PCSK9 Expression Research Articles

Diallyl disulfide improves lipid metabolism by inhibiting PCSK9 expression and increasing LDL uptake via PI3K/Akt-SREBP2 pathway in HepG2 cells

Background and aimDiallyl disulfide (DADS), a volatile sulfide extracted from garlic, has been suggested as a chemical of anti-atherosclerotic drugs, while its molecular mechanism for this benefit has not fully been understood. The aim of the present study was to investigate the effects of DADS on lipid metabolism and its potential mechanisms in HepG2 cells induced by lipopolysaccharides (LPS). Methods and resultsHepG2 cells were treated with LPS with or without different concentrations of DADS (0, 20, 40, 80, 160 μg/ml) for 24 h. The cell activity was detected by CCK8, and Dil-LDL uptake assay was used to examine the LDL uptake. Real-time PCR and Western blot were used to detect the expression of LDLR, PCSK9 SREBP2 and HMGCR. In addition, we examined the effect of the combination of DADS with atorvastatin on PCSK9 expression. The results showed that LPS significantly increased PCSK9 and SREBP2 expressions in a dose-dependent manner in HepG2 cells. DADS attenuated PCSK9, SREBP2 and HMGCR expressions and up-regulated the expression of LDLR. Moreover, DADS reversed the expressions of PCSK9, SREBP2, HMGCR and LDLR induced by LPS and DADS could promote the LDL uptake in HepG2 cells. Furthermore, DADS decreased the expression of PCSK9 by activating the PI3K/Akt-SREBP2 signal pathway. Notably, DADS could reduce PCSK9 expression induced by atorvastatin in HepG2 cells. ConclusionDADS could significantly attenuated PCSK9 expression in a dose-dependent manner induced by LPS and increased the LDLR expression in HepG2 cells, which was associated with the activation of PI3K/Akt-SREBP2 signaling pathway.

Abstract 1856: Effects of different ethanol elutefractions of Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang on expression of secretory proteins in esophageal carcinoma EC9706cell

Abstract Purpose: To investigate the effect of different concentration of ethanol elution of Chinese medicine prescriptions(Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang) on the proliferation, cell cycle and MMP1, MMP11, PCSK9, TREM2, STX1A secretory proteins expression in esophageal cancer EC9706 cells line, so as to explore the connotation of TCM syndrome pathogenesis and molecular biology. Methods: Water extracts of Qigesan, Tongyoutang, Shashenmaidong tang and Buqiyunpitang were made gradient elution and got different concentration of ethanol elution fractions by macroporous resin, ultrapure water, 30% ethanol, 50% ethanol, 70% ethanol, and anhydrous ethanol. MTT methods was used to screen the the above four prescriptions for inhibiting the proliferation of EC9706 cell. The effects of most obvious elution fractions of esophageal four prescriptions on the proliferation, cell cycle and secretory proteins expression were investigated by MTT assay, flow cytometry and Western blot analysis, respectively. Results: Different concentration of ethanol elution fractions of Qigesan, Tongyoutang, Shashenmaidong tang and Buqiyunpitang were got through macroporous resin D101 gradient elution with different concentrations of alcohol. 30% ethanol elution fraction of Qigesan, 50% ethanol elution fraction of Tongyoutang and Sashenmaidongtang, anhydrous ethanol elution fractions of Buqiyunpitang had obviously inhibitory effect on the proliferation of EC9706 cell in dose-dependent and time-dependent manner, changed morphology of EC9706 cell, which appeared as round with rough edges, karyopyknosis, and karyorrhexis. Ic50 values of Qigesan 30% ethanol elution fraction, Tongyoutang 50% ethanol elution fraction, Sashenmaidongtang 50% ethanol elution fraction, Buqiyunpitang anhydrous ethanol elution fractions for EC9706 were 825.09ug·ml-1,860.66 ug·ml-1, 270.43 ug·ml-1, 634.60 ug·ml-1, which all induced G0/G1 phase arrest in EC9706 cells. Qigesan 30% ethanol elution fractions could inhibit the expression of MMP1, MMP11, TREM2, IGFPB3; Tongyoutang 50% alcohol fractions could inhibit STX1A expression; Sashenmaidongtang 50% ethanol elution fractions could inhibit MMP1, PCSK9, TREM2, IGFPB3, STX1A expression; Buqiyunpitang anhydrous ethanol fractions could inhibit MMP1, MMP11, PCSK9, TREM2, IGFPB3 and STX1A expression. Conclusion: Qigesan 30% ethanol elution fractions, Tongyoutang and Sashenmaidongtang 50% ethanol elution fractions, Buqiyunpitang anhydrous ethanol elution fractions can inhibit the proliferation of EC9706 cell, which have close relationship with MMP1, MMP11, PCSK9, TREM2, IGFPB3, STX1A proteins expression. This study provides new basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine. Key words: esophageal carcinoma; Qigesan; Tongyoutang; Shashenmaidongtang; Buqiyunpitang; secretory protein Citation Format: Fuchun Si. Effects of different ethanol elutefractions of Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang on expression of secretory proteins in esophageal carcinoma EC9706cell [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1856.

