Study Of Drug-modified Endothelial Progenitor Cells On Reendothelialization And Related Mechanisms

Objectives Nowadays percutaneous coronary intervention (PCI) is claimed to be most effective treatment for coronary heart disease which threatens human lives. However, the contribution of PCI has been limited by restenosis. Deendothelialization has been considered as a trigger point for the formation of restenosis, and reendothelialization is presumed to be the next target for restenosis prevention. Yet the reparative ability of mature endothelial cells is quite limited. Recently, endothelial progenitor cells(EPCs) have been found to be the promising seed cells of repairing endothelium, which have ability of proliferation, adherence, secretion and to differentiate towards endothelial cells. The number of EPCs in the peripheral circulation of healthy adults are scanty. Especially under the pathologic state (like coronary heart disease and hypercholesteremia, etc.), the number and function of EPCs will be further impaired. Therefore, it is really need to explore the effective methods to enhance the number and function of EPCs. PPAR-γactivators and statins are widely used in diabetic patients with hypercholesteremia. We suppose whether combination of the two drugs is more effective in increasing the number and function of EPCs. Whether drug-modified EPCs contribute to the accelerating reendothelialization and preventing restenosis is not clear. In addition, the precise mechanisms of risk factor negatively regulating the EPCs characteristics are poorly understood. Oxidative stress plays an important role in restenosis and atherosclearosis. We suppose that oxidative stress may take part in downregulating the activity of EPCs. But this is rarely reported.Part One Study of hypercholesteremia on characteristics of EPCs in rabbits and related mechanismsMethods 12 New Zealand White rabbits were randomly divided into normal group (n=6) and control group (n=6). Rabbit peripheral blood samples were obtained from a central ear artery. The mononuclear cells were isolated by density-gradient centrifugation and cultured for 3 to 4 weeks in EBM-2 medium with supplements of 20% FCS and EGM-2MV Single Quots. The EPCs were characterized as adherent cells by immunofluorescence, immunocytochemistry and flow cytometry. EPCs proliferation was measured by MTT assay. EPCs adhesion assay was performed by counting the adherent cells number when EPCs were replated on fibronectin-coated dishes. Nitric oxide (NO) level were evaluated by Griess reaction. Superoxide anion (O2.-), the activity of Endothelial cell NO synthase (eNOS) and NADPH oxidase were measured by chromatography. eNOS and NADPH oxidase subunit p22phox(NADPH p22phox) mRNA were determined by RT-PCR.Results The EPCs were characterized as cells by high expression of CD34 and the specific endothelial mark, vWF, and double positive for Dil-acLDL-uptaking and lectin-binding. Compared with control group, the number(30.43±1.76 vs 50.18±6.83/fields×200, P<0.05) and function(proliferation, 0.43±0.05 vs 0.65±0.01; adhesion, 22.33±3.51 vs 45.33±2.52/fields×200; NO, 9.89±1.51 vs 33.37±6.99μmol/L, P<0.05) of EPCs were impared in hypercholesteremia group significantly. The concentration of O2.- (108.68±15.07 vs 61.74±6.23 U/L)was lower than that in control group. eNOS mRNA expression (0.563±0.091 vs 0.998±0.267,P<0.05)was downregulated in hypercholesteremia group, while NADPH p22phox mRNA expression(1.730±0.071 vs1.016±0.070,P<0.05) was upregulated in hypercholesteremia group. The activity of eNOS and NADPH oxidase was the same way as their mRNA expression.Conclusions Hypercholesteremia contributes to the reduction of EPCs with impaired functional activity. Oxidative stress might negatively influence the characteristics of EPCs.Part Two Effects of Rosiglitazone and Atorvastatin on the Number and Function of EPCsMethods 12 rabbits were randomly divided into control group and hypercholesteremia group. Each rabbit was drawn 30 ml blood, from which mononuclear cells were isolated and cultured in EBM-2 medium with supplements of 20% FCS and EGM-2MV Single Quots. Cells were identified by immunofluorescence, immunohistochemistry and flow cytometry. Cells were enrolled in the following groups: control group, hypercholesteremia group, rosiglitazone group(in a series of final concentrations of 0.01μM,0.1μM , 1μM and 10μM), atorvastatin group(in a series of final concentrations of 0.01μM,0.1μM,1μM and 10μM) and the combination treatment of 1μM rosiglitazone and 1μM atorvastatin group. The number and function of EPCs were measured by the same methods as that used in the Part One experiment. eNOS, NADPH p22phox and PPARγmRNA were determined by RT-PCR. Results 1. 0.01μM rosiglitazone showed no improvement on the number and function of EPCs of rabbit with hypercholesteremia. Compared with 0.01μM rosiglitazone, 0.1μM rosiglitazone made no significant difference of EPC number, VEGF and NO levels. But 0.1μM rosiglitazone increased the proliferating(0.50±0.04 vs0.42±0.04, P<0.05 ) and adhering(28.33±1.58 vs 22.33±2.52, P<0.05) activities significantly. 1μM rosiglitazone futher improved the EPCs'number and function. However, 10μM rosiglitazone did not show superiority to 1μM rosiglitazone on EPCs'number and function.2. Atorvastatin showed improvement on EPCs'number, proliferating, adhering and secreting functions in a concentration manner from 0.01μM to 1μM. There was no statistical difference between the effects of 1μM and 10μM atorvastatin on the number and function of EPCs.3. Except NO levels, the combination of 1μM rosiglitazone and 1μM atorvastatin increased the number and function of EPCs statistically, compared with single drug treatment at the same concentration.4. 