The Effects Of Simvastatin On Inhibiting Neointimal Hyperproliferation And Promoting Reendothialization After Vascular Injury

BACKGROUNDThe injury and dysfunction of endothelial cells play a key role in the starting events of atherosclerasis and harmful renovation and induce vascular injuries, proliferation and migration of smooth muscle cells(SMCs). To inhibit smooth SMCs proliferation and accelerate reendothelialization is the key for the repairment of injured vessel. In the past decades, intense efforts has been applied to discern the mechanisms that regulate SMCs proliferation after angioplasty and to develop therapies to inhibit SMCs overgrowth. Drug-eluting stents that employing cytostatic/immunomodulatory compound rapamycin and the chemotherapeutic paclitaxcel have been show to induce neointimal lesion formation. These therapies can also perturb endothelial recovery, and lead to incomplete, delayed reendothelialization or late stent thrommosis. Accordingly, identification of compounds that select to inhibit neointimal formation, as well as do not adversely affect endothelial regrowth could be of substantial clinical usefulness and is regarded as the main target for evolutionary concepts toward the development of next generation drug-eluting stents.In traditional opinions, it is thought that vascular SMCs and ECs were the predominant cells which take part in repairment of injured vessel. Recent studies found that vascular function not only depends on cells within the vessels, but also significantly modulated by circulating smooth muscle progenitor cells(SPCs) and endothelial progenitor cells(EPCs) derived from the bone marrow. Following vascular injuries, vascular progenitor cells can be mobilized into the peripheral blood flow, home to the site of vascular injury, and SPCs can differentiate into smooth muscle cells and accelerate neointima formation and lead to luminal stenosis; and EPCs differentiate into endothelial cells and rebuild luminal endothelial cells layer and inhibit bad vascular remodeling. Recent animal and clinic studies both showed that the most of neointima SMCs were derived from bone marrow and about 25% ECs at the vascular injury site came from the differentiated EPCs. These data indicate that SPCs and EPCs derived from bone marrow are important sources of repairing cells after vascular injury. Therefore, inhibition of SPCs proliferation can induce neointima formation, and improvement of EPCs proliferation can accelerate reendothelialization of injured vessel and inhibit restenosis.Statins can effectively modulate the lipid profile of an individual patient. Moreover, there is compelling evidence that statins may also exhibit non cholesterol dependent pleiotropic effects, such as inhibiting SMCs poliferation, promoting EPCs proliferation, improving endothelial function, inhibiting platelet function, anti-inflammation and stabilizing atherosclerotic plaques. These biological properties of statins suggest that it can be a perfect candidate for coat compound of next generation drug-eluting stents. However, systemic statin therapy is not effective for the limitation of human coronary artery neointima formation because bioactive concentrations were neither achieved nor maintained at the site of the potential lesion. Local delivery of vasculature facilitates the achievement of high regional drug concentration, with prolonged retention at therapeutic doses less likely to product systemic toxicity. Nevertheless, it remains unknown whether local statin therapy can effectively inhibit restenosis and accelerate endothelial regrowth.OBJECTIVES1. To investigate effects of sirolimus on the differentiation, proliferation, adhension, and migration of EPCs and SPCs.2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury.METHODS1. Effects of sirolimus on EPCs and SPCs differentiation, proliferation, adhension, and migration: (1) The bone marrow mononuclear cells(MNCs) were isolated from the bone marrow of rats by density gradient centrifugation with Ficoll. MNCs were cultured in fibronectin-coated dishes in endothelial progenitor cells growth medium or smooth muscle progenitor cells growth supplements with or without sirolimus(final concentrations: 0.01, 0.1, 1, 10, 100 ng/ml) for 12 days. (2) After 8 days primarily cultured, attached cells were