Low Density Lipoprotein Receptor Pathway Research Articles

Possible Pivotal Role of Latent Chronic T. gondii Infection in the Pathogenesis of Atherosclerosis

Cardiovascular diseases such as atherosclerosis, atherothrombosis, coronary artery disease and stroke are the main causes of death worldwide . Atherosclerosis is a chronic, progressive immunoinflammatory and fibroproliferative disease of medium and large sized arteries with increased blood lipoprotein/cholesterol and their disposition in the arterial wall, important T H 1 type proinflammatory reaction, and thrombogenic status. Elevated plasma levels of proinflammatory cytokines have been demonstrated in patients with established atherosclerotic disease, and therefore it is believed that cytokines are key players in all stages of disease and have a profound influence on the pathogenesis of the disease. Several authors suggested that the number of different pathogens, including T. gondii , may promote synergistic inflammatory responses that are capable of triggering and exacerbating atherosclerotic process. Latent chronic T. gondii infection may be a frequent cause of vascular endothelial cell dysfunction because this pathogen attacks all nucleated cells, and endothelium cells have enhanced susceptibility to infection with T. gondii tachyzoites. Oxidative stress characteristic for atherosclerosis may be caused by the host infection with the parasite. T. gondii is unable to synthesize sterol and acquires cholesterol from the host LDL receptor pathway. The accumulation of foamy transformed macrophages in the aortic intima characteristic for atherosclerotic lesion is due to acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) increased expression in macrophages and other cells infected with T. gondii . ACAT2 is crucial in foam cell formation and development of atherosclerosis. The parasite also expresses two cholesteryl ester(CE)-synthesizing enzymes Tg ACAT1 and Tg ACAT2 that contribute to the CEs formation for storage in lipid bodies, but at the same time ACAT and CEs play a crucial role in replication of the pathogen. It must be emphasized that the increased expression of ACAT1 and ACAT2 normally present in macrophages may thus be further enhanced by the superimposing Tg ACAT1 and Tg ACA2 activities available in the host cells infected with T. gondii . In addition, p roinflammatory cytokines increase foam cell formation and latent chronic T. gondii infection persistently generates these biomediators and thus play an important role in foam cell biogenesis. P atients with atherosclerosis had increased plasma levels of TGF- b and this cytokine increased T. gondii replication in the host cells, and participated in development of fibrotic changes in atherosclerotic lesions. Atherogenesis involved platelets activation with subsequent serotonin release. Hyperserotoninemia was also reported in autistic and mentally retarded children, and recently, a significantly higher seroprevalence of chronic toxoplasmosis was found in autistic children as compared with controls. T. gondii infection caused also increased leptin levels, and a marked association between T gondii seropositivity and obesity has been demonstrated. Cysteine cathepsins (Cat) play an important role of in foam cell formation and generation of amyloid in atherosclerotic arteries. Infection of vascular endothelial cells with T. gondii tachyzoites provide an additional source of cysteine cathepsins superimposing on the enzymes normally present in host cells, because the parasite expresses few members of the cathepsins, such as CatL-like, CatB-like, and CatC-like proteases . Finally, vitamin D exerted beneficial effects in both atherosclerosis and T. gondii infection, especially that it improved host immunity and decreased proliferation of T. gondii tachyzoites in macrophages and reduced tissue pathology caused by the pathogen. All together, it seems that latent chronic toxoplasmosis play a pivotal but so far neglected role in the pathogenesis of atherosclerosis.

Lipid disorder and intrahepatic renin-angiotensin system activation synergistically contribute to non-alcoholic fatty liver disease.