Secretion of the epithelial sodium channel chaperone PCSK9 from the cortical collecting duct links sodium retention with hypercholesterolemia in nephrotic syndrome

The proprotein PCSK9 functions as a chaperone for the epithelial sodium channel in the cortical collecting duct (CCD), is highly expressed in the liver, and plays a significant role in the pathogenesis of hypercholesterolemia. Lower levels of PCSK9 expression also occur in the normal kidney and intestine. Here, we found increased PCSK9 expression in the CCD of biopsies of patients with primary glomerular disease and explored a possible relationship with hypercholesterolemia of nephrotic syndrome. Significantly elevated serum PCSK9 and cholesterol levels were noted in two models of focal and segmental glomerulosclerosis, the Rrm2b-/- mouse and the Buffalo/Mna rat. Increased expression of PCSK9 in the kidney occurred when liver expression was reduced in both models. The impact of reduced or increased PCSK9 in the CCD on hypercholesterolemia in nephrotic syndrome was next studied. Mice with selective deficiency of PCSK9 expression in the collecting duct failed to develop hypercholesterolemia after injection of nephrotoxic serum. Blocking epithelial sodium channel activity with Amiloride in Rrm2b-/- mice resulted in increased expression of its chaperone PCSK9 in the CCD, followed by elevated plasma levels and worsening hypercholesterolemia. Thus, our data suggest that PCSK9 in the kidney plays a role in the initiation of hypercholesterolemia in nephrotic syndrome and make a case for depletion of PCSK9 early in patients with nephrotic syndrome to prevent the development of hypercholesterolemia.

PCSK9 Deficiency Modulates Glucose Homeostasis and Insulin Secretion Via Pancreatic Ldl Receptors And Cholesterol Accumulation

Intracellular lipid accumulation contributes to β-cell dysfunction in type 2 diabetes. How PCSK9 interacts with lipid metabolism to alter insulin availability/resistance is not well studied. Genetic deficiency of PCSK9 mice was fed a high fat (HFD; 45% Kcal from fat) for 14 weeks, and the metabolic phenotype including glucose, insulin, GTT, ITT, and cholesterol was examined. In addition, the expression of PCSK9 and LDLR was were examined by RT-qPCR, and immunohistochemistry. We provide data supporting a role for PCSK9 in pancreatic islets. In HFD mice, PCSK9 deletion led to impaired glucose tolerance and decreased insulin secretion. Pancreatic islets of HFD PCSK9-/ mice showed impaired insulin production and increased LDLR mRNA. Cholesterol was higher in islets of both HFD-fed WT and PCSK9 KO mice compared to low-fat diet controls. Glucose intolerance in PCSK9 deficiency was reversed when the LDLR was also lacking. Further, islets isolated from PCSK-/-LDR-/- mice showed improved insulin secretion. Our findings support a tissue-specific role for PCSK9 in pancreatic cholesterol homeostasis such that PCSK deficiency may accentuate LDLR-dependent β-cell dysfunction.

Genetic and In Vitro Inhibition of PCSK9 and Calcific Aortic Valve Stenosis

The authors investigated whether PCSK9 inhibition could represent a therapeutic strategy in calcific aortic valve stenosis (CAVS). A meta-analysis of 10 studies was performed to determine the impact of the PCSK9 R46L variant on CAVS, and the authors found that CAVS was less prevalent in carriers of this variant (odds ratio: 0.80 [95% confidence interval: 0.70 to 0.91]; p=0.0011) compared with noncarriers. PCSK9 expression was higher in the aortic valves of patients CAVS compared with control patients. In human valve interstitials cells submitted to a pro-osteogenic medium, PCSK9 levels increased and a PCSK9 neutralizing antibody significantly reduced calcium accumulation.

