Pharmacology Of Blood Vessel And Genetic And Proteomic Study Of Severe Cadiovascular Disease

Part-I Pharmacology of vasomotion and coronary artery bypass graftingBackground and Objectives:Graft spasm remains challenging in coronary artery bypass grafting (CABG) surgery. Calcium antagonists are commonly used in patients with coronary artery disease. We investigated the inhibitory effect of a new third-generation dihydropyridine calcium antagonist azelnidipine on the vasoconstriction in human internal mammary artery (IMA) from patients undergoing CABG.Intracellular calcium regulation in endothelial cells depends on transient receptor potential channels (TRPs). Canonical TRPs (TRPCs) are now recognized as the most important Ca2+-permeable cation channels in vascular endothelium and TRPC3channel is reported to play a role in vasodilation in animal vessels. However, little is known about the role of TRPCs in the human arteries. We therefore tested the hypothesis that TRPCs plays a role in the human arteries.Methods:Isolated IMA rings (n=68, taken from28patients) were studied in myograph in two ways:the relaxing effect of azelnidipine on vasoconstrictor-induced precontraction by KCl and U46619and the depressing effect of azelnidipine at plasma concentrations on the contraction.Cumulative concentration-relaxation curves to acetylcholine (-11to-4.5log M) were established in the human internal mammary artery (IMA) rings (n=42) taken from28patients undergoing coronary artery bypass grafting in pre-contraction induced by U46619(-8log M) in the absence or presence.of SKF96365(10μmol/L) or Pyr3(3μmol/L). Protein expressions of TRPC3were determined by Western Blot and Immunohistochemistry Staining.Results:Azelnidipine caused full relaxation in KCl-contracted (96.5%±0.7%) and in U46619-contracted (96.5%±1.4%) IMA rings (n=8) with28.8-fold higher potency to KC1than to U46619(EC50:-7.49±0.21vs-6.03±0.11log M, p<0.01). Pretreatment of IMA with plasma concentrations of azelnidipine (-6.1log M) significantly depressed subsequent contraction to KCl (from25.8±2.2mN to16.0±1.5mN, p<0.01) and U46619(from30.6±4.0mN to16.5±2.2mN, p<0.05).The maximal relaxation induced by acetylcholine was significantly attenuated by the non-specific cation channels inhibitor, SKF96365(48.2±3.7vs.66.0±0.9%in control, P<0.01) or the selective TRPC3blocker, Pyr3(58.4±2.3%vs.67.7±1.1%in control, P<0.01). The protein expression of TRPC3was detected in human IMA.Conclusions:We conclude that in human IMA azelnidipine has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors. Thus, use of azelnidipine in CABG patients is favored in treating and preventing graft spasm.TRPC3exists and plays a role in the acetylcholine-induced endothelium-dependent relaxation in the human IMA. This study suggests that TRPC3may have the potential to be a new target in endothelial protection in patients with endothelial dysfunction such as in patients with coronary artery disease in order to improve the long term patency of the grafting vessels.Part-Ⅱ Genetic and proteomic study on heart diseaseBackground and Objectives:The genetic basis and proteomics of common valvular heart disease (VHD) are important to provide information for diagnosis and treatment of these diseases. However, little is known about biomarkers in VHD including rheumatic (RVD) and degenerative (DVD) valvular disease. The present proteomic study examined the hypothesis that certain proteins may be related to the pathological changes in the plasma of VHD.Ventricular septal defect (VSD) is the most common congenital heart disease (CHD). A number of genetic studies have linked the gene oiPLAGLl to the etiology of CHD. The present study aimed to identify potential pathogenic mutations for PLAGL1and to provide insights into the etiology of isolated VSD. Methods:Differential protein analysis was performed in the plasma of patients with RVD, DVD and normal controls by using two-dimensional electrophoresis (2-DE) and mass spectrometry. Candidate proteins that might relate to disease processes were further confirmed by enzyme-linked immunosorbent assays (ELISA) in enlarging samples.Case-control mutational analysis was performed in300patients with isolated VSD and300healthy controls. Two protein-coding extons of PLAGL1and their partial flanking intron sequences were amplified by polymerase chain reaction and sequenced on an ABI3730Automated Sequencer. CLC workbench software was used to compare the conservatism of PLAGL1protein with other multiple species.Results:Eighteen differentially expressed protein spots and the14corresponding proteins or polypeptides were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry. Further, two up-regulated (complement C4A and carbonic anhydrase1) and three down-regulated proteins (serotransferrin, alpha-1-antichymotrypsin, and vitronectin) as candidate proteins for validation were measured by ELISA in the above and new samples. The plasma levels (n=40in each group) of complement C4A in RVD (715.8±35.6vs.594.7±28.2ng/ml, P=0.009) and carbonic anhydrase1(237.70±15.7vs.184.7±10.8U/L, P=0.007) in DVD patients were significantly higher than those in normal controls. The plasma levels of serotransferrin (2.36±0.20vs.2.93±0.16mg/ml, P=0.025) and alpha-1-antichymotrypsin (370.0±13.7vs.413.0±11.6μg/ml, P=0.019) in RVD patients were significantly lower than those in normal controls. In addition, the plasma vitronectin level in both RVD (281.3±11.0vs.323.2±10.0μg/ml, P=0.006) and DVD (283.6±11.4vs.323.2±10.0μg/ml, P=0.011) groups were significantly lower than those in normal controls.Neither missense nor frame-shift mutations were detected in two protein-coding extons of PLAGL1. But a novel synonymous variation (c.486A>G, p. E162E) was detected in protein-coding exon-2. The glutamic that translated with the mutational codon is conservative when compared with other species. Conclusions:The elevation of plasma complement C4A in RVD and carbonic anhydrase1in DVD and the decrease of serotransferrin and alpha-1-antichymotrypsin in RVD patients may be useful biomarkers for these valvular diseases. In addition, the decreased plasma level of vitronectin-a protein related to the formation of valvular structure-in both RVD and DVD patients might indicate the possible genetic deficiency in these patients.We detected a synonymous variation in the protein-coding exon-2of PLAGL1in isolated VSD patients. It is possible that the etiology of isolated VSD might not be directly linked with this mutation, but might be associated with other patterns of gene expression regulation in PLAGL1, such as in the methylation-dependent manner.