This study aimed to investigate the possible synergistic effects of lipid disorder with renin-angiotensin system (RAS) activation in non-alcoholic fatty liver disease (NAFLD). Apolipoprotein E gene-knockout mice, angiotensin II (Ang II) type 1 receptor (AT1) gene-knockout mice and human hepatoblastoma cell line (HepG2) were used for experiments. Lipid accumulation was examined by Filipin staining and intracellular cholesterol quantitative assay. The gene and protein expression of molecules involved in RAS and low-density lipoprotein receptor (LDLr) pathway was examined by real-time PCR, immunofluorescent staining and Western blot. There was significantly increased expression of RAS components and extracellular matrix (ECM) in livers of high-fat-diet-fed apolipoprotein E gene-knockout mice compared with controls. Upregulation of RAS components was positively associated with increased plasma levels of lipid profile. The in vitro study further confirmed that cholesterol loading increased supernatant renin activity and Ang II level of HepG2 cells, accompanied by increased ECM production that was positively associated with increased expression of intracellular RAS components. Interestingly, Ang II treatment increased lipid accumulation in livers of C57BL/6 mice and HepG2 cells. Furthermore, Ang II treatment increased gene and protein expression of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), SREBP-2 and LDLr, which were mediated by enhanced SCAP/SREBP-2 complex translocation from endoplasmic reticulum to Golgi. However, LDLr pathway was accordingly downregulated in livers of AT1 gene-knockout C57BL/6 mice or in HepG2 cells treated by telmisartan. These findings demonstrate that lipid disorder and intrahepatic RAS activation synergistically accelerate NAFLD progression.

Abstract 394: ApoE3-containing High Density Lipoprotein Serves as a Multifunctional Platform for Delivery of Gold Nanoparticles for Hyperthermic Treatment

We have developed a novel HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties. We exploit the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor (LDLr) to gain entry into glioblastoma cells. The issue of poor aqueous solubility of AuNP was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing 3, 10, or 10 nm AuNP. UV-Vis spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm, a signature feature of AuNP. Transmission electron microscopy (TEM) revealed discoidal geometry for rHDL with 3, 10 and 17 nm particles. Co-immunoprecipitation assay using a soluble form of the LDLr indicated robust binding of rHDL-AuNP to the receptor. Immunofluorescence analysis indicated that all 3 rHDL particles were internalized by glioblastoma cells, as revealed by the presence of punctate, peri-nuclear endocytic/lysosomal vesicles; this suggests cellular uptake of rHDL-AuNP by receptor-mediated endocytosis. Cellular uptake was further confirmed by TEM, in which aggregated AuNP were found in the endosomal-lysosomal compartments. Finally, cytotoxicity studies demonstrated that more than 50% of cells were viable with rHDL-AuNP treatment of up to 0.1 mg/ml for 24 h. The findings that apoE3: (i) acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, and, (ii) facilitates cellular uptake of rHDL-AuNP by the LDLr pathway, are significant since they offer an effective means of delivering AuNP across the cell membrane. This is particularly relevant in tumor cells since they overexpress LDLr to meet the high demand for cholesterol that is required for rapid proliferation and membrane biogenesis.

Dysregulation of the Low-Density Lipoprotein Receptor Pathway Is Involved in Lipid Disorder-Mediated Organ Injury.

The low-density lipoprotein receptor (LDLR) pathway is a negative feedback system that plays important roles in the regulation of plasma and intracellular cholesterol homeostasis. To maintain a cholesterol homeostasis, LDLR expression is tightly regulated by sterol regulatory element-binding protein-2 (SREBP-2) and SREBP cleavage-activating protein (SCAP) in transcriptional level and by proprotein convertase subtilisin/kexin type 9 (PCSK9) in posttranscriptional level. The dysregulation of LDLR expression results in abnormal lipid accumulation in cells and tissues, such as vascular smooth muscle cells, hepatic cells, renal mesangial cells, renal tubular cells and podocytes. It has been demonstrated that inflammation, renin-angiotensin system (RAS) activation, and hyperglycemia induce the disruption of LDLR pathway, which might contribute to lipid disorder-mediated organ injury (atherosclerosis, non-alcoholic fatty liver disease, kidney fibrosis, etc). The mammalian target of rapamycin (mTOR) pathway is a critical mediator in the disruption of LDLR pathway caused by pathogenic factors. The mTOR complex1 activation upregulates LDLR expression at the transcriptional and posttranscriptional levels, consequently resulting in lipid deposition. This paper mainly reviews the mechanisms for the dysregulation of LDLR pathway and its roles in lipid disorder-mediated organ injury under various pathogenic conditions. Understanding these mechanisms leading to the abnormality of LDLR expression contributes to find potential new drug targets in lipid disorder-mediated diseases.