Molecular mechanism linking a novel PCSK9 copy number variant to severe hypercholesterolemia

Background and aimsA 42 year-old male with premature atherosclerosis, severe dyslipidemia and resistance to treatment with high dose statin and a recommended dose of a PCSK9 inhibitor, was found to have a duplication of the PCSK9 gene. However, the clinical phenotype, which included a more than 15-fold elevation in circulating PCSK9, was unexpected given that he had one additional gene copy. MethodsHere we have carried out whole genome sequencing and transcriptional reporter assays to investigate the molecular mechanism leading to this unusual FH phenotype. ResultsThe PCSK9 duplication was found to contain the full coding sequence but with an 829 bp shorter 3′-untranslated region (UTR) sequence. All possible rearrangements include a head-to-head fusion between a completely duplicated PCSK9 and a chromosomal region, normally situated ~80 kb away, that includes HNF4 and USF1 binding sites that could promote transcription of the PCSK9 gene. Transcriptional reporter assays demonstrated that a construct harboring the HNF4 binding site significantly increased the promoter activity by 2.5-fold with a smaller effect noted for a USF1 construct. ConclusionsHere we describe, in a patient with resistant hypercholesterolemia, a novel PCSK9 gene rearrangement that enables upregulation of PCSK9 expression by allowing proximity to an active enhancer binding to HNF4A.

CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation.

CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)-based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).

SAT-576 Possible Involvement of Thyroid Function in PCSK9 Inhibitor Therapy

INTRODUCTIONProprotein convertase subtilisin kexin type 9 (PCSK9) inhibition is an effective strategy for lowering plasma LDL-cholesterol and enhancing the LDL-cholesterol lowering ability of statins. PCSK9, a serine protease that binds to the LDL receptor promoting its degradation, is an important regulator of LDL metabolism. In addition, LDL-cholesterol is also controlled by TSH and thyroid hormones via PCSK9. TSH has received increasing attention as being closely associated with increased LDL-cholesterol level and higher atherosclerotic risks. In vitro study, the effects of TSH on hepatic PCSK9 expression in HepG2 cells were reported (1). I here report a case of transient hyperthyroidism secondary to PCSK9 inhibitor therapy. This case highlights the involvement of thyroid function in PCSK9 Inhibitor therapy.CLINICAL CASEA 65-year-old man had a weight loss of 6 kg (13 lbs.) in 4 months, accompanied with fatigue. He had a past history of myocardial infarction and his LDL was 83 mg/dL by 2.5mg of rosuvastatin and heart rate was controlled by 10mg of carvedilol. Six months ago, he started a PCSK9 Inhibitor therapy with 140mg of evolocumab every 2 weeks for 6 weeks. He had no preceding viral illness and denied anterior neck pain or tenderness. His height was 1.53 m, weight 52.6 kg (115 lbs.), and body mass index (BMI) 22.46 kg/m2. His thyroid was not enlarged and non-tender without clear palpable thyroid nodules or neck lymph nodes. Hyperthyroidism was suspected and confirmed by thyroid function tests: TSH was less than 0.0005 μIU/mL (normal 0.35–4.94), and free T4 1.830 ng/dL (0.70–1.48). Graves’ disease was considered, and thyroid antibody tests performed. Thyroid peroxidase (TPO) antibody titer was less than 9 IU/mL (<9), and TSI 141% (<120%). To confirm the diagnosis of this hyperthyroid patient, Technetium-99m uptake and scan was done which showed uptake of 0.8% (0.5–7%). After careful observation for 2 months with 5mg of carvedilol, he turned asymptomatic and free T4 lowered to 1.480 ng/dL and TSH remained less than 0.0005 μIU/mL.CLINICAL LESSONSI here report a case of transient hyperthyroidism secondary to PCSK9 inhibitor therapy. There has been no report of hyperthyroidism induced by PCSK9 inhibitors. Immunological influence of anti-PCSK9 therapy on thyroid is unknown. In this case, the decrease of TSH due to hyperthyroidism was considered to reduce hepatic PCSK9 expression, leading to additive effect to PCSK9 inhibitor. PCSK9 inhibitors may modify the effects of hyperlipidemia treatment by causing changes in thyroid function. When using PCSK9 inhibitors, follow-up of thyroid function should be considered. This case highlights the involvement of thyroid function in PCSK9 inhibitor therapy.Reference(1) Gong, Y., Ma, Y., et al. Thyroid stimulating hormone exhibits the impact on LDLR/LDL-c via up-regulating hepatic PCSK9 expression. Metabolism. 2017;76;32–41

The systemic implication of novel non-statin therapies in cardiovascular diabetology: PCSK9 as a case model.

