| Background and objective:Vascular endothelial injury is the main pathophysiological basis of atherosclerotic diseases and in stent restenosis.In recent years,the role of endothelial progenitor cell(EPC)in vessel recovery,angiogenesis,and maintenance of normal endothelial function has attracted widespread attention.But EPC number decreased in patients with cardiovascular diseases and related risk factors,as well as their impaired function.Therefore,it is a useful therapeutic strategy to improve EPC biological function and increase EPC number.Statins has multiple cardiovascular protective effects,independent of their capability of lowering cholesterol,including the improvement of biological function of EPC.However,in order to achieve these benefits,a long time and intensive statin therapy are necessary.Pitavastatinhas strong therapeutic effects in regulation of cholesterol,improvement of endothelial functions,and reverse of plaque.However,higher local concentration of pitavastatin and longer time treatment may induce a series of systemic adverse reactions.The emergence of nanotechnology provides a new method for transportation of drugs in vivo.Biodegradable molecular materials serving as carrier can improve therapeutic effect and reduce adverse reactions via altering pharmacokinetic characteristics.In recent years,pitavastatin nanoparticles have presented a huge advantage in promoting angiogenesis in ischemic tissues,reversing/stabilizing atherosclerotic plaque and treating pulmonary arterial hypertension.Our aims are as follows:1.To obtain pitavastatin nanoparticles.2.To observe the influences and mechanism of pitavastatin nanoparticles on EPC function.3.To observe the effect of pitavastatin nanoparticles treated-EPC on repair of injured vessels.Methods:1.Preparation and evaluation of pitavastatin nanoparticlesPitavastatin nanoparticles were obtained by ultrasonic emulsion solvent evaporation method with poly lactic-co-glycolic acid(PLGA)as a carrier which encapsulated pitavastatin.Morphology of the nanoparticles was observed by electron microscope,and particle size was detected by laser nanometer analyzer.High performance liquid chromatography(HPLC)was used to evaluate drug loading capacity and encapsulation efficiency.Also,the drug release experiment was performed to observe drug release characteristics of nanoparticles.2.Culture and characterization of spleen-derived EPC from ratsMononuclear cells were isolated from rat spleen and cultured in low sugar DMEM containing fetal bovine serum and vascular endothelial growth factor(VEGF).DiI-ac LDL and FITC-UEA-I double staining was used to identify their ECs function.Meanwhile,flow cytometry was used to detect specific antigens indicating endothelial cells and stem cells,respectively.3.Intake of nanoparticles by EPCEPC was incubated with FITC-PLGA nanoparticles which were prepared via loading FITC fluorescence probe,and the intake of nanoparticles was observed under laser confocal microscope.4.Influences of carrier material(PLGA)on viability of EPCEPC cultured for 7 days was incubated with various concentration of blank PLGA nanoparticles(5?25?50?100?200?g/mL).And Impact of blank nanoparticles on the viability of endothelial progenitor cells was tested by CCK8.5.Effects of pitavastatin nanoparticles on of EPC functionEPC cultured for 7 days was treated with various concentration of pitavastatin nanoparticles containing pitavastatin(0.001?M,0.01?M and 0.1?M).Medium was exchanged after 24 h.Meanwhile,blank control,blank nanoparticles and pitavastatin group(0.001?M,0.01?M and 0.1?M)were evaluated as well.After 3 days,EPC proliferation,migration,adhesion and apoptosis were evaluated by CCK8 analysis,transwell,cell counting and flow cytometry.6.The role of PI3K/Akt signaling pathway in pitavastatin nanoparticles-mediated EPC proliferationEPC was pretreated with an inhibitor of PI3 K signaling pathway(LY294002)and then with pitavastatin nanoparticles.Western blot was used to detect influences of pitavastatin nanoparticles on Akt phosphorylation.7.Studies of pitavastatin nanoparticles-treated EPC on injured vesselsCarotid artery injury models in rats were established.DiI labeled EPC were injected via tail vein.3 days after surgery,homing of EPC was evaluated.Furthermore,endothelial cell phenotype of transplanted EPC was detected under fluorescence microscope.7 days after surgery,reendothelialization was evaluated by Evans blue staining.14 days after surgery,intimal hyperplasia was evaluated by HE staining.Results:1.Characterization of pitavastatin nanoparticlesPitavastatin-loaded PLGA nanoparticles exhibited spherical shape structure with smooth surface and average diameter of(230.1±45)nm.The drug loading capacity and encapsulation efficiency were(10.00±1.83)% and(35.54±5.40)% respectively.These nanoparticles prepared in vitro displayed sustained release,acumulative release of 2 weeks was(83.20 + 5.63)%(n=3).2.Characterization of cultured EPCAfter 7 days culture in endothelial medium,most adherent cells showed double staining positive(yellow,87.25±2.03%)of DiI-LDL-uptaking(red)and FITC-UEA-I binding(green),(n=3).The expression of VEGFR-2(79.00±5.21%)and CD34 were(80.07±4.70%)were detected by flow cytometry(n=3).3.Intracellular uptake of nanoparticles by EPCLaser scanning confocal microscopy showed FITC-nanoparticles were mostly distributed in the cytoplasm,showing granular green fluorescence.4.Influences of carrier material(PLGA)on viability of EPCWith the increase of blank PLGA nanoparticles concentration,there is on difference of optic density(P = 0.495).5.Effects of pitavastatin nanoparticles on EPC biological functions5.1 Effects of pitavastatin nanoparticles on EPC proliferation: CCK8 analysis showed that the proliferation of EPC could be promoted by the pitavastatin nanoparticles(0.001?M pitavastatin nanoparticles vs control,1.52±0.04 vs 1.41±0.02,P(27)0.05;0.01?M pitavastatin nanoparticles vs control,1.64±0.06 vs 1.41±0.02,P(27)0.01;0.1?M pitavastatin nanoparticles vs control,1.86±0.05 vs 1.41±0.02,P(27)0.01).Compared with pitavastatin group,pitavastatin nanoparticles(0.01?M,0.l ?M)had better effect on the proliferation of EPC(0.01?M pitavastatin nanoparticles vs 0.01?M pitavastatin,1.64±0.06 vs 1.53±0.06,P(27)0.05;0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin,1.86±0.05 vs 1.73±0.06,P(27)0.01)(n=9).5.2 Effects of pitavastatin nanoparticles on EPC migration: the number of migrated cells increased after pitavastatin nanoparticles treatment(0.01?M pitavastatin nanoparticles vs control,89.28±4.25 vs 77.44±7.42,P(27)0.05;0.1?M pitavastatin nanoparticles vs control,108.22±5.85 vs 77.44±7.42,P(27)0.01).The number of migrated EPC in pitavastatin nanoparticles were higher than that of the same concentration of pitavastatin(0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin,108.22±5.85 vs 94±5.86,P(27)0.01)(n=18).5.3 Effects of pitavastatin nanoparticles on EPC adhesion: the number of adherent cells increased after pitavastatin nanoparticles treatment(0.001?M pitavastatin nanoparticles vs control,37.00±3.06 vs 30.72±4.17,P(27)0.05;0.01?M pitavastatin nanoparticles vs control,43.06±2.01 vs 30.72±4.17,P(27)0.01;0.1?M pitavastatin nanoparticles vs control,50.28±3.08 vs 30.72±4.17,P(27)0.01).Compared with pitavastatin group,pitavastatin nanoparticles increase adhesion EPC(0.01?M pitavastatin nanoparticles vs 0.01?M pitavastatin,43.06±2.01 vs 38.28±4.03,P(27)0.05;0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin,50.28±3.08 vs 44.33±0.93,P(27)0.05)(n=18).5.4 Effects of pitavastatin nanoparticles on EPC apoptosis:Flow cytometry analysis indicated that the apoptosis EPC decreased after pitavastatin nanoparticles treatment(0.001?M pitavastatin nanoparticles vs control,14.07±2.69 %vs 20.31±2.09%,P(27)0.01;0.01?M pitavastatin nanoparticles vs control,10.37±2.04% vs 20.31±2.09%,P(27)0.01;0.1?M pitavastatin nanoparticles vs control,3.28±1.34% vs 20.31±2.09%,P(27)0.01).Effects of pitavastatin nanoparticles on EPC apoptosis inhibition is better than that of pitavastatin group(0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin,3.28±1.34% vs 8.54±2.64%,P(27)0.01)(n=3).6.The role of PI3K/Akt signaling pathway in pitavastatin nanoparticles meidated EPC proliferationpitavastatin nanoparticles could promote the proliferation of EPC(0.1?M pitavastatin nanoparticles vs control nanoparticles,1.99±0.10 vs 1.43±0.06,P(27)0.01)(n=9)and Akt(Ser473)protein phosphorylation(0.1?M pitavastatin nanoparticles vs control,0.85±0.13 vs 0.55±0.14,P(27)0.01;0.1?M pitavastatin nanoparticles vs 0.1 pitavastatin,0.85±0.13 vs 0.70±0.17,P(27)0.05)(n=4).When LY294002 treatment inhibited the effect of pitavastatin nanoparticles-induced EPC proliferation(0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin nanoparticles +LY294002,1.99±0.10 vs 1.57±0.09,P(27)0.01)(n=9),and phosphorylation of Akt(Ser473)(0.1?M pitavastatin nanoparticles vs 0.1?M pitavastatin nanoparticles +LY294002,0.85±0.13 vs 0.66±0.15,P(27)0.01)(n=4).7.Effects of EPC intaking Pitavastatin nanoparticles on injured vessels7.1 EPC homing: 3 days after EPC transplantation,red fluorescence cells were found at sites of injured sites,also these cells were UEA-I staining positive.Compared with pitavastatin group,the number of homed EPC in pitavastatin nanoparticles was higher(pitavastatin nanoparticles vs pitavastatin,16.67±4.47 vs 12.33±1.53,p<0.05)(n=3).7.2 Effect of Pitavastatin nanoparticles treated-EPC on re-endothelialization: Re-endothelialization was evaluated by Evans blue staining.The re-endothelialized area in the pitavastatin nanoparticles group increased compared to pitavastatin group(pitavastatin nanoparticles vs pitavastatin,0.48±0.05 vs 0.41±0.02,p<0.05)(n=3).7.3 Effect of Pitavastatin nanoparticles treated-EPC on intimal hyperplasia: HE staining showed Pitavastatin nanoparticles treated-EPC significantly inhibited the ratio of intima/Medial(pitavastatin nanoparticles vs pitavastatin,0.79±0.23 vs 0.98±0.16,p<0.05)(n=6).Conclusions:1.Emulsion solvent diffusion method is an ideal method to obtain pitavastatin-loaded PLGA nanoparticles.2.The nanoparticles can be intaked by EPC.The carrier materials have good biocompatibility with cells.3.Pitavastatin nanoparticles can significantly improve EPC proliferation,adhesion,migration function and inhibit apoptosis.4.Pitavastatin nanoparticles promote EPC proliferation through PI3K/Akt signaling pathway.5.Pitavastatin nanoparticles show a significant enhancement in the therapeutic effects on re-endothelialization and inhibition of intimal hyperplasia. |
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