Lower the Low-density Lipoprotein Cholesterol to the Level When You Born

Lower the Low-density Lipoprotein Cholesterol to the Level When You Born

The Deubiquitylase USP2 Regulates the LDLR Pathway by Counteracting the E3-Ubiquitin Ligase IDOL.

The low-density lipoprotein (LDL) receptor (LDLR) is a central determinant of circulating LDL-cholesterol and as such subject to tight regulation. Recent studies and genetic evidence implicate the inducible degrader of the LDLR (IDOL) as a regulator of LDLR abundance and of circulating levels of LDL-cholesterol in humans. Acting as an E3-ubiquitin ligase, IDOL promotes ubiquitylation and subsequent lysosomal degradation of the LDLR. Consequently, inhibition of IDOL-mediated degradation of the LDLR represents a potential strategy to increase hepatic LDL-cholesterol clearance. To establish whether deubiquitylases counteract IDOL-mediated ubiquitylation and degradation of the LDLR. Using a genetic screening approach, we identify the ubiquitin-specific protease 2 (USP2) as a post-transcriptional regulator of IDOL-mediated LDLR degradation. We demonstrate that both USP2 isoforms, USP2-69 and USP2-45, interact with IDOL and promote its deubiquitylation. IDOL deubiquitylation requires USP2 enzymatic activity and leads to a marked stabilization of IDOL protein. Paradoxically, this also markedly attenuates IDOL-mediated degradation of the LDLR and the ability of IDOL to limit LDL uptake into cells. Conversely, loss of USP2 reduces LDLR protein in an IDOL-dependent manner and limits LDL uptake. We identify a tri-partite complex encompassing IDOL, USP2, and LDLR and demonstrate that in this context USP2 promotes deubiquitylation of the LDLR and prevents its degradation. Our findings identify USP2 as a novel regulator of lipoprotein clearance owing to its ability to control ubiquitylation-dependent degradation of the LDLR by IDOL.

Abstract 17868: Role of Lysosomal Acid Lipase in LDL-C Regulation in Familial Hypercholesterolemia and Metabolic Syndrome

Lysosomal acid lipase (LAL), coded by LIPA gene, is key role player of LDL receptor pathway, and LIPA is a susceptibility gene for CAD in many GWAS studies. LAL hydrolyzes cholesterol-ester in hepatocytes, and is essential for M2 activation of macrophages. Furthermore, LAL-deficiency, with increased LDL-C and premature CAD, has been focused as underdiagnosed disease similar to familial hypercholesterolemia (FH). On the other hand, little is known about the role of physiological LAL activity on atherosclerosis. We investigated LIPA gene as genetic cause of FH, and LAL activity in CAD high-risk group. Methods: This study consisted of two parts. First part was genetic analysis of LIPA gene. From 940 clinically diagnosed hetero-FH patients in Japan, pathogenic mutations were identified in 788 patients through screening in LDLR, PCSK9, and APOB genes. All coding regions of LIPA gene were investigated in 143 hetero-FH patients without pathogenic mutation in those genes. Identified variants were also evaluated in 200 genetically confirmed hetero-FH, 200 non-FH dyslipidemia, and 250 general population samples. Second part was LAL acclivity measurement with very recently developed fluorometric assay method using dried blood spots. A total of 122 CAD high-risk patients (M/F: 67/55, age: 65 ± 15 yrs, established CAD: 34%, DM: 46%, hetero-FH: 25%) were investigated their LAL activity. Results: Analysis in LIPA gene revealed no patients with LAL-deficiency among FH without confirmed mutations. All variants identified were considered as benign. Intriguingly, common LIPA T16P carriers showed lower pre-treatment LDL-C in genetically confirmed hetero-FH (LDL-C 215±61 vs. 242±70 mg/dL, p <0.05). In activity analysis, LAL activity correlated positively with BMI (R 2 : 10%, p <0.0005), γGTP (R 2 : 11%, p <0.0005), and HbA1c (R 2 : 5%, p <0.05), and negatively with LDL-C (R 2 : 4%, p <0.05). Exclusion of FH did not change these results. LAL might have a protective role in obesity, like metabolic syndrome does not necessarily reveal high LDL-C. Conclusion: LAL-deficiency was not common genetic cause of FH in Japan. LAL activity increased with obesity, and negatively correlated with LDL-C. Further study will clarify the role of LAL in atherosclerosis.

MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis

RationaleA strong risk factor for atherosclerosis– the leading cause of heart attacks and strokes– is the elevation of low-density lipoprotein cholesterol (LDL-C) in blood. The LDL receptor (LDLR) is the primary pathway for LDL-C removal from circulation, and their levels are increased by statins –the main treatment for high blood LDL-C. However, statins have low efficiency because they also increase PCSK9 which targets LDLR for degradation. Since microRNAs have recently emerged as key regulators of cholesterol homeostasis, our aim was to identify potential microRNA-based therapeutics to decrease blood LDL-C and prevent atherosclerosis. Methods and resultsWe over expressed and knocked down miR-27a in HepG2 cells to assess its effect on the expression of key players in the LDLR pathway using PCR Arrays, Elisas, and Western blots. We found that miR-27a decreases LDLR levels by 40% not only through a direct binding to its 3′ untranslated region but also indirectly by inducing a 3-fold increase in PCSK9, which enhances LDLR degradation. Interestingly, miR-27a also directly decreases LRP6 and LDLRAP1, two other key players in the LDLR pathway that are required for efficient endocytosis of the LDLR-LDL-C complex in the liver. The inhibition of miR-27a using lock nucleic acids induced a 70% increase in LDLR levels and, therefore, it would be a more efficient treatment for hypercholesterolemia because of its desirable effects not only on LDLR but also on PCSK9. ConclusionThe results presented here provide evidence supporting the potential of miR-27a as a novel therapeutic target for the prevention of atherosclerosis.

Role of PCSK9 beyond liver involvement.

Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9. The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons. Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors.

The life cycle of the low-density lipoprotein receptor: insights from cellular and in-vivo studies.

Long-term exposure to elevated concentrations of LDL cholesterol increases the risk of cardiovascular events. The main player in clearing LDL cholesterol is the LDL receptor (LDLR) trafficking pathway; however, our fundamental knowledge about the mechanisms regulating this pathway is still incomplete. The LDLR pathway is very complex and involves multiple proteins. Endocytosis is regulated by two different adaptor proteins, that is, autosomal recessive hypercholesterolemia and Disabled-2. The proteolysis of the LDLR is regulated by inducible degrader of the LDLR and proprotein convertase subtilisin/kexin type 9. However, only a few proteins have been identified that provide insights into the endosomal sorting and recycling of the LDLR. Since the discovery of LDLR, knowledge about its function has greatly expanded. As a result of its importance in maintaining homeostatic LDL levels, the LDLR pathway has emerged as a key therapeutic target to reduce circulating cholesterol. In order to be able to treat and diagnose individuals with hypercholesterolemia in the future, it is important to learn more about the LDLR trafficking pathway, as we still lack a full mechanistic understanding of how LDLR trafficking is controlled.

Clinical experience of scoring criteria for Familial Hypercholesterolaemia (FH) genetic testing in Wales

Background/ObjectiveFamilial Hypercholesterolaemia (FH) is caused by mutations in genes of the Low Density Lipoprotein (LDL) receptor pathway. A definitive diagnosis of FH can be made by the demonstration of a pathogenic mutation. The Wales FH service has developed scoring criteria to guide selection of patients for DNA testing, for those referred to clinics with hypercholesterolaemia. The criteria are based on a modification of the Dutch Lipid Clinic scoring criteria and utilise a combination of lipid values, physical signs, personal and family history of premature cardiovascular disease. They are intended to provide clinical guidance and enable resources to be targeted in a cost effective manner. Methods623 patients who presented to lipid clinics across Wales had DNA testing following application of these criteria. ResultsThe proportion of patients with a pathogenic mutation ranged from 4% in those scoring 5 or less up to 85% in those scoring 15 or more. LDL-cholesterol was the strongest discriminatory factor. Scores gained from physical signs, family history, coronary heart disease, and triglycerides also showed a gradient in mutation pick-up rate according to the score. ConclusionThese criteria provide a useful tool to guide selection of patients for DNA testing when applied by health professionals who have clinical experience of FH.