PCSK9, like other novel non-statin drugs were primarily developed to help patients achieve low-density lipoprotein cholesterol targets, especially in patients with dyslipidemia not achieving lipid goals with statins due to poor tolerance or inadequate response. PCSK9 inhibitors, in addition to modulating lipid metabolism, improve mortality outcomes in cardiovascular disease. These benefits are markedly pronounced in patients with type 2 diabetes mellitus. However, these benefits do not come without associated risk. Multiple trials, studies, and case reports have attempted to explain observed outcomes with PCSK9 expression and administration of PCSK9 inhibitors from multiple perspectives, such as their effects on insulin sensitivity and glucose tolerance, changes in renal physiology, thyroid physiology, vascular tone, intestinal regulation of lipids, and improved cardiovascular function. These agents represent an opportunity for physicians to exercise prudence by using appropriate clinical judgement when managing comorbidities in the hyperglycemic patient, a concept that extends to other novel non-statin drugs.

A stilbene dimer and flavonoids from the aerial parts of Chromolaena odorata with proprotein convertase subtilisin/kexin type 9 expression inhibitory activity.

Investigation of components of the chloroform-soluble and ethyl acetate-soluble extracts of the aerial parts of Chromolaena odorata L. selected by PCSK9 mRNA expression monitoring assay in HepG2 cells led to the isolation of a new stilbene dimer, (+)-8b-epi-ampelopsin A (1), and 30 known compounds (2-31). The structures of the isolates were established by interpretation of NMR spectroscopic data and the stereochemistry of the new stilbene (1) was proposed based on ECD and NMR calculations. Among the isolates, 1, 5,6,7,4'-tetramethoxyflavanone (6), 5,6,7,3',4'-pentamethoxyflavanone (7), acacetin (18), and uridine (21) were found to inhibit PCSK9 mRNA expression with IC50 values of 20.6, 21.4, 31.7, 15.0, and 13.7µM, respectively. Furthermore, the most abundant isolate among the selected compounds, 6, suppressed PCSK9 and low-density lipoprotein receptor protein expression in addition to downregulating the mRNA expression of HNF-1α.

Etiology of disrupted cholesterol metabolism in the liver of young rats submitted to obesogenic diets

The purpose of the study was to test the hypothesis that a short period (2 weeks) of a high fat (HFD) and a western (WD) diet leading to an increase in liver fat accumulation will disrupt key markers of liver cholesterol metabolism mainly targeting the LDL‐receptor (LDL‐R) pathway. Rats were submitted to either a standard (SD), HFD (60%) or a WD (40% fat + 35% sucrose) diet for 2 and 6 weeks. Body weight gain and body fat values were higher (p &lt; 0.01) in rats fed the HFD and WD for 6 weeks although food intakes in kcal/d were not different for all groups throughout the experimental period. Liver fat accumulation was significantly (P &lt; 0.01) higher in HFD and WD rats as compared to the SD diet for both feeding durations. On the other hand, liver fat accumulation was higher in WD rats (P &lt; 0.05) than HFD animals after two weeks of treatment. In addition, WD animals displayed the highest plasma cholesterol levels. After 2 and 6 weeks of treatment, hepatic gene expression of LDL‐R, Pcsk9 and SREBP‐2 was lower (p &lt; 0.01) in WD animals than in HFD and SD rats. In addition, mRNA levels of Lrp1 and Acat2 were lower (p &lt; 0.01) in WD rats as compared to those fed with SD. Gene expression of HMGCoA‐r, the main enzyme involved in the regulation of cholesterol synthesis, was lower in HFD (2 weeks) and even more so in rats fed the WD diet (2 and 6 weeks). The WD was associated with a higher (p &lt; 0.01) gene expression of markers involved in the mediation of de novo lipogenesis (ACC and ChREBP). It is concluded that a short‐term WD promptly increased fat accumulation while disrupting cholesterol metabolism in the liver. The present results provide new insights on the mechanisms through which westernized diets rapidly promote increases in plasma cholesterol levels.Support or Funding InformationNSERC, FRQS, CFI

