Long Noncoding RNA-GAS5:a Novel Regulator Of Hypertension-Induced Vascular Remodeling

Objective:This study aimed to investigate the role of LncRNA-GAS5 in hypertension-induced vascular remodeling.Method:1.In situ hybridization was performed to detect the expression of GAS5 in vascular endothelial cells(ECs)and vascular smooth muscle cells(VSMCs)in the thoracic aorta of WKY rats.RNA-fluorescent in situ hybridization was conducted to detect GAS5 expression in human umbilical vein endothelial cells(HUVECs)and VSMCs.Quantitative polymerase chain reactions(qPCRs)were conducted to detect GAS5 expression in the nuclei and cytoplasm of HUVECs and VSMCs.qRT-PCRs were performed to compare GAS5 expression difference in the retinas,caudal arteries,renal arteries,thoracic arteries,carotid arteries and plasma between SHR and age-matched WKY rats.qRT-PCRs were conducted to detect GAS5 expression in the plasma from 50 patients with hypertension and 30 age-matched healthy controls.2.The experiment included four groups:Wistar-Kyoto rats(WKY),spontaneously hypertensive rats(SHR),Scr shRNA SHR and GAS5 shRNA SHR.Retinal trypsin digestion was performed to detect the change of acellular capillaries.Evans blue was used to detect capillary leakage of the retinas of SHR.The corneal neovascularization models were built up in ICR mice,and the corneal neovasculature was observed by slit-lamp.Systolic,diastolic,and mean arterial blood pressure were measured noninvasively using a Coda 6 Blood Pressure System.Hematoxylin/eosin-stained was used to detect the vascular morphology of caudal artery,renal artery,carotid artery and thoracic artery.3.We built up oxidation stress models using HUVECs and VSMCs.The experiment included four groups:normal group,50 pmol/L H2O2 treatment,scrambled siRNA with 50 μmol/L H2O2 treatment and GAS5 siRNA with 50 pmol/L H2O2 treatment.We used MTT,tube formation and transwell assay to detect cell viability,Hoechst 33342 and PI staining to detect cell apoptosis,and Ki67 staining to detect cell proliferation.Immunofluorescence was performed to detect the proliferation of ECs and VSMCs in the carotid arteries.4.VSMCs was incubated with the medium from GAS5-overexpressed HUVECs to observe its activity.HUVECs was incubated with the medium from GAS5-overexpressed VSMCs to observe its activity.We exposed the medium to deoxyribonuclease,ribonclease,or proteinase K and observed its changes.5.We also investigated whether GAS5 knockdown affected the expression of c-Myc,cyclinDl,and PPARδin the HUVECs and VSMCs.To investigate whether GAS5 regulates the function of ECs and VSMCs through β-catenin signaling pathway.Result:1.GAS 5 was expressed in vascular ECs and VSMCs of WKY rats.Compared with age-matched wild-type controls,plasma GAS5 level was significantly downregulated in SHRs.GAS5 expression was also significantly downregulated in the plasma of hypertensive patients.2.GAS 5 knockdown further increased the number of neovascular in retinas of SHR,further aggravated capillary degeneration and capillary leakage.GAS5 knockdown further aggravated alkali-burn-induced angiogenesis.GAS5 knockdown SHRs had a significant higher blood pressure compared with the control SHRs.3.Oxidative stress can significantly reduced the viability and proliferation of HUVECs and VSMCs.GAS5 knockdown could further up-regulate the viability and proliferation.Moreover,GASS knockdown further decreased the death and apoptosis.GAS5 knockdown increased the proliferation of ECs and VSMCs in the carotid arteries.4.Incubation of VSMCs with the medium from GAS5-overexpressed HUVECs significantly reduced the activity of VSMCs.Incubation of HUVECs with the medium from GAS5-overexpressed VSMCs significantly reduced the activity of HUVECs.The ability of medium to decrease proliferation and migration was partially abolished by ribonclease A or proteinase K but not affected by deoxyribonuclease treatment.5.GASS knockdown significantly upregulated the expression of c-Myc,cyclinD1,and PPARδ in the HUVECs and VSMCs.Conclusion:LncRNA-GAS5 regulates ECs and VSMCs function through the β-catenin signaling,which is involved in the regulation of vascular remodeling in hypertension.GAS5 may represent an interesting pharmacological target for the treatment of hypertensive conditions.