Reduced Necrosis and Content of Apoptotic M1 Macrophages in Advanced Atherosclerotic Plaques of Mice With Macrophage-Specific Loss of Trpc3

In previous work we reported that ApoeKO mice transplanted with bone marrow cells deficient in the Transient Receptor Potential Canonical 3 (TRPC3) channel have reduced necrosis and number of apoptotic macrophages in advanced atherosclerotic plaques. Also, in vitro studies with polarized macrophages derived from mice with macrophage-specific loss of TRPC3 showed that M1, but not M2 macrophages, deficient in Trpc3 are less susceptible to ER stress-induced apoptosis than Trpc3 expressing cells. The questions remained (a) whether the plaque phenotype in transplanted mice resulted from a genuine effect of Trpc3 on macrophages, and (b) whether the reduced necrosis and macrophage apoptosis in plaques of these mice was a manifestation of the selective effect of TRPC3 on apoptosis of M1 macrophages previously observed in vitro. Here, we addressed these questions using Ldlr knockout (Ldlr−/−) mice with macrophage-specific loss of Trpc3 (MacTrpc3−/−/Ldlr−/− → Ldlr−/−). Compared to controls, we observed decreased plaque necrosis and number of apoptotic macrophages in MacTrpc3−/−/Ldlr−/− → Ldlr−/− mice. Immunohistochemical analysis revealed a reduction in apoptotic M1, but not apoptotic M2 macrophages. These findings confirm an effect of TRPC3 on plaque necrosis and support the notion that this is likely a reflection of the reduced susceptibility of Trpc3-deficient M1 macrophages to apoptosis.Fil: Solanki, Sumeet. University of Toledo; Estados UnidosFil: Dube, Prabhatchandra R.. University of Toledo; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo; Estados Unido

Hyperglycemia and Hyperlipidemia Act Synergistically to Induce Renal Disease in LDL Receptor-Deficient BALB Mice

Diabetic nephropathy is the leading cause of end-stage renal disease in Western countries, but only a portion of diabetic patients develop diabetic nephropathy. Dyslipidemia represents an important aspect of the metabolic imbalance in diabetic patients. In this study, we addressed the impact of combined hyperlipidemia and hyperglycemia on renal pathology. Kidneys from wildtype (WT) or LDL receptor-deficient BALB/cBy mice (BALB. LDLR -/-) were examined at 22 weeks of age. Diabetes was induced by administration of streptozotocin and mice were randomly assigned to either standard chow or Western diet. Chow fed BALB. LDLR -/- mice did not demonstrate renal abnormalities, whereas BALB. LDLR -/- mice fed a Western diet showed occasional glomerular and tubulointerstitial foam cells. Diabetic WT mice had modestly increased glomerular cellularity and extracellular matrix. Hyperlipidemic and diabetic BALB. LDLR -/- mice exhibited an increase in glomerular cellularity and extracellular matrix, accumulation of glomerular and tubulointerstitial foam cells and mesangial lipid deposits. The tubular epithelium demonstrated pronounced lipid induced tubular degeneration with increased tubular epithelial cell turnover. Hyperlipidemia and hyperglycemia seem to act synergistically in inducing renal injury in the BALB. LDLR-/- mouse. This model of diabetic nephropathy is unique in its development of tubular lesions and may represent a good model for hyperlipidemia-exacerbated diabetic nephropathy. Copyright (C) 2004 S. Karger AG, Basel

The E3 ubiquitin ligase c-IAP1 regulates PCSK9-mediated LDLR degradation: Linking the TNF-α pathway to cholesterol uptake

