Local Application Of ShRNA Targeting Pik3cb Modulates Phosphatidylinositol 3-kinase Signaling Pathway And Reduces Intimal Hyperplasia In Rat Vein Grafts

Objective: To construct rat Pik3cb(phosphatidylinositol 3-kinase, catalytic, betapolypeptide)shRNA eukaryon plasmid express vector targeting on PI3K p110βsubunit,then exam its antiproliferative and apoptotic effects of vascular smooth muscle cells(VSMCs) from rats aorta.Methods: The thoracic aorta of rat was separated carefully and cut into small tissue pieces.The explants were seeded onto culture flasks. Cell was observed through phase contrastmicroscope and immunohistochemical method. VSMCs from 5-8 generations are applied toexperiment. Six shRNA of rat Pik3cb were designed and synthesized according to thesequence of Pik3cb in the Genbank, then they were annealed to form double strands andthen cloned into pGenesil-1, named pU6-Pik3cb-shRNA-1 to pU6-Pik3cb-shRNA-6,respectively. The sequences were examined. After the initial experiment, two effectiveplasmid express vectors named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 wereselected for the following experiments. Both pU6-Pik3cb-shRNA-1 and pU6-Pik3cbshRNA-2 were transfected into rat thoracic VSMC through METAFECTENETM, and sevengroups were selected for the study: Group A: the normal cultured VSMCs; Group B:pU6-Pik3cb-shRNA-1 plasmid vector; Group C: pU6-Pik3cb-shRNA-2 plasmid vector;Group D: pU6-Pik3cb-shRNA-1and pU6-Pik3cb-shRNA-2 half for each; Group E: thenegative control scramble plasmid; Group F: the negative control empty plasmid; Group G:the positive control wortmannin. The expression of Pik3cb mRNA was detected by realtime quantitative PCR, the protein expression of phospho-Akt(Thr308), phospho-Akt(Ser473) and phospho-mTOR(Ser2448) were evaluated by Western blot, the anti-proliferativeeffects of VSMC were tested by CCK-8, cell apoptosis were obtained through flow cytometry and TUNEL, the expression of phospho-Akt(Thr308), phospho-Akt(Ser473) andphospho-mTOR(Ser2448) were studies through immunohistochemical method, and theexpressions of PCNA by immunofluorescence.Results: The passaged VSMC developed typically"peak and valley"growth pattern andexpressed smooth muscle-α-actin, the smooth muscle specific differentiation marker. RatPik3cb shRNA eukaryon express plasmid vectors were correctly constructed and thensuccessfully transfected to VSMCs. The 48 h and 72 h transfection rate were 15.7% and10.1%, respectively. The expression of Pik3cb mRNA and phospho-Akt(Thr308),phospho-Akt(Ser473) and phospho-mTOR(Ser2448) were significantly decreased in theshRNA groups (P < 0. 05) as demonstrated by real time quantitative PCR and Western bolt,respectively; the proliferation of VSMC was inhibited (P < 0. 05)as demonstrated byCCK-8 and the expression of PCNA through immunofluorescence, respectively; theapoptosis cells were increased as revealed through flow-cytometry and TUNEL in theshRNA groups than the control groups (P < 0. 05), respectively; the expression ofphospho-Akt(Thr308), phospho-Akt(Ser473) and phospho-mTOR(Ser2448) weresignificantly reduced in the shRNA groups (P < 0. 05) as demonstrated byimmunohistochemical method.Conclusion: The study presented here reports the simple and effective methods to isolateand purify smooth muscle cells from rat thoracic artery. Rat Pik3cb shRNA eukaryonexpress plasmid vectors were successfully constructed and transfected into and rat smoothmouse cells, then effectively downregulated the expression of Pik3cb mRNA and the downstream effective proteins of phospho-Akt(Thr308), phospho-Akt(Ser473) and phosphomTOR(Ser2448). As a result, effectively inhibits the proliferation and spur the apoptosis ofrat smooth mouse cells. Targeting Pik3cb to silence PI3K p110βsubunit through RNAitechnique in the treatment of restenosis is suggested by this study. Objective: To construct shRNA eukaryon plasmid expression vector targeting on ratPik3cb gene, then determine its antiproliferative effects on vascular smooth muscle cell andthe intimal hyperplasia. Construct rat jugular vein-to-artery interposition modals, observesthe origin of neointimal cells in autologous vein graft; evaluates its downregulative effectson PI3K-Akt-mTOR signal pathway and the antiproliferative effects on smooth musclecells, which as a results, reduces intimal hyperplasia.Methods: Six of the sense and antisense RNA oligonucleotide strands targeting Pik3cbmRNA were designed and synthesized individually according to the sequence of the ratPik3cb, then they were annealed to form double strands and then cloned into pGenesil-1,the sequences were examined all corrected as design. Two effective shRNA namedpU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were selected for this study. Modified ratvein-to-artery interposition modals using the autologous branch of jugular vein wereconstructed. Jugular vein grafts were treated with 25% Pluronic F-127 only (group A, n =30), plasmid encoding shRNA targeting Pik3cb p110βsubunit (pU6-Pik3cb-shRNA-1 andpU6-Pik3cb-shRNA-2, group B and C, n = 30, respectively), half pU6-Pik3cb-shRNA-1and pU6-Pik3cb-shRNA-2 (group D, n = 30), pGenesil-1 scramble shRNA (group E, n =30), or wortmannin (Group F, n = 30). Specimens were harvested at 1, 3, 7, 14 and 28 dayspost surgery to assessed jugular vein graft neointimal hyperplasia. Immunohistochemicalstaining was performed with primary antibodiesα-smooth actin, CD-34, phosphor-Akt(Thr308) , phosphor-mTOR(Ser2448) and PCNA, as well as TUNEL method to evaluatethe antiproliferative effects of shRNA. A subset (n = 18, 3 for each group) received thesame treatment as above were killed on postoperative day 3 for real time polymerase chainreaction and immunoblotting assays to evaluate expression of Pik3cb mRNA and phospho Akt (Ser308), phosphor-mTOR(Ser2448) and PCNA. Another subset (n = 9) receivedjugular vein grafts treated with a pGenesil-1 scramble shRNA were killed on postoperativedays 1, 2 and 3 (n = 3, respectively) to confirm the transfecive efficency of shRNAeukaryon plasmid expression vector.Results: Two shRNAs targeting rat Pik3cb were pricked off and synthesized according tothe sequence of Pik3cb. The transfective efficiency is about 60% for vascular smoothmuscle cell, and more than 90% for endothelial cell. Modified rat vein-to-artery interpositionmodal was successfully constructed, and the results suggesting vein graft neointimal cellshad an intimate association with endothelial cells or circulating progenitor cells by theimmunohistochemical usingα-SM-actin and CD-34.The Pik3cb mRNA and its down stream effective molecules phospho-Akt(Thr308),phospho-Akt(Ser473), phospho-mTOR(Ser2448), as well as PCNA were effectively downregulated by pU6-Pik3cb-shRNA and wortmannin, the mRNA level of transfective groupsand the positive control group were reduced 70.3%, 51.8%, 59.9% and 74.8%, respectively(P < 0.05); the phospho-Akt(Thr308) protein level of transfective groups and the positivecontrol group were reduced 88%, 80.4%, 85.6% and 90.5%, respectively (P < 0.05); thephospho-Akt(Ser473) protein level of transfective groups and the positive control werereduced 77.8%, 70.8%, 74.7% and 84.1%, respectively (P < 0.05); the phospho-mTOR(Ser2448) protein level of transfective groups and the positive control were decreased73.4%, 62%, 70.5% and 80.6%, respectively (P < 0.05); the PCNA protein level of transfectivegroups and the positive control were reduced 73.4%, 62%, 70.5% and 80.6%, respectively (P< 0.05). There has no difference between the untreated and pGenesil-1 scramble shRNAgroups (P > 0.05).The cell expression of phospho-Akt(Thr308) were significantly reduced between thetransfective groups and the positive control in all stages by means of immunohistochemical,especially at 7-14 days; the masculine cells in untreated and pGenesil-1 scramble shRNAwere significantly more than the transfective groups (P < 0.05). The percentage ofmasculine cells in transfective groups and positive control were 0.57%,0.71%, 0.63% and0.69% at 14 days, respectively (P < 0.05), the untreated and pGenesil-1 scramble shRNA were 1.72% and 1.66%, respectively; the percentage of masculine cells in transfectivegroups and positive control were 0.54%, 0.68%, 0.65% and 0.78% at 28 days, respectively(P < 0.05), the untreated and pGenesil-1 scramble shRNA were 1.52% and 1.47%respectively; it has no difference between the untreated and pGenesil-1 scramble shRNAgroups (P > 0.05).The cell expression of phospho-mTOR(Ser2448) were significantly reduced betweenthe transfective groups and the positive control in all stages by means of immunohistochemical,especially at 7-14 days; the masculine cells in untreated and pGenesil-1 scramble shRNAwere significantly more than the transfective groups (P < 0.05). The percentage ofmasculine cells in transfective groups and positive control were 0.65%,0.82%, 0.74% and0.86% at 14 days, respectively (P < 0.05), the untreated and pGenesil-1 scramble shRNAwere 1.70% and 1.67%, respectively, the percentage of masculine cells in transfectivegroups and positive control at 28 days were 0.56%, 0.72%, 0.64% and 0.78%, respectively(P < 0.05), the untreated and pGenesil-1 scramble shRNA were 1.52% and 1.48%,respectively. There has no difference between the untreated and pGenesil-1 scrambleshRNA groups (P > 0.05).The cell expression of PCNA were significantly reduced between the transfectivegroups and the positive control in all stages by means of immunohistochemical; themasculine cells in untreated and pGenesil-1 scramble shRNA were significantly more thanthe transfective groups (P < 0.05). The percentage of masculine cells in transfective groupsand positive control at 28 days were 0.64%, 0.83%, 0.72% and 1.24%, respectively, whilethe untreated and pGenesil-1 scramble shRNA were 2.31% and 2.53%, respectively; therehas no difference between the untreated and pGenesil-1 scramble shRNA groups (P >0.05).The apoptosis cells were significantly increased in the transfective groups and thepositive control by TUNEL in all stages. The percentage of masculine cells in transfectivegroups and positive control at 14 days were 3.13%, 2.56%, 2.93% and 3.64%, respectively,while, the untreated and pGenesil-1 scramble shRNA were 1.45% and 1.33%, respectively;the percentage of masculine cells in transfective groups and positive control at 28 dayswere 2.62%, 1.93%, 2.28% and 2.45%, respectively, while the untreated and pGenesil-1 scramble shRNA were 0.66% and 0.84%, respectively; the apoptosis cells has no differencebetween the untreated and pGenesil-1 scramble shRNA groups (P > 0.05).The proliferation of new vascular endothelium cells was significantly higher at 1~2weeks after operation, the intimal thickness and neointimal area of transfective groups weredecreased than the untreated and pGenesil-1 scramble shRNA (P < 0.05). The intimalthickness of the untreated and pGenesil-1 scramble shRNA at 14 days were thicken 6.8 and6.6 fold than 1 days, while the transfective groups were thicken 3.8, 4.5 and 4.1 fold merely,respectively. The neointimal area of the untreated and pGenesil-1 scramble shRNA at 14days were increased 5.2 and 5.1 fold than 1 day, while the transfective groups were onlyincreased 2.8, 4.6 and 3.1 fold, respectively. The intimal thickness and neointimal area ofpositive control were low before 14 days in positive control, but it increased gradually after14 days because of the weaken drug effect.Conclusions: The shRNA eukaryon express plasmid vectors targeting rat Pik3cb weresuccessfully constructed. Both pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 werecapable of suppressing intimal hyperplasia through down regulating the PI3K-Akt-mTORpathway by knockdown Pik3cb, then depressing smooth muscle cells proliferation andpromoting their apoptosis. Thr study presented here provides a new gene therapy strategy toprevent vein graft intimal hyperplasia after bypass grafting.