Poorly-Controlled Preoperative Blood Glucose Imbalances The Native Excellular Matrix Genes Expression In Saphenous Vein Of Type2Diabetic Patients Undergoing Coronary Surgery

Although percutaneous coronary intervention (PCI) has made significant inroads in the revascularization of patients with coronary artery disease (CAD), The choice of coronary artery bypass grafting (CABG) conduit still plays a critical role in the prognosis of surgical revascularization, which does significantly better in terms of survival and reduced need for further intervention with coronary artery bypass surgery. Although many studies have shown that arterial conduits have the superiority in promoting improved long-term survival and reduced recurrence of angina after CABG in all patients, including diabetic patients, saphenous vein (SV) conduits remains the backbone in graft conduits of CABG. However, only65to80percent of SV grafts remain patent five years after the operation,50to60percent at7to10years, and50percent at15years. Type2diabetes mellitus (DM) is a well-established risk factor to reduce benefits of CABG. It plays an important role in the development and modulation of vein graft disease. Vein grafts failure occur secondary to the deposition of excellular matrix (ECM). The aims of this study were, therefore, to survey the genes expression profile of ECM in SV conduits of diabetic patients with different preoperative glucose profile.Part1Confirm Study Subjects and Obtain Tissue SamplesObjective Patients were divided into3groups according to the preoperative level of fasting blood glucose (FBG) and glycated hemoglobin (HbAlc). Get the demographics of the studied population to make sure it did not differ among the3groups except for preoperative diabeticprof ile. Methods One hundred and thirty patients with coronary artery disease undergoing elective CABG at our institute were enrolled for the study. They were divided into3groups according to the preoperative level of fasting blood glucose and glycated hemoglobin. Fifty four subjects with type2DM had long-term FBG>7.0mmol/L and HbAlc>6.5%were in HG group, regardless of whether getting current treatment with insulin or oral hypoglycemic agents. In LG group,36subjects with type2DM had well-controlled FBG<7.0mmol/L and HbAlc<6.5%. The control group comprised the other40patients who had no preoperative history of Type2DM. Get the demographics of the studied population and obtain the SV tissue samples in CABG surgerys. Results The demographics of the population studied did not differ between the3groups except for diabetic history and Albuminuria. The3groups were well matched for age, sex, and presentation profiles. Conclusion The demographics of the studied population did not differ among the3groups. Part2cDNA Gene Expression Array Analysis in SV of HG, LG and Control GroupsObjective To analyzed ECM genes expression profile in SV obtained at the time of CABG of3groups by microarray. Methods The groups and SV obtained was same as the counterparts in Part1. Total RNA was isolated from sample tissues using TRIzol. Total RNA was used as a template for biotinylated probe synthesis. Biotinylated cDNA probes were denatured and hybridized to ECM and adhesion molecules gene-specific cDNA fragments spotted on the membranes. The array membrane was hybridized with denatured cDNA probes overnight. After washing, the membrane was blocked with GEAblocking Solution and incubated with alkaline phosphatase-conjugated streptavidin. Chemiluminescent detection was performed using CDP-Star substrate. The results were analyzed with ScanAlyze and GEArray Analyzer. Signals were quantitated by scanning the film, and the intensity of the spots was analyzed. Results Of the84ECM-focused genes in this microarray,30genes demonstrated at least a3-fold difference in expression between the HG and control group. Upregulation was observed in24genes, while6genes appeared to be down-regulated. However, only21genes appeared a3-fold difference in expression between the LG and control group, with16genes up-regulated and5genes down-regulated. Conclusion Before exposed to increased blood flow and pressure in the arterial system, the pathological remodeling of SV characterized by imbalanced ECM-related gene expression in diabetic patients has begun. ECM-related gene expression was the most significantly disordered in SV conduits of diabetic patients with uncontrolled level of FBG. However, compared with non-diabetic patients, noticeable differences still exist even though FBG level could be well controlled for a long time before surgery. Part3Western-blot Analysis of Tissue MMP-2, MMP-9, TIMP-2and TIMP-3Levels in SV of HG, LG and Control GroupsObjective To confirm the outcomes of DNA microarray analysis and evaluate the tissue MMP-2, MMP-9, TIMP-2and TIMP-3protein levels in SV by western-blot. Methods The groups and SV obtained was same as the counterparts in Part1. Total protein was fractionated and collected from each group. The samples were run SDS-PAGE electrophoresis and after that transferred to a PVDF membrane. Each membrane was blocked with blocking buffer and then incubated with specific antibodies with secondary antibody. Immune complexes were visualized with the enhanced chemiluminescence detection system. Results The highest tissue MMP-2and MMP-9protein levels in SV were noted in SV of HG group, lower MMPs protein levels were in LG group, and the lowest were in control group, respectively, P<0.05. In contrast, TIMP-2and TIMP-3expression levels of LG group were respectively higher than that of HG group, but lower than that of control group, P<0.05. Conclusion The tissue MMP-2, MMP-9, TIMP-2and TIMP-3protein levels in SV relates to, but not only to the levels of preoperative blood glucose. Besides, it confirmed the outcomes of DNA microarray analysis. Part4Immunocytochemistry Evaluation of MMP-2, MMP-9, TIMP-2and TIMP-3Activity in SV of HG, LG and Control GroupsObjective To evaluate the tissue MMP-2, MMP-9, TIMP-2and TIMP-3protein activity in SV by immunocytochemistry. Methods The groups and SV obtained was same as the counterparts in Part1. The optimal dilutions for each primary antibody were found. The sections were incubated with goat anti-mouse biotinylatted second antibody IgGs. Dehydrate and seal after using DAB substrate. Quantitative evaluation was performed by computer-assisted image analysis. Results Compared with the HG group, MMP-2and MMP-9expression were significantly decreased in LG group, but still increased than that of control group, respectively, P<0.05. Besides, TIMP-2and TIMP-3expression of the LG group were noticeably higher than that of HG group, however, still lower than that of control group, respectively P<0.05. Conclusion The tissue MMP-2, MMP-9, TIMP-2and TIMP-3protein activity in SV relates to the level of preoperative blood glucose. However, noticeable differences still exist even though FBG level could be well controlled for a long time before surgery.