Ext1 heterozygosity causes a modest effect on postprandial lipid clearance in humans

Elevated nonfasting TG-rich lipoprotein levels are a risk factor for CVD. To further evaluate the relevance of LDL-receptor (LDLr) pathway and heparan sulfate proteoglycans (HSPGs) in TG homeostasis, we analyzed fasting and postprandial TG levels in mice bearing combined heterozygous mutations in both Exostosin (Ext) 1 and Ldlr, in subjects with hereditary multiple exostosis (HME) due to a heterozygous loss-of-function mutation in EXT1 or EXT2 (N = 13), and in patients with heterozygous mutations in LDLR [familial hypercholesterolemia (FH)] and SNPs in major HSPG-related genes (n = 22). Mice bearing a homozygous mutation in hepatic Ext1 exhibited elevated plasma TGs similar to mice lacking other key enzymes involved in HSPG assembly. Compound heterozygous mice lacking Ldlr and Ext1 showed synergy on plasma TG accumulation and postprandial clearance. In human subjects, a trend was observed in HME patients toward reduced postprandial TG clearance with a concomitant reduction in chylomicron clearance [area under the curve (AUC)-retinyl ester (RE) HME, 844 ± 127 vs. controls, 646 ± 119 nM/h, P = 0.09]. Moreover, in FH subjects with a high HSPG gene score, retinyl palmitate excursions were higher (AUC-RE, 2,377 ± 293 vs. 1,565 ± 181 nM/h, P < 0.05). Incremental AUC-apoB48 was similar between the groups. In conclusion, the data are supportive for a minor yet additive role of HSPG in human postprandial TG clearance, and further studies are warranted.

Activation of mTORC1 disrupted LDL receptor pathway: A potential new mechanism for the progression of non-alcoholic fatty liver disease

Our previous studies demonstrated that inflammation exacerbates the progression of non-alcoholic fatty liver disease (NAFLD) by disrupting cholesterol homeostasis. This study aimed to investigate the role of mammalian target of rapamycin complex 1 (mTORC1) in NAFLD under conditions of inflammation. Chronic inflammation was induced by using subcutaneous injections of 10% casein in apolipoprotein E knockout (ApoE KO) mice in vivo and interleukin-1β stimulation of the HepG2 hepatoblastoma cell line in vitro. Results demonstrated that inflammation increased lipid accumulation in HepG2 cells and in livers of apolipoprotein E knockout mice. These effects were correlated with an increase in low density lipoprotein receptor (LDLR) gene transcription, which was mediated through the up-regulation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), SREBP-2, and through enhanced translocation of the SCAP/SREBP-2 complex from endoplasmic reticulum (ER) to Golgi. In addition, our data indicated that inflammation down-regulated the expression of proprotein convertase subtilisin kexin 9 (PCSK9) and prevented the degradation of LDLR protein via posttranscriptional mechanisms. Further analysis showed that inflammation increased the protein phosphorylation of mTOR, eukaryotic initiation factor 4E-binding protein 1, and p70 S6 kinase. Interestingly, blocking mTORC1 activity inhibited the translocation of SCAP/SREBP-2 complex from the ER to the Golgi and decreased the expression of LDLR, SCAP, and SREBP-2. These effects were accompanied by an increase in the expression of PCSK9 and accelerated LDLR degradation. Our findings demonstrated that increased mTORC1 activity exacerbated the progression of NAFLD by disrupting LDLR expression via transcriptional and posttranscriptional mechanisms.

Hypercholesterolemia, low density lipoprotein receptor and proprotein convertase subtilisin/kexin-type 9.