Maternal diet intervention before pregnancy primes offspring lipid metabolism in liver

Nonalcoholic fatty liver disease (NAFLD) has a developmental origin and is influenced in utero. We aimed to evaluate if maternal diet intervention before pregnancy would be beneficial to reduce the risk of offspring NAFLD. In our study, female mice were either on a normal-fat diet (NF group), or a high-fat diet for 12 weeks and continued on this diet throughout pregnancy and lactation (HF group), or switched from HF-to-NF diet 1 week (H1N group), or 9 weeks (H9N group) before pregnancy. Compared with the NF offspring, the H1N and HF, but not the H9N offspring, displayed more severe hepatic steatosis and glucose intolerance. More specifically, an abnormal blood lipid panel was seen in the H1N offspring and abnormal hepatic free fatty acid composition was present in both the HF and H1N offspring, while the H9N offspring displayed both at normal levels. These physiological changes were associated with desensitized hepatic insulin/AKT signaling, increased expression of genes and proteins for de novo lipogenesis and cholesterol synthesis, decreased expression of genes and proteins for fatty acid oxidation, increased Pcsk9 expression, and hypoactivation of 5′ AMP-activated protein kinase (AMPK) signaling in the HF and H1N offspring. However, these effects were completely or partially rescued in the H9N offspring. In summary, we found that early maternal diet intervention is effective in reducing the risk of offspring NAFLD caused by maternal HF diet. These findings provide significant support to develop effective diet intervention strategies and policies for prevention of obesity and NAFLD to promote optimal health outcomes for mothers and children.

A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α.

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates hepatic low-density lipoprotein receptor (LDLR) by binding and shuttling LDLR to lysosomes for degradation. The development of therapy that inhibits PCSK9 has attracted considerable attention for the management of cardiovascular disease risk. However, only monoclonal antibodies of PCSK9 have reached the clinic use. Oral administration of small-molecule transcriptional inhibitors has the potential to become a therapeutic option.MethodsHere, we developed a cell-based small molecule screening platform to identify transcriptional inhibitors of PCSK9. Through high-throughput screening and a series of evaluation, we found several active compounds. After detailed investigation on the pharmacological effect and molecular mechanistic characterization, 7030B-C5 was identified as a potential small-molecule PCSK9 inhibitor.FindingsOur data showed that 7030B-C5 down-regulated PCSK9 expression and increased the total cellular LDLR protein and its mediated LDL-C uptake by HepG2 cells. In both C57BL/6 J and ApoE KO mice, oral administration of 7030B-C5 reduced hepatic and plasma PCSK9 level and increased hepatic LDLR expression. Most importantly, 7030B-C5 inhibited lesions in en face aortas and aortic root in ApoE KO mice with a slight amelioration of lipid profiles. We further provide evidences suggesting that transcriptional regulation of PCSK9 by 7030B-C5 mostly depend on the transcriptional factor HNF1α and FoxO3. Furthermore, FoxO1 was found to play an important role in 7030B-C5 mediated integration of hepatic glucose and lipid metabolism.Interpretation7030B-C5 with potential suppressive effect of PCSK9 expression may serve as a promising lead compound for drug development of cholesterol/glucose homeostasis and cardiovascular disease therapy.FundThis work was supported by grants from the National Natural Science Foundation of China (81473214, 81402929, and 81621064), the Drug Innovation Major Project of China (2018ZX09711001-003-006, 2018ZX09711001-007 and 2018ZX09735001-002), CAMS Innovation Fund for Medical Sciences (2016-I2M-2-002, 2016-I2M-1-011 and 2017-I2M-1-008), Beijing Natural Science Foundation (7162129).

A New Liver Expression Quantitative Trait Locus Map From 1,183 Individuals Provides Evidence for Novel Expression Quantitative Trait Loci of Drug Response, Metabolic, and Sex-Biased Phenotypes.

Expression quantitative trait locus (eQTL) studies in human liver are crucial for elucidating how genetic variation influences variability in disease risk and therapeutic outcomes and may help guide strategies to obtain maximal efficacy and safety of clinical interventions. Associations between expression microarray and genome-wide genotype data from four human liver eQTL studies (n=1,183) were analyzed. More than 2.3 million cis-eQTLs for 15,668 genes were identified. When eQTLs were filtered against a list of 1,496 drug response genes, 187,829 cis-eQTLs for 1,191 genes were identified. Additionally, 1,683 sex-biased cis-eQTLs were identified, as well as 49 and 73 cis-eQTLs that colocalized with genome-wide association study signals for blood metabolite or lipid levels, respectively. Translational relevance of these results is evidenced by linking DPYD eQTLs to differences in safety of chemotherapy, linking the sex-biased regulation of PCSK9 expression to anti-lipid therapy, and identifying the G-protein coupled receptor GPR180 as a novel drug target for hypertriglyceridemia.

The atheroprotective roles of heart-protecting musk pills against atherosclerosis development in apolipoprotein E-deficient mice.

Heart-protecting musk pill (HMP), derived from Chinese herbal medicines, has been found to possess protective roles against atherosclerosis-related cardiovascular diseases (CVDs), however, the anti-atherosclerotic mechanisms of HMP are still unclear. Here, we investigated the effects of HMP on alleviating atherosclerotic lesion severity in mice and explored the molecular mechanisms. Apolipoprotein E-deficient mice were fed western-type diet supplemented with HMP (25 mg/kg/day) or normal saline gavage for 20 weeks. Then histopathological staining was performed to assess the atheromatous plaque burden. Biochemical kits were used to detect levels of lipid profiles. Moreover, effector factors associated with lipid metabolism in liver and intestinal tissues were investigated by western blot and real-time PCR assays. Levels of signal molecules participating in the mitochondrial-mediated apoptosis pathway were detected by Western blot. We found that HMP notably reduced atherosclerotic lesion size (P<0.05) and improved plaque stability (P<0.05). HMP treatment decreased circulating TC (P<0.01), LDL-C (P<0.01) and TG (P<0.05) levels and increased HDL-C (P<0.05) content. HMP was found to suppress SREBP2, HMGCR and PCSK9 expressions (P<0.05), yet promote LDLR expression (P<0.05) in hepatocytes. Moreover, HMP was discovered to activate PPARα/CPT-1A cascade (P<0.05) and inhibit contents of SREBP1c and the lipogenic genes FAS and ACCα (P<0.05). The LBK1/AMPK cascade was also activated after HMP administration (P<0.05). Additionally, HMP was found to facilitate transintestinal cholesterol excretion by increasing ABCG5 and ABCG8 levels and reducing NPC1L1 content (P<0.05). In terms of vasoprotective activities, we observed that HMP decreased cleaved caspase-3 content (P<0.05) in the vascular intima, which might be due to inhibition of mitochondrial-related signaling pathway. Altogether, our study indicates that HMP plays anti-atherosclerotic roles via regulating lipid metabolism and improving vascular intimal injury.

P4667Loss of function in PCSK9, atherogenic lipoprotein concentrations, and calcific aortic valve stenosis

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition reduces plasma concentrations of most atherogenic lipoproteins such as low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (apoB) and lipoprotein(a) [Lp(a)]. Atherogenic lipoprotein concentrations have also been linked with calcific aortic valve stenosis (CAVS). Purpose 1) To determine the association between genetic variants in PCSK9 and lipoprotein-lipid levels, 2) to determine whether loss of function (LOF) in PCSK9 is associated with CAVS and 3) to evaluate if PCSK9 could be implicated in aortic valve interstitial cells (VICs) calcification. Methods We built a weighted genetic risk score (wGRS) using 10 single nucleotide polymorphisms at the PCSK9 locus associated with LDL-C in the Global Lipids Genetics Consortium. We determined the association between the wGRS and LDL-C, apoB and Lp(a)] in 9692 participants of the EPIC-Norfolk study using linear regression. We investigated the association between the LOF PCSK9 R46L variant and CAVS risk in a meta-analysis of published (three Copenhagen studies, 1463 cases and 101,620 controls) and unpublished studies (UK Biobank, 1350 cases and 349,043 controls, Malmö Diet and Cancer study, 682 cases and 5963 controls and EPIC-Norfolk, 508 cases and 20,421 controls) prospective, population-based studies using logistic regression adjusted for age and sex. We evaluated PCSK9 expression and localization in explanted aortic valves by capillary Western blot and immunohistochemistry in patients with and without CAVS. Von Kossa staining was used to visualize aortic leaflet calcium deposits. We also assessed VICs calcification potential under oxidative stress condition. Results In EPIC-Norfolk, the wGRS was significantly associated with TC, LDL-C, and apoB (all p&lt;0.0001), but not with VLDL-C, HDL-C, triglycerides apoA-I, or Lp(a). Carriers of the R46L variant were at lower CAVS risk (odds ratio=0.71 (95% CI, 0.57–0.88, p&lt;0.001)). Aortic valves of patients with aortic sclerosis (n=12) and CAVS (n=8) presented elevated PCSK9 levels (log2 fold change [FC]=+28.6±5.1, p=0.008 and FC=+39.3±15.2, p=0.02, respectively) compared to controls (n=4).In calcified leaflets, PCSK9 expression co-localized with calcium deposits. PCSK9 expression in VICs was induced by oxidative stress (FC=+2.3±0.4, p=0.02), and subsequent increment in calcification potential was observed. Conclusion PCSK9LOF variants are associated with lifelong reductions in non-Lp(a) apoB-containing lipoprotein levels and a lower risk of coronary artery disease and CAVS. PCSK9 is abundant in fibrotic and calcified aortic leaflets. Oxidative stress increases PCSK9 expression in VICs. These results support randomized clinical trials of PCSK9 inhibition in the prevention of CAVS.

