The Genetic Association Of ORMDL3 Gene With Atherosclerosis And The Mechanisms Involved

BackgroundAtherosclerosis (AS) is uniformly conceived as a lipid-induced chronic inflammatory reaction of arterial wall. The complications of AS, such as cardiovascular and cerebrovascular diseases, are amongst the most prevalent causes of mortality and morbidity worldwide. As well, AS represents the common pathological basis for coronary artery disease (CAD), stroke, aneurysm and peripheral artery disease.AS is a multifactorial disease in which environmental and genetic factors orchestrate a complex interplay in disease pathogenesis. The pathogenesis of AS remains largely undermined and the detailed elucidation of its genetic basis is just in the preliminary phase. With the aid of genome-wide association study (GWAS) more than fifty susceptibility genes have been noted associated with AS. However, most findings await robust verification and functional investigation in the future. In 2007, one GWAS verified ORMDL3 gene associated with bronchial asthma risk and this finding has been subsequently replicated in multiple ethnicities worldwide. Then ORMDL3 gene has also been found associated with several other immune-mediated chronic inflammatory diseases that include inflammatory bowel disease (IBD) and type I diabetes. Besides, accumulating evidence has suggested the functional relevance of ORMDL3 particularly in endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), inflammation and lipid metabolism-mechanisms also closely involved in the pathogenesis of AS. Given that chronic inflammatory disorders possess genetic overlaps and mechanistic similarities, it is tempting to explore whether ORMDL3 might contribute to AS susceptibility and the underlying mechanism involved in it.For complex diseases, subsequent to the detection of statistical correlation of susceptibility genes by genetic association studies, deciphering the major regulatory networks involved still remains a challenging issue. Autophagy refers to an evolutionarily conserved catabolic process mediating lysosomal degradation of intracellular constitutes for cellular hemostasis maintenance.ORMDL3 promotes ERS and UPR, exacerbates airway inflammation in the context of asthma and dysregulates ceramide metabolism. Interestingly, potential factors that include ER stress, inflammation and dyslipidemia stimulate autophagy, suggesting a potential role of ORMDL3 in autophagy regulation. Evidence is accumulating that autophagy deregulation participates centrally in multiple pathologies such as tumorigenesis, AS, viral infection, neurodegeneration and autoimmune diseases. How autophagy mediates atherogenesis is indeed to a large extent elusive due to the technical limitations for detection and inherent complexity of this process. Mostly likely in AS autophagy represents a protective mechanism against inflammation, oxidative stress and apoptosis, with evidence also supporting its detrimental effects meanwhile. Hence, it is of significance to confirm novel autophagy-associated genes to unveil the essence of autophagy and its role in complex diseases. Besides, accurate observation and exquisite control of autophagy is potentially applied as a promising intervening method in treating AS. Hence, it is reasonable to postulate the functional relevance of ORMDL3 gene as an important autophagy-associated gene in AS. Besides, the modulation of inflammation and apoptosis remains the hotspots in AS research. Given the relevance of the known functions of ORMDL3 with inflammation and ERS/UPR that potently promote apoptosis in AS, it is essential to investigate that whether ORMDL3 might confer AS predisposition potentially via the regulation of inflammation and apoptosis.In the present study, endothelial cells (ECs) were chosen for the investigation of the association of ORMDL3 gene with AS susceptibility and the mechanisms involved. Several questions arise as follow:first, whether or not ORMDL3 gene is statistically associated with AS susceptibility in Chinese Han population; second, whether or not ORMDL3 mediates EC autophagy in the context of AS; third, whether or not ORMDL3 has a role in EC apoptosis and inflammation in AS.Objectives1. To investigate whether or not two single nucleotide polymorphisms (SNPs) in 17q12 that harbors ORMDL3 gene are associated with AS risk in Chinese Han population; in the meanwhile, to explore the difference in ORMDL3 gene expression in peripheral blood mononuclear cells (PBMCs) between AS cases and healthy controls; to clarify the relationship of the genotypes of the 2 SNPs above-mentioned with gene expression in PBMCs.2. To confirm that oxidized low-density lipoprotein (ox-LDL) can modulate ORMDL3 gene expression and induce autophagy in ECs; and with the stimulation of ox-LDL and knockdown of ORMDL3 gene, to elucidate the role of ORMDL3 in ox-LDL-induced autophagy in ECs.3. With the treatment of ox-LDL and silencing of ORMDL3 gene, to verify the effects of ORMDL3 in ox-LDL-induced apoptosis and inflammation in ECs.Material and Methods1. In genetic association study, CAD cases and healthy controls from Chinese Han population were recruited. PBMCs were collected from the samples and DNA was extracted for genotyping two SNPs (rs7216389 and rs9303277). In the meantime, total RNA and protein were extracted from PBMCs and underwent real-time PCR and immunoblot to determine the differences in the gene expression at the mRNA and protein levels between cases and controls. Besides, the differences in the gene expression between carriers of different genotypes were also explored at the mRNA level.2. Human umbilical vein endothelial cell (HUVEC) and human aortic endothelial cell (HAEC) were selected for investigation. Real-time PCR and immunoblot was applied for measuring the mRNA and protein levels of ORMDL3 gene and autophagy markers including MAPLC3B, SQSTM1/P62 and BECN1, and immunofluorescence assay for observing the formation of LC3 puncta. With the stimulation of ox-LDL and short hairpin RNA (shRNA)-mediated knockdown of ORMDL3 gene, the responsiveness of this gene expression to ox-LDL and its effects on ox-LDL-induced autophagy in ECs were explored.3. HUVEC was chosen for investigation in which immunoblot was used for determining the protein levels of apoptotic markers including Cleaved Caspase 3 and Cleaved PARP, flow cytometry for measuring the proportions of apoptotic cells, CCK-8 for determining the cell viability and real-time PCR for measuring the mRNA levels of inflammatory markers including TNF, IL-6 and IL-17A. With the stimulation of ox-LDL and shRNA-mediated knockdown of ORMDL3 gene, the effects of ORMDL3 on ox-LDL-induced apoptosis and inflammation in ECs were explored.ResultsPart One The association of ORMDL3 gene with atherosclerosis risk in Chinese Han populationIn order to explore the association of ORMDL3 genetic polymorphisms (rs7216389 and rs9303277) with AS risk in Chinese Han population, a genetic association study with case-control design was conducted in this section. We recruited a sum of 1829 cases and 1806 healthy controls from Chinese Northern Han and Chinese Southern Han populations and extracted the genomic DNA from PBMCs for genotyping. No significant difference was noted between the cases and controls in respect to age and gender.However, body mass index values, proportion of cigarette smoking, and triglyceride, total cholesterol and low-density lipoprotein cholesterol levels were notably higher whereas high-density lipoprotein cholesterol levels evidently lower in cases, compared to controls. The distributions of the 2 SNPs were in Hardy-Weinberg Equilibrium (HWE) HWE in cases and controls from both the northern and southern Chinese cohorts (P>0.05).In the northern Chinese cohort, the frequencies of rs7216389 T allele and rs9303277 C allele were higher in cases than in controls (rs7216389:71.77% versus 67.07%; rs9303277:68.50% versus 64.66%). Then we found that rs7216389 and rs9303277 were significantly associated with AS after adjustment for age, sex, BMI, serum lipid levels and tobacco smoking in the nouthern Chinese population(rs7216389:allelic P=0.003; rs9303277:allelic P=0.017). The subjects carrying the rs7216389 T allele and rs9303277 C allele showed an increased risk of AS (rs7216389:odds ratio [OR] 0.80,95% confidence interval [CI] 0.69-0.93; rs9303277:OR 0.84,95% CI 0.73-0.97).Similarly in the southern Chinese population, higher frequencies of rs7216389 T allele and rs9303277 C allele were observed in cases than in controls (rs7216389: 75.22% versus 71.38%; rs9303277:75.72% versus 72.28%) and the associations were replicated after adjustment for the covariates above-mentioned (rs7216389:allelic P=0.009; rs9303277:allelic P=0.022). Also the subjects carrying the rs7216389 T allele and rs9303277 C allele showed an increased risk of AS (rs7216389:OR 0.82, 95% CI 0.71-0.95; rs9303277:OR 0.84,95% CI 0.72-0.97).Subsequently, for examining the relationship between ORMDL3 genetic variants and gene expression, we extracted the total RNA of PBMCs from 89 cases. We observed that the mRNA expression of ORMDL3 gene was significantly increased in cases carrying the risk alleles of rs7216389 and rs9303277 in comparison with homozygotes carriers of reference alleles (rs7216389, P=0.012; rs9303277, P=0.004). Besides,ORMDL3 gene mRNA levels were significantly increased in cases compared to controls (P=0.003) and protein levels in accordance with the trend (P=0.034).Part Two ORMDL3 mediates endothelial autophagy in atherosclerosisStimulation with ox-LDL in vitro upregulated the mRNA and protein levels of ORMDL3 gene in a dose-dependent (0,50 and 100μg/ml for 12h) and a time-dependent manner (100μg/ml for 0,4,8 and 12 h). Next, we further explored the details of ox-LDL-induced autophagy in ECs.We demonstrated that in HUVECs and HAECs, ox-LDL incubation resulted in pronounced protein expression of autophagy markers MAPLC3B-II in a dose-dependent (0,50 and 100 μg/ml for 12h) and a time-dependent manner (100μg/ml for 0,4,8 and 12 h); ox-LDL also led to the accumulation of SQSTM1/P62; in HUVECs fluorescence microscopy showed the redistribution of LC3 to form puncta in ox-LDL-incubated HUVECs; and in HUVECs co-stimulation with ox-LDL and chloroquine (CQ,50μM) led to further accumulation of LC3-II and SQSTM1/P62. The above-mentioned results supported that ox-LDL could up-regulate autophagy in endothelial cells.To investigate whether or not ORMDL3 could mediate autophagy, we applied shRNA-mediated knockdown of ORMDL3 which has been proved to have high interference efficiency. We showed that shRNA-mediated silencing of ORMDL3 alleviated not only ox-LDL-induced autophagy but also basal autophagy. Besides, serum starvation.for 6h could efficiently induce autophagy in HUVEC and knockdown of ORMDL3 also suppressed serum starvation (6h)-induced autophagy in ECs. These results indicated that ORMDL3 might play a vital role in dyslipidemia-induced autophagy in AS and also basal and starvation-induced autophagy.BECN1 is a crucial modulator for autophagy. We noted that knockdown of ORMDL3 not only suppressed ox-LDL-induced BECN1 protein expression but also basal and starvation-induced BECN1 protein expression. This suggested that ORMDL3 might regulate autophagy via BECN1. However, no alteration in phosphorylated mTOR level was noted due to ORMDL3 knockdown, as was the level of phosphorylated AMP-activated protein kinase (AMPK).To sum up,ORMDL3 is implicated in an ox-LDL-ORMDL3-BECNl-autophagy regulatory pathway in AS. Additionally,ORMDL3 might play a role in both physiological and pathological autophagy.Part Three The effects of ORMDL3 on endothelial apoptosis and inflammation in atherosclerosisOx-LDL potently triggers apoptotic process in ECs. Our findings demonstrated that stimulation with ox-LDL (100μg/ml for 12h) led to enhanced protein levels of Cleaved Caspase 3 and Cleaved PARP. Under basal condition knockdown of ORMDL3 promoted Caspase 3 and PARP cleavage. With ox-LDL stimulation knockdown of ORMDL3 markedly exacerbated apoptosis, demonstrated by augmented Caspase 3 and PARP cleavage, cell viability impairment and apoptotic cell rates. Furthermore, ox-LDL is an effective inflammation inducer for ECs and here we presented the evidence that silencing ORMDL3 in part suppressed ox-LDL-induced mRNA expression of TNF and IL-17A, but not IL-6. In summary,ORMDL3 might function as an apoptosis and inflammation mediator in the pathogenesis of AS.Conclusions1. Two SNPs rs7216389 and rs9303277 in 17q12 that harbors ORMDL3 gene are associated with AS susceptibility and gene expression in Chinese Han population. Besides, the expression of ORMDL3 gene in CAD cases was significantly enhanced as compared to controls.2.ORMDL3 participates in not only ox-LDL-induced, but also basal and starvation-induced autophagy in ECs, which indicates that ORMLD3 might represent a vital autophagy-associated gene. However, the detailed mechanisms remain to be further clarified.3.ORMDL3 might possess anti-apoptotic property, but how it regulates apoptosis and its relationship with autophagy is largely elusive. Moreover,ORMDL3 is highly likely to be involved in inflammation in AS.