| Acute lung injury (ALI) is a devastating disease, which is characterized by diffuse endothelium, epithelial damage, and increased pulmonary capillary permeability. Recent data has suggested that the circulating endothelial progenitor cells (EPCs) play an important role in endothelial repairing after vascular injury. This study was undertaken to investigate possible endothelial repairing effects of EPC transplantation in rats with lipopolysaccharide (LPS)-induced ALI. Using Y-chromosome in situ hybridization and RT-PCR assay, we detected the expression of sex-determining region y in the injured lungs of female model rats, suggesting that allogenic EPCs can migrate to the injured lung tissues. Rats that have received the EPC treatment had a reduced pulmonary edema level, inflammation, hemorrhage and hyaline membrane formation, as well as an increased survival rate from 44% to 81%. Furthermore, anti-inflammatory cytokine, interleukin-10 level was dramatically increased in the EPC-treated rats, compared with the phosphate buffered saline-treated rats. On the contrary, endothelin-1 and inducible nitric oxide synthase were down-regulated in the EPC-treated group. These findings provide evidence that intravenous EPC treatment results in engraftment of EPCs to the injured lung tissue, which can significantly attenuate lung injury and improve survival in rats with ALI. The beneficial effects of EPCs engraftment is likely to come from maintaining the integrity of pulmonary alveolar–capillary barrier, reestablishing the endothelial function in vessels and ameliorating inflammatory state.METHODS1. Isolation, culturing and Characterization of EPCs: EPCs were isolated from rat bone marrow. Briefly, mononuclear cells were separated from the tibia and femur of Sprague-Dawley rats (4wk old, male) by density gradient centrifugation with 1.083g/ml Histopaque. Isolated cells were seeded on fibronectin-coated culture flasks and maintained in DMEM containing 20% fetal bovine serum, 100units/ml penicillin, and 100 units/ml streptomycin. After 7~10 days in culture, Characterization of EPCs By immunofluorescence method to detect the capacities of acetyl-LDL and FITC-labeled lectin uptake of EPCs and its surface antigens of CD133, CD34, VEGFR-2 and von Willebrand Factor.2. The expression of Y-chromosome detected by RT-PCR and ISH: The section of animal experiments in vivo: acute lung injury model was performed by using LPS. EPCs isolated from male rat bone marrow were transplanted intravenously into female rats that had been challenged with LPS. Signals of Y-chromosome gene sry were evaluated using RT-PCR and ISH in lung tissues of rats after EPC transplantation for 3, 7 and 14 days.3. The beneficial effects of EPC transplantation: In another experiment, Rats in EPC-treated group and PBS-treated group were respectively received corresponding EPCs or the same amount of vehicle (200μl PBS) by the previous described method after LPS treated. Animals in PBS or EPC control group were received either PBS or EPC transplantation without LPS injection. Seven days later, The beneficial effects of EPC treatment were evaluated by HE staining and Immunohistochemistry,measuring Dry / wet and MVD, and TNF-αand IL-10 expression levels were examined with ELISA, and ET-1 and iNOS levels of ALI biomarkers were evaluated using western blot. At the same time, we compare survival rate between EPC-treated group and the PBS-treated group after EPC or PBS transplantation 7 days.4.Statistical analysis:Data are expressed as mean±SD. SPSS13.0 software was used for statistical analysis. Student-t-tests were used to compare 2 groups, and one way ANOVA was used with the Tukey's multiple comparison tests for multiple groups. Statistical significance was accepted at a level of P < 0.05.RESULTS1. Characterization of EPCs:Seven days after cultivation, early EPCs showed incorporation of acetyl-LDL and binding to isolectin. EPCs also displayed expression of endothelial markers CD133, CD34, vWF and VEGFR-2. Formation of round or fusiform shape appearance was also observed 1 week after culturing in DMEM containing 20% fetal bovine serum.2. Allogenic EPCs from male origin were detected in the female injured lung tissues 7 days after transplantation. Sry signals were detected in lung tissue endothelium of EPC-treated group.3. The EPC treatment significantly reduced pulmonary edema level, inflammation, hemorrhage and hyaline membrane formation in the lung, and significantly increased the microvessel density in rats with LPS induced ALI. Rats'survival rate was markedly increased as well. Anti-inflammatory cytokine, IL-10 level was increased in the EPC-treated rats, compared with phosphate buffered saline (PBS)-treated rats. TNF-α, ET-1 and iNOS levels were down-regulated in the EPC-treated group.CONCLUSIONSThis study has provided three major findings : 1) EPCs was successfully isolated from rat bone marrow by density gradient centrifugation method; 2) intravenously administrated EPCs can successfully engraft to the LPS-induced lung injury area and differentiate toward endothelial cell phenotypes; 3) there are significant improvements in the lung injury, as well as improved survival following the EPC treatment in the ALI model rats; the beneficial effects of the EPC treatment is likely to come from restoring damaged pulmonary endothelium, maintaining the integrity of pulmonary alveolar–capillary barrier and improving pulmonary inflammation.
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