): (I) control group received saline; (II) MCT group received MCT (60 mg/kg); (III) SIL-Low group received MCT + SIL at 10 mg/kg/day; (IV) SIL-high group received MCT + SIL at 30 mg/kg/day; (V) XMA-High group received MCT + XMA at 251.6 mg/kg/day; (VI) SIL (Low)+XMA (Low) group received SIL (10 mg/kg) + XMA at 62.9 mg/kg/day; (VII) SIL (Low)+XMA (Medium) group received SIL (10 mg/kg) + XMA at 125.8 mg/kg/day; (VIII) SIL (Low)+XMA (High) group received SIL (10 mg/kg) + XMA at 251.6 mg/kg/day. Both XMA and SIL were given by gavage and were maintained daily for 2 weeks. RESULTS: XMA could improve SIL's efficacy in the treatment of PAH by decreasing cell viability more effectively at non-cytotoxic concentrations (250 µg/mL) and reducing Right Ventricular Systolic Pressure (RVSP) in PAH rat. Potential mechanisms might at least in part be through activating the MAPK signalling pathway. DISCUSSION AND CONCLUSIONS: The combination of XMA and SIL can improve the efficacy of pulmonary hypertension and reduce the dosage of SIL.

Effects of carotid baroreceptor stimulation on aortic remodeling in obese rats.

BACKGROUND AND AIM: Our previous study found carotid baroreceptor stimulation (CBS) reduces body weight and white adipose tissue (WAT) weight, restores abnormal secretion of adipocytokines and inflammation factors, decreases systolic blood pressure (SBP) by inhibiting activation of sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in obese rats. In this study, we explore effects of CBS on aortic remodeling in obese rats. METHODS AND RESULTS: Rats were fed high-fat diet (HFD) for 16 weeks to induce obesity and underwent either CBS device implantation and stimulation or sham operation at 8 weeks. BP and body weight were measured weekly. RAS activity of WAT, histological, biochemical and functional profiles of aortas were detected after 16 weeks. CBS effectively decreased BP in obese rats, downregulated mRNA expression of angiotensinogen (AGT) and renin in WAT, concentrations of AGT, renin, angiotensin II (Ang II), protein levels of Ang II receptor 1 (AT1R) and Ang II receptor 2 (AT2R) in WAT were declined. CBS inhibited reactive oxygen species (ROS) generation, inflammatory response and endoplasmic reticulum (ER) stress in aortas of obese rats, restrained vascular wall thickening and vascular smooth muscle cells (VSMCs) phenotypic switching, increased nitric oxide (NO) synthesis, promoted endothelium-dependent vasodilatation by decreasing protein expression of AT1R and leptin receptor (LepR), increasing protein expression of adiponectin receptor 1 (AdipoR1) in aortic VSMCs. CONCLUSION: CBS reduced BP and reversed aortic remodeling in obese rats, the underlying mechanism might be related to the suppressed SNS activity, restored adipocytokine secretion and restrained RAS activity of WAT.

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Identification of securinine as vascular protective agent targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice.

BACKGROUND: Atherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. PURPOSE: The purpose of the present study was to identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque. METHODS: Differentially expressed genes (DEGs) were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein-E-deficient (ApoE KO) mice for 18 weeks. RESULTS: Securinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase-2/-9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF-κB activation. Growth curve assays using the real-time cell analyzer showed that securinine significantly decreased TNF-α-induced aortic smooth muscle cell proliferation and migration in a dose-dependent manner. CONCLUSION: Securinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.

Combination of Dichloroacetate and Atorvastatin Regulates Excessive Proliferation and Oxidative Stress in Pulmonary Arterial Hypertension Development via p38 Signaling.

Pulmonary arterial hypertension (PAH) is a lethal disease generally characterized by pulmonary artery remodeling. Mitochondrial metabolic disorders have been implicated as a critical regulator of excessively proliferative- and apoptosis-resistant phenotypes in pulmonary artery smooth muscle cells (PASMCs). Dichloroacetate (DCA) is an emerging drug that targets aerobic glycolysis in tumor cells. Atorvastatin (ATO) is widely used for hyperlipemia in various cardiovascular diseases. Considering that DCA and ATO regulate glucose and lipid metabolism, respectively, we hypothesized that the combination of DCA and ATO could be a potential treatment for PAH. A notable decrease in the right ventricular systolic pressure accompanied by reduced right heart hypertrophy was observed in the DCA/ATO combination treatment group compared with the monocrotaline treatment group. The DCA/ATO combination treatment alleviated vascular remodeling, thereby suppressing excessive PASMC proliferation and macrophage infiltration. In vitro, both DCA and ATO alone reduced PASMC viability by upregulating oxidative stress and lowering mitochondrial membrane potential. Surprisingly, when combined, DCA/ATO was able to decrease the levels of reactive oxygen species and cell apoptosis without compromising PASMC proliferation. Furthermore, suppression of the p38 pathway through the specific inhibitor SB203580 attenuated cell death and oxidative stress at a level consistent with that of DCA/ATO combination treatment. These observations suggested a complementary effect of DCA and ATO on rescuing PASMCs from a PAH phenotype through p38 activation via the regulation of mitochondrial-related cell death and oxidative stress. DCA in combination with ATO may represent a novel therapeutic strategy for PAH treatment.

