| Background and PurposeThe technique of percutaneous coronary intervention (PCI) is regarded as one of usefultreatments for coronary atherosclerosis heart disease, however, restenosis following PCI, whichoccurs in 30-40% of patients undergoing this procedure, limites its longer-term benefits.Although drug-eluting stents can reduce the incidence of RS, the existences of thrombosis in thelate threaten the life of patient. Neointimal hyperplasia is the major pathogenesis of RS, whichresults from proliferation, migration of vascular smooth muscle cells (VSMCs) and extracellularmatrix (ECM) deposition intrigued by vascular injury. In 2002, renin/prorenin receptor wascloned. Schefe et al indicated that rennin stimulation of rat H9c2 cardiomyoblasts induced anincrease of cell number and a decrease of apoptosis. Ichihara et al reported that a synthesized"handle region peptide" (HRP) could competitively bind to (P)RR as a decoy peptide and thusprevented the development of diabetic nephropathy and the progression of cardiac fibrosis inanimal models. All of these studies suggest that (P)RR plays an important role in physiologicaland pathophysiological situations, especially in cardiovascular and renal diseases.The uptakes of (pro)renin into vascular tissue are more than directly into the bloodcirculation, so the vessel may be main parts where (pro)renin converted to renin. the existence of(P)RR on vascular smooth muscle cells has been confirmed, and Mariyo SAKODA et al haveconfirmed that (pro)renin and renin can stimulate vascular smooth muscle cells proliferation by(P)RR. Therefore, we presume that the receptor-associated prorenin system (RAPS) will playimportant role in the occurrence of RS. The purposes of our study were to observe the effectof (P) PR on vascular smooth muscle cells proliferation in vitro and neointimaproliferation in arterial injury model.Reactive oxygen species (ROS) represent a class of oxygen-derived molecules that act asphysiological or pathophysiological signaling molecules in various vascular functions anddiseases. Although there are several ways to produce ROS in cells multicomponent nonphagocyticNADPH oxidase seems to be especially important and be the one takes charge of theROS generation in vascular smooth muscle cells. The catalytic moieties of NADPH oxidases arehomologs of the flavin-and NADPH-binding protein gp91phox(Nox2), termed Nox1, Nox3,Nox4, Nox5, Duox1, and Duox2. VSMCs express predominantly Nox1 and Nox4 isoforms,which are differentially distributed in the cellular compartments and direct several redoxdependentprocesses.5 It was postulated that Nox1 associates with and promotes VSMCs proliferation, whereas Nox4 is required for the maintenance of differentiated phenotype.ThereforeTherefore, this study will observe the role of NOX1-ROS in the (P)RR-mediated vascularsmooth muscle cell proliferation in vitro and the neointimal proliferation after carotidinjury.MethodsOur study was divided into the following four parts:(1) Experiment one: to identify the pair of miRNA with the best efficiency to inhibit theexpression of (P)RR, microRNA (miRNA) interference nucletides of (P)RR were chemicallysynthesized and inserted into pcDNATM6·2-GW/EmGFPmiR vector, which were confirmed bysequencing, then the recombinant miRNA vectors were transfected into A7r5 cell byLipofectamineTM 2000. The mRNA expression of (P)RR was detected by real time-PCR andwestern blot.(2) Experiment two: to identify the effects of renin and renin/prorenin receptor onproliferation of vascular smooth muscle cells(VSMCs), on expression of Cyclin D1 andproliferating cell nuclear antigen (PCNA), then the cultured VSMCs were divided into thefollowing two parts:a) Part one: to identify the effects of renin on proliferation of vascular smooth musclecells(VSMCs), on expression of Cyclin D1 and proliferating cell nuclear antigen (PCNA),the cultured VSMCs were divided into five groups:①blank group;②control group;③renin10-10 group;④renin 10-9 group;⑤renin 10-8 group;b) Part two: to further study the function of (P)RR in the VSMCs, we analyzed the effectsof RNA interference on the activities of the VSMCs. the cultured VSMCs were divided intofollowing groups:①negative group;②control group (negative +Los+ PD123319);③reningroup;④(P)RR-miRNA + renin group;⑤PDGF-BB group;⑥(P)RR-miRNA + PDGF-BBgroup;To observe the effect of renin/prorenin receptor on the proliferation of VSMCs , losartan(AT1 receptor antagonist) and PD123319 (AT2 receptor antagonist) were given before to blockAngⅡinduced by renin.The cell proliferation was determined by CCK8.The cell cycle was detected by flowcytometry. The mRNA expression of PCNA and CyclinD1 was measured by real time-PCR; Theprotein expression of PCNA and CyclinD1 were detected by Western blot. (3) Experiment three: to observe the effect of oxidative stress on proliferation of vascularsmooth muscle