ABSTRACT
<p><b>BACKGROUND</b>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).</p><p><b>METHODS</b>Mononuclear cells isolated from rat bone marrow were treated with rapamycin (0.01, 0.1, 1, or 10 µg/L) or 3-MA (1.25, 2.5, 5, or 10 mmol/L) for 24 hours. Expression of the autophagy marker protein LC3-II was analyzed by Western blotting. Apoptosis and cell-cycle progression were analyzed by flow cytometry. Cell proliferation was measured using the MTT assay.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Subject(s)
Animals , Rats , Adenine , Pharmacology , Apoptosis , Autophagy , Cell Cycle , Cell Proliferation , Cells, Cultured , Sirolimus , PharmacologyABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>