Nalp3 inflammasome is activated and required for vascular smooth muscle cell calcification

Background

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. Increased inflammasome activation has been shown to play an important role in the pathogenesis of atherosclerosis. However, the contribution of inflammasome activation on the development of vascular calcification has not been investigated.

Methods

β-Glycerophosphate (β-GP) was used as a procedure to induce extensive artery calcification in primary vascular smooth muscle cells (VSMCs). Analysis of the levels of Nalp3 inflammasome complex was performed by quantitative real-time PCR and western blotting. The effect of Nalp3 deficiency on VSMC calcification was examined after transfecting Nalp3 siRNA into cultured VSMCs.

Results

We demonstrated for the first time that the mRNA levels of Nalp3 inflammasome complex including Nalp3, ASC and caspase1 were upregulated in calcifying VSMCs, resulting in increased IL-1β secretion. Inhibition of inflammasome activation by Nalp3 RNA interference reduced IL-1β secretion and inhibited VSMC calcification. Further analysis of clinical popliteal artery specimens showed an upregulation of inflammasome complex mRNA levels (4/5) and caspase1 activity (5/5) compared with their non-calcified adjacent tissues, indicating that Nalp3 inflammasome was tightly correlated with arterial calcification disease.

Conclusion

Our findings indicate that activation of the Nalp3-mediated inflammatory response pathway is an important venue associated with host response and pathogenesis of VSMC calcification.     

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim

To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods

Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results

Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion

p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.     

PI3K/Akt在肺炎衣原体感染诱导血管平滑肌细胞迁移中的作用

目的研究PI3K/Akt在肺炎衣原体(Chlamydia pneumoniae,C.pn)感染诱导血管平滑肌细胞(vascularsmooth muscle cell,VSMC)迁移中的作用。方法利用透射电镜鉴定C.pn成功感染VSMC;采用PI3K特异性抑制剂LY294002预处理VSMC后,wound-healing实验和Transwell实验分别观察VSMC迁移能力的改变;Western blot实验检测VSMC内Akt的磷酸化水平。结果透射电镜可观察到感染的VSMC内出现典型的C.pn原体;C.pn感染VSMC 24 h后,细胞迁移实验结果显示C.pn感染组VSMC的迁移能力增强且明显高于正常对照组(P<0.05);Western blot结果显示C.pn感染组Akt的磷酸化表达水平明显上调且高于正常对照组(P<0.05);PI3K特异性抑制剂LY294002可有效抑制C.pn感染诱导的VSMC迁移及Akt的磷酸化。结论 C.pn感染通过激活PI3K/Akt促进VSMC迁移。     

Activation of Notch-mediated protective signaling in the myocardium

The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. These processes are regulated via Notch-mediated activity that involves hepatocyte growth factor (HGF)/c-Met receptor and phosphatidylinositol 3-kinase/Akt signaling cascades. The impact of HGF on Notch signaling was assessed following myocardial infarction as well as in cultured cardiomyocytes. Notch1 is activated in border zone cardiomyocytes coincident with nuclear c-Met following infarction. Intramyocardial injection of HGF enhances Notch1 and Akt activation in adult mouse myocardium. Corroborating evidence in cultured cardiomyocytes shows treatment with HGF or insulin increases levels of Notch effector Hes1 in immunoblots, whereas overexpression of activated Notch intracellular domain prompts a 3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function in comparison with control mice after 4 weeks, implicating Notch signaling in a cardioprotective role following cardiac injury. These results indicate Notch activation in cardiomyocytes is mediated through c-Met and Akt survival signaling pathways, and Notch1 signaling in turn enhances Akt activity. This mutually supportive crosstalk suggests a positive survival feedback mechanism between Notch and Akt signaling in adult myocardium following injury.     

The Mammalian Target of Rapamycin Signalling Pathway Is Involved in Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Background

Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated.

Methods

Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors.

Results

The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner.

Conclusions

mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.     