A Genetic Approach to the Association Between PCSK9 and Sepsis

Whether the PCSK9 gene is associated with the progress from infection to sepsis is unknown to date. To test the associations between PCSK9 genetic variants, a PCSK9 genetic risk score (GRS), or genetically estimated PCSK9 expression levels and the risk of sepsis among patients admitted to a hospital with infection. This retrospective cohort study used deidentified electronic health records to identify patients admitted to Vanderbilt University Medical Center, Nashville, Tennessee, with infection. Patients were white adults, had a code indicating infection from the International Classification of Diseases, Ninth Revision, Clinical Modification, or the International Statistical Classification of Diseases, Tenth Revision, Clinical Modification, and received an antibiotic within 1 day of hospital admission (N = 61 502). Data were collected from January 1, 1993, through December 31, 2017, and analyzed from April 1, 2018, to March 16, 2019. Four known PCSK9 functional variants, a GRS for PCSK9, and genetically estimated PCSK9 expression. The primary outcome was sepsis; secondary outcomes included cardiovascular failure and in-hospital death. Of patients with infection, genotype information was available in 10 922 white patients for PCSK9 functional variants (5628 men [51.5%]; mean [SD] age, 60.1 [15.7] years), including 7624 patients with PCSK9 GRS and 6033 patients with estimated PCSK9 expression. Of these, 3391 developed sepsis, 835 developed cardiovascular failure, and 366 died during hospitalization. None of the 4 functional PCSK9 variants were significantly associated with sepsis, cardiovascular failure, or in-hospital death, with or without adjustment for (1) age and sex or (2) age, sex, and Charlson-Deyo comorbidities (in model adjusted for age, sex, and comorbidities, odds ratios for any loss-of function variant were 0.96 [95% CI, 0.88-1.04] for sepsis, 1.05 [95% CI, 0.90-1.22] for cardiovascular failure, and 0.89 [95% CI, 0.72-1.11] for death). Similarly, neither the PCSK9 GRS nor genetically estimated PCSK9 expression were significantly associated with sepsis, cardiovascular failure, or in-hospital death in any of the analysis models. For GRS, in the full model adjusted for age, sex, and comorbidities, the odds ratios were 1.01 for sepsis (95% CI, 0.96-1.06; P = .70), 1.03 for cardiovascular failure (95% CI, 0.95-1.12; P = .48), and 1.05 for in-hospital death (95% CI, 0.92-1.19; P = .50). For genetically estimated PCSK9 expression, in the full model adjusted for age, sex, and comorbidities, the odds ratios were 1.01 for sepsis (95% CI, 0.95-1.06; P = .86), 0.96 for cardiovascular failure (95% CI, 0.88-1.05; P = .41), and 0.99 for in-hospital death (95% CI, 0.87-1.14; P = .94). In this study, PCSK9 genetic variants were not significantly associated with risk of sepsis or the outcomes of sepsis in patients hospitalized with infection.

