| Background and objectivesAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) are progressive acute respiratory failure caused by various etiological factor, and they are common critical diseases in clinical practice. The mortality of ALI/ARDS was up to 70% in 1970's, and the current mortality is still about 40% despite the advances in supportive and pharmacologic treatment. Researchers have conducted a lot of studies and made some progress, but the treatment strategy can only reduce the injury and cannot totally change its poor outcomes. The injury of pulmonary capillary endothelial cells and alveolar epithelial cells mediated by inflammation is the characteristic pathological changes of ALI/ARDS, so the ideal treatment strategy of ALI/ARDS is the repairing of injuryed lungs and the reconstructing of function. Adult stem cells have important values in the treatment of ALI/ARDS.Endothelial progenitor cells (EPCs) are immature cells capable of differentiating into mature endothelial cells. EPCs have been shown to display a higher proliferative potential and may migrate to regions of the circulatory system with injured endothelia. Since the EPCs have important role in maintaining the integrity of endothelial cells and the injury of endothelial cells is the characteristic lesion in early period of ALI/ARDS, EPCs could become a novel cell-based therapeutic strategy for prevention and treatment of ALI/ARDS. The aims of this study include observing the variation of peripheral blood endothelial progenitor cells in the early stage of ALI induced by lipopolysaccharide (LPS) in rabbit, exploring the protective effects of autotransplantation of circulating EPCs on lung injury and the underling mechanisms, and investigateing the therapeutic potential of EPC-derived conditioned medium (EPC-CM) on ALI.Methods and results1 Isolation, Culture and identification of EPC Peripheral blood was obtained from New Zealand white rabbits via ear artery (10 ml/kg). Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque Plus. Mononuclear cells were then washed and planted on culture dish coated with human plasma fibronectin and supplemented with endothelial growth medium 2 (EGM-2). Seven days after isolation, incorporation of acLDL and binding of isolectin were detected in early EPCs, and the percentage of double positive cell was nearly 100%. Expressions of VEGFR2 and CD34 of those cells were detected by immunofluorescent staining, and the percentage of double positive cell was over 95%. Formation of round or fusiform shape appearance of those cells was also observed 7 days after culturing in EGM-2.2. Develpoment of animal model of ALIConscious rabbits were rapidly injected intravenously via one marginal ear vein with 500μg/Kg or 700μg/Kg body weight of purified LPS endotoxin (from Escherichia coli O55.B5), and the lung of two group rabbits were harvested for HE staining at 72h. In both groups of rabbits, HE staining showed typical ALI pathological traits, such as size decrease in the pulmonary alveolus cavity, thickening of alveolar wall, expanded mesenchyme with increased numbers of polymorphonuclear cells (PMNs), formation of hyaline membrane, and hemorrhage. The lung injury of 700μg/Kg group was more severe than 500μg/Kg group, but its mortality was too high and may produce the adverse effect to the subsequent analysis. We decided to use 500μg/Kg LPS to produce the rabbit model of ALI.3. The changes of quantity and function of peripheral blood EPCs in early stage of ALIRabbit model of ALI was induced by LPS (500μg/Kg), and control group was injected with normal sodium (NS). Peripheral blood was drawn at 12h,24h, 48h,72h and 5d. The expression rate of VEGFR-2+/CD34+EPCs in rabbit peripheral blood was counted by flow cytometry. The proliferation, adherenee and metabasis function of EPCs were measured after in vitro culture. The rate of EPC in control group maintain stable at each time. The rate of EPC in ALI group immediately reduced after the injection of LPS. The rate reduced to the lowest point at 24h and gradually rised after that time, but it still lower than the rate in control group at 5d. The proliferation, adherenee and metabasis function of EPCs were impaired after the injection of LPS. The functional parameters reduced to the lowest point at 48h and gradually rised after that time, but it still lower than the functional parameters in control group at 5d. The differences between the control and ALI group were significant at each time point.4. Protective effects of EPC autotransplantion on ALI and the possible mechanismConscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. Autologous early EPCs (105 cells in 200μL PBS; EPC group) or 200μL PBS alone (control group) were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. To track the homing of EPCs in the pulmonary, a fluorescent cell tracker CM-DiI was used to label early EPCs immediately before transplantation. Sham-operated animals (sham group) received PBS without injection of LPS.1,3 and 5 days after EPCs transplantation, rabbits were killed and samples of lung tissue, serum and bronchoalveolar lavage fluid (BALF) were collected.The CM-DiI labeled cells were observed in frozen lung sections of the rabbit treated with EPCs, although in less amount. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of control group showed typical ALI pathological traits. At each time point, EPC treatment improved the lung injury compared with control. However, the lung injury of EPC group was still apparent compared with sham group at all time points. Autotransplantation with EPCs lowered the increased lung wet/dry ratio and BAL protein at all time point, and the differences were significant at 3d and 5d (P<0.05). In LPS-treated rabbit, a significant reduction in MPO activity was found following autotransplantation with EPCs compared with the group treated with LPS alone at 1 and 3d (P<0.05). Neutrophils in the BALF were reduced in LPS-treated rabbit that received autologous transplantation of EPCs compared with the group treated with LPS alone, and the differences were significant at 3d (P<0.05). Administration of EPCs abated the increase of IL-1βand TNF-a, and the differences were significant at 3d and 5d for IL-1βand at 24h and 3d for TNF-a (P<0.05). Administration of EPCs magnified the increasing amplitude of IL-10, and the difference beteween control group and EPC group was statistically significant by 3d (P<0.05). Rabbit given EPC showed a trend toward lower sICAM-1, and there was significant difference beteween control group and EPC group at 1d (P<0.05). The changes of P-selectin mRNA expression were similar with sICAM-1, and the differences were significant at 3d (P<0.05). Administration of EPCs decreased the number of TUNEL-positive cells and prevented LPS-mediated apoptosis.5. Protective effects of EPC-derived conditioned medium autotransplantion on ALIEPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC for three days.15 rabbits were divided into three group:sham group (n=5), control group (n=5) and EPC-CM group (n=5). Conscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. EPC-CM or PBS with equal volume were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. Rabbits were killed and samples of lung tissue and BALF were collected 48h after EPC-CM injection. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of lung sections showed that EPC-CM treatment slightly improved the lung injury compared with control. Compared with the control group, lung wet/dry ratio, BAL protein, MPO activity and neutrophils in the BALF of the EPC-CM group were all improved, but the differences were not significant between control and EPC-CM group (P>0.05).Conclusions1. EPCs can be successfully isolated from peripheral blood by density gradient centrifugation and adherence selection, and can be cultured in vitro.2. In rabbit model of ALI, the quantity and proliferation, adherenee and metabasis function of EPCs were significantly reduced in early stage of ALI. The quantity and fuctions of EPCs partly restored with the resolution of inflammation in lungs.3. autotransplantation of EPCs from peripheral blood can home to injuryed lung and attenuate LPS-induced ALI in rabbit model. EPCs transplantation can reduce neutrophil recruitment by decreasing ICAM-1 and P-selectin in initial phase of ALI and subsequently prevent lung injury. Proinflammatory cytokine responses to LPS partly altering to anti-inflammatory response and decreased apoptosis of lung cells also make contribution to the therapeutic benefits of EPCs. Treatment with EPC-CM can slightly improve the lung injury induced by LPS, suggesting that paracine of EPCis one of the mechanisms.
|
PDF Full Text Request