LASER Regulates Hepatic Cholesterol Homeostasis By HNF-1?-PCSK9 Pathway & Zonulin Facilitates Enteric Bacteria Permeation In Atherosclerosis

Part 1 LASER Regulate Cholesterol Homeostasis by HNF-1?-PCSK9 pathway LASERBackgroundAtherosclerotic cardiovascular disease(ASCVD)and stroke cause immense health and economic burdens worldwide.Elevated low-density lipoprotein(LDL)cholesterol level is a significant risk factor for cardiovascular diseases.Many factors,including the exercise behavior,dietary habits,disease status,and gene variants are all contribute to the variance of lipid levels.Genetic variations are heritable,non-modifiable risk factors and gene polymorphism study helps to identify new targets for cholesterol therapy.Large-scale genome-wide association studies analyses the naturally occurring genetic variations from unrelated individuals and have gained great research interest in recent years,which show that many single nucleotide polymorphisms loci are associated with blood lipids,accounting for ~10–12% of total trait variance,especially the loci in 11q12 region was closely correlated with lipid levels in humans.However,the dilemma,when trying to explain the GWAS results,is that many of these variants(43%)lie in intergenic regions and hence remain largely unexplained,moreover,some loci whose nearby genes are not annotated in classical lipid metabolism pathways.Recent studies revealed that gene regions,previously thought to be silent,can transcribe into a wide-range of functional transcripts namely as non-coding RNAs.Long(>200 nt)non-coding RNAs(lnc RNAs)are more likely to locate within disease or trait-associated regions from published GWAS catalog.For example,the chromosome 9p21 locus was highlighted as the strongest genetic susceptibility locus for atherosclerosis,type 2 diabetes,cancer and glaucoma.This region encodes a novel long non-coding RNA,ANRIL(antisense non coding RNA in the INK4 locus).ANRIL specifically binds to two polycomb proteins(CBX7 in PRC1 and SUZ12 in PRC2)and play a diverse role in gene regulation.Whether or not there is lnc RNA with effect on cholesterol metabolism is not known.We hypothesize that genes in lipid associated GWAS regions could also encode an lnc RNA and regulate lipid metabolism.ObjectivesTo investigate whether and how lnc RNAs regulate cholesterol homeostasis in hepatocytes.MethodsThe expression of LASER in peripheral mononuclear cells(PBMCs)was determined by q RT-PCR methods.RNA interference in cultured human liver hepatocytes(Hep G2)were used to investigate the role of lnc RNAs in cholesterol metabolism.The intracellular cholesterol levels were determined by enzymatic methods and Filipin stain.Microarrays were used to reveal the m RNA profiles involved in cholesterol metabolis m in Hep G2 cells treated with si RNA against LASER.The RNA-protein interaction was determined by RNA immunoprecipitation.The histone methylation status covering the promotor region of the HNF-1? gene was analyzed by Chromatin immunoprecipitation(Ch IP)methods.ResultsWe identified a novel long non-coding RNA in Lipid Associated Single nucleotide polymorphism g Ene Region(LASER)by bioinformatic analysis.We report that LASER is highly expressed in both hepatocytes and peripheral mononuclear cells(PBMCs).Clinical studies showed that LASER expression is positively related with that of cholesterol containing apolipoprotein levels.In particular,we found that LASER is positively correlated with plasma PCSK9 levels in statin free patients.si RNAs mediated knock down of LASER dramatically reduces intracellular cholesterol levels and affects the expression of genes involved in cholesterol metabolism.Transcriptome analyses show that knockdown of LASER affects the expression of genes involved in metabolic pathways.We found that HNF-1? and PCSK9 was reduced after LASER knock-down.Interestingly,the reduction of PCSK9 can be blocked by the treatment of berberine,a natural cholesterol-lowering compound which functions as a HNF-1? antagonist.Mechanistically,we found that LASER binds to LSD1(lysine-specific demethylase 1),a member of Co REST/REST complex,in nucleus.LASER knock-down interferes with LSD1 targeting to genomic loci,resulting in increased histone H3 lysine 4 di-methylation at the promoter regions of HNF-1? gene.Conversely,LSD1 knock-down abolished the effect of LASER on HNF-1? and PCSK9 expressions.Finally,we found that statin treatment increased LASER expression,accompanied with increased PCSK9 expression,suggesting a feedback regulation of cholesterol on LASER expression by activating the LXR receptors.This observation may partly explain the statin escape in clinics.ConclusionThese findings identified a novel lnc RNA in cholesterol homeostasis.Therapeutic targeting LASER might be an effective approach to augment the effect of statins on cholesterol levels in clinics.Part 2 Zonulin Facilitates Enteric Bacteria Permeation in AtherosclerosisBackgroundInspite of great advances in the prevention and treatment of ASCVD,it remains to be a major cause of death worldwide.The occurrence and development of ASCVD involves multiple factors of which inflammation activation plays an important role in the pathogenesis of atherosclerotic ASCVD.It is known that immune cells are not only involved into the pathogenesis of atherosclerosis,but also the major factor in initiating plaque vulnerability that subsequently leads to acute coronary syndrome.Besides immune cells,infectious agents have gained a growing research interest in recent decades.Epidemiological and experimental studies have shown a linkage between ASCVD and several pathogens,including Chlamydia pneumoniae,Helicobacter,and Cytomegalovirus.Intestinal microbiota comprise of trillions of typically non-pathogenic commensal organisms.However,several studies have shown that intestinal bacteria could influence cholesterol metabolism by gut flora-dependent metabolism of dietary phosphatidylcholine or carnitine and accelerate atherosclerosis.There is evidence that bacterial 16 S r RNA genes can be detected in as high as 95% of atherosclerotic plaque biopsies,among which Enterobacteriacea(e.g.Serratia sp.and Klebsiella sp.)are the most frequently found family,indicating that,besides an indirect effect,bacteria themselves might be directly engaged in the pathogenesis of ASCVD.The intestines harbor the largest number of bacterial colony in the body,which vastly outnumber the whole body cells.However,how intestinal bacteria get into the blood and reside in atherosclerotic plaques is not known.Intestinal epithelia forms a continuous barrier between the intestinal microbiota and the host.Intestinal intercellular tight junctions,which determine the IP and control the entrance of bacteria from intestine to host blood,are the key structures.As the major component of intercellular tight junction,zonulin,a recently discovered protein,originally found in the eukaryotic counterpart of Vibrio cholerae zonula occludens toxin,was identified as the major factor determining the degree of IP.Recent researches revealed that circulating zonulin levels are significantly elevated in patients with diabetes,polycystic ovary syndrome,obesity,nonalcoholic fatty liver disease,all of which are regarded as traditional risk factors of atherosclerosis.Our previous work have confirmed the presence of high diversity bacteria in blood samples from ASCVD patients by 16 S r RNA gene amplification,most of them(99.4%)belong to Enterobacteriaceaes.We hypothesize therefore,that zonulin might be engaged in the pathogenesis of ASCVD by controlling IP and facilitate intestinal bacteria translocation to the host blood.ObjectivesTo investigate whether zonulin could regulates intestinal permeability and facilitates enteric bacteria permeation in the pathogenesis of atherosclerosis.MethodsThe circulation zonulin levels in patients with coronary heart disease were determined with enzyme-linked immunosorbent assay.The human intestinal barrier model was established by culturing human colon adenocarcinoma-derived cells(Caco-2)to form a cells monolayer in transwell inserts.Pseudomonas fluorescens were cultured in brain heart infusion broth and bacterial suspension were applied to the apical surface of the Caco-2 cells.The medium was withdrawn from the lower and upper chambers of the transwell units and plated onto HE agar plates after bacterial exposure.Plates were incubated at 37°C for 24 hrs,at which time the appearance of bacterial colonies were recorded by photography.The Caco-2 cell monolayer morphology after bacteria exposure were stained and observed using a transmission electron microscope.The localization of zonulin in Caco-2 cell monolayer was examined by laser confocal microscope.ResultsThe plasma zonulin levels were significantly higher in ASCVD patients.Pseudomonas fluorescens exposure significantly increased zonulin expression a in a time dependent manner.The elevated zonulin increase IP and may facilitate enteric translocation by disassembling the tight junctions,which might explain the observed high diversity of bacterial 16 S r RNA genes in blood samples.ConclusionOur results revealed that the Pseudomonas fluorescens could induce zonulin secrection and faciliate enteric bacteria permeation in atherosclerosis.