Effects Of The Conditioned Medium From Autophagy And Apoptosis Enhanced Umbilical Cord Mesenchymal Stem Cells On Angiogenesis

BackgroundUmbilical Cord-derived Mesenchymal Stem Cells(UCMSCs)are a kind of adult stem cells with high proliferation and multidirectional differentiation potential.UCMSCs have become one of the seed cells in research and application of regenerative medicine,due to their wide source,easy avlaible and stable genetic background.It has been found that the transplanted UCMSCs can not compensate for the loss of functional cells in the damaged tissue and organs,and its effect to repair tissue and organ mainly attributs to its strong exocrine.Some studies have shown that vascular growth factor-A can be detected in the conditioned medium of UCMSCs,which can promote the proliferation and angiogenesis of endothelial cells.Autophagy maintains cell homeostasis and plays a key role in cell metabolism and function.However,the effect of the conditioned medium of UCMSCs on angiogenesis is still not clear.ObjectiveTo investigate effects of the conditioned medium from autophagy-enhanced UCMSCs on angiogenesis in vitro and in vivo.Method1.UCMSCs were cultured in growth medium with different concentrations(0 nM,100 nM,1 ?M and 10 ?M)of rapamycin for 6 h,and the autophagy level of each group of UCMSCs was detected by immunofluorescent staining.2.UCMSCs were cultured with the medium with above-mentioned concentrations ofrapamycin for 6 h.And then the medium was discarded,the cells were washed with PBS for 3 times,the fresh medium was replaced,and the conditioned medium was collected after 24 h of culture.3.Human Umbilical Vein Endothelial Cells(HUVECs)were culture in 96-well plates coated with Matrigel using the conditioned medium for 3 h,the tube formation of each group of HUVECs was viewed and recorded undera invert microscope.The most obvious group of tube formation was screened out,and was set as the optimized conditional medium.4.Mice were anaesthetized by intraperitoneal injection of 4% chloralhydrate(10m L/kg),and 200 ?L Matrigel with or without HUVECs was injected subcutaneously into the groin each mouse.The conditioned medium(no treatment with rapamycin or 0 nM group)and optimized conditional medium daily injected into mice around the implanted Matrigel for 1 week.The mice were anaesthetized and sacrificed,Matrigel plaques were collected,and then paraffin embedding,sectioning,HE staining and immunofluorescence staining performed to idenfy the effect the optimized conditional medium on angiogenesis in vivo.5.UCMSCs were cultured with fresh medium and the medium with 100 nM rapamycin for 6 h.Total RNA was extracted and q-PCR was performed to analyze the expression of VEGF-A,FGF-2,MMP-9,HIF-1? and PDGF-? in the autophagy-enhanced UCMSCs6.Statistical analysis using Graph Pad Prism 5 software.P < 0.05,which represents statistical significance,where *P < 0.05 and **P < 0.01.All data represents the results of at least three independent experiments.Result1.The immunofluorescence showed that 100 nM,1 u M and 10 u M rapamycin all could enhance autophagy of UCMSCs in a dose-dependent manner.2.The in vitro data of tube formation from HUVECs showed that the conditioned medium of UCMSCs without treatment with rapamycin(0 nM group)and treatments with100 nM,1 u M and 10 u M rapamycin(autophagy enhanced)all could promote the tube formation from HUVECs as compared with the control group(the fresh medium),in which,100 nM rapamycin group of conditioned medium was with the most obvious proangiogenetic effect,and was set as the optimized conditional medium.3.The in vivo tube formation data showed that the autophay-enhanced(with 100 nM rapamycin)conditional medium(the optimized conditional medium)also promoted angiogenesis in the matrigel plaques as compared with the conditioned medium(without autophagy-enhanced group;0 nM group).4.q-PCR data showed that the expressions of angiogenesis-related factors FGF-2,VEGF,MMP-9,HIF-1? and PDGF-? were significantly increased in UCMSCs of the autophay-enhanced group(100nM rapamycin group)as compared with without autophagyenhanced group(0 nM group).ConclusionThe conditional medium from the autophagy-enhanced UCMSCs could facilitate angiogenesis in vitro and in vivo.Background Autophagy is an important physiological factor