Cholesterol Transport Research Articles

Star1 gene mutation reveals the essentiality of 11-ketotestosterone and glucocorticoids for male fertility in Nile Tilapia (Oreochromis niloticus)

Steroidogenic acute regulatory protein (Star) plays an essential role in the biosynthesis of corticosteroids and sex steroids by mediating the transport of cholesterol from the outer to the inner membrane of mitochondria. Two duplicated Star genes, namely star1 and star2, have been identified in non-mammalian vertebrates. To investigate the roles of star genes in fish steriodogenesis, we generated two mutation lines of star1−/− and star1−/−/star2−/− in Nile tilapia (Oreochromis niloticus). Previous studies revealed that deficiency of star2 gene caused delayed spermatogenesis, sperm apoptosis and sterility in male tilapia. Our present data revealed that mutation of star genes impaired male fertility. Disordered seminiferous lobules and spermatic duct obstruction were found in the testis of both types of mutants. Moreover, significant decline in semen volume, sperm abnormality and impaired fertility were also detected in star1−/− and star1−/−/star2−/− males. In star1−/− male fish, lipid accumulation, up-regulation of steroidogenic enzymes, and significant decline of androgens were found. Additionally, hyperplasic interrenal cells, elevated steroidogenic gene expression level and decline of serum glucocorticoids were detected in star1 mutants. Intriguingly, either 11-KT or cortisol supplementation successfully rescued the impaired fertility of the star1−/− mutants. Taken together, these results further indicate that Star1 might play critical roles in the production of both 11-KT and glucocorticoids, which are indispensable for the maintenance of male fertility in fish.

Cholesterol uptake in the intestine is regulated by the LASP1-AKT-NPC1L1 signaling pathway.

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolemia and atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake, and excretion were studied in mice deficient for low-density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high-fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial CaCo-2 cells. When compared with diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdown human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared with Ldlr-/- mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.NEW & NOTEWORTHY We here uncovered LASP1 as a novel regulator of the shuttling of the sterol transporter NPC1L1 to the cell surface in enterocytes to control cholesterol absorption. Accordingly, LASP1-deficient mice displayed lowered serum cholesterol levels under dietary cholesterol supplementation.

Exosomes in Atherosclerosis: Role in the Pathogenesis and Targets for Therapy.

Atherosclerotic cardiovascular disease (ASCVD) is an advanced chronic inflammatory disease and the leading cause of death worldwide. The pathological development of ASCVD begins with atherosclerosis, characterised by a pathological remodelling of the arterial wall, lipid accumulation and build-up of atheromatous plaque. As the disease advances, it narrows the vascular lumen and limits the blood, leading to ischaemic necrosis in coronary arteries. Exosomes are nano-sized lipid vesicles of different origins that can carry many bioactive molecules from their parental cells, thus playing an important role in intercellular communication. The roles of exosomes in atherosclerosis have recently been intensively studied, advancing our understanding of the underlying molecular mechanisms. In this review, we briefly introduce exosome biology and then focus on the roles of exosomes of different cellular origins in atherosclerosis development and progression, functional significance of their cargoes and physiological impact on recipient cells. Studies have demonstrated that exosomes originating from endothelial cells, vascular smooth muscle cells, macrophages, dendritic cells, platelets, stem cells, adipose tissue and other sources play an important role in the atherosclerosis development and progression by affecting cholesterol transport, inflammatory, apoptotic and other aspects of the recipient cells' metabolism. MicroRNAs are considered the most significant type of bioactive molecules transported by exosomes and involved in ASCVD development. Finally, we review the current achievements and limitations associated with the use of exosomes for the diagnosis and treatment of ASCVD.

Abstract PO4-15-02: ApoE polymorphism is associated with aggressive tumor phenotypes for breast cancer patients

Abstract Introduction: Apolipoprotein E (APOE) is implicated in several biological processes, such as protein synthesis, cell growth and differentiation, cholesterol transport, lipid metabolism, and tissue repair. APOE has different isoforms (E2, E3, and E4) that combine conflicting relations with cancer risk. This study aimed to evaluate the distribution of different isoforms of APOE among suspected HBOC (Hereditary Breast and Ovarian Cancer Syndrome) patients. Methodology: DNA was extracted from the blood cells of 108 patients. The detection was performed using qPCR after amplifying specific regions containing the polymorphisms. It was considered that all the amplicons containing Cq amplification < 35. The melting curve for each patient was analyzed to determine the frequency of the isoforms E2, E3, E4, and genotypes E2/E2, E2/E3, E2E4, E3/E3, and E3/E4. The allele and genotype were also correlated with hereditary variants previously identified by genetic tests for germline mutations and with tumoral phenotype. The data were expressed in frequency and associated using chi-square tests or Fisher's exact test (SPSS v20.0, p< 0.05). Results A total of 108 patients filling criteria for HBOC syndrome were evaluated. Among them, 15 (13.9%) had a mutation in BRCA1, 5 (4.6%) in BRCA2, and 2 (1.9%) in TP53. Most patients were under 45 years old (n=76, 70.4%), female (n=104, 96.3%), had a high school education (n=44, 44.0%), were non-smokers (n=79, 80.6%) or drinkers (n=76, 78.4%), and had no comorbidities (n=66, 64.1%). Overweight at diagnosis was observed in 61 (68.5%) individuals. The most frequent tumor phenotype and stage were luminal B (n=46, 43.4%) and stage IIIA (n=22, 26.2%), respectively. Neoadjuvant chemotherapy was performed in 40 (47.1%) with a high rate of complete and partial response 18 (36.7%) and 22 (44.9%), respectively. Regarding ApoE polymorphisms, the E2 allele showed the lowest frequency (n=7, 6.5%), E3 was present in 103 (95.4%) cases, while E4 was present in 39 (36.1%). The most frequent genotype was E3/E3 (n=64, 58.3%), followed by E3/E4 (n=34, 31.5%), E2/E3 (n=6, 5.6%), E4 (n=4, 3.7%), and E2/E4 (n=1, 0.9%). No cases of E2/E2 genotype were observed. The presence of E2 was inversely associated with comorbidity frequency (p=0.040) but directly associated with TP53 mutation (p=0.012) and triple-negative tumor phenotype (p=0.027). E3 was not associated with clinical characteristics, but E4 was associated with low Ki-67 immunoreactivity (p=0.033). The E2/E3 phenotype was directly associated with triple-negative tumors (p=0.044) and heterozygous tumors (p=0.011). Conclusion: The ApoE polymorphism, particularly the presence of the E2 allele in heterozygosity, is associated with TP53 mutation and a more aggressive tumor phenotype. Citation Format: Maria Claudia Luciano, Rosane Sant' Ana, Valeska Lima, Paulo Goberlanio Silva, Clarissa Albuquerquye, Flavio Bitencourt, Maria Julia Bezerra, Francisca Fernanda Oliveira, José Fernando De Moura, Isabelle Joyce Fernandes, Fernanda Leite. ApoE polymorphism is associated with aggressive tumor phenotypes for breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-02.

Abstract PO3-08-07: Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers

Abstract Background: Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Further, they do not prevent ER- breast cancer. Effective alternative strategies with lower toxicity are needed. Previously, we have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. These data led us to hypothesize that LicA creates a tumor preventive environment in the breast by reprogramming metabolism and antioxidant/anti-inflammatory responses in the breast leading to decreased proliferation and tumor suppression. We now report on the breast tumor preventive effects of LicA in xenograft models, its oral bioavailability, and its biologic effects on human breast microstructures from women at increased risk of breast cancer. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified and analyzed by gene ontology and pathway membership. The RNA-seq data was also utilized to conduct metabolism flux analysis. Combined enrichment scores > 4 and FDR < 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Xenograft models of MCF-7aro and MDA-MB-231 tumors were established in female nude mice and the animals treated for 28 days with vehicle or LicA (80 mg/kg.day, s.c.). We measured the rate of tumor growth. We also conducted a PK/PD study with oral LicA (100 mg/kg) in intact BALB/c female mice. Results: We observed significant (FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses < 10 µM. LicA significantly reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) in vivo models (unpaired t-test with Welch’s correction for unequal variances). Promising serum and breast bioavailability, equivalent to low micromolar concentrations sufficient to show efficacy was demonstrated as well. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention in both ER+ and ER- breast cancers through reprogramming metabolism and antioxidant pathways leading to decreased proliferation. We will study LicA in intraductal models of ER+ and ER- precancer lesions in immunocompetent mice and will monitor their progression to invasive breast cancer to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-07.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Heat-killed Lactiplantibacillus plantarum Shinshu N-07 exerts antiobesity effects in western diet-induced obese mice.

The aim of this study was to evaluate the antiobesity effects of heat-killed Lactiplantibacillus plantarum Shinshu N-07 (N-07) isolated from fermented Brassica rapa L. Male mice were divided into three groups (n=10/group); normal diet, western diet (WD), or WD+N-07 (N-07) group and administered each diet for 56 days. The N-07 group showed significant suppression of body weight gain and epididymal fat, perirenal fat, and liver weights compared with the WD group. Higher levels of fecal total cholesterol, triglyceride (TG), and free fatty acid (FFA) were observed in the N-07 group than in the WD group. The mRNA expression of the cholesterol transporter ATP-binding cassette transporter G5 (ABCG5) was significantly increased in the small intestine of N-07-fed mice compared with WD-fed mice. Moreover, N-07 supplementation significantly increased the mRNA expression of ABCG5 and ABCG8 in Caco-2 cells. Furthermore, the TG- and FFA-removal ability of N-07 was confirmed to evaluate its soybean oil- and oleic acid-binding capacities in in vitro experiments. The antiobesity effects of N-07 might be due to its ability to promote lipid excretion by regulating cholesterol transporter expression and lipid-binding ability.

Abstract 2073: StarD4 Is Required For Efficient Clearance Of Extracellular Free Cholesterol Generated From Digestive Exophagy Of Aggregated Low-density Lipoprotein.

In the development of atherosclerotic plaques, macrophages infiltrate the intima of the aortic wall to digest modified lipoproteins. Over time, these macrophages become overloaded with lipids and become plaque-associated foam cells. Most modified lipoproteins are aggregated and cross-linked to the extracellular matrix of the vessel wall, which prevents them from being phagocytosed by macrophages. Our lab has described how macrophages catabolize aggregated LDL extracellularly in a process termed digestive exