Role Of MicroRNA-130A In Proliferation Of Vascular Smooth Muscle Cell And Vascular Remodeling

Chapter1Role and mechanism of microRNA-130a in hypertensive vascular remodelingBackgroundHypertension is the most common disease and causes of morbidity and mortality in China. Vascular remodeling caused by long-term high blood pressure can lead to cardiovascular complications, which is the direct cause of death of hypertensive patients. Vascular remodeling is the most important pathological factors during the development of hypertension. And prevention and treatment of hypertension vascular remodeling is the one of the important goals of treatment of hypertension.Proliferation of vascular smooth muscle cells (VSMCs) is essential for vascular remodeling. Many factors, such as nitric oxide (NO), angiotensin Ⅱ (Ang Ⅱ), platelet-derived growth factor (PDGF), endothelial growth factor (VEGF) and so on, can regulate proliferation of VSMCs. Recently, the role of microRNAs (miRNAs) in proliferation of VSMCs begins to be focused.It has been demonstrated that Gax is a target gene of miR-130a. Gax, which is restrictively expressed in cardiovascular system, has remarkable inhibitory effects on proliferation, differentiation, and migration of VSMC. Gax is found to be downregulated in some vasculopathy such as balloon injury, pulmonary hypertension, and neurovascular dysfunction in Alzheimer disease or by some growth factors including Ang II and PDGF. And overexpression of Gax alleviates vascular dysfunction. It is likely that miR-130a may be a new modulator of proliferation of VSMCs, and may play a key role in vascular remodeling of hypertension.MethodIn vitro study:VSMCs were prepared from the thoracic aorta of male10-week-old Sprague-Dawley rats using explant method. The mimic of miR-130a was used to overexpression of it. Proliferation of VSMCs was determined by BrdU incorporation method. MiR-130a and mRNA of Gax were performed by qRT-PCR (Syber green method), proten level of Gax were determined by Western blot.In vivo study:Male26-week-old spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) were used. Systolic blood pressure of rats was measured by tail-cuff method2days before operation. Blood (2ml) was collected for Ang II assay. Thoracic and aorta and mesenteric artery were collected for RNA, protein and morphological analysis.Results25or50nmol/1significantly promoted proliferation of VSMCs, and the expression of Gax mRNA and protein was markedly reduced. Ang Ⅱ dose-dependently (10-10～10-6mol/L) and time-dependently (1,3,6,12,24h) promoted proliferation of cells concomitantly with an increase of miR-130a expression, and the proliferative effect of Ang Ⅱ was attenuated by the inhibitor of miR-130a (100,200nmol/L).Systolic blood pressure and plasma Ang Ⅱ level and was significantly higher in SHRs than that of WKYs. Thoracic aorta and mesenteric artery in SHRs were significantly thicker than that of WKYs. The mRNA and protein expression of Gax in the remodeled thoracic aorta and superior mesenteric artery of SHRs decreased concomitantly with an increase miR-130a expression.ConclusionMiR-130a is a novel regulator of proliferation of VSMCs via inhibiting the expression of Gax, which contribute to vascular remodeling in hypertension. Chapter2Role and mechanism of microRNA-130a in hypertensive vascular remodeling of pulmonary arterial hypertensionBackgroudPulmonary arterial hypertension (PAH) is one of type of pulmonary hypertension, has increased pulmonary arterial blood pressure and normal pulmonary venous pressure. It was characterized by progressive increase in pulmonary vascular resistance, and ultimately leading to right heart failure and death.PAH has poor prognosis, and lack effective treatment. The etiology involving environmental and genetic factors, its pathogenesis has not been fully elucidated. The predominant cause of increased pulmonary vascular resistance is loss of vascular luminal cross section due to vascular remodeling. Many factors involve in development of pulmonary vascular remodeling, such as transforming growth factor beta (TGFβ)、5-hydroxy tryptamine (5-HT)、endothelin (ET-1)、nitric oxide (NO)、 platelet-derived growth factor (PDGF)、fibroblast growth factor2(FGF2). Recent studies show that microRNAs also have an important role in process of pulmonary vascular remodeling in PAH.It has been reported that miR-130a mediates proliferation, migration and angiogenesis of vascular endothelial cells. Moreover we have testified that miR-130a can promote proliferation of vascular smooth muscle cells by inhibiting Gax, and was increased in remodeling vessels in hypertension. But, the role of miR-130a in PAH is unclear. It has been shown that Gax, the target of miR-130a, was down-regulated in lung of PAH rat and PASMCs treated with hypoxia. These results imply that miR-130a-Gax pathway may also involve in proliferation of PASMCs and process of vascular remodeling in PAH.Method1. We used two types PAH rat model:hypoxia indued and monocrotaline (MCT) induced PAH. Sprague-Dawley (SD) rats (180-200g) was placed in camber with10%O2, or injected with2%MCT60mg/kg. After3weeks,2. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were prepared from the pulmonary artery of male10-week-old Sprague-Dawley rats using explant method. The mimic of miR-130a was used to overexpress it. Down-regulation of miR-130a and Ddx6was used miR-130a inhibitor and Ddx6siRNA, respectively. Proliferation of VSMCs was determined by BrdU incorporation method. MiR-130a and mRNA were performed by qRT-PCR (Syber green method), proten level of Ddx6was determined by Western blot.3. Construction and determination of luciferase reporter gene vector: About200bp fragment from the precdict target gene3’-untranslated region (3’-UTR) containing miR-130a targeting site cloned to pGL3-Control vector. The vector was then transfected with pRL-SV40vector, as an internal control, and miR-130a mimic into human smooth muscle cell line. Luciferase activity was determined after trasfection24h.Results1. Right ventricular pressure and pulmonary artery pressure of PAH rats induced by hypoxia and MCT were higher than that of normal rats. PAH rats have thicker pulmonary artery than control. Expression of miR-130a and Gax was up-regulated in pulmonary artery from PAH rats, and expression of pri-miR-130a was not changed. Immunohistochemistry experiment showed that Gax expressed mainly in pulmonary vessels.2. Hypoxia produced proliferation of PASMCs. And expression of miR-130a was increased time-dependently in PASMCs treated with hypoxia, while pri-miR-130a was not changed. Inhibitor of miR-130a alleviated the pro-proliferaion effect of hypoxia.3. Overexpression of miR-130a directly produced proliferation of PASMCs, but did not affect expression of SMA-the marker of phenotype of smooth muscle cells.4. Ddx6, a target of miR-130a, was decreased in pulmonary artery from PAH rats. Overexprssion of miR-130a inhibited the expression of Ddx6. Downreglation of Ddx6produced proliferation of PASMCs.5. We predict three potential miR-130a target genes:BMPR2(Bone morphogenetic protein receptor, type Ⅱ), SMAD5(SMAD family member5) and ACVR1(Activin A receptor, type Ⅰ).3’-UTR of these genes containing miR-130a targeting site were cloned to pGL3-Control vector. But miR-130a has no influence on luciferase activity in PASMCs transfected with these vectors.Conclusion1. MiR-130a has a direct role in promoting proliferation of PASMCs and mediates pulmonary vascular remodeling in PAH by inhibition of Ddx6.2. Ddx6has an inhibitory effect on proliferation of PASMCs. Downregulation of Ddx6can produce proliferation of PASMCs.3. Increae of miR-130a in PAH occurs at post-transcriptional level. Hypoxia can promote accelerated synthesis of miR-130a, thereby induce cell proliferation.4. Gax is mainly expressed in pulmonary vessels in lung and has a low level at physiological conditions, but is upregulated in PAH.5. BMPR2、SMAD5and ACVR1is not targets of miR-130a.