Combined Use of Transmyocardial Laser Revascularization and Endothelial Progenitor Cells Enhances Neovascularization and Regional Contractility in a Canine Model of Ischemic Hearts

The purpose of this study was to determine the combined effect of transmyocardial laser re- vascularization （TMLR） and the implantation of endothelial progenitor cells （EPCs） on cardiac function of ischemic hearts in canines. The left anterior descending artery （LAD） was occluded to establish the canine model of acute myocardial infarct （AMI）. Four weeks later, the animals were randomly divided into four groups： TMLR group, in which transmyocardial laser-induced channels were established at the ischemic region; EPCs＋TMLR group, in which EPCs were locally transplanted into laser-induced chan- nels at the ischemic region; EPCs group, in which the EPCs were injected into the ischemic region; con- trol group, in which the AMI animals received neither TMLR nor EPCs. The peripheral blood （50 mL） was sampled in all groups. Mononuclear cells from the peripheral blood were separated and cultured to obtain spindle-shaped attaching （AT） cells in vitro. AT cells were labeled with 1, 1 ＇-dioctadecyl-1 to 3,3, 3＇,3＇-tetramethyl-indocarbocyanine perchlorate （DiI） before injecting into the laser-induced channels or ischemic region. Four weeks after the first operation, TMLR was performed in the TMLR group and EPCs＋TMLR group, and at the same time, the EPCs originating from the AT cells were mixed with cal- cium alginate （CA）. Then the EPCs-CA composites were implanted into myocardial channels induced by laser in the EPCs＋TMLR group, and into the myocardial infarct area in the EPCs group. All dogs underwent echocardiography at second month after LAD occlusion. Finally the samples of myocardium around the LAD were subjected to histochemical and immunohistologic examinations. The results showed there was no significant difference in the diameter of left atrium and ventricle before treatment among all groups （P〉0.05）. Eight weeks after modeling, the regional contractility in the LAD territory in the EPCs＋TMLR group was increased as compared with control group and TMLR group, but there was no significant difference between control group and TMLR group. Neoangiogenesis was observed in the EPCs＋TMLR group, and the fibrosis was seen in the TMLR group. There was no significant dif- ference in neoangiogenesis around the channels induced by laser among EPCs＋TMLR, EPCs and TMLR groups. It was concluded that TMLR combined with EPCs could improve the regional and global cardiac function in AMI, and augment neovascularizaiton in channels of ischemic myocardium induced by laser.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Rapamycin and 3-methyladenine regulate apoptosis and autophagy in bone-derived endothelial progenitor cells

Background Mammalian target of rapamycin （mTOR） is involved in a caspase independent form of programmed cell death called autophagy. The aim of this research was to investigate the effects of rapamycin and 3-methyladenine （3-MA） on autophagy, proliferation, apoptosis, and cell-cycle parameters of rat bone marrow-derived endothelial progenitor cells （EPCs）. Methods Mononuclear cells isolated from rat bone marrow were treated with rapamycin （0.01, 0.1, 1, or 10 pg/L） or 3-MA （1.25, 2.5, 5, or 10 mmol/L） for 24 hours. Expression of the autophagy marker protein LC3-11 was analyzed by Western blotting. Apoptosis and cell-cycle progression were analyzed by flow cytometry. Cell proliferation was measured using the MTT assay. Results Rapamycin treatment of EPCs induced apoptosis and autophagy and inhibited proliferation and cell-cycle progression in a dose-dependent manner. Treatment with 5 mmol/L 3-MA promoted cell proliferation; in contrast, treatment with 10 mmol/L 3-MA promoted apoptosis and induced S-phase arrest. Conclusions Rapamycin treatment of EPCs induced apoptosis and autophagy. Low concentrations of 3-MA had no significant effect on the proliferation and apoptosis of EPCs; The 5 mmol/L group promoted cell proliferation, but had no effect on the apoptosis; the 10 mmol/L group inhibited the proliferation and promoted apoptosis through the cell cycle.

Resveratrol-induced augmentation of telomerase activity delays senescence of endothelial progenitor cells

Background Previous studies have shown that resveratrol increases endothelial progenitor cell （EPC） numbers and functional activity. Increased EPC numbers and activity are associated with the inhibition of EPC senescence. In this study, we investigated the effect of resveratrol on the senescence of EPCs, leading to potentiation of cellular function. Methods EPCs were isolated from human peripheral blood and identified immunocytochemically. EPCs were incubated with resveratrol （1, 10, and 50 pmol/L） or control for specified times. After in vitro cultivation, acidic 13-gatactosidase staining revealed the extent of senescence in the cells. To gain further insight into the underlying mechanism of the effect of resveratrol, we measured telomerase activity using a polymerase chain reaction （PCR）-enzyme-linked immunosorbent assay （ELISA） technique. Furthermore, we measured the expression of human telomerase reverse transcriptase （hTERT） and the phosphorylation of Akt by immunoblotting. Results Resveratrot dose-dependently inhibited the onset of EPC senescence in culture. Resveratrol also significantly increased telomerase activity. Interestingly, quantitative real-time PCR analysis demonstrated that resveratrol dose-dependently increased the expression of the catalytic subunit, hTERT, an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase （PI3-K） blockers （wortmannin）. The expression of hTERT is regulated by the PI3-K/Akt pathway; therefore, we examined the effect of resveratrol on Akt activity in EPCs. Immunoblotting analysis revealed that resveratrol led to dose-dependent phosphorylation and activation of Akt in EPCs. Conclusion Resveratrol delayed EPCs senescence in vitro, which may be dependent on telomerase activation.

Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats

Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D （P〈0.05）; the VEGF mRNA levels in groups B and C were significantly higher than those in group D （P〈0.05）, and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C （P 〈0.05） and D （P 〈0.01）; the recanalization capillary densities in groups B and C were significantly higher than that in group D （P 〈0.05）. Moreover, there was no statistical significant difference between the values for groups B and C. Conclusions The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.