Study On The Mechanism Of GALNT3, A Candidate Gene Of Atherosclerosis, In Vascular Endothelial Cell Injury

Part 1:GALNT3, a Novel Candidate Gene for Atherosclerosis and its Function in Endothelial DysfunctionBackground and Objectives:Coronary artery disease (CAD) is one of leading causes of death worldwide. Atherosclerosis is a major pathogenic mechanism of CAD, characterized by plaque formation and subsequent fissure, erosion, or rupture of the plaque with thrombosis of the plaque surface. The GALNT3 gene encoding UDP-GalNAc:polypeptide GalNAc-transferases3 (GalNAc-T3), which transfers O-linked N-acetyl galactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in human. Emerging studies have linked GALNTs to cardiovascular disease or susceptibility to CAD. However there were no reports about the linkage of GALNT3 with CAD. This study examined the relationship of common variants in GALNT3 gene with CAD, and ascertained its function in endothelial dysfunction.Methods:1. Single-marker association and gene-based analysis were conducted in CAD patients (n=1515) and control individuals (n=5019), and 13 SNPs in and around the GALNT3 gene were tagged and analyzed. Association of the SNPs with CAD was tested with multiple logistic regression analysis in an additive genetic model (with 1 degree of freedom) after adjusting for age and sex. Association analysis were performed using PLINK and R software packages.2. Real-time PCR was used to detect the GALNT3 mRNA levels in the PBMCs of an independent cohort (CAD group:58 cases, control group:120 cases).3. Gain- and loss-of-function experiments were used for further study of the function of GALNT3 in HUVECs. MTT assay and Annexin-V conjugated Flow cytometry (FACS) analysis were employed to measure HUVEC proliferation and apoptosis. The mRNA and protein levels of GALNT3, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-14 (MMP-14) were detected with real-time PCR and Western Blot.Results:1. Genetic analysis found the association of two independent SNPs rs13427924 (P=0.008) and rs4621175 (P=0.033) with CAD. Furthermore, gene-based analysis confirmed the association of GALNT3 gene with CAD (P=0.011).2. Compared with the control group, the mRNA levels of GALNT3 were significantly descreased in CAD patients. The PBMCs GALNT3 mRNA level in CVD group was 58.5% of that in control group(P<0.001).2. Expression of GALNT3 was decreased in HUVECs under hypoxia. GALNT3 knockdown exaggerated HUVECs apoptosis and increased the expression of MMP-2 and MMP-14. Conversely, Overexpression of GALNT3 inhibited HUVECs apoptosis, as well as the expression of MMP-2 and MMP-14 in mRNA and protein levels (P<0.05). Under hypoxic condition, GALNT3 overexpression attenuated hypoxia-induced apoptosis and blocked the expression of MMP-2 and MMP-14 genes. Further studies found that GALNT3 suppressed p38 phosphorylation.Conclusions:Our findings suggest for the first time that GALNT3 gene is involved in CAD. The GALNT3 mRNA levels were decreased in PBMCs from CAD patients and in HUVECs under hypoxia. Decreased GALNT3 expression in HUVECs might contribute to endothelial dysfunction by promoting apoptosis and up-regulating the expression of MMP-2 and MMP-14 genes. Furthermore, the effect of GALNT3 on HUVECs appeared to be associated with its inhibition of p38 phosphorylation.Part 2:Hypertension Susceptibility SNP rs820430 Regulated the Expression of Blood Pressure Salt-sensitive Related SLC4A7 Gene Background and Objectives:Hypertension is a common, heritable cause of cardiovascular disease worldwide. Genome-wide association studies (GWASs) have identified multiple chromosomal regions associated with blood pressure (BP). Our previous meta-analysis of GWAS of blood pressure and hypertension detected a Chinese-specific variant rs820430 at 3p24.1 near SLC4A7 with systolic blood pressure (P=1.36×10-12). However, whether rs820430 is a functional variant is unknown. In this