Genome-wide identification of lncRNAs and mRNAs differentially expressed in human vascular smooth muscle cells stimulated by high phosphorus.

Background: Cardiovascular events are the primary cause of death for chronic kidney disease patients, which occurred via vascular calcification evolving pathogenically. Although a high level of phosphorus contributes to the induction of osteogenic differentiation of vascular smooth muscle cells (VSMCs), the role of lncRNA in this process awaits further study.Methods: In this study, we systematically investigated the variation of gene expression in human VSMCs induced by high phosphorus. LncRNAs and mRNAs expression were revealed by microarray analyses of the control group and high-phosphorus (HP) group. LncRNA-mRNA co-expression network was established based on the specific lncRNA-mRNA relationships. Hierarchical clustering was used to identify a common set of regulated genes. In addition, Gene Ontology enrichment, Kyoto Gene-Encyclopedia and genomic analyses were conducted for the mRNAs differentially expressed under high phosphorus.Result: RT-qPCR results confirmed that the expression of RUNX2, BMP2 and osteocalcin in HP group exhibited significant increases than in control group (p < .05). VSMC in HP group also showed higher intracellular calcium content. Volcano plots results show that 379 mRNAs and 728 lncRNAs different expressed in HP group. LncRNA-mRNA co-expression networks analysis revealed that 8 lncRNAs were the most highly connected lncRNAs. Quantitative analysis indicated that two lncRNAs were confirmed to increase significantly in the HP group. The mRNA expression of NT5E and ICAM1 were higher in group HP, while MAP3K7CL was lower than CON group (p < .05).Conclusion: This study provided a working list of lncRNAs that may be relevant to osteogenic differentiation, which presents a new insights into the mechanism of vascular calcification induced by high phosphorus in VSMCs.

Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice.

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

Medial calcification in the arterial wall of smooth muscle cell-specific Smpd1 transgenic mice: A ceramide-mediated vasculopathy.

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)-derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1trg /SMcre mice with SMC-specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1trg /SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1trg /SMcre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome-MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1trg /SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1trg /SMcre mice, increased Pi concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome-MVB interaction. However, amitriptyline prevented these changes in Pi -treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.

Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway.

BACKGROUND AND AIM: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, which is mainly caused by inflammation. Inhibiting inflammation can relieve PAH. Grape seed procyanidin (GSP) possesses remarkable anti-inflammatory property and vascular protective function. In this experiment, we verified the anti-inflammatory property of GSP in cigarette smoke-exposed PAH rats and revealed its molecular mechanism. METHODS AND RESULTS: In vivo, 45 Sprague Dawley (SD) rats were divided into 5 groups randomly, treated with normoxia/cigarette smoke (CS)/GSP + CS/CS + solvent/GSP. After GSP + CS administration, a decrease in mPAP, PVR, RVHI, WT%, and WA% was detected in the rats as compared to those treated with CS. In vitro, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) caused by cigarette smoke extract (CSE) was effectively attenuated with GSP + CSE administration. Furthermore, GSP significantly increased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) together with the lowered expression level of cyclooxygenase 2 (COX-2) in PASMCs co-incubated with CSE. CONCLUSION: These findings indicate that GSP ameliorates inflammation by the PPAR-γ/COX-2 pathway and finally inhibits the proliferation of PASMCs, which leads to pulmonary vascular remodeling.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Osteogenic transdifferentiation of vascular smooth muscle cells isolated from spontaneously hypertensive rats and potential menaquinone-4 inhibiting effect.

Vascular calcification (VC) is an active and cell-mediated process that shares many common features with osteogenesis. Knowledge demonstrates that in the presence of risk factors, such as hypertension, vascular smooth muscle cells (vSMCs) lose their contractile phenotype and transdifferentiate into osteoblastic-like cells, contributing to VC development. Recently, menaquinones (MKs), also known as Vitamin K2 family, has been revealed to play an important role in cardiovascular health by decreasing VC. However, the MKs' effects and mechanisms potentially involved in vSMCs osteoblastic transdifferentiation are still unknown. The aim of this study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of MKs family, in the modulation of the vSMCs phenotype. To achieve this, vascular cells from spontaneously hypertensive rats (SHR) were used as an in vitro model of cell vascular dysfunction. vSMCs from Wistar Kyoto normotensive rats were used as control condition. The results showed that MK-4 preserves the contractile phenotype both in control and SHR-vSMCs through a γ-glutamyl carboxylase-dependent pathway, highlighting its capability to inhibit one of the mechanisms underlying VC process. Therefore, MK-4 may have an important role in the prevention of vascular dysfunction and atherosclerosis, encouraging further in-depth studies to confirm its use as a natural food supplement.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway.

