The Study Of The Correlation Between Peripheral Blood Smooth Muscle Progenitor Cells And Type 2 Diabetes

Background and ObjectiveAs the economy of China is developing and the quality of life has improved, the incidence of type 2 diabetes is rapidly increasing. Diabetic patients often have the metabolic disorders of glucose and lipid. However, the most important cause of disability and mortality in diabetic patients is complicated by large vessel diseases. It has been shown that the incidence of coronary heart disease in diabetes is significantly higher than non-diabetic patients. Recent studies have found that SPCs plays an important role in the development of atherosclerosis.Circulating-derived SPCs express CD14/CD105 double positive, which can proliferate and differentiate into smooth muscle cells. In addition, SPCs can also be found in the bone marrow and adventitia. Studies have shown that a variety of cardiovascular risk factors cause the vascular endothelium damage. The chemokines and cytokines released from local vascular cells mobilize the bone marrow derived-SPCs into the peripheral circulation, and SPCs home in the damaged vessels involving in the formation of neointima, leading to the development of atherosclerosis.The aim of the current study is to observe the changes of circulating derived-SPCs in type 2 diabetes, and find the factors to these changes in the body. It will help explore the mechanism of the cardiovascular complications in diabetic patients.MethodsPart one: Isolation ,culture and differentiation of adult human circulating- derived SPCs in vitro. Mononuclear cells were isolated from adult human peripheral blood by density gradient centrifugation. The isolated cells subsequently were plated onto dishes in medium supplemented with PDGF-BB and bFGF. After 12 days of culture in vitro, SPCs were (identified)characterized as adherent cells and sorted by flow cytometry. SPCs were then induced to differentiate in response to TGF-β1. Inverted microscope was used to observe morphological changes of SPCs. Smooth muscle cells specific markers (α-SMA, Calponin, SM-MHC) were then checked with immunofluorescent staining and Western blotting.Part two: To investigate the correlation within the number of circulating- derived SPCs and factors in the patients with type 2 diabetes mellitus. we selected 55 type 2 diabetic patients enrolled in Xijing Hospital from January 2010 to January 2011 and 20 healthy volunteers. They were divided into 3 groups: 20 type 2 diabetic patients without coronary artery disease (DM group), 15 type 2 diabetes patients with coronary artery disease (DM+CAD group) and 20 healthy volunteers (Con group). We detected peripheral blood CD14⁺/CD105⁺ cells by flow cytometry. In addition, we detected other parameters (FPG, HbA1c, SBP, DBP, TC, TG, LDL-C, HDL-C).The relationship between the number of SPCs and the parameters were analyzed by multivariate regression analysis. Part three: Effects of high glucose on biological characteristics of smooth muscle progenitor cells. Mononuclear cells were isolated from adult human peripheral blood by density gradient centrifugation and cultured. After 12 days of culture in vitro, SPCs were sorted by flow cytometry. They were randomly divided into 5 groups (control group, 11 mmol/L group , 22 mmol/L group , 44 mmol/L group and osmotic pressure control group). The SPCs were incubated with glucose in a series of concentrations for 6 days. Proliferation and migration of SPCs were assayed by MTT assay and Transwell chamber assay respectively. SPCs adhesion assay was performed by replating the cells on fibronectin-coated dishes and the adherent cells were then counted. In addition, the SPCs were incubated with glucose (22mmol/L) for different durations (0 , 2 , 4 , 8 days). The SPCs proliferation and migration ability were abserved and adhesion assay was performed.Results1. Four days later, cell colonies were presented. Twelve days later, most adherent cells were showing spindle-shape. CD14/CD105 double positive cells were SPCs, which accounted for (71.8±7.2)% of adherent cells. Twenty-eight days later, cells arranged in order, showing whirlpool-shape. On day 28, immunostaining of adherent cells was positive forα-SMA. Expression of calponin and SM-MHC was found at 14 d and 21 d with Western blotting respectively, and they gradually enhanced.2. The number of circulating-derived SPCs in patients of DM group was significantly increased compared with controls (26.20±11.36 vs. 14.30±7.61,P<0.05), and it was higher in patients of DM+CAD group than in the DM group (42.00±8.93 vs. 26.20±11.36,P<0.05).Multiple regression analysis showed that HbA1c, HDL-C and SBP were independent related factors influencing circulating-derived SPCs levels. The equation was y=13.433+2.435 x₂-21.625 x₈+0.188 x₃ . Linear correlation analysis show that the number of SPCs in peripheral blood was correlated positively with HbA1c and SBP (r=0.684, r=0.379, P<0.01), and negatively with HDL-C (r=-0.654,P<0.01).3. Compared with the control group, high glucose accelerated proliferation , migration and adhesion ability. Especially when the SPCs were incubated with glucose in the final concentration of 22mmol/L for 8 days, the effects were the most prominent.ConclusionsOur results demonstrated that:1. SPCs could be harvested through culturing mononuclear cells from adult human peripheral blood. They could differentiate into smooth muscle cells in vitro.2. The number of circulating-derived SPCs was associated with metabolism disorder. And the increasing SPCs may contribute, in part, to the pathogenesis of atherosclerosis in type 2 diabetic patients. As the independent related factors, the level of HbA1c,SBP and HDL-C can predict the number of circulating SPCs.3. Within a certain range, high glucose could significantly improve the function of SPCs in a concentration and time dependent manner. It can be speculated that long-term hyperglycemia in patients could stimulate the function of SPCs, which plays an important role in the the development of cardiovascular disease.