to maintain cell homeostasis.Usually,autophagy can protect cells from apoptosis.However,under pathological conditions,excessive enhancement of autophagy can induce cell apoptosis,and then affect cell activity and various physiological functions.Studies have shown that autophagy of cardiac myocytes in hypertensive patients can lead to a large number of cell death at the time of over-enhancement of autophagy,thus accelerating the progress of heart failure.The results of the first part of this study show that UCMSCs conditioned medium with enhanced autophagy can promote angiogenesis in vitro and in vivo.However,compared with the conditioned medium with low concentration(100 n M)rapamycin for enhanced autophagy of UCMSCs,the angiogenesis promoting effect of 10 ?M rapamycin conditioned medium with enhanced autophagy(over autophagy)of UCMSCs is relatively weak.These results suggest that over-autophagy may cause other pathophysiological reactions of UCMSCs,and then inhibit the angiogenesis of endothelial cells.Objective To investigate the effects of the conditioned medium from apoptosis-enhanced UCMSCs on angiogenesis..Method1.Based on the first part of work,we firtly measured the apoptotic status of UCMSCs cultured with 10 ?M of rapamycin using flow cytometry assay as the instructions of the apoptosis determination kit.2.UCMSCs were cultured with CCCP(100 ?M)for different times(0 min,30 min and 60 min).The apoptotic rate of UCMSCs in each group was measured by flow cytometry assay.3.UCMSCs were cultured with 100 ?M CCCP for different times,and then the medium was discarded.The cells were washed with PBS for 3 times,and the fresh medium was replaced.After additional 24 h of culture,the conditioned medium was collected.4.HUVECs were seeded in 96-well plates coated with Matrigel and cultured in the above conditioned medium for 3 h,and tube formation of each group were viewed and recorded under an invert microscope.5.Mice were anaesthetized by intraperitoneal injection of 4% chloralhydrate(10m L/kg),and 200 ?L Matrigel with HUVECs was injected subcutaneously into the groin each mouse.The control conditioned medium(UCMSCs was not treated with CCCP)and the conditional medium(UCMSCs was treated with CCCP for 60 min)were daily injected daily into mice around the implanted Matrigel for 1 week.The mice were anaesthetized and sacrificed,Matrigel plaques were collected,and then paraffin embedding,sectioning,HE staining and immunofluorescence staining performed to idenfy the effect the optimized conditional medium on angiogenesis.6.UCMSCs were cultured with fresh medium and the medium with CCCP(100 ?M)for 60 min.RNA was extracted,and q-PCR was performed to analyze the expression of VEGF-A?FGF-1?FGF-2 ?TGF-??MMP-3?MMP-9?PDGF-??PDGF-??HIF-1?and Angiopoietin angiogenesis related factors in the CCCP-treated UCMSCs.7.Statistical analysis using Graph Pad Prism 5 software.P< 0.05,which represents statistical significance,where * P< 0.05 and **P< 0.01.All data represents the results of at least three independent experiments.Result1.Flow cytometry data showed that the apoptotic rate of UCMSCs was markedly increased as compared with the control(non-rapamycin treatment)group.At different treatment time,the apoptotic rates of UCMSCs were all increased markedlyin a timedependent manner.2.The in vitro data of tube formation from HUVECs showed that compared with the control group(fresh medium),the conditioned medium from the UCMSCs not treated with CCCP(0 min group)could promote the formation of HUVECs,while the conditioned mediums from the UCMSCs treated with CCCP for 30 minutes and 60 minutes both inhibited the tube formation from HUVECs,in which,the conditioned medium from the UCMSCs treated with CCCP for 60 min had the most effective to promote angiogenesis.3.The in vivo tube formation data showed that the conditional medium from the UCMSCs treated with CCCP for min could significantly inhibit angiogenesis as compared with the conditional medium from UCMSCs without CCCP treatment.4.The q-PCR data showed that the expressions of angiogenesis-related genes including FGF-1,FGF-2,TGF-?,MMP-3 and HIF-1? were markedly decreased in apoptosis-enhanced UCMSCs.Conclusion In the in vitro and in vivo conditions,the conditional medium from apoptosis-enhancd UCMSCs could inhibit angiogenesis.