OBJECTIVE: To test the hypothesis that the inhibition of endoplasmic reticulum (ER) stress-induced apoptosis in oxidized low-density lipoproteins (ox-LDL)-induced human aortic-vascular smooth muscle cells (HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway by Pollen Typhae total flavone (PTF). METHODS: Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group (70 µg/mL high ox-LDL), an HPTF group (70 µg/mL high ox-LDL+500 µg/mL PTF), an MPTF group (70 µg/mL high ox-LDL+250 µg/mL PTF), and a LPTF group (70 µg/mL high ox-LDL+100 µg/mL PTF) in the first part; and a normal control group, an ox-LDL group (70 µg/mL high ox-LDL), an MPTF group (70 µg/mL high ox-LDL+250 µg/mL PTF), a shRNA group (transducted with PERK shRNA lentiviral particles), a scramble shRNA group (transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group (250 µg/mL PTF+70 µg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group (70 µg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability, and the level of apoptosis was monitored by flow cytometry. RESULTS: The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and shRNA groups. Moreover, the ox-LDL group had increased protein and mRNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, eIF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and shRNA groups. CONCLUSIONS: The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

Sclerostin/Receptor Related Protein 4 and Ginkgo Biloba Extract Alleviates ß-Glycerophosphate-Induced Vascular Smooth Muscle Cell Calcification By Inhibiting Wnt/ß-Catenin Pathway.

BACKGROUND: Abnormal mineral metabolism in patients with chronic kidney disease (CKD) may lead to vascular calcification, which is markedly associated with adverse events, including ischemic cardiac diseases and all-cause cardiovascular mortality. Thus, preventing and treating vascular calcification play an important role in improving the prognosis of CKD patients. OBJECTIVES: To investigate the potential functions of sclerostin and low-density lipoprotein receptor-related protein 4 (Lrp4) in alleviating the ß-glycerophosphate (ß-GP)-induced vascular smooth muscle cell (VSMC) calcification, and the protective effect of Ginkgo biloba extract (GBE). METHODS: VSMC were extracted from Sprague-Dawley rat aorta and cultured in medium. The VSMCs were divided into 3 groups: (1) Negative control group, (2) ß-GP group, in which the VSMCs were treated with ß-GP, and (3) GBE and ß-GP group, where the VSMCs were treated with both ß-GP and GBE. The calcium nodules within the cells were examined by using Alizarin red S staining. The mRNA expression levels of ß-catenin and bone gamma-carboxyglutamic-acid-containing proteins (BGP) were detected by real-time PCR. The protein levels of sclerostin and Lrp4 were determined by Western blot. RESULTS: Alizarin red S staining showed that the VSMCs in ß-GP group had a distinct orange-red precipitate when compared with VSMCs in the negative control group, while the orange-red precipitate of the GBE and ß-GP group was significantly reduced compared to the ß-GP group. Real-time PCR showed that the mRNA levels of ß-catenin and BGP in VSMCs of ß-GP group were significantly higher than those of the negative control group (p < 0.05); while they were significantly reduced in VSMCs of the GBE and ß-GP group (p < 0.05). Western blot results showed that the expression of sclerostin in the ß-GP group was significantly higher than that in the control group (p < 0.05), whereas Lrp4 was significantly lower than in control group (p < 0.05). Sclerostin in GBE and ß-GP group was significantly reduced (p < 0.05), but Lrp4 was significantly elevated when compared with that of the ß-GP group (p < 0.05). CONCLUSION: ß-GP induced VSMC calcification by activating the Wnt/ß-catenin signaling pathway. Sclerostin and Lrp4 were involved in ß-GP-induced VSMC calcification and play an important role. GBE could alleviate VSMC calcification induced by ß-GP through inhibiting the Wnt/ß-catenin signaling pathway.

The G-protein coupled receptor ChemR23 determines smooth muscle cell phenotypic switching to enhance high phosphate-induced vascular calcification.