treated with sirolimus(final concentrations: 0.1, 1, 10, 100, 200 ng/ml) or vehicle for various time points(0h, 12h, 24h, 48h, 96h). SPCs were identified as adherent cells positive forα-SMA by indirect immunofluorescent staining. And EPCs were characterized as adherent cells double positive stained for DiI-acLDL-uptake and FITC-UEA-Ⅰ(lectin) binding under immunofluence microscope by direct fluorescent staining. EPCs and SPCs proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively. Adhension assay of EPCs and SPCs were performed by replating them on fibronectin-coated dishes, and the adhension cells were then courted.2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs: Rat aorta SMCs were cultured in high-glucose DMEM medium and Rat aorta ECs were cultured in M199 medium. EPCs and SPCs were cultured based on above meathod. Treatment with simvastatin was performed in fully supplemented media for the indicated time(0h, 6h, 12h, 24h, 48 h) and concentration ranges(0,0. 01,0.1,1,10μmol/L). The proliferation of SMCs, ECs, EPCs and SPCs were assayed with 3H-TdR incorporation assay. Migration and adhension assay of SMCs, ECs, EPCs and SPCs were performed as same as above procedure. In addition, western blot was performed for the assessment of effects of a series concentration of simvastatin(0,0. 01,0.1,1,10μmol/L) on cyclin-dependent kinase inhibitor p27 protein expression of ECs, SMCs, EPCs and SPCs for 24 hours.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression: SMCs were treated with a range concentration of simvastatin(0,0. 01,0.1,1,10μmol/L) for 24h or with 1μmol/L simvastatin for 0h, 6h, 12h, 24h, 48 h. SDF-1αmRNA expression was detected by RT-PCR.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury: the model of vascular restenosis established by balloon injury of rat carotid arteries was used. 400μg of simvastatin suspended in 100μL of Pluronic F-127 gel was administrated to the perivascular area of injured vessel, and the same volume of Pluronic gel without simvastatin was used in control group. Two weeks after surgery, rats were sacrificed. The common carotid arteries were embedded in paraffin, and cross sections were made. Sections were stained with hematoxylin-eosin for assessement of Neointima thickness and with vWF antibody for observation of endothelial coverage.RESULTS1. Effects of sirolimus on EPCs and SPCs differentiation, proliferation, adhension, and migration: EPCs and SPCs were successfully cultured in vitro. EPCs were doubly stained by DiI-acLDL and FITC-UEA-Ⅰwith"line"structure. SPCs were positive forα-SMA and CD34 and mRNA forα-SMA was detected in adherent SPCs. The number ofα-SMA-positive SPCs and DiI-acLDL and FITC-UEA-Ⅰ-double-positve EPCs differentiated from MNCs at 12 days was significantly lower in sirolimus treated cells than that of vehicle-treated cells in dose-depended manner. At a concentration as low as 0.1 ng/mL, sirolimus dramatically reduced the number of SPCs to 59.3±5.8% of control(0.1 ng/mL sirolimus versus control: 23±3 versus 78±5, n=5,P<0.01) and reduced the number of EPCs to 70.5±34.5%(0.1 ng/mL sirolimus versus control: 37±5 versus 90±7, n=5,P<0.01). Sirolimus significantly inhibited the proliferative capacity of EPCs and SPCs in a time and dose dependent manner. There was no significant effects on EPCs and SPCs proliferation by sirolimus at 0.1 ng,mL, whereas at higher concentration(≥1 ng/mL), sirolimus inhibited the proliferation of EPCs(1 ng/mL sirolimus versus control: 0.414±0.019 versus 0.580±0.034, n=5,P<0.01) and SPCs(1 ng/mL sirolimus versus control: 0.476±0.016 versus 0.687±0.043, n=5,P<0.01). Moreover, sirolimus also significantly dose-dependently inhibited the migratory and adhensive capacity of EPCs and SPCs.1ng/mL sirolimus significantly inhibited EPCs adhension(1 ng/mL sirolimus versus control: 34±5 versus 52±6, n=5,P<0.01) and SPCs adhension(1 ng/mL sirolimus versus control: 27±2 versus 51±3, n=5,P<0.01).2. To study effects of simvastin on EPCs and SPCs differentiation, in addition, on proliferation, adhension, and migration of ECs, SMCs, EPCs and SPCs: Rat SMCs proliferation was inhibited by simvastatin at concentration as low 0.01μmol/L(0.01μmol/L simvastatin versus control: 5647±268 versus 6038±218 , n=5,P<0.05), the same concentration had no significant effect on rat ECs