Effect of atorvastatin and rosuvastatin on the PCSK9 blood concentration in statin-naive patients with atherosclerosis

Statins are currently the main group of lipid-lowering drugs used in clinical practice. However not all patients manage to achieve ‘target’ levels of LDL. A possible cause is statin-induced increase of expression of hepatocyte PCSK9 which regulates LDL uptake. Aim. The aim of the study was to determine whether the changes of parameters of lipid spectrum depend on the changes of PCSK9 concentration in blood during treatment with rosuvastatin or atorvastatin. Material and methods . 20 patients (9 men), median age 63 (57;72) years referred for the examination at NMRCC with coronary and carotid atherosclerosis and with the indications for statin therapy were included. In 9 patients the therapy with atorvastatin (40-80 mg/day) was initiated. 11 patients started taking rosuvastatin (10-40 mg/day). The parameters of lipid spectrum, Lp(a) and PCSK9 blood levels were determined at baseline and after 1 month after the initiation of statin therapy. Results. Atorvastatin and rosuvastatin intake were accompanied by the decrease in the levels of total cholesterol, triglycerides, LDL. The level of Lp(a) did not change. In patients receiving rosuvastatin a significant increase in the PCSK9 serum concentration was achieved while atorvastatin therapy was associated with much less pronounced and not statistically significant increase in PCSK9 levels. Conclusion. We suggest that the lipid-lowering activity of statins does not depend on their effect on PCSK9 expression. The limitation of this study is the small sample size and short observation time.

Maternal Diet Transition Before Pregnancy Plays a Transgenerational Effect on Offspring Lipid Homeostasis in Liver (P11-143-19)

ObjectivesIt is now clear that non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, has a developmental origin. Thus, we aims on to evaluate if a maternal diet intervention before pregnancy would be beneficial to reduce the risk for offspring NAFLD. MethodsIn our study, female mice were either on an NF diet (NF group), or a HF diet (HF group), for 9 weeks and continued on such diet through pregnancy and gestation, or switched from the 9-week HF to an NF diet for 1 week (H1N group) or 9 weeks (H9N group) before pregnancy. After weaning, the male offspring were given the HF diet for 12 weeks to promote NAFLD. ResultsComparing to the NF offspring, the H1N and HF but not the H9N offspring, displayed more severe hepatic steatosis and glucose intolerance. More specifically, the H1N and HF offspring had abnormal blood lipid panel and abnormal free fatty acid composition in liver, while the H9N offspring displayed normal. These physiological changes were associated with a desensitized hepatic insulin/AKT signaling, an increased expression of genes and proteins for de novo lipogenesis, a decreased expression of genes and proteins for fatty acid oxidation, increased Pcsk9 expression and the AMPK signaling hypoactivation in the HF and H1N offspring. However, these effects were either completely or partially avoided in the H9N offspring. ConclusionsIn summary, we showed that an early maternal diet intervention is efficient to reduce the risk for offspring NAFLD caused by the maternal HF diet. These information will provide significant support for setting up guidelines that women at childbearing age could follow to select the ideal diets, prior to and during pregnancy, which not only immediately benefit the pregnant outcome but also give a long-term beneficial effect on offspring health. Funding SourcesThis project is supported by grants from the National Institutes of Health (NIDDK 1R01DK112368-01 to LX and KZ) and the USDA National Institute of Food and Agriculture, [Hatch] project [1010406] to LX.

Regulation of Cholesterol Homeostasis by a Novel Long Non-coding RNA LASER

Genome-wide association studies (GWAS) have identified many genetic variants in genes related to lipid metabolism. However, how these variations affect lipid levels remains elusive. Long non-coding RNAs (lncRNAs) have been implicated in a variety of biological processes. We hypothesize lncRNAs are likely to be located within disease or trait-associated DNA regions to regulate lipid metabolism. The aim of this study was to investigate whether and how lncRNAs in lipid- associated DNA regions regulate cholesterol homeostasis in hepatocytes. In this study, we identified a novel long non-coding RNA in Lipid Associated Single nucleotide polymorphism gEne 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. siRNAs 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 metabolism pathways. We found that HNF-1α and PCSK9 were 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-1α antagonist. Mechanistically, we found that LASER binds to LSD1 (lysine-specific demethylase 1), a member of CoREST/REST complex, in nucleus. LASER knock-down enhance LSD1 targeting to genomic loci, resulting in decreased histone H3 lysine 4 mono-methylation at the promoter regions of HNF-1α gene. Conversely, LSD1 knock-down abolished the effect of LASER on HNF-1α 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. This observation may partly explain the statin escape during anti-cholesterol treatment. These findings identified a novel lncRNA in cholesterol homeostasis. Therapeutic targeting LASER might be an effective approach to augment the effect of statins on cholesterol levels in clinics.