Effects And Mechanisms Of Suv39h1on Vascular Repair After Injury In Diabetic Rat Artery

Objectives:1. To construct the recombinant adenoviral vector carrying the rat Suv39h1gene (Ad-Suv39h1), and observe the overexpression effect of Suv39hl on rat vascular smooth muscle cells (VSMCs).2. To construct recombinant lentiviral vector carrying RNA interference sequences of rat Suv39h1gene (LV-Suv39hl shRNA), and observe silencing effect of Suv39h1on VSMCs.3. To evaluate the effect of Suv39h1on VSMCs proliferation and migration induced by angiotensin ? (Ang ?) or high glucose (HG), and explore its possible molecular mechanism.4. To estimate the role of Suv39hl in neointimal hyperplasia and endothelialization in diabetic rat carotid artery after balloon injury.Methods:1. The shuttle plasmid pDC316-Suv39hl and skeleton plasmid co-transfected into293cells to construct recombinant adeno viral vector carrying Suv39h1gene. After amplification, purification and PCR identification, the titer of acquired recombinant vector was determined. VSMCs were infected with adenovirus at multiplicity of infection (MOI) from0to30, and then the flow cytometry method was used to detect infection efficiency. Following Ad-Suv39hl transfecting into VSMCs in logarithmic phase at the optimal MOI, the protein level of Suv39h1was measured using Western blot.2. Four siRNA target sequence were designed according to the Suv39hl mRNA. DNA fragment containing shRNA sequence was connected with linearized vector named GV118. After transformation, the positive clones were screened out by PCR and then sequenced. The correct recombinant vector, GV118-Suv39h1shRNA, together with lentiviral packaging plasmids co-transfected into293T cells. Fluorescence method was used to measure the titer of collected lentiviral particles. Four kinds confirmed LV-Suv39hl shRNA transfected into VSMCs. The knock-down effect on Suv39h1expression was evaluated through Real-time PCR assay and the best was chosen for the subsequent experiments.3. VSMCs isolated from rat thoracic aorta were cultured using tissue explants adherent method. The third to fifth passage cells were used for experiment. Under stimulation with Ang ? at concentration of0.2?g/ml, Suv39h1and ID3expression in VSMCs were detected by Western blot. Induced by glucose at concentration of5mmol/L (NG) or30mmol/L (HG), Suv39h1and complement C3protein levels in VSMCs were measured. The recombinant Ad-Suv39h1, LV-Suv39hl shRNA and the corresponding control virus transfected into VSMCs as described above. After Ang II stimulation, VSMCs migration, Suv39hl, ID3, p21, p27KIPl and PCNA protein levels were evaluated. After HG stimulation, VSMCs migration, as well as Suv39hl, complement C3, p-p38, p-ERK1/2and PCNA protein expression were detected.4. Fed with high-fat diet four weeks, male SD rats were intraperitoneal injected with streptozocin at a dose of40mg/kg to induce DM models. All experimental animals were randomly divided into six groups:normal saline (Nor-NS), DM-NS, DM-Ad-Suv39h1, DM-Ad-Null, DM-LV-Suv39hl shRNA and DM-LV-NC shRNA. Carotid artery balloon injury model was established and the injured arteries were local transfected with the appropriate virus vector. The saline group got an equal volume of saline as the control. The injured arteries were collected for DNA microarray analysis. Suv39h1, complement C3and ID3mRNA levels were detected by Real-time PCR analysis. Vessel segments were stained with hematoxylin and eosin to assess intimal hyperplasia. Immunohistochemistry method was used to evaluate proliferating cell nuclear antigen (PCNA) and CD31expression in vessel wall.Results:1. The results of PCR assay confirmed recombinant adenovirus vector Ad-Suv39hl was successfully constructed as expected. The optimal MOI of Ad-Suv39hl in VSMCs was15. After infection with Ad-Suv39h1for72h, Suv39hl protein expression was almost twice as Ad-Null group (p<0.05).2. PCR analysis and sequencing showed the recombinant GV118-Suv39hl shRNA was constructed successfully. The titers of four kinds recombinant LV-Suv39h1shRNA (KD1, KD2, KD3and KD4) were ranging from108to109TU/ml. When MOI was40, approximately85%VSMCs were infected. Compared to the NC group, the level of Suv39hl mRNA was decreased by52%in KD2group, more than60%in KD3and KD4group, and more than80%in KD1group after transfection for96h (all p<0.05).3. Knockdown of Suv39hl expression significantly inhibited Ang II-induced VSMCs migration and proliferation. Compared with Ad-Null group, VSMCs transfected with LV-Suv39hl shRNA after Ang II stimulation for1h, the protein level of Suv39h1and ID3was obviously reduced, accompanied with significant increase of p21and p27KIPl (all p<0.05). Suv39hl silencing also antagonized HG-induced VSMCs migration and proliferation. In response to HG for16h, complement C3content, as well as p-ERKl/2level in VSMCs was dramatically decreased attributing to depletion of Suv39h1expression in LV-Suv39hl shRNA group (all p<0.05). However, the decline of p-p38was with no significance (p>0.05).4. Overexpression of Suv39h1resulted in233genes up-regulation and221genes down-regulation in diabetic injured arteries. Knockdown of Suv39h1induced849genes increase and658genes decrease compared to the control group (relative content>1.5, p<0.05). Diabetes mellitus mediated inhibition of Suv39h1, and promotion of complement C3and ID3expression in injured arteries. Suv39hl gene silencing contributed to remarkable decline of complement C3and ID3mRNA level in diabetic arteries. Compared to the control group, arteries in DM-LV-NC shRNA group displayed less neointimal formation at28days after injury. Consistent with the morphological result, there was a decrease in number of cells positive to PCNA and one-fold increase of CD31-positive endothelial coverage in LV-Suv39h1shRNA-transfected vessels (all p<0.05).Conclusions:1. The adenovirus vector carrying rat Suv39hl gene has been successfully constructed, which has obvious effect on Suv39h1overexpression in cultured VSMCs.2. The lentiviral vector mediating Suv39h1gene silencing has been successfully constructed, which could induce significant depletion of Suv39h1in rat VSMCs.3. Suv39h1gene silencing could attenuate VSMCs proliferation and migration induced by Ang ? or HG. Its molecular mechanism may be partly ascribed to downregulation of ID3and complement C3, leading to enhancement of cell cycle inhibitor expression, including p21and p27KIP1, and inhibition of ERK1/2phosphorylation.4. Knockdown of Suv39h1could inhibit neointimal hyperplasia after balloon injury in DM rat carotid artery. The anti-proliferative effect may be related to downregulation of multiple genes participating in biological processes, like cell proliferation, migration, differentiation and DNA replication. The decline of complement C3and ID3are also involved.5. Suv39h1knockout could promote vascular endothelialization in diabetic rats, which is possibly associated with reduction of complement C3.