Atherosclerotic cardiovascular disease is the main cause of mortality and morbidity in the world. Plasma levels of low density lipoprotein cholesterol (LDL-C) are positively correlated with the risk of atherosclerosis. High plasma LDL concentrations in patients with hypercholesterolemia lead to build-up of LDL in the inner walls of the arteries, which becomes oxidized and promotes the formation of foam cells, consequently initiating atherosclerosis. Plasma LDL is mainly cleared through the LDL receptor (LDLR) pathway. Mutations in the LDLR cause familiar hypercholesterolemia and increase the risk of premature coronary heart disease. The expression of LDLR is regulated at the transcriptional level via the sterol regulatory element binding protein 2 (SREBP-2) and at the posttranslational levels mainly through proprotein convertase subtilisin/kexin-type 9 (PCSK9) and inducible degrader of the LDLR (IDOL). In this review, we summarize the latest advances in the studies of PCSK9.

Activation of mTOR contributes to foam cell formation in the radial arteries of patients with end-stage renal disease.

Our previous in-vivo and in-vitro studies demonstrated that inflammation accelerated the progression of atherosclerosis via the dysregulation of the low-density lipoprotein receptor (LDLr) pathway. The current study aimed to investigate the effects and their underlying mechanisms of inflammation on lipid accumulation in the radial arteries of endstage renal disease (ESRD) patients with arteriovenostomy. 30 ESRD patients with arteriovenostomy were included. The patients were divided into two groups based on their plasma levels of C-reactive protein: a control (n = 16) and an inflamed group (n = 14). The expression of tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein-1 of the radial arteries were increased in the inflamed group. Foam cell formation and lipid droplet accumulation were examined by hematoxylin and eosin (H & E) and Oil Red O staining. Intracellular cholesterol trafficking-related proteins were examined by immunohistochemistry and immunofluorescent staining. There was significant lipid accumulation in the radial arteries of the inflamed group compared with the control. Further analysis demonstrated that this accumulation was correlated with the increased protein expression of LDLr, sterol regulatory element-binding protein-2 (SREBP-2), and SREBP cleavageactivating protein (SCAP). Confocal microscopy showed that inflammation enhanced the translocation of SCAP escorting SREBP-2 from the endoplasmic reticulum to the Golgi, thereby activating LDLr gene transcription. Interestingly, upregulated LDLr expression was positively associated with the increased protein expression of mammalian target of rapamycin (mTOR), which had enhanced coexpression with SREBP-2. This finding suggests that the activation of mTOR may be involved in LDLr pathway disruption through the upregulation of SREBP-2 expression. Inflammation contributed to foam cell formation in the radial arteries of ESRD patients via the dysregulation of the LDLr pathway, which could be modulated by the activation of the mTOR pathway.

Lipoprotein(a)

Lipoprotein(a)

Activation of mTOR modulates SREBP-2 to induce foam cell formation through increased retinoblastoma protein phosphorylation

Our previous studies demonstrated that inflammation contributes to atherosclerosis through disruption of the low density lipoprotein receptor (LDLr) pathway. However, this effect is overridden by rapamycin, which is an inhibitor of mammalian target of rapamycin (mTOR). This study investigated the role of the mTOR pathway in atherosclerosis in vivo and in vitro. To induce inflammation, we used subcutaneous injection of 10% casein in apolipoprotein E knockout (ApoE KO) mice and lipopolysaccharide stimulation in rat vascular smooth muscle cells (VSMCs). Results showed that inflammation increased lipid accumulation in aortas of ApoE KO mice and in VSMCs, which were correlated with increased expressions of LDLr, sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), and SREBP-2 as well as with enhanced translocation of SCAP/SREBP-2 complex from the endoplasmic reticulum (ER) to the Golgi. Furthermore, inflammation increased both the percentage of cells in the S phase of cell cycle and protein expressions of the phosphorylated forms of retinoblastoma tumour suppressor protein (Rb), mTOR, eukaryotic initiation factor 4E-binding protein 1 (4EBP1), and P70 S6 kinase. After treatment with rapamycin or mTOR siRNA, the activity of the mTOR pathway was blocked. Interestingly, the expression levels of LDLr, SCAP, and SREBP-2 and the translocation of SCAP/SREBP-2 complex from the ER to the Golgi in treated VSMCs were decreased even in the presence of inflammatory stress. Our findings demonstrate for the first time that inflammation disrupts LDLr feedback regulation through the activation of the mTOR pathway. Increased mTORC1 activity was found to up-regulate SREBP-2-mediated cholesterol uptake through Rb phosphorylation.