Effects of extracellular acid stimulation on rat vascular smooth muscle cell in Gas6/Axl or PI3K/Akt signaling pathway

Recent studies have indicated that extracellular acid stimulation inhibited the calcification of vascular smooth muscle cells (VSMCs). Cell apoptosis played an important role in the occurrence and development of vascular calcification. We further explored the effects of Gas6/Axl or PI3K/Akt signaling pathway on the inhibition of rat VSMCs calcification in response to extracellular acid stimulation. Our study demonstrated that a high concentration of phosphorus induced apoptosis and calcification of VSMCs, decreased expression of Axl, and reduced phosphorylation of Akt. Stimulation of extracellular acid counteracted the effects as above by increasing the expression of Axl and Akt phosphorylation and decreasing the expression of activated Caspase3, which thereby decreased cell apoptosis and calcification. Moreover, the effects can be attenuated by PI3K inhibitor. Our study proved that extracellular acid stimulation played a vital role in the inhibition of rat VSMCs calcification and apoptosis in Gas6/Axl or PI3K/Akt signaling pathway.     

Arterial calcification in chronic kidney disease: key roles for calcium and phosphate

Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteogenic/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and extracellular matrix degradation. These studies suggest a major role for elevated P in promoting osteogenic/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic, providing a major stimulus for vascular calcification in CKD. Unraveling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.     

Novel role of protein kinase C-delta Tyr 311 phosphorylation in vascular smooth muscle cell hypertrophy by angiotensin II

We have shown previously that activation of protein kinase C-delta (PKC delta) is required for angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC delta phosphorylation at Tyr(311) plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC delta phosphorylation at Tyr(311), and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC delta was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr(311) by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC delta wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC delta Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC delta K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC delta kinase activation but also the Src-dependent Tyr(311) phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.     

内质网应激介导了高糖引起的血管平滑肌细胞钙化

目的探讨内质网应激是否介导了高糖引起的血管平滑肌细胞(VSMC)钙化。方法体外高糖(35μmol/L D-葡萄糖)处理VSMC模拟糖尿病环境,观察高糖是否引起VSMC内质网应激反应和凋亡,探索高糖是否引起VSMC表型转化(收缩型转变为成骨样细胞),观察内质网应激诱导剂和抑制剂对VSMC钙化的影响,碱性磷酸酶(ALP)活性、钙沉积和骨分化转录因子(Runx2和Osterix)通过比色法、O-cresolphthalein法和Western blot测定。结果应用35μmol/L D-葡萄糖分别处理VSMC不同时间,可以上调VSMC内质网应激标志蛋白表达、ALP活性、钙沉积和骨分化标志蛋白;然而,4-PBA预处理抑制VSMC内质网应激反应的同时,也能阻断高糖引起的VSMC钙化,表现为ALP活性、钙沉积和骨分化标志蛋白下降。结论高糖可以激活VSMC的内质网应激和凋亡,进而促VSMC钙化的发生,提示可能内质网应激介导了此激活过程。     

Dopamine d1-like receptors suppress proliferation of vascular smooth muscle cell induced by insulin-like growth factor-1

Objective: Proliferation of vascular smooth muscle cells (VSMCs) participates in the pathogenesis and development of cardiovascular diseases, including essential hypertension and atherosclerosis. Our previous study found that stimulation of D₁-like dopamine receptors inhibited insulin-induced proliferation of VSMCs. Insulin-like growth factor-1 (IGF-1) and insulin share similar structure and biological effect. However, whether or not there is any effect of D₁-like receptors on IGF-1-induced proliferation of VSMCs is not known. Therefore, we investigated the inhibitory effect of D₁-like dopamine receptors on the IGF-1-induced VSMCs proliferation in this study.

Method: VSMC proliferation was determined by [³H]-thymidine incorporation, the uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell number. Phosphorylated/non-phosphorylated IGF-1 receptor, Akt, mTOR and p70S6K expressions were determined by immunoblotting. The oligodeoxynucleotides were transfected to A10 cells to identify the effect of D₁ and D₅ receptors, respectively.

