Effects Of Adiponectin On Proliferation And Differentiation Of Endothelial Progenitor Cells And The Possible Mechanism

Improvement in people's living standards,long-term existence of some adverse living and dietary patterns have caused the number of patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes increased greatly in the recently years in China.This has become a public health problem and drawn the attentions of endocrine,cardiovascular and cerebrovascular physicians,and researchers in public health.Clinical researches and epidemiological investigations have confirmed that cardiovascular diseases are closely related with metabolic disorders such as diabetes.Metabolic syndromes and type 2 diabetes have been considered as equivalent to the risk of coronary heart disease(CAD).The increasing of inflammatory cytokines caused by obesity leads to insulin resistance,which also leads to endothelial cell damage and functional disorders.High blood glucose in patients with type 2 diabetes causes vascular endothelial damage,especially microcirculation vascular endothelial cells,which is also an important factor for the development of atherosclerosis and CAD.Adiponectin is an adipocyte-specific protein and hypoadiponectinemia is often observed in patients with obesity,impaired glucose tolerance,insulin resistance,and type 2 diabetes.Recent studies have showed that adiponectin's overexpression could decrease atherosclerosis by attenuating the endothelial inflammatory response and macrophage-to-foam cell transformation in vivo.Therefore,adiponectin has anti-atherogenic properties through anti-inflammatory effects.Vascular endothelial functions are closely related to cardiovascular diseases.Endothelial function's disorders happen in the situation of high blood glucose and endothelial injury.Low plasma adiponectin level was also observed in patients with cardiovascular diseases such as hypertension and CAD.In a recent study,adiponectin affected plaque components,and low plasma adiponectin,a well-known independent risk factor for CAD,might enhance the vulnerability of atherosclerotic plaques and vessels,which could lead to acute coronary syndrome(ACS).Therefore,plasma adiponectin level is now considered as one of the indicators of its prognosis prediction.Endothelial progenitor cells(EPCs) are a cell population that have the capacity to circulate,proliferate,and differentiate into mature endothelial cells,but have neither acquired characteristic mature endothelial markers nor formed a lumen.EPCs have been shown to be incorporated into sites of physiological and pathological neovascularization in vivo.More recently,it has been suggested that circulating EPCs may also be a marker of endothelial function and cardiovascular risk.Endothelial dysfunction is usually one of the earlier markers of atherosclerosis,and predisposes the vasoconstriction and thrombosis.EPCs may exert an important function as an endogenous repair mechanism to maintain the intergrity of the endothelial monolayer by replacing denuded parts of the artery.These beneficial propertities of EPCs are attractive for cell therapy that targets endothelial regeneration for treatment of ischemic diseases and wound healing,Therefore,how to promote expansion,proliferation,and differentiation of EPCs effectively is a current research hotspot.Recent research has shown that adiponectin receptors expressed in mouse hematopoietic stem cells(HSCs) and adiponectin could promote HSCs proliferation in vitro /vivo and improve their functions.EPCs are also precursor cells and have some biological characteristics of both endothelial cells and progenitors cells.Whether adiponectin receptors are expressed in EPCs or adiponectin affects proliferation and differentiation of EPCs and its mechanism is unclear.On the basis of these considerations,we hypothesized that adiponectin affected EPCs number and functional activity,thus influenced endothelial repair process and maintained the balance between the magnitide of injury and the capacity for repair, which prevented atherosclerotic lesion developing.Therefore,the aim of this study was to investigate the expression of adiponectin receptors in EPCs,and whether adiponectin affect the proliferation and differentiation of EPCs and the possible mechanism.The following 2 Parts would describe the methods,results and conclusions of the study briefly.Part 1 Isolation,Culture and Identification of EPCs from Human Umbilical Blood and Expression of Adiponectin Receptors in EPCs.Objective:To isolate,culture and identify EPCs from human umbilical blood,and investigate the expression of adiponectin receptors in EPCs.Methods:Total mononuclear cells(MNCs) were isolated from human umbilical blood by Ficoll density gradient centrifugation,and plated on fibronection-coated culture dishes.EPCs were characterized as adherent cells double positive for DiI-Ac-LDL-uptake and FITC-UEA-I lectin binding by direct fluorescent staining under a fluorescent microscope.EPCs were further documented by demonstrating the expression of VEGFR2,CD34,and AC133 with flow cytometry.The expression of adiponectin receptors were measured using RT-PCR,Western blot and immunocytochenistry.Results:Adherent cells isolated and cultured from human umbilical blood DiI-Ac-LDL uptake and FITC-UEA-I lectin binding were assessed under direct fluorescent microscope,and the percentage of double positive cells were 81.4±11.7%. Analysis of VEGFR2,CD34 and AC133-expression by flow cytometry.Ex vivo EPCs were positive by 76.6±7.0%for VEGFR2,42.9±8.5%for CD34,and 19.8±3.9%for AC133.Adoponectin receptors' mRNA and protein were expressed in EPCs. Conclusions:EPCs can be isolated from human umbilical blood and cultured by Ficoll density gradient centrifugation and adherence to fibronectin-coated culture dishes. Both AdopR1 and AdopR2 are expressed in EPCs.Part 2 Effects of Adiponectin on Proliferation and Differentiation of Endothelial Progenitor Cells and the Possible MechanismObjective:To investigate the effects of adiponectin on proliferation and differentiation of EPCs and the possible signaling pathway.Methods:After isolated EPCs were cultured 15-20 days,EPCs' proliferation was assayed with MTT assay.EPCs' differentiation was determined by Real-time PCR by measureing relative expression of surface markers including vWF,CD31,CD34,and AC133.The expression of p-JNK,JNK,p-p38,p38,ERK,p-ERK were measured by Western blot.MAPK's inhibitor SB203580 and SP600125 were also used for testing the signaling pathway.Results:Adiponectin increased the number of EPCs with dose and time dependence, with maximum at 30μg/ml,48h.Adiponectin significantly increased vWF and CD31 expression of EPCs with maximum at 30μg/ml,48h,but decreased CD34 and AC133 expression.Western blot analysis showed that adiponectin induced activation of p38 and JNK,but not activation of ERK1/2 in EPCs.These effects were blocked by JNK inhibitor(SB600125) and p38 inhibitor(SB203580).Pretreatment of EPCs with p38 inhibitor(SB203580) and JNK inhibitor(SB600125) partly diminished adiponectin-induced cell proliferation and the expression of vWF,CD31,CD34,and AC133. Conclusions:Adiponectin can promote EPCs proliferation and differentiation by dose-and time-dependent manner.Adiponectin enhances EPCs proliferation and differentiation through MAPK signaling pathway.