study, we evaluated the functionality of rs820430 using multiple experimental methods. Salt sensitivity of BP is an independent risk factor for cardiovascular morbidity, so we also examine the associations of five Na-coupled bicarbonate transports (NCBT) genes, containing SLC4A7 gene and BP responses to dietary sodium intervention.Methods:1. All of the 274 healthy subjects were genotyped for rs820430 using TaqMan assays. The relationship between rs820430 and SLC4A7 mRNA levels in peripheral blood mononuclear cells (PBMCs) was analyzed by real time PCR.2. Luciferase reporter assays were conducted to test allele specific transcriptional activation activity of rs820430. Electrophoretic mobility shift assay (EMSA), supershift-EMSA and CMP were conducted to determine the transcription factors specifically binding to different alleles, respectively in vitro and in vivo.3. A total of 1906 Han Chinese were participated in the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study, which received a 3-day baseline observation, and 7-day low-sodium intake (51.3 mmol per day), followed by a 7-day high-sodium intake (307.8 mmol per day) intervention. Nine BP measurements were obtained at baseline and each intervention using a random-zero sphygmomanometer. The mixed-effect model was used to assess the additive, dominant, codominant and rective associations between 76 tag single nucleotide polymorphisms (SNPs) in five NCBTs genes, including SLC4A4, SLC4A5, SLC4A7, SLC4A8 and SLC4A10, and salt-sensitivity phenotypes. The Bonferroni method was used to adjust for multiple testing in all analyses. These analyses were conducted using SAS statistical software.Results:1. In healthy subjects, SLC4A7 mRNA levels were found to be significantly higher in homozygotes of minor allele T in comparison with homozygotes of major allele C of rs820430.2. The luciferase reporter activity assay showed that transcriptional activity of rs820430-TT was 1.3 fold of rs820430-CC. EMSA demonstrated that T allele of rs820430 had greater binding affinity of nuclear protein than C allele. Supershift-EMSA and ChIP assay certified the cFOS binding to the SNPs with T allele specific binding affinity.3. SLC4A4 marker rs4254735 was significantly associated with diastolic BP (DBP) responses to low-sodium intervention in additive model. Carriers with allele A of rs4254735 had more significant reductions in DBP (-2.91 mmHg vs -0.40 mmHg, =5.05x10’4) in comparision with homozygotes of G allele. In addition, SLC4A7 marker rs13077400 was significantly associated with systolic BP (SBP) responses to low-sodium intervention both in codominant and rective model, for example, Mean SBP responses were AA+AG vs GG (-5.50 mmHg vs -0.57 mmHg, P=1.15×10-6)in rective model. Duing high-sodium intervention, BP responses to high-sodium intervention significantly increased with the number of minor C allele of SLC4A4 marker rs10022637 all in additive, dominant and codominant model. For example, among individuals with genotypes TT, CT and CC in additive model, mean SBP response was 4.62 (4.29,4.99),5.94 (5.31,6.58) and 6.00 (3.57,8.43) mmHg (P=1.14×10-4), mean DBP responses were 1.72(1.41,2.03),3.22(2.52,3.92) and 3.94 (1.88,5.99) mmHg (P=2.26×10-5), and mean arterial pressure (MAP) responses were 2.69 (2.40,2.97),4.13 (3.57,4.70) and 4.61 (2.51,6.71) mmHg (P=2.07×10-6), respectively, during high-sodium intervention.Conclusions:We presented evidences that the hypertension risk locus containing rs820430 has a transcription enhancer property in vitro and in vivo. We propose that this risk locus act as part of a cis-regulatory enhancer element for the SLC4A7, mediating hypertension in part through its allelic differential binding with cFOS. Also we indicate that common variants of the SLC4A4 and SLC4A7 gene may contribute to the variation of BP response to dietary sodium intake in Han Chinese. Further studies are warranted to identify functional variants.