Results: IGF-1 increased the proliferation of VSMCs, while in the presence of fenoldopam, IGF-1-mediated stimulatory effect was reduced. Use of either antisense for D₁ or D₅ receptor partially inhibited the fenoldopam-induced antiproliferation effect of VSMCs. Use of both D₁ and D₅ receptor antisenses completely blocked the inhibitory effect of fenoldopam. In the presence of PI3k and mTOR inhibitors, the IGF-1-mediated proliferation of VSMCs was blocked. Moreover, IGF-1 increased the phosphorylation of PI3k and mTOR. The inhibitory effect of fenoldopam on VSMC proliferation might be due to the inhibition of IGF-1 receptor expression and IGF-1 phosphorylation, because in the presence of fenoldopam, the stimulatory effect of IGF-1 on phosphorylation of IGF-1 receptor, PI3k and mTOR is reduced, the IGF-1 receptor expression was reduced in A10 cells.

Conclusion: Activation of the D₁-like receptors suppressed the proliferative effect of IGF-1 in A10 cells via the inhibition of the IGF-1R/Akt/mTOR/p70S6K pathway and downregulated the expression of IGF-1 receptor.     

Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis

AIMS: Low shear stress (LSS) plays a significant role in vascular remodelling during atherogenesis, which involves migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). The aim of the present study is to elucidate the molecular mechanisms by which LSS induces vascular remodelling. METHODS AND RESULTS: Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague-Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm(2), were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDIalpha) in the LSS vessels. Rho-GDIalpha signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDIalpha in the LSS-induced vascular remodelling. A decrease in Rho-GDIalpha expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDIalpha expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDIalpha siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis. CONCLUSION: The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDIalpha. The effect of Rho-GDIalpha on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.     

Notch Signaling in Cardiovascular Disease and Calcification

Recent increase in human lifespan has shifted the spectrum of aging-related disorders to an unprecedented upsurge in cardiovascular diseases, especially calcific aortic valve stenosis, which has an 80% risk of progression to heart failure and death. A current therapeutic option for calcified valves is surgical replacement, which provides only temporary relief. Recent progress in cardiovascular research has suggested that arterial and valve calcification are the result of an active process of osteogenic differentiation, induced by a pro-atherogenic inflammatory response. At molecular level, the calcification process is regulated by a network of signaling pathways, including Notch, Wnt and TGFbeta/BMP pathways, which control the master regulator of osteogenesis Cbfa1/Runx2. Genetic and in vitro studies have implicated Notch signaling in the regulation of macrophage activation and cardiovascular calcification. Individuals with inactivating Notch1 mutations have a high rate of cardiovascular disorders, including valve stenosis and calcification. This article reviews recent progress in the mechanism of cardiovascular calcification and discusses potential molecular mechanisms involved, focusing on Notch receptors. We propose a calcification model where extreme increases in vascular wall cell density due to inflammation-induced cell proliferation can trigger an osteogenic differentiation program mediated by Notch receptors. Key Words: Calcification, cardiac valve, inflammation, Notch signaling, mesenchymal stem cells, atherosclerosis.     

Local Delivery of Pravastatin Inhibits Intimal Formation in a Mouse Vein Graft Model

Background

Pravastatin can reduce atherosclerotic progression in patients after coronary artery bypass graft. However, it is unknown whether pravastatin has a direct effect on intimal hyperplasia of grafted vessels in vivo or what the underlying mechanisms may be. In this study, a murine vein graft model was applied to deal with these issues.

Methods

Vein grafting was performed between C57BL/6J mice. Immediately after operation, pravastatin (30 μM) or phosphate-buffered saline in 50 μL 20% pluronic F-127 gel was delivered to the adventitia of grafted vessels.

Results

Compared with the vehicle, pravastatin significantly reduced intimal hyperplasia 4 weeks after the surgical procedure. Immunohistochemical studies revealed that vascular smooth muscle cells (VSMCs) are a major component of the neointima. The percentage of cells positive for proliferating cell nuclear antigen and Mac-3-positive immunostaining intensity within the intima of vein grafts was significantly lower in the pravastatin-treated group than in the control group. We separated VSMCs from mouse inferior vena cava and collected peritoneal macrophage from mice injected intraperitoneally with 4% thioglycollate. Pravastatin significantly decreased VSMC proliferation and platelet-derived growth factor–induced VSMC migration and, in a dose-dependent manner, inhibited macrophage migration induced by monocyte chemotactic protein-1.

Conclusions

Local delivery of pravastatin at the time of vein-graft surgery directly suppresses subsequent neointimal formation of grafted vessels in a vein graft model of normocholesterolemic mice. These beneficial effects are associated with inhibitory actions on VSMC and macrophage functions.     

Interaction between monocytes and vascular smooth muscle cells induces expression of hepatocyte growth factor

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF)--a multifunctional factor implicated in tissue regeneration, wound healing and angiogenesis--that is induced by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs), using coculture of human VSMCs and cells of the human monocytoid cell line, THP-1. METHODS: We collected supernatant from the coculture medium and measured HGF concentrations with an enzyme-linked immunosorbent assay. Northern blot analysis of HGF mRNA was performed using a specific cDNA. To explore which types of cells produce HGF, we performed immunohistochemistry. RESULTS: Coculture of VSMCs with THP-1 cells for 24 h caused a fivefold increase in HGF concentrations over that in control VSMC culture. Northern blot analysis showed an induction of HGF mRNA in the coculture with a peak at 3 h. Separated cocultures demonstrated that both direct contact and soluble factors contribute to the production of HGF. Immunohistochemistry demonstrated that both types of cells in the coculture produce HGF. Neutralizing antibodies against tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 inhibited the HGF production in THP-1 cells and VSMCs that was induced by the coculture conditioned medium. The protein kinase C inhibitors H-7, calphostin C and K252b, and the tyrosine kinase inhibitor, genistein, significantly inhibited the production of HGF in the coculture. CONCLUSIONS: Cell-to-cell interactions between monocytes and VSMCs induced HGF synthesis in both types of cells, suggesting that local HGF production induced by this cell-to-cell interaction has an important role in the pathogenesis of hypertension, atherosclerosis or vascular remodelling.     

Melatonin alleviates vascular calcification and ageing through exosomal miR-204/miR-211 cluster in a paracrine manner

In the elderly with atherosclerosis, hypertension and diabetes, vascular calcification and ageing are ubiquitous. Melatonin (MT) has been demonstrated to impact the cardiovascular system. In this study, we have shown that MT alleviates vascular calcification and ageing, and the underlying mechanism involved. We found that both osteogenic differentiation and senescence of vascular smooth muscle cells (VSMCs) were attenuated by MT in a MT membrane receptor-dependent manner. Moreover, exosomes isolated from VSMCs or calcifying vascular smooth muscle cells (CVSMCs) treated with MT could be uptaken by VSMCs and attenuated the osteogenic differentiation and senescence of VSMCs or CVSMCs, respectively. Moreover, we used conditional medium from MT-treated VSMCs and Transwell assay to confirm exosomes secreted by MT-treated VSMCs attenuated the osteogenic differentiation and senescence of VSMCs through paracrine mechanism. We also found exosomal miR-204/miR-211 mediated the paracrine effect of exosomes secreted by VSMCs. A potential target of these two miRs was revealed to be BMP2. Furthermore, treatment of MT alleviated vascular calcification and ageing in 5/6-nephrectomy plus high-phosphate diet-treated (5/6 NTP) mice, while these effects were partially reversed by GW4869. Exosomes derived from MT-treated VSMCs were internalised into mouse artery detected by in vivo fluorescence image, and these exosomes reduced vascular calcification and ageing of 5/6 NTP mice, but both effects were largely abolished by inhibition of exosomal miR-204 or miR-211. In summary, our present study revealed that exosomes from MT-treated VSMCs could attenuate vascular calcification and ageing in a paracrine manner through an exosomal miR-204/miR-211.