AIMS: Vascular calcification, a marker of increased cardiovascular risk, is an active process orchestrated by smooth muscle cells. Observational studies indicate that omega-3 fatty acids protect against vascular calcification, but the mechanisms are unknown. The G-protein coupled receptor ChemR23 transduces the resolution of inflammation induced by the omega-3-derived lipid mediator resolvin E1. ChemR23 also contributes to osteoblastic differentiation of stem cells and bone formation, but its role in vascular calcification is unknown. The aim of this study was to establish the role of ChemR23 in smooth muscle cell fate and calcification. METHODS AND RESULTS: Gene expression analysis in epigastric arteries derived from patients with chronic kidney disease and vascular calcification revealed that ChemR23 mRNA levels predicted a synthetic smooth muscle cell phenotype. Genetic deletion of ChemR23 in mice prevented smooth muscle cell de-differentiation. ChemR23-deficient smooth muscle cells maintained a non-synthetic phenotype and exhibited resistance to phosphate-induced calcification. Moreover, ChemR23-deficient mice were protected against vitamin D3-induced vascular calcification. Resolvin E1 inhibited smooth muscle cell calcification through ChemR23. Introduction of the Caenorhabditis elegans Fat1 transgene, leading to an endogenous omega-3 fatty acid synthesis and hence increased substrate for resolvin E1 formation, significantly diminished the differences in phosphate-induced calcification between ChemR23+/+ and ChemR23-/- mice. CONCLUSION: This study identifies ChemR23 as a previously unrecognized determinant of synthetic and osteoblastic smooth muscle cell phenotype, favouring phosphate-induced vascular calcification. This effect may be of particular importance in the absence of ChemR23 ligands, such as resolvin E1, which acts as a calcification inhibitor under hyperphosphatic conditions.

Magnesium ion leachables induce a conversion of contractile vascular smooth muscle cells to an inflammatory phenotype.

Phenotype switching is a characteristic response of vascular smooth muscle cells (vSMCs) to the dynamic microenvironment and contributes to all stages of atherosclerotic plaque. Here, we immersed pure magnesium and AZ31 alloy in the completed medium under cell culture condition, applied the resultant leaching extracts to the isolated contractile rat aortic vSMCs and investigated how vSMCs phenotypically responded to the degradation of the magnesium-based stent materials. vSMCs became more proliferative and migratory but underwent more apoptosis when exposed to the degradation products of pure magnesium; while the AZ31 extracts caused less cell division but more apoptosis, thus slowing cell moving and growing. Noticeably, both leaching extracts dramatically downregulated the contractile phenotypic genes at mRNA and protein levels while significantly induced the inflammatory adhesive molecules and cytokines. Exogenously added Mg ions excited similar transformations of vSMCs. With the liberation or supplementation of Mg2+ , the expression patterns of the pro-contractile transactivator myocardin and the pro-inflammatory transcriptional factor kruppel-like factor 4 (KLF4) were reversed. Overall, the degradation of the Mg-based materials would evoke a shift of the contractile vSMCs to an inflammatory phenotype via releasing Mg ions to induce a transition from the phenotypic control of vSMCs by the myocardin to that by the KLF4. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 988-1001, 2019.

Restoring mitochondrial biogenesis with metformin attenuates ß-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of PDK4/oxidative stress-mediated apoptosis.

Mitochondrial abnormalities have long been observed in the development of vascular calcification. Metformin, a member of the biguanide class of antidiabetic drugs, has recently received attention owing to new findings regarding its protective role in cardiovascular disease. Since the precise control of mitochondrial quantity and quality is critical for the survival and function of vascular smooth muscle cells (VSMCs), maintaining mitochondrial homeostasis may be a potential protective factor for VSMCs against osteoblast-like phenotypic transition. However, limited studies have been reported in this area. Here, we investigated the role of metformin in the phenotypic transformation of VSMCs, as well as its intracellular signal transduction pathways. We demonstrated that supplementation with metformin restored the ß-glycerophosphate (ß-GP)-mediated impairment of mitochondrial biogenesis in VSMCs, as evidenced by an increased mitochondrial DNA copy number, a restored mitochondrial membrane potential (MMP), and upregulated mitochondrial biogenesis-related gene expression, whereas the AMP-activated protein kinase (AMPK) inhibitor compound C suppressed these effects. We also observed that overexpression of pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular energy metabolism, exacerbated ß-GP-induced oxidative stress and subsequent apoptosis in VSMCs but that these effects were suppressed by dichloroacetate, a widely reported PDK4 inhibitor. More importantly, enhanced mitochondrial biogenesis attenuated the ß-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype through inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, whereas disruption of mitochondrial biogenesis by zidovudine aggravated ß-GP-induced apoptosis in VSMCs. In addition, inhibition of autophagy by small interfering RNA targeting Atg5 reduced mitochondrial biogenesis in VSMCs. In summary, we uncovered a novel mechanism by which metformin attenuates the phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, and mitochondrial homeostasis is involved in this process.