The Effects And Mechanism Of Sphingosine 1-Phosphate Protecting Endothelial Progenitor Cells From Serum-deprived Apoptosis

【background】Endothelial cells (ECs) in adults are mature cells, whose proliferation capacity are limited. Compensatory response to tissue ischemia and re-endothelialization after endothelial injury, such as tissue damage, ischemia, systemic inflamatory response syndrome and multiple organ dysfunction syndrome, may not be achieved only by the reproduction of ECs themselves nearby the sites. Extensive data supported the existence of endothelial proenitors cells (EPCs), their contribution to the formation of new blood vessels in adults and re-endothelialization in endothelial injury sites. Emerging researches on isolation, culture, identification, and derivation, distribution, differentiation, migration, homing, function and potential usage were launched in the recent years. In the first part of present study, we cultured EPCs from bone marrow-derived mononuclear cells (BMNCs), and identified them.Postnatal vasculogenesis and neo-angiogenesis had been shown to be closely correlated with the number and properties of EPCs recruited to injure sites. Repair processes often had to take place under ischemia conditions which were unfavorable for the survival and proliferative potential and the ability to expand of EPCs. Thus, protecting EPCs in such unfavorable conditions was very important for the prognosis of diseases. In the present study, we treated EPCs by serum deprivation (culturing EPCs in the media lack of serum and growth factors), and then examined the survival of EPCs. Pretreated EPCs under such conditions with S1P, and then examined the effects.Kimura, T et al investigated the role of the lipoprotein-associated lipids in the lipoprotein-induced cytoprotective actions in human umbilical vein endothelial cells (HUVECs). They concluded that the lipoprotein-associated S1P and its receptors mediated cytoprotective actions. Furthermore exogenous S1P mimicked cytoprotective actions. The study suggested that S1P had some favorable effects on cells and potentially useful means. There is no report showing whether this material has the same favorable effects on EPCs. Thus, we postulated that S1P had the same cytoprotective effects on EPCs under the unfavorable conditions lack of serum and growth factors. And it reduced the apoptosis of EPCs and protected cells so that there were enough EPCs in the injury sites. Furthermore, we explored the mechanism of it. 【objective】To investigate the methods of isolation and culture of bone marrow-derived endothelial progenitor cells (BM-EPCs). To demonstrate the anti-apoptotic effects of sphingosine 1-phosphate on EPCs under serum deprivation conditions, and then further explore the mechanism.【methods】1. Isolation, culture and identification of bone marrow-derived EPCs from rats: Bone marrow-derived mononuclear cells (BMNCs) was collected from rats by density gradient centrifugation, and EPCs were obtained and explanded by means of differential attachment technique from the BMNCs.2. The serum deprived media (contrasted to full culture media, it was lack of calf serum and growth factors) induced apoptosis of EPCs. However, sphingosine 1-phospahte exerted protective effects on EPCs under such conditions: EPCs was exposed to serum deprived media, and other groups was pretreated with different concentration of S1P (0.1, 1, 10, 20μM). Changes of cellular morphology were observed at regulated time, and detected the apoptosis rate by Annexin V-FITC apoptosis detection kit.3. The mechanism of the protective effects of S1P on EPCs under serum deprived conditions: RT-PCR and western blot were aimed to examined the S1P receptors expressed on EPCs. The block agents of the receptors and the downstream elements of the signal pathway were also emplored to investigate the mechanism. The qualitation and quantitation of phosphated Akt in EPCs treated by agents were detected. The mount of NO and apoptosis of EPCs were also detected by the kits. And then the signal pathway activated though the efffects of S1P on EPCs were discussed.【results】1. EPCs could be cultured and expanded from bone marrow-derived cells of rats. Bone marrow-derived monounclear cells were isolated from the bone merrow-derived cells by density gradient centrifugation, and EPCs could be expanded by means of differential attachment technique. The cells exhibited a spindle-shape morphology, and form endothelial colonies which is one of qualities of stem/progenitor cells. They showed the shape of"slabstone"after culture for 20 days in vitro, which was a typical characterization of endothelial cells. EPCs were characterized by their phonotypical expression of CD34, KDR, vWF. But CD133 only could be detected in a few days after isolated cells. There were only about 5 percent cells expressing the phaenotype after cells cultured 9 days in vitro. EPCs up-took Dil-acLDL and binded UEA-1, and also expressed endothelial-specific genes such as CD31, vWF, eNOS.2. Serum deprivation could induce apoptosis of EPCs, and the apoptotic rate was increased in a time-dependent manner. The apoptotic effects on EPCs were suppressed by the addition of S1P. Serum deprivation leaded to a singnificant increase of apoptotic EPCs, which resulted subsequently in a significant decline of EPCs munber in vitro. 27.7±1.08% of EPCs undergo apoptosis after 24h in the serum deprived conditions, and 49.3±1.12% after 48h. EPCs treated with S1P in different concentrations (0.1, 1, 10, 20μM) resulted in decreasing apoptosis respectively, and in a concentration-dependent manner. The protective effects of addition of 20μM S1P almost reversed the apoptosis induced by serum deprivation after 24h.3. S1P exerts apoptotic effects on EPCs by activating the G-coupled S1P receptors, and involving the upregulation of NO. The results of RT-PCR (Reverse Transcription-Polymerase Chain Reaction experiments) and western blot showed EPCs expressed mRNA transcripts and the corresponding proteins for three S1P receptors, S1P1, S1P2, S1P3 dominantly, whereas these cells almost do not expressed mRNA for S1P4, S1P5. S1P increased NO release in supernatant and derease the apoptosis of EPCs in a concentration-dependent manner. S1P-induced anti-apoptotic effects on EPCs was significantly suppressed when the cells was pretreated with pertussis toxin (PTX), an inhibitor of G proteins receptors. The apoptotic rates and the levels of NO were of no significant difference compared to control group. The results suggest that S1P-induced NO is responsible for S1P protection from apoptosis.4. S1P exerted the anti-apoptotic effected on EPCs was involved with activating phospholipase C (PLC) and Src kinase. The anti-apoptotic effects of S1P on EPCs was partly attenuated by the pretreatment of U73122, a specific inhibitor of PLC or PP2, a specific inhibitor of Src kinase, and NO production was downragulated accordingly. And the effects of both of them were significantly different in comparison to the control. Pretreating EPCs with both U73122 and PP2 was mimiced the effects of PTX or serum deprivation.5. Pretreatment with U73122 did not inhibit S1P-induced phosphorylation of Akt, but PTX and PP2 significantly diminished it. The posphorylation of Akt induced by S1P and the inhibitors of element downstream of S1P was examined by western blot. The results showed that pretreatment with PTX or PP2 efficiently blocked S1P-induced activation of Akt, but U73122 not. It indicated that there are two isolated signal pathways activated by S1P/S1P receptors.【conclusion】BMNCs of rates can be collected by density gradient centrifugation. EPCs can be obtained from BMNCs by means of differential attachment technique. We show EPCs underwent apoptosis in the media absence of serum and growth factors. S1P significantly depresses the apoptosis of EPCs under the adverse conditions. S1P exerts the favorable effects on EPCs associating with S1P receptors, and activates the anti-apoptosis signal pathways in EPCs. S1P-induced NO synthesis protects EPCs from apoptosis, and both S1P/S1P receptors/PLC/NO and S1P/S1P receptors/Src kinaes/Akt/NO pathways have essential roles in S1P-induced anti-apoptosis singaling. It is quite conceivable that the anti-apoptotic effects are favorable for EPCs cultured in vitro for transplantation. It may also be favorable for angiogenesis and vascularization of EPCs under unfavorable conditions in vivo.