Proprotein convertase subtilisin/kexin type 9 (PCSK9), in addition to LDLR (low-density lipoprotein receptor) and APOB (apolipoprotein B), is one of three loci implicated in autosomal dominant hypercholesterolaemia (ADH)^1^. A number of PCSK9 gain-of-function mutations and loss-of-function mutations have been identified from families afflicted with ADH with hypercholesterolaemia or hypocholesterolaemia, respectively^1-4^. In humans, the main function of PCSK9 appears to be the post-transcriptional regulation of the number of cell-surface LDL receptors^5-7^. To date, only LDLR and its closest family members VLDLR and ApoER2 have been shown to bind with PCSK9^8,9^. To find new binding partners for PCSK9, we used a shotgun proteomic method to analyse the protein complex pulled down by immunoprecipitation against FLAG-tagged PCSK9 protein. Among 22 potential novel binding proteins identified, we found that the cellular inhibitor of apoptosis protein 1 (c-IAP1^10^) and the TNF receptor-associated factor 2 (TRAF2^11^) complex are regulated differently in different dominant PCSK9 mutations that occur naturally. Further immunoprecipitation analysis showed that c-IAP1 is a direct binding partner for PCSK9. One of the "gain-of-function" mutants, PCSK9-S127R, which has impaired autocatalytic activity, is defective in binding to c-IAP1. The other dominant mutation, PCSK9-D374Y^12^, which is 10-fold more potent in degrading the LDLR protein than wild-type PCSK9, can be significantly ubiquitinated by c-IAP1 in vitro. The ubiquitinated PCSK9-D374Y is unable to degrade LDLR, which is its main cause of hypercholesterolaemia in patients. These results indicate that there is a novel cholesterol uptake regulation pathway linking PCSK9/LDLR to the E3 ubiquitin ligase c-IAP1 in a TNF-[alpha] response pathway. This highlights the possibility of developing new treatments for human cardiovascular diseases through ubiquitin ligase-mediated ubiquitination of target proteins in cholesterol metabolism

Site-Specific Effects of PECAM-1 on Atherosclerosis in LDL Receptor-Deficient Mice

Objective—Atherosclerosis is a vascular disease that involves lesion formation at sites of disturbed flow under the influence of genetic and environmental factors. Endothelial expression of adhesion molecules that enable infiltration of immune cells is important for lesion development. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31) is an adhesion and signaling receptor expressed by many cells involved in atherosclerotic lesion development. PECAM-1 transduces signals required for proinflammatory adhesion molecule expression at atherosusceptible sites; thus, it is predicted to be proatherosclerotic. PECAM-1 also inhibits inflammatory responses, on which basis it is predicted to be atheroprotective. Methods and Results—We evaluated herein the effect of PECAM-1 deficiency on development of atherosclerosis in LDL receptor– deficient mice. We found that PECAM-1 has both proatherosclerotic and atheroprotective effects, but that the former dominate in the inner curvature of the aortic arch whereas the latter dominate in the aortic sinus, branching arteries, and descending aorta. Endothelial cell expression of PECAM-1 was sufficient for its atheroprotective effects in the aortic sinus but not in the descending aorta, where the atheroprotective effects of PECAM-1 also required its expression on bone marrow–derived cells. Conclusion—We conclude that PECAM-1 influences initiation and progression of atherosclerosis both positively and negatively, and that it does so in a site-specific manner. (Arterioscler Thromb Vasc Biol. 2008;28:1996-2002

The adaptor protein PID1 regulates receptor-dependent endocytosis of postprandial triglyceride-rich lipoproteins.

ObjectiveInsulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins.MethodsLocalization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr-/-) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL.ResultsPID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr-/- background reduced lipoprotein uptake into liver and caused plasma TRL accumulation.ConclusionsBy acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver

Three patients with homozygous familial hypercholesterolemia: Genomic sequencing and kindred analysis.