Correction: Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients

Correction: Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients

The extended abnormalities in lipoprotein metabolism in familial hypercholesterolemia: Developing a new framework for future therapies

Familial hypercholesterolemia (FH) is a dominantly inherited disorder characterized by marked elevation of plasma low-density lipoprotein (LDL) cholesterol concentrations and premature coronary artery disease (CHD). In addition to impaired LDL receptor-mediated clearance of LDL particles, in vitro and in vivo studies suggest that hepatic oversecretion of apolipoprotein (apo) B may contribute to the hypercholesterolemia in FH. This may be due to an effect of the expanded hepatic pool of cholesterol (a consequence of increased receptor-independent uptake of LDL) and/or a direct effect of the LDL receptor on apoB secretion. Hepatic oversecretion of apoB may depend on the type and severity of the genetic mutation causing FH. FH can also increase plasma Lp(a) concentration by an undefined mechanism that may not directly involve the LDL receptor pathway. Decreased catabolism of triglyceride-rich lipoproteins could also be due to deficient LDL receptor function, accounting for postprandial dyslipidemia in FH. The metabolism of high-density lipoprotein (HDL) in FH is poorly understood, but preliminary data suggest abnormal HDL composition and functionality, as well as altered transport of apoA-I. Beyond effects related to specific genetic defects in the LDL pathway, co-existing secondary causes, particularly obesity and insulin resistance, and other genetic variants may also perturb lipoprotein metabolism in individuals with FH. Furthermore, residual risk remains high in statin-treated FH. Knowledge of an extended metabolic framework will, therefore, provide the basis for judiciously selecting new pharmacotherapies to treat FH, including apoB antisense oligonucleotides, microsomal transfer protein (MTP) inhibitors and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors.

Abstract 63: Post-transcriptional Control of the LDLR Pathway by Cholesterol-responsive microRNAs.

Despite the success of statins to reduce levels of LDL-cholesterol (LDL-C), cardiovascular disease remains the leading cause of mortality in westernized countries. PCSK9 inhibition is emerging as an exciting new therapy to further lower LDL and holds tremendous promise to reduce the significant residual cardiovascular risk in many individuals. Recent advances from our group and others have shown that microRNAs (miRNAs) have emerged as critical components in the regulation of cholesterol metabolism. In addition to miR-33, which we discovered as a key regulator of metabolic programs including cholesterol and fatty acid homeostasis, we identified two new cholesterol responsive miRNAs, miR-520d-5p (miR-520d) and miR-224. Here we show that miR-520d and miR-224 target the 3’UTR of both PCSK9 and Idol, “accessory” proteins that regulate cell surface expression of the LDL receptor (LDLR). Moreover, miR-520d and miR-224 simultaneously target the 3’UTR of HMGCR, which codes for the limiting enzyme in cholesterol synthesis and is the target of statins. In mouse and human cells, the overexpression of miR-520d inhibits PCSK9, Idol and HMGCR mRNA levels by 30-50% compared to a control miRNA, while overexpression of miR-224 results in an even more potent 75% decrease in PCSK9, Idol and HMGCR mRNA compared to controls. In human hepatocytes (HepG2/Huh7) and mouse peritoneal macrophages, miR-520d and miR-224 overexpression caused a repression of PCSK9, Idol and HMGCR protein levels, and a decrease in PCSK9 protein secretion. Importantly, these miR-224 and miR-520d-induced changes in PCSK9, Idol and HMGCR expression were associated with a concomitant increase in LDLR protein expression, increased cell surface expression of an LDLR-GFP fusion protein and enhanced LDL binding to the cell surface. Notably, these increases in LDLR were above and beyond those achieved with statin treatment. Together, these findings suggest a role of miR-224 and miR-520d in contributing to the post-transcriptional regulation of LDLR expression, and represent the first evidence of miRNA control of the LDLR pathway. This highlights the therapeutic promise of miR-224/miR-520d for reducing LDL cholesterol beyond statin therapy alone.