Significant Cholesterol Accumulation Research Articles

Proteomic analysis of s-acylated proteins in human retinal pigment epithelial cells and the role of palmitoylation of Niemann-Pick type C1 protein in cholesterol transport.

Palmitoylation is a dynamic process that regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in the visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 55 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant enrichment of palmitoylated proteins in cell-matrix adhesion, cell-cell recognition, protein cellular localization, and translation, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900, and 816) of Niemann-Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys799 and 800 caused significant cholesterol accumulation in the late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. However, the mutation did not alter the cellular localization of the protein. Conclusion: PRE cells have an abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol export, but not the intracellular localization of NPC1.

Master regulators in the foam cell formation; the role of phagocytosis

Abstract Background Foam cell formation caused by modified LDL is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. 10 inflammation-related master regulators, which were involved in the cholesterol accumulation in cultured macrophages induced by the incubation with modified LDL, have been identified. Objective We hypothesised that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. Methods Cholesterol accumulation was evaluated in primary macrophages with master regulator genes knock-downed by siRNA for either IL15, EIF2AK3, F2RL1, TSPYL2, or ANXA1. Analysis of enriched transcription factor binding sites in promoters of differentially expressed genes and identification of master regulators in the signal transduction network were performed with TRANSFAC and TRANSPATH databases. Results Genes which were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads were determined. Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages, whereas atherogenic naturally occurring LDL caused significant cholesterol accumulation in the control cells. The ANXA1 gene knock-down caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. Conclusions The results, showing that inflammatory response and the cholesterol accumulation are related, may confirm our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Russian Science Foundation

Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis.

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.

Abstract 442: Generation of an Abcg1 Knock Out Mouse on the Reversa Background

Background: Clinically HDL mediated reverse cholesterol transport (RCT) from macrophages has been shown to be inversely associated with carotid intima media thickness. Cholesterol efflux to mature HDL is mediated by ATP binding cassette transporter G1 (Abcg1). Abcg1 pays a key role in cholesterol transport with loss of function in macrophages and endothelial cells associated with significant cholesterol accumulation. However, mechanistic studies into the role of Abgc1 in plaque regression have been restricted due to the limitations of current regression models. Aims: To use TALENS mediated genomic editing to generate an Abcg1 knockout mouse on the REVERSA background to enable the investigation of its role in plaque regression. Methods and results: TALENs constructs were targeted to exon 3 upstream of the phosphate binding Walker A domain. TALEN mRNA was injected into REVERSA oocytes which were then implanted into foster mice. Founders were screened by Cel1 nuclease assay and sequencing. Three independent alleles were identified two of which create frameshift mutations (predicted to be null alleles) and one which resulted in a 3 amino acid deletion and a one amino acid substitution near the Walker A domain (potential hypomorphic allele). The two founder lines with frame shift mutations (KO 145 and 171) were taken forward for additional analysis. RNA extracted from primary macrophages from WT (REVERSA) and homozygous Abcg1 knockout mice was used to confirm the mutation was transcribed to RNA. Intron-spanning primers were designed and a product of the expected size was obtained and sequence analysis confirmed the insertion (KO-145) and deletion (KO-171) within the WALKER A domain of Abcg1. To ensure the mutations resulted in loss of function, a radioactive RCT assay was carried out in bone marrow derived primary macrophages. A significant decrease in RCT to HDL was observed in macrophages from both the KO-145 and KO-171 lines as expected there was no difference in RCT to ApoA1-I. Conclusions: Using genomic editing we have generated a gene specific knockout on the REVERSA background which will enable, for the first time, the investigation of the role of Abcg1 in plaque regression.

Cholesterol accumulation inhibits ER to Golgi transport and protein secretion: studies of apolipoprotein E and VSVGt

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.

Replacing dietary carbohydrate with protein and fat decreases the concentrations of small LDL and the inflammatory response induced by atherogenic diets in the guinea pig

Guinea pigs resemble humans in cholesterol and lipoprotein metabolism; however, there is limited information on the vascular inflammatory response with induction of atherosclerosis in this animal model. The purpose of this study was to document a vascular inflammatory response associated with dietary-induced atherosclerosis in the guinea pig and determine the effect of replacing dietary carbohydrate with protein and fat on this response. Thirty male Hartley guinea pigs were randomly assigned to a high dietary cholesterol, high-carbohydrate (HC); a high-cholesterol, low-carbohydrate (LC) or a control (CON) diet for 12 weeks. Analysis of cytokine protein expression [interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and GM-CSF) and m RNA expression (IFN-γ, TNF-α, IL-1β, MCP-1 and IL-8] were performed along with the measurement of cholesterol concentration in the aorta, plasma lipids and plasma low-density lipoprotein subfractions. There was a similar and significant accumulation of cholesterol in the thoracic aorta in the HC and LC groups compared to the CON group. Aortic cytokine protein expression (TNF-α, IFN-γ and IL-6) and m RNA expression (TNF-α and IFN-γ) were significantly elevated in both high-cholesterol fed groups (HC and LC) ( P<.05) compared to the CON group. Compared to the HC group, animals fed the LC diet had reduced protein and m RNA TNF-α expression, as well as a reduced concentration of small LDL particles in the plasma. This study is the first to document a dietary cholesterol-induced vascular inflammatory response in guinea pigs that is partially regulated by the macronutrient content of the diet. Guinea pigs may be a useful animal model to evaluate the cellular and molecular components of atherosclerosis.

A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by progressive accumulation of cholesterol, gangliosides, and other lipids in the central nervous system and visceral organs. In the NPC1 mouse model, neurodegeneration and neuronal cell loss occur before postnatal day 21. Whether neuronal cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss has not been demonstrated. In this report, we used the NPC1 mouse model and employed a novel cholesterol binding reagent, BC theta, that enabled us to visualize cellular cholesterol accumulation at a level previously unattainable. The results demonstrate the superiority of BC theta staining over conventional filipin staining in confocal microscopy and highlight several new findings. We show that at postnatal day 9, although only mild signs of neurodegeneration are detectable, significant neuronal cholesterol accumulation has already occurred throughout the NPC1 brain. In addition, although NPC1 Purkinje neurons exhibit a normal morphology at day 9, significant cholesterol accumulation within their extensive dendritic trees has occurred. We also show that in the thalamus and cortex of NPC1 mice, activated glial cells first appear at postnatal day 9 and heavily populate by day 22, suggesting that in NPC1 mice, neuronal cholesterol accumulation precedes neuronal injury and neuronal cell loss.

Hexachlorocyclohexane (HCH) induced changes in microsomal drug metabolizing enzymes and liver lipids in rats fed different levels of protein

The interaction of HCH (50 mg/kg) and dietary protein levels on microsomal drug metabolizing enzymes system and liver lipids were studied in the rats for 90 days. The results indicated that rats fed a lower protein diet and HCH has a higher rate of mortality, lower rate of growth and an increased liver weight. A significant induction in the hepatic microsomal aminopyrine‐N‐demethylase, p‐nitroanisole‐O‐dealkylase, benzo(a)pyrene hydroxylase and glutathione‐S‐transferase activity was observed in pesticide treated animals as compared to control animals. The pathological changes observed in liver of HCH treated animals consisted mainly of necrosis and fatty degeneration of hepatocytes. HCH also induced the significant accumulation of cholesterol, triglycerides, phospholipid and total lipid in liver in low protein diet animals. Protein accelerates the metabolism of HCH, resulting in a decrease of HCH concentration with the increase of dietary protein level. A close correlation existed between lipid accumulati...

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells.

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.

Effect of prolactin on tissue cholesterol levels of male albino rats

Prolactin modulated tissue cholesterol distribution of male albino rats. Testis and epididymis had elevated cholesterol levels which was correlated to increased androgenesis. Both prostate and seminal vesicles had depleted levels, might be due to elevated seminal plasma secretion. Since heart and dorsal aorta recorded significant accumulation of cholesterol in response to prolactin administration, involvement of prolactin in cardiovascular disorders in rats was envisaged.

Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters on a lithogenic diet.

The activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme for liver cholesterol biosynthesis, has been determined in young hamsters given a diet known to produce cholesterol gallstones in this species and compared to the activity found in chow-fed hamsters. None of the hamsters fed the lithogenic diet for 15 days or less developed gallstones but 74 percent of those on the diet for 26 to 49 days had cholesterol gallstones. None of the chow-fed animals developed gallstones. The mean HMG CoA reductase activity of hamsters on the lithogenic diet at 4 to 6 hours after the onset of the dark period was 20 times greater than in hamsters in the fasting state and 12 times that of fed hamsters on the chow diet. These greatly elevated enzyme activities were found as early as 2 to 15 days on the lighogenic diet and thus long before the gallstones appeared. The percentage of the bile acid pool represented by chenodeoxycholic acid was significantly higher, and that cholic acid correspondingly lower on the gallstone-inducing diet. This shift in bile acid composition may have contributed to gallstone formation. No significant accumulation of cholesterol was noted in liver or carcass. Increased synthesis of cholesterol induced by the greatly stimulated enzyme activity in hamsters on the lithogenic diet could account for the increased secretion of cholesterol in bile noted by others with subsequent supersaturation of gallbladder bile with respect to cholesterol, and then gallstone formation.