BackgroundHomozygous Familial Hypercholesterolemia (HoFH) is an inherited recessive condition associated with extremely high levels of low-density lipoprotein (LDL) cholesterol in affected individuals. It is usually caused by homozygous or compound heterozygous functional mutations in the LDL receptor (LDLR). A number of mutations causing FH have been reported in literature and such genetic heterogeneity presents great challenges for disease diagnosis.ObjectiveWe aim to determine the likely genetic defects responsible for three cases of pediatric HoFH in two kindreds.MethodsWe applied whole exome sequencing (WES) on the two probands to determine the likely functional variants among candidate FH genes. We additionally applied 10x Genomics (10xG) Linked-Reads whole genome sequencing (WGS) on one of the kindreds to identify potentially deleterious structural variants (SVs) underlying HoFH. A PCR-based screening assay was also established to detect the LDLR structural variant in a cohort of 641 patients with elevated LDL.ResultsIn the Caucasian kindred, the FH homozygosity can be attributed to two compound heterozygous LDLR damaging variants, an exon 12 p.G592E missense mutation and a novel 3kb exon 1 deletion. By analyzing the 10xG phased data, we ascertained that this deletion allele was most likely to have originated from a Russian ancestor. In the Mexican kindred, the strikingly elevated LDL cholesterol level can be attributed to a homozygous frameshift LDLR variant p.E113fs.ConclusionsWhile the application of WES can provide a cost-effective way of identifying the genetic causes of FH, it often lacks sensitivity for detecting structural variants. Our finding of the LDLR exon 1 deletion highlights the broader utility of Linked-Read WGS in detecting SVs in the clinical setting, especially when HoFH patients remain undiagnosed after WES

Vitamin D3 supplementation of a high fat high sugar diet ameliorates prediabetic phenotype in female LDLR–/–and LDLR+/+mice

© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd. INTRODUCTION: Fatty liver disease is prevalent in populations with high caloric intake. Nutritherapeutic approaches are being considered, such as supplementary Vitamin D 3 , to improve aspects of metabolic syndrome, namely fatty liver disease, hyperlipidemia, and insulin resistance associated with obesity. METHODS: We analyzed female LDLR -/- and LDLR +/+ mice on a 10-week diabetogenic diet for markers of fatty liver disease, metabolic strain, and inflammation. RESULTS: The groups on a high fat high sugar diet with supplementary Vitamin D 3 , in comparison with the groups on a high fat high sugar diet alone, showed improved transaminase levels, significantly less hypertriglyceridemia and hyperinsulinemia, and histologically, there was less pericentral hepatic steatosis. Levels of non-esterified fatty acids and lipid peroxidation products were significantly lower in the group supplemented with additional Vitamin D 3 , as were systemic markers of inflammation (serum endotoxin and IL-6). M2 macrophage phenotype predominated in the group supplemented with additional Vitamin D 3 . Beneficial changes were observed as early as five weeks’ supplementation with Vitamin D 3 and extended to restoration of high fat high sugar diet induced decrease of bone mineral density. CONCLUSION: In summary, Vitamin D 3 was a significantly beneficial dietary additive to blunt a prediabetic phenotype in diet-induced obesity of female LDLR -/- and LDLR +/+ mice

LDLR-Gene therapy for familial hypercholesterolaemia: Problems, progress, and perspectives

Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency

An integrated mathematical model of cellular cholesterol biosynthesis and lipoprotein metabolism

Cholesterol regulation is an important aspect of human health. In this work we bring together and extend two recent mathematical models describing cholesterol biosynthesis and lipoprotein endocytosis to create an integrated model of lipoprotein metabolism in the context of a single hepatocyte. The integrated model includes a description of low density lipoprotein (LDL) receptor and cholesterol synthesis, delipidation of very low density lipoproteins (VLDLs) to LDLs and subsequent lipoprotein endocytosis. Model analysis shows that cholesterol biosynthesis produces the majority of intracellular cholesterol. The availability of free receptors does not greatly effect the concentration of intracellular cholesterol, but has a detrimental effect on extracellular VLDL and LDL levels. We test our model by considering its ability to reproduce the known biology of Familial Hypercholesterolaemia and statin therapy. In each case the model reproduces the known biological behaviour. Quantitative differences in response to statin therapy are discussed in the context of the need to extend the work to a more {\it in vivo} setting via the incorporation of more dietary lipoprotein related processes and the need for further testing and parameterisation of {\it in silico} models of lipoprotein metabolism

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis