Ghrelin blunted vascular calcification in vivo and in vitro in rats

Ghrelin is a new peptide with regulatory actions in growth hormone secretion in the anterior pituitary gland and in energy metabolism. Currently, ghrelin has potently protective effects in cardiovascular diseases. We used an in vivo model of rat vascular calcification induced by vitamin D3 and nicotine and one of cultured rat vascular smooth muscular cells (VSMCs) calcification induced by beta-glycerophosphate to study the possible mechanism in the regulatory action of ghrelin in vascular calcification. Calcification increased total Ca2+ content and 45Ca2+ deposition in aortas and VSMCs and alkaline phosphatase (ALP) activation in plasma, aortas and VSMCs. However, calcified aortas and VSMCs showed a significant decrease in osteopontin (OPN) mRNA expression and a marked reduction of ghrelin levels in plasma and its mRNA expression in aortas. The aortic calcification was significantly attenuated by subcutaneous administration of ghrelin 30 and 300 nmol kg(-1) day(-1) for 4 weeks, and the latter dosage was more potent than the former. Ghrelin treatment at the two dosages reduced the total aorta Ca2+ content by 24.4% and 28.1%, aortic 45Ca2+ deposition by 18.4% and 24.9%, plasma ALP activity by 36.6% and 76.7%, and aortic ALP activity by 10.3% and 47.6% (all P < 0.01 or 0.05), respectively. Ghrelin at 10(-8)-10(-6) mol/L attenuated the calcification in cultured VSMCs, with decreased total Ca2+ content, 45Ca2+ deposition and ALP activity and increased OPN mRNA expression, in a concentration-dependent manner. In addition, endothelin levels in plasma and aortas and its mRNA expression in aortas significantly increased with calcification, but ghrelin treatment significantly decreased endothelin levels and mRNA expression, with the high dosage being more potent than the lower dosage. These results indicate that local ghrelin in vascular was down-regulated during vascular calcification, whereas administration of ghrelin effectively attenuated vascular and VSMCs calcification.     

补镁对大鼠血管钙化的影响

目的:在大鼠血管钙化模型上,观察外源性补充硫酸镁对大鼠血管钙化的影响,以探讨硫酸镁在血管钙化中作用及机制。方法:用维生素D3加尼古丁诱导大鼠血管钙化,von Kossa染色、钙含量测定及碱性磷酸酶活性测定判断血管钙化程度;用半定量RT-PCR方法测定血管钙化标志分子骨桥蛋白mRNA水平;用生物化学方法测定血管一氧化氮(NO)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量。结果:钙化组大鼠血压升高,收缩压较对照组高27%(P<0.05);血管von Kossa染色见血管中膜弹性纤维间可见大量棕黑色颗粒沉积,主动脉钙含量及碱性磷酸酶活性分别较对照高3.9倍和3.4倍(P<0.01),钙化血管组织骨桥蛋白mRNA表达上调40%(P<0.01),血管钙化后可加重血管组织过氧化损伤;而诱导钙化后外源性补充硫酸镁可减轻血管钙化程度,与钙化组比较,低、高剂量硫酸镁组均明显缓解上述指标的变化。结论:诱导血管钙化后外源性补充硫酸镁可以减轻大鼠血管钙化和血管损伤程度。     

Changes of heme oxygenase-carbon monoxide system in vascular calcification in rats

The aim of the present study was to investigate the change in heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification. Vascular calcification model was established in rats by using vitamin D(3) and nicotine. Vascular calcium content, alkaline phosphatase (ALP) activity, HO activity, HbCO formation and content of cGMP in vessels were measured. Immunochemistry (IH) for HO 1 expression and in situ hybridization (ISH) for HO 1 mRNA were observed. Compared to those of control rats, the aortic calcium content and vascular ALP activity in rats of the calcified group (VDN group) were obviously increased, but HO 1 activity, CO concentration and cGMP content in vessels of rats in VDN group were markedly decreased. Expressions of HO-1 protein and mRNA were significantly decreased compared to control rats. Vascular calcification might induce a down regulation in vascular HO-CO-cGMP pathway.     

Changes in amount of ADM mRNA and RAMP2 mRNA in calcified vascular smooth muscle cells

This work was aimed to explore the changes and significance of adrenomedullin (ADM) mRNA and receptor activity modifying protein 2 (RAMP2) mRNA in calcified vascular smooth muscle cells (VSMCs). Calcification of cultured rat VSMCs was produced by incubation with beta-glycerophosphate. Content of ADM released by VSMCs was measured by radioimmunoassay (RIA). The amount of ADM mRNA and RAMP2 mRNA was determined by competitive quantitative RT-PCR. The intracellular calcium content, alkaline phosphatases activity and cellular (45)Ca(2+)-uptake were determined. The results showed that the content of calcium, (45)Ca(2+)-uptake and alkaline phosphatases activity in calcified VSMCs were increased by 118%, 174% and seven-fold (all P<0.01), respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (P<0.01). Furthermore, it was found that the amount of ADM mRNA and RAMP2 mRNA in calcified cells was elevated by 78 and 56% (all P<0.05), respectively, compared with control. The elevated levels of RAMP2 mRNA were in positive correlation with ADM mRNA (r=0.76, P<0.05) in calcified VSMCs. In conclusion, calcified VSMCs generated an increased amount of ADM, and up-regulated gene expressions of ADM and RAMP2.     

高同型半胱氨酸血症促进大鼠血管钙化

目的:在大鼠血管钙化模型上,探讨高同型半胱氨酸血症对血管钙化的影响及其作用机制.方法:用维生素D3加尼古丁诱导大鼠血管钙化模型,并给以高蛋氨酸饮食六周诱导大鼠高同型半胱氨酸血症,用高效液相色谱法检测血浆总同型半胱氨酸(Hcy)水平;采用血管组织vonKossa染色、钙含量测定、碱性磷酸酶(ALP)活性和骨钙素(OC)含量测定以判断血管钙化程度,同时测定血浆脂质共轭烯(Diene键)含量反映其脂质过氧化水平.结果:钙化组大鼠血管yon Kossa染色可见大量黑色颗粒沉积,其血管的钙含量,碱性磷酸酶活性及骨钙素含量分别较对照组增加8.09倍、45.57%和2.81倍(P＜0.01).高蛋氨酸饮食的钙化组大鼠血管内钙含量较单纯钙化组增高了34.29%,而碱性磷酸酶活性及骨钙素含量则较单纯的钙化组降低29.13%和74.69%(P＜0.01).钙化组大鼠血浆脂质共轭烯含量与对照组比较无显著性差异,单纯高蛋氨酸饮食和钙化加高蛋氨酸饮食大鼠血浆脂质共轭烯含量较对照组增加了1.93和2.89倍(P＜0.01),而钙化加高蛋氨酸饮食大鼠血浆脂质共轭烯含量较单纯高蛋氨酸饮食大鼠又增加了32.90%(P＜0.01).结论:高同型半胱氨酸血症可以促进血管的钙化,可能与其所致的脂质过氧化程度增强有关.     

Homocysteine potentiates calcification of cultured rat aortic smooth muscle cells

Aortic calcification was demonstrated in experimental animal models of hyperhomocysteinemia. Mild hyperhomocysteinemia was associated with aortic calcification, suggesting a relationship between homocysteine (HCY) and the pathogenesis of aortic calcification. In the present study, the effect of HCY on vascular calcification was examined in calcifying and non-calcifying vascular smooth muscle cells (VSMCs). Cell calcification was induced by incubation of VSMCs with beta-glycerophosphate. Proliferation of VSMCs was studied by cell counting, 3H-thymidine (3H-TdR) and 3H-leucine (3H-Leu) incorporation. 45Ca accumulation, cell calcium content, and alkaline phosphatase (ALP) activity were measured as indices of calcification. The results showed that the proliferation of calcifying VSMCs, which was indicated by cell counting, 3H-TdR and 3H-Leu incorporation in calcifying VSMCs, was enhanced as compared with that of non-calcifying VSMCs. HCY promoted increases in cell number, 3H-TdR and 3H-Leu incorporation in both calcifying and non-calcifying VSMCs, but with more prominent effect in calcifying VSMCs. The stimulating effects of HCY on the three parameters in calcifying VSMCs were antagonized by PD98059, a specific inhibitor of mitogen activated protein kinase kinase (MAPKK). The ALP activity, 45Ca uptake, and calcium deposition in the calcifying VSMCs were greater than those in non-calcifying VSMCs. PD98059 had no effect on ALP activity, 45Ca uptake, and calcium deposition in calcifying VSMCs. HCY caused marked increases in 45Ca uptake and calcium deposition both in calcifying and non-calcifying VSMCs. HCY, however, enhanced ALP activity in the calcified VSMCs but not in the non-calcifying VSMCs. The non-calcifying VSMCs treated with HCY showed the same low ALP activity, as did the control VSMCs. In calcifying VSMCs, the HCY-induced increases in 45Ca uptake, calcium deposition, and ALP activity were also attenuated by PD98059. The results demonstrated that HCY potentiated VSMC calcification probably through the mechanisms by which HCY promotes atherosclerosis.     

Carvacrol modulates instability of xenobiotic metabolizing enzymes and downregulates the expressions of PCNA,MMP-2, and MMP-9 during diethylnitrosamine-induced hepatocarcinogenesis in rats

Vascular calcification (VC) is highly associated with increased morbidity and mortality in patients with advanced chronic kidney disease. Paracrine/autocrine factors such as vasoactive peptides are involved in VC development. Here, we investigated the expression of the novel peptide C-type natriuretic peptide (CNP) in the vasculature, tested its ability to prevent VC in vivo and in vitro, and examined the mechanism involved. Rat aortic VC was induced by vitamin D3 plus nicotine (VDN). CNP (500 ng/kg/h) was administered by mini-osmotic pump. Calcification was examined by von Kossa staining; CNP and cyclic guanosine monophosphate (cGMP) contents were detected by radioimmunoassay, and mRNA and protein levels were examined by real-time PCR and Western blot analysis in aortas and calcified vascular smooth muscle cells (VSMCs). VDN-treated rat aortas showed higher CNP content and decreased expression of its receptor natriuretic peptide receptor B, along with increased vascular calcium deposition and alkaline phosphatase (ALP) activity. Low CNP levels were accompanied by increased vascular calcium deposition and ALP activity in VDN-treated rats when compared to vehicle treatment, which was further confirmed in cultured VSMCs. Administration of CNP greatly reduced VC in VDN-treated aortas compared with controls, which was confirmed in calcified VSMCs. The decrease in alpha-actin expression was ameliorated by CNP in vitro. Moreover, protein expression levels of osteopontin (OPN) were significantly up-regulated in calcified aortas, and CNP increased OPN expression in calcified aortas. Furthermore, CNP downregulated OPN and bone morphogenic protein 2 (BMP-2) expression in calcified aortas and VSMCs. Modulation of OPN and BMP-2 expression by CNP and the beneficial effects of CNP on calcified VSMCs were blocked significantly by protein kinase G inhibitor H7. Impaired local endogenous CNP and its receptor system may be associated with increased mineralization in vivo in rat aortas with VC, and administration of CNP inhibits VC development in vivo and in vitro, at least in part, via a cGMP/PKG pathway.     

Cortistatin attenuates vascular calcification in rats

Cortistatin (CST) is a newly discovered polypeptide with multiple biological activities that plays a regulatory role in the nervous, endocrine and immune systems. However, the role of CST in the pathogenesis of cardiovascular diseases remains unclear. In this study, we investigated in rats whether CST inhibits vascular calcification induced by vitamin D3 and nicotine treatment in vivo and calcification of cultured rat vascular smooth muscular cells (VSMCs) induced by beta-glycerophosphate in vitro and the underlying mechanism. We measured rat hemodynamic variables, alkaline phosphatase (ALP) activity, calcium deposition and pathological changes in aortic tissues and cultured VSMCs. CST treatment significantly improved hemodynamic values and arterial compliance in rats with vascular calcification, by decreasing systolic blood pressure, pulse pressure, left ventricular end-systolic pressure and left ventricular end-diastolic pressure. CST also significantly decreased ALP activity and calcium deposition, alleviated pathological injury and down-regulated the mRNA expression of type III sodium-dependent phosphate co-transporter-1 (Pit-1) in aortic tissues. It dose-independently inhibited the calcification of VSMCs by decreasing ALP activity and calcium deposition, alleviating pathologic injury and down-regulating Pit-1 mRNA expression. As with CST treatment, ALP activation and calcium deposition were decreased significantly on treatment with ghrelin, the endogenous agonist of growth hormone secretagogue receptor 1a (GHSR1a), but not significantly with somatostatin-14 or proadrenomedullin N-terminal 20 peptide in VSMCs. Further, growth hormone-releasing peptide-6[D-lys], the endogenous antagonist of GHSR1a, markedly reversed the increased ALP activity and calcium deposition in VSMCs. CST could be a new target molecule for the prevention and therapy of vascular calcification, whose effects are mediated by GHSR1a rather than SSTRs or Mrg X2.     

Cholestane-3beta, 5alpha, 6beta-triol promotes vascular smooth muscle cells calcification

Oxysterols found in atherosclerotic plaque may be associated with vascular calcification. We investigated the effect of oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) on in vitro calcification of rat vascular smooth muscle cells (VSMCs). In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. Calcifying nodule formation, calcium deposition in extracellular matrix, and alkaline phosphatase (ALP) activity were measured as indices of calcification. Because apoptotic bodies can serve as nucleation sites for calcification, apoptosis of calcifying VSMCs was determined by Hoechst 33258 staining, TUNEL, and FITC-labeled annexin V/PI double staining. The calcium deposition and ALP activity in calcifying VSMCs were much higher than those in non-calcifying VSMCs. Triol increased calcifying nodule formation, calcium deposition, ALP activity, and apoptosis of nodular cells in calcifying VSMCs. As determined by 2,7-dichlorofluorescein fluorescence, Triol induced the generation of reactive oxygen species (ROS) in calcifying VSMCs dose- and time-dependently. Triol-induced increases in calcium deposition, ALP activity, apoptosis, and ROS generation were all attenuated by antioxidant vitamin C plus vitamin E (VC + VE). The results demonstrated that Triol promoted VSMCs calcification through direct increase of ALP activity and apoptosis, probably by ROS-related mechanism.     

Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells

Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components—CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, ⁴⁵Ca²⁺ uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (p<0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r=0.83, 0.92 and 0.93, respectively, all p's<0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components—CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification.     

Fibroblast growth factor 21 ameliorates vascular calcification by inhibiting osteogenic transition in vitamin D3 plus nicotine-treated rats

FGF21, a special member of FGF superfamily, has been proven to have pleiotropic metabolic effects and many potential therapeutic action in various metabolic disorders. Vascular calcification (VC), a perplexing clinical issue, is a major risk factor for many cardiovascular diseases, especially for patients with some metabolic diseases. However, the role of FGF21 on VC in vivo remains unclear. Thus, in this study, we observed the effect and mechanism of FGF21 on VC induced by vitamin D3 plus nicotine (VDN) treated rats. After four weeks' treatment, the calcium overload is mainly manifested in the increased blood pressure, aortic calcium content and ALP activity. Also, the HE and Alizarin-red S staining showed the structural damage of calcified vessel walls. In addition, the level of endogenous FGF21/β-Klotho/FGFR1 axis was up-regulated in the aortas of VC rats. Furthermore, exogenous FGF21 treatment significantly ameliorated the aortic injury and calcification in VC rats, and the level of β-Klotho and FGFR1 were furtherly increase. Moreover, FGF21 inhibited the osteogenic transition of VSMCs by down-regulating the expression of bone-associated proteins such as osteopontin (OPN), osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2), together with restored the expression of SM22α and SM α-actin, which are two of lineage markers in VSMCs. We provide the first evidence that FGF21 can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs in rats. FGF21 might be an efficient endogenous vasoprotective factor for calcification.     

Irisin protects against vascular calcification by activating autophagy and inhibiting NLRP3-mediated vascular smooth muscle cell pyroptosis in chronic kidney disease

Irisin protects the cardiovascular system against vascular diseases. However, its role in chronic kidney disease (CKD) -associated vascular calcification (VC) and the underlying mechanisms remain unclear. In the present study, we investigated the potential link among Irisin, pyroptosis, and VC under CKD conditions. During mouse vascular smooth muscle cell (VSMC) calcification induced by β-glycerophosphate (β-GP), the pyroptosis level was increased, as evidenced by the upregulated expression of pyroptosis-related proteins (cleaved CASP1, GSDMD-N, and IL1B) and pyroptotic cell death (increased numbers of PI-positive cells and LDH release). Reducing the pyroptosis levels by a CASP1 inhibitor remarkably decreased calcium deposition in β-GP-treated VSMCs. Further experiments revealed that the pyroptosis pathway was activated by excessive reactive oxygen species (ROS) production and subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in calcified VSMCs. Importantly, Irisin effectively inhibited β-GP-induced calcium deposition in VSMCs in vitro and in mice aortic rings ex vivo. Overexpression of Nlrp3 attenuated the suppressive effect of Irisin on VSMC calcification. In addition, Irisin could induce autophagy and restore autophagic flux in calcified VSMCs. Adding the autophagy inhibitor 3-methyladenine or chloroquine attenuated the inhibitory effect of Irisin on β-GP-induced ROS production, NLRP3 inflammasome activation, pyroptosis, and calcification in VSMCs. Finally, our in vivo study showed that Irisin treatment promoted autophagy, downregulated ROS level and thereby suppressed pyroptosis and medial calcification in aortic tissues of adenine-induced CKD mice. Together, our findings for the first time demonstrated that Irisin protected against VC via inducing autophagy and inhibiting VSMC pyroptosis in CKD, and Irisin might serve as an effective therapeutic agent for CKD-associated VC.Subject terms: Calcification, Chronic kidney disease     

筛选大鼠血管钙化消退差异表达基因及初步分析

目的：探讨大鼠在血管钙化消退过程中基因表达的变化。方法：选取6周龄SPF级雄性SD大鼠24只,随机分成3组（n=8）,分别为对照组、钙化组和消退组。钙化组和消退组制作血管钙化模型（vitamin D3 plus nieotine,VDN）,对照组生理盐水和花生油灌胃。钙化组和对照组于实验第8周处死,消退组继续饲养至16周处死,测定各组大鼠动脉组织钙含量并作病理检查。采用抑制性消减杂交方法将消退组和钙化组大鼠血管cDNA作差减杂交,分离消退组较钙化组高表达或低表达基因的cDNA片段,建立消退组和钙化组的差异表达文库,扩增、鉴定文库并测序阳性克隆,BLAST比对测序序列。随机挑选4个基因进行RT-PCR验证和DNA条带半定量分析。结果：①血管组织钙含量测定显示钙化组（（15．34±2．51）mg／g）较对照组（（5．20±0．75）mg／g）血管组织钙含量明显升高（P〈0．01）;消退组（（12．73±1．89）mg／g）较钙化组降低（P〈0．05）;②构建了血管钙化的差减文库,对所获阳性克隆进行测序和BLAST比对分析,获得28个表达上调基因和22个表达下调基因。RT-PCR验证示Prdx3,Ank2,Ror2,Abcel等基因在消退组和钙化组间差异表达,消退组较钙化组表达增高,平均约为后者的1．7倍。结论：VDN模型诱导的大鼠血管钙化可以发生主动消退。钙化消退过程中焦磷酸合成相关基因、谷氨酸信号相关基因、还原及凋亡调节基因表达上调,同时见较多骨化相关基因及氧化活性等基因表达下调。钙化抑制基因的表达增多而钙化促进基因表达的下降可能是钙化发生主动消退的内源性机制。     

Superoxide production: A procalcifying cell signalling event in osteoblastic differentiation of vascular smooth muscle cells exposed to calcification media

Recent studies showed that hydrogen peroxide (H₂O₂) enhanced bone markers expression in vascular smooth muscle cells (VSMCs) implicated in osteoblastic differentiation. This study aimed at investigating the role of NAD(P)H oxidase in vascular calcification processes. A7r5 rat VSMCs were incubated with β-glycerophosphate (10 mm) or uremic serum to induce a diffuse mineralization. H₂O₂ production by VSMCs was determinated by chemiluminescence. NAD(P)H oxidase sub-unit (p22^phox), Cbfa-1, ERK phosphorylation and bone alkaline phosphatase (ALP) expressions were measured by Western blotting. VSMCs exhibited higher production of H₂O₂ and early expression of p22^phox with β-glycerophosphate or uremic serum within 24 h of treatment. β-glycerophosphate-induced oxidative stress was associated with Cbfa-1 expression followed by ALP expression and activity, meanwhile the VSMCs expressing ALP diffusely calcified their extracellular matrix. Interestingly, diphenyleneiodonium partly prevented the osteoblastic differentiation. Results from this model strongly suggest a major implication of vascular NAD(P)H oxidase in vascular calcification supported by VSMCs osteoblastic differentiation.     

Endoplasmic reticulum stress-mediated apoptosis is activated in vascular calcification

Apoptosis of vascular smooth muscle cells plays an important role in vascular calcification (VC). However, the potential mechanism remains poorly understood. Previous studies showed that apoptosis mediated by endoplasmic reticulum stress (ERS) participates in several diseases with VC. We prepared two rat models of calcification, vitamin D₃ plus nicotine (VDN) and rapid calcification (RC), to investigate whether ERS-mediated apoptosis is activated in VC. TUNEL staining and cleaved caspase 3 protein levels illustrated enhanced apoptosis in calcification groups. Western blot analysis revealed the ERS hallmarks GRP78 and GRP94 increased by 43.9% and 91.7%, respectively, in the VDN group and GRP78 elevated by 84.0% in the RC group (all P < 0.05) as compared with controls. Moreover, two molecules of ERS-induced apoptosis, caspase 12 and C/EBP homologous protein, were up-regulated nearly 3-fold (P < 0.05) in the VDN group and 10-fold (P < 0.01) in the RC group. Our results indicated that ERS-induced apoptosis may be involved in VC, and amelioration of ERS could be a novel strategy to prevent and treat the related diseases.