cells(VSMCs) induced by renin , then the cultured VSMCs were dividedinto the following two parts:a) Part one: to study the effects of DPI, the inhibitor of NADPH oxidase , on proliferationof vascular smooth muscle cells(VSMCs), on expression of Cyclin D1 and proliferating cellnuclear antigen induced by renin(PCNA), the cultured VSMCs were divided into followinggroups:①blank group;②control group;③renin 10-10 group;④renin 10-9 group;⑤renin 10-8group;⑥renin 10-9+DPI group;⑦DPI group;b) Part two: to further study the function of (P)RR in the VSMCs, we analyzed the effectsof RNA interference on the activities of the VSMCs. the cultured VSMCs were divided intofollowing groups:①negative group;②control group (negative +Los+ PD123319);③reningroup;④(P)RR-miRNA + renin group;⑤PDGF-BB group;⑥(P)RR-miRNA + PDGF-BBgroup;To observe the effect of renin/prorenin receptor on the proliferation of VSMCs , losartan(AT1 receptor antagonist) and PD123319 (AT2 receptor antagonist) were given before to blockAngⅡinduced by renin.The average flourescence value of ROS , activity of SOD and level of MDA were detectedafter experiments. The cell proliferation was determined by CCK8. The cell cycle was detectedby flow cytometry. The mRNA expression of PCNA, CyclinD1 and NOX1 was measured byreal time-PCR; The protein expression of PCNA, CyclinD1 and NOX1 were detected byWestern blot.(4) Experiment four: to observe the effects of renin and renin/prorenin receptor on theneointimal hyperplasia after acute arterial injury, the rats were divided into Sham group(PBS, n=10, intragastric administration), injury group (PBS, n=10, intragastric administration),Low HRP group(10μg/kg/d, n=10, Caudal Vein Injection), High HRP group(100μg/kg/d, n=10,Caudal Vein Injection), and losartan group (100 mg/kg/d, n=10, intragastric administration). Rattail artery blood pressure were measured daily. Injured and control arteries were harvested at 14days. The activity of SOD and level of MDA on Injured and control arteries were detected afterexperiments. The mRNA expression of PCNA, CyclinD1 and NOX1 was measured by realtime-PCR; The protein expression of PCNA, CyclinD1 and NOX1 were detected by Westernblot. ResultResults1 Experiment one: construction and identification of miRNA recombinant eukaryoticexpression vectors of (P)RR(1) Sequencing suggested that miRNA eukaryotic expression vectors targeting (P)RR possessecorrect read frame and nucleotide sequence;(2) Plasmids:transfection agent (μg:μl) 3.0:7.5 was the optimized transfected condition.(3) Real time–PCR and western blot results showed that the sequence of X2-2-3 couldeffectively knowdown the level of mRNA and protein of (P)RR.2 Experiment two: the effects of renin and renin/prorenin receptor on proliferation ofvascular smooth muscle cells(VSMCs), on expression of Cyclin D1 and proliferating cellnuclear antigen (PCNA)Part one: the effects of renin on proliferation of vascular smooth muscle cells(VSMCs), onexpression of Cyclin D1 and proliferating cell nuclear antigen (PCNA)(1) The results of CCK-8 assay showed that renin could stimulate VSMCs proliferation indose-dependant manner (P<0.05).(2) Shown by flow cytometry, constituent ratio of VSMCs S cell cycle and proliferation indexof renin group were higher obviously than those of control group (P<0.05).(3) The real time-PCR and Western blot assay showed that renin could stimulate the mRNAand protein expression of CyclinD1 and PCNA in dose-dependant manner (P<0.05).Part two: The effects of renin/prorenin receptor on proliferation of vascular smoothmuscle cells(VSMCs), on expression of Cyclin D1 and proliferating cell nuclear antigen(PCNA)(1) The results of CCK-8 assay showed that renin and PDGF-BB could stimulate VSMCsproliferation (P<0.05). However, (P)RR knockdown with micro interfering RNA (miRNA)significantly inhibited VSMCs proliferation induced by renin and PDGF-BB (P<0.05).(2) The flow cytometry assay showed that constituent ratio of VSMCs S cell cycle andproliferation index of renin and PDGF-BB group were higher obviously than those ofcontrol group (P<0.05). However, (P)RR knockdown with micro interfering RNA (miRNA)significantly inhibited the increased constituent ratio of VSMCs S cell cycle andproliferation index induced by renin and PDGF-BB (P<0.05). (3) The real time-PCR and Western blot assay showed that the mRNA and protein expressionof CyclinD1 and PCNA were elevated by renin and PDGF-BB (P<0.05), however, (P)RRknockdown with micro interfering RNA (miRNA) significantly inhibited the increasedmRNA and protein expression of CyclinD1 and PCNA induced by renin and PDGF-BB(P<0.05).3. Experiment three: the effects of oxidative stress on VSMCs proliferation induced by reninPart one :the effects of DPI, the inhibitor of NADPH oxidase , on proliferation of vascularsmooth muscle cells(VSMCs), on expression of Cyclin D1 and proliferating cell nuclearantigen induced by renin(PCNA)(1) Renin could stimulate VSMCs to produce ROS in dose-dependant manner (P<0.05).theROS level in VSMCs were significantly suppressed by addition of DPI, the inhibitor ofNADPH oxidase(P<0.05), which means NADPH oxidase is implicated and may beactivated by renin to produce O2-.(2) The MDA concentration in the renin group was significantly higher than that in the controlgroup(P<0.05), whereas, conversely, the SOD activity in the former group is considerablylower than that in the latter(P<0.05), suggesting an imbalance the oxidation and antioxidationstates in VSMCs, a hallmark of oxidative stress. Compared to the renin group,the renin + DPI + Los+PD123319 group showed a decreased MDA but increased SODactivity(P<0.05).(3) Shown by flow cytometry, constituent ratio of VSMCs S cell cycle and proliferation indexof renin group were higher obviously than those of control group (P<0.05). In comparisonwith renin group, renin + DPI + Los+PD123319 group could remarkably cut down theconstituent ratio of S cell cycle and proliferation index(P<0.05).(4) The real time-PCR and Western blot assay showed that the mRNA and protein expressionof CyclinD1 and PCNA were elevated by renin(P<0.05), however, DPI could significantlydown-regulated the increased mRNA and protein expression of CyclinD1 and PCNAinduced by renin(P<0.05).Part two: the effects of renin/prorenin receptor on oxidative stress of vascular smoothmuscle cells(VSMCs)(1) The MDA concentration in the renin and PDGF-BB group was significantly higher thanthat in the control group(P<0.05), whereas, conversely, the SOD activity in the formergroups is considerably lower than that in the latter(P<0.05), the (pro)renin receptor waspresent in VSMCs, and its knockdown with micro interfering RNA (miRNA) significantly inhibited the increased MDA concentration induced by renin and PDGF-BB(P<0.05),whereas, conversely, heightened the SOD activity inhibited by renin and PDGFBB(P<0.05).(2) The real time-PCR and Western blot assay showed that the mRNA and protein expressionof NOX1 were elevated by renin and PDGF-BB (P<0.05), however, (P)RR knockdownwith micro interfering RNA (miRNA) significantly inhibited the increased mRNA andprotein expression of NOX1 induced by renin and PDGF-BB (P<0.05).4 Experiment four: the effects of (P)RR on neointimal proliferation after carotid injury(1) Morphometric analysis of the injured artery revealed that losartan and HRP(100μg/kg/d)treatment group reduced the neointima area compared to injury group(P<0.05). Theintima/media ratio of losartan and HRP(100μg/kg/d) treatment group were also decreasedcompared to injury group(P<0.05). However, The intima/media ratio was not markedlychanged by HRP(10μg/kg/d) compared to injury group(P>0.05).(2) The real time-PCR and Western blot assay showed that the mRNA and protein expressionof CyclinD1 and PCNA were elevated in the neointimal area after carotid injury(P<0.05).The mRNA and protein expression of CyclinD1 and PCNA were significantly downregulatedby losartan treatment and HRP(100μg /kg/d) (P<0.05), but those of CyclinD1and PCNA were not markedly changed by HRP(10μg/kg/d) (P>0.05).(3) The MDA concentration in the injury group was significantly higher than that in thecontrol group(P<0.05), whereas, conversely, the SOD activity in the former groups isconsiderably lower than that in the latter(P<0.05), The MDA concentration weresignificantly down-regulated by losartan treatment and HRP(100 mg/kg/d) (P<0.05) ,however, the SOD activity significantly up-regulated by losartan treatment and HRP(100μg/kg/d) (P<0.05) .(4) The real time-PCR and Western blot assay showed that the mRNA and protein expressionof NOX1 were elevated in the neointimal area after carotid injury(P<0.05). The mRNAand protein expression of NOX1 were significantly down-regulated by losartan treatmentand HRP(100μg/kg/d) (P<0.05) , but those of NOX1 were not markedly changed byHRP(10μg/kg/d) (P>0.05).Conclusions1 The miRNA eukaryotic expression vectors targeting (P)RR were successfully constructedand the effectively interference RNA were identified, which may be used for understandingthe effect of (P)RR in the vascular smooth muscle cells .2 Renin could stimulate VSMCs proliferation and up-regulate the mRNA and protein expression of CyclinD1 and PCNA through the (P)RR-mediated activation,independently of the generation of angiotensin II or the activation of its receptor.3 Renin could activate Nox1 through (P)RR, leading to VSMCs proliferation and up-regulatethe mRNA and protein expression of CyclinD1 and PCNA.4 HRP could reduce neointima proliferation, decrease the production of MDA and incrasethe activity of SOD, and down-regulate the elevated mRNA and protein expression ofCyclinD1, PCNA and NOX1, suggesting the (P)RR plays an important role in theneointima proliferation. |
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