Effect And Mechanism Of Complement Activation In Diabetes On The Survival Of Mesenchymal Stem Cells

BackgroundDiabetes is a chronic metabolic disease characterized by hyperglycemia that last a lifetime.The pathogenesis of this disease may involve genetic and environmental factors,as well as immune imbalance.At present,over 422 million people around the world suffer from diabetes,accounting for 8.5% of the whole population,and the incidence is still on the rise,making it a great public health problem.China's incidence of diabetes is higher than the global average,and one in four diabetic patients in the world is from China,making it a serious threat to health of Chinese people and China's economic and social development.Diabetes itself doesn't cause much harm,it is the over a hundred diabetic complications that cause the real damage.Thus,prevention and treatment of the diabetic complications are the most important in the treatment of diabetes.However,the mechanism of diabetic complications has not been fully elucidated,and the current first-line treatment for diabetes,i.e.intensive glucose control,failed to prevent the occurrence of diabetic complications.Several major clinical trials have demonstrated that intensive glycemic control does not reduce the incidence of the macrovascular complications,and one of the studies even reported increased incidence of macrovascular complications after intensive glycemic control.It is also observed during clinical practice that some patients stably controlled their blood sugar,as well as blood lipids and blood pressure at a normal level,but they eventually had one or more complications.Therefore,there must be some other abnormalities beside hyperglycemia and hyperlipidemia that promote the development and progression of diabetic complications.Glucose control alone would be far from sufficient to prevent,treat,or even reverse diabetic complications,and we have to identify the other abnormalities of the diabetic patients and elucidate their correlation with diabetic complications.Most of the diabetic complications can be attributed to damage to micro-and macrovessels due to hyperglycemia-related metabolic disorders and immune abnormalities.This may lead to structural damage and/or dysfunction of multiple tissues and organs,including peripheral nerve,fundus,heart,brain,kidney,foot,etc.Damage to the tissue and organs due to hyperglycemia and related metabolic disorders can be repaired by various seed cells of the body,but when such self-repair mechanism fails to do so or is depleted,the affected tissue or organ would manifest obvious structural damage and dysfunction,and diabetic complications occur.If not being properly treated,the complications would grow more severe and more organs would be affected.Therefore,intensive glucose control must be combined with measures to enhance the body's self-repair mechanism,so as to effectively prevent and treat diabetic complications.Diabetes-related tissue damage may occur at every corner of the body,and so are mesenchymal stem cells?MSCs?that effectively regenerate and repair the damaged tissue;in addition,the MSCs possess direct and indirect immunomodulatory properties that may antagonize immune disorders-related tissue damage in diabetic patients.MSCs are the most important repairing cells of the body and have been used in treatment of several diabetic complications with good efficacy.The repairing capacity of MSCs depends on their quality and quantity.Previous studies have revealed diabetes-related damage to quality of the MSCs;however,the changes in quantity of MSCs in diabetic patients have not been clarified.An animal study revealed that the abundance of MSCs in bone marrow of diabetic rats decreased,but whether this also occurs in human and the mechanism of such decrease have not been clarified.In addition,the MSCs for tissue repair are recruited directly from the peripheral circulation.As such,quantity of circulating MSCs may represent a key factor in the body's self-repair capacity.We therefore speculate there may be a decrease in number of circulating MSCs in diabetic patients.If so,whether the diabetic microenvironment affect the self-renew and survival of the MSC? What is its specific mechanism?AimThe aim of this study was to investigate whether there is a deficiency of circulating MSCs in patients with diabetes,to determine the effect of diabetic microenvironment on proliferation and survival of MSCs,and to explore its specific molecular mechanism through in vivo and in vitro experiments.MethodsThe abundance of MSC-like cells in peripheral blood was evaluated by using flow cytometry.Selected diabetic and non-diabetic serum?DS and NDS,respectively?samples were used to mimic diabetic and non-diabetic microenvironments,respectively.The viability and proliferation of MSCs under different serum conditions were analyzed using MTT assay and Real-time Cell Analysis?RTCA?,respectively.The survival of MSCs under different serum conditions were analyzed using flow cytometry following Annexin V-FITC/PI staining and TUNEL apoptosis detection kit.The apoptosis-related proteins in cells were detected using Western blotting.The survival of MSCs in diabetic microenvironments was also investigated in vivo by using leptin receptor mutant(Lepr^db/db)mice.ResultsIn the present study,we found that the abundance of circulating MSC-like cells in patients with T2 DM was significantly lower when compared with non-diabetic individuals.Moreover,the number of diabetic complications in patients was negatively correlated with the number of circulating MSC-like cells.DS strongly impaired the proliferation and survival of cultureexpanded MSCs through the complement system but not through exposure to high glucose levels.DS-induced MSC apoptosis was mediated,at least in part,by the complement C5 adependent up-regulation of Fas-associated protein with death domain?FADD?and the Bax/Bcl-2 ratio,which was significantly inhibited by neutralizing C5 a or by the pharmacological or genetic inhibition of C5 a receptor?C5aR?on MSCs.Moreover,blockade of the C5a/C5 aR pathway significantly inhibited the apoptosis of transplanted MSCs in Lepr^db/db recipient mice.ConclusionBased on the results of this study,we believe that there is a relative lack of circulating MSCs in patients with diabetes,which results in a deficiency of self-healing MSCs at the sites of damaged target tissues,eventually leading to the occurrence and development of diabetic complications.Diabetic microenvironment restricts the self-renew and survival of MSCs and induces MSC apoptosis,which may be one of the important reasons for the relative lack of circulating MSCs in patients.The pro-apoptotic effect of diabetic microenvironment on MSCs is independent of hyperglycemia,but mainly achieved through the over-activated complement system,and among which,the biological effects triggered by C5 a are the most critical.Targeted blockade of C5a/C5 aR pathway can significantly improve the survival of implanted MSCs in diabetic microenvironment,in vitro and in vivo,which may represent an effective strategy to improve the abundance of circulating MSCs.