proliferation. Treatment with simvastatin at dosages of 1μmol/L or higher led to very prominent growth arrest in SMCs but not ECs. Remarkably, 3H-TdR incorporation was constantly maintained at significantly higher levels in ECs with increasing relative differences at higher simvastatin concentrations. (10μmol/L simvastatin versus control: 4310±132 versus 4321±133, n=5,P>0.05)In addition, simvastatin led to dose dependent inhibition of SMCs migration(0.01μmol/L simvastatin versus control: 41±3 vs 45±3, n=5,P<0.05). In contrast to SMCs, simvastatin had no detectable effect on migration of ECs at concentrations of 0.01 to 10μmol/L in vitro(10μmol/L simvastatin versus control: 35±5 versus 37±5, n=5,P>0.05). EPCs and SPCs are also important tools for assessment of stent-coated compounds. Incubation of isolated rat MNCs with simvastatin dose-dependently increased the number of EPCs differentiated from MNCs and decreased the number of SPCs. The number of differentiating EPCs significantly increased 1.2±0.1 fold(1μmol/L simvastatin versus control: 87±5 versus 39±4, n=5,P<0.01) at concentration of 1μmol/L simvastatin and the number of differentiating SPCs dramatically reduced 62.1±3.5% of control(1μmol/L simvastatin versus control: 32±5 versus 85±4, n=5,P<0.01) at the same concentration. In addition, simvastatin time- and dose-dependently promoted EPCs proliferation, while reached the maximum 24 hours after the simulation at 1μmol/L. In contrast with EPCs, SPCs proliferation was significantly inhibited by simvastatin in a time and dose dependent manner. The number of EPCs increased 2.2±0.1 fold at concentration of 1μmol/L simvastatin for 24 hours(1μmol/L simvastatin versus control: 3762±138 versus 1249±146, n=5,P<0.01), and that of SPCs decreased 62.1±5.6% of control at the same concentration for the same time(1μmol/L simvastatin versus control: 1962±145 versus 4070±184, n=5,P<0.01). Moreover, simvastatin also dose-dependently promoted EPCs adhension and migration, while dose-dependently inhibited SPCs adhension and migration. Consistent with above results, simvastatin dose-dependently promoted the p27 expression of SMCs and SPCs, and inhibited the p27 expression of EPCs, and had no effect on the p27 expression of ECs.3. To explore effects of simvasation on SMCs SDF-1αm RNA expression: simvastatin significantly reduced the SDF-1αm RNA expression of SMCs at a time and dose dependent manner.4. To study effects of locally delivered simvastatin on neointima formation and reendothelialization after vascular injury: local delivery of simvastatin significantly reduced neointimal formation(neointimal area 0.58±0.13 mm~2 versus 0.27±0.12 mm~2; intima/media ratio 1.95±0.27 versus 0.97±0.24,n=6,P<0.01) . Immunohistochemical assessment of injured segments with an endothelial cell marker(von Willebrand factor) revealed no appreciable difference between animals receiving simvastatin administration and control.CONCLUSIONS1. Inhibition of bone marrow-derived endothelial progenitor cells maybe reslt in delayed reendothelialization, which may lead to fatal late thrombosis. The clinical efficacy of sirolimus-eluting stent against restenosis might be achieved, at least in part, through its inhibitory effect on smooth muscle progenitor cells derived from bone marrow. It is necessary that think about the effects of new generation drug-eluting stent-coated compounds on the bone marrow-derived EPCs and SPCs.2. Simvastatin of equal dosage displayed a differential effect on SMCs and SPCs as compared to ECs and EPCs with regard to differentiation, proliferation, migration and adhension, inhibiting SPCs differentiation from the bone marrow MNCs, promoting EPCs differentiation from the bone marrow MNCs, limiting proliferation, migration and adhension of SMCs and SPCs, promoting proliferation, migration and adhension of EPCs, but having no effect on proliferation and migration of ECs.3. Simvastatin significantly reduced the SDF-1αm RNA expression of SMCs at a time and dose dependent manner, which might indirectly inhibiting neointimal hyperplasia by reducing mobilization and recruitment of the bone marrow smooth muscle progenitor cells.4. Local delivery of simvastatin can significantly reduce neointima hyperproliferation in injured artery, together without adverse interfere of reendothelialization.