1μM rosiglitazone and 1μM atorvastatin increased eNOS mRNA expression 23% and 60%, respectively. The combination treatment raised nearly 94% of level and showed no difference with normal group. In contrast, NADPH p22phox mRNA expression were decreased by the treatment of 1μM rosiglitazone and 1μM atorvastatin. The level was further downregulated by combination treatment, but was still higher than that in normal group. There was on difference of PPARγmRNA expression between hypercholestermia and normal group. Treatment of 1μM atorvastatin did not influence its expression. 1μM rosiglitazone and combination treatment equally increased the PPARγmRNA expression.Conclusions Rosiglitazone and atorvastatin could synergistically increase the number and functional activity of EPCs.Part Three Effects of Rosiglitazone and Atorvastatin- pretreated EPCs Transplantation on RendothelializationMethods 25 New Zealand White rabbits were randomly divided into 4 groups: normal group(N, n=10), injured group (n=8), EPCs transplantation group(E, n=8) and 1μM rosiglitazone plus 1μM atorvastatin pretreated-EPCs transplantation group (ME, n=8). Except normal group, each rabbit were fed with high cholesterol diet and underwent carotid injury. Immediately after balloon denuation, Dil-acLDL-labeled EPCs were delivered by 30 minutes of local dwelling. Animals of the injured group were given saline as control. 4 weeks after operation, the ratio of reendothelialization , the intima/media andα-SMA expression of the vessel wall were measured. Results 1. Labeled EPCs could be identified within the neointima, media, and adventitia of injured segments. No labeled cells were identified in uninjured control arteries.2.Compared with injured group, EPCs transplantation increased the reendothelialization(75.57±4.03% vs 51.66±1.69%, P<0.05). Drug-pretreated EPCs transplantation was more effective in accelerating reendothelialization(93.38±2.41% vs 75.57±4.03%, P<0.05).3. The Intima to media ratio (I/M)was significantly increased in injured group than that in normal group. It demonstrated that neointimal hyperplasia formed. EPCs transplantation alleviated I/M obviously(1.31±0.22 vs 1.80±0.16,P<0.05). Drug-pretreated EPCs transplantation was more effective in preventing restenosis(0.51±0.08 vs 1.31±0.22,P<0.05).4. Compared with normal group,α-SMA expression increased significantly in injured group. EPCs transplantation reduced theα-SMA expression and drug-pretreated EPCs transplantation brought the reduction further.Conclusions Transplantation of EPCs to balloon–injured arteries is associated with accelerated reendothelialization, reduced neointimal hyperplasia. Drug-modified EPCs can more effectively accelerate reendothelialization. This delivery represents a novel approach to prevent restenosis.Part Four Study of Rosiglitazone and Atorvastatin on Reendothelialization by Mobilization of EPCs and Related MechanismsMethods 40 male New Zealand white rabbits were randomly divided into 5 groups: normal group(N, n=8), injured group(C, n=8), rosiglitazone group(R, n=8, 1mg/kg/d), atorvastatin group(A, n=8, 1mg/kg/d) and rosiglitazone (1mg/kg/d) combined with atorvastatin (1mg/kg/d) group(R+A, n=8). After 2 weeks of high cholesterol diet, except normal group, all animals underwent carotid balloon denudation injuries. 4 weeks of operation, CD34+VEGFR-2+ cell number characterized as EPCs was measured by flow cytometry. NO level and O2.- were evaluated by Greiss reaction and chromatography. The ratio of reendothelialization , the intima to media andα-SMA expression of the vessel wall were detected. Endothelial recovery was observed under the scanning electron microscope. eNOS and NADPH p22phox mRNA of injured vessel wall were determined by RT-PCR.Results 1. The level of blood lipid and glucose showed no difference between rosiglitazone and injured group. The level of TC and LDL decreased in atorvastatin group, but TG, HDL and Glu did not change. The combination treatment only reduced the level of TC and LDL, but did not influence the level of TG, HDL and Glu.2. The number of EPCs was significantly reduced 40% of injured group. Rosiglitazone and atorvastatin treatment respectively increased the counts of EPCs 57% and 65% of control. The combination treatment further raised one fold.3. The endothelial cells were largely denudated in injured group. There remained small patch of endothelial denudation in rosiglitazone and atorvastatin treatment group, respectively. The combination treatment made the endothelium intact, but endothelial cells aligned in a little disorder manner.4.Rosiglitazone treatment and atorvastatin treatment all decreased I/M, respectively. Combination treatment brought the ratio to normal level.5.Rosiglitazone treatment and atorvastatin treatment reduced theα-SMA expression, respectively. Combination treatment decreased the expression more.6. NO in rosiglitazone treatment and atorvastatin treatment groups was increased about 30% and 38% of injured group. Combination treatment raised NO nearly 62%, but NO was still lower than normal group. In the same way, rosiglitazone treatment and atorvastatin treatment enhaunced clearance of superoxide 1 fold and 87% of injured group. Combination treatment brought the level to normal level.7.Rosiglitazone treatment and atorvastatin treatment upregulated eNOS mRNA expression, respectively. Combination treatment further increased the eNOS expression, but did not restore to normal level. In contrast, rosiglitazone treatment and atorvastatin treatment caused a downregulation of NADPH oxidase subunit p22phox mRNA expression. Combination treatment brought it to normal level.Conclusions Rosiglitazone could mobilize EPCs in vivo independently of its metabolic effects. Atorvastatin could mobilize EPCs on reduction of lipid level. Combination treatment can enhance mobilization of EPCs. The underlying mechanism may in part in a reduction of NADPH oxidase and an increase in eNOS mRNA expression. It may contribute to reduced production of ROS and increase in availability of NO. The combination treatment can accelerate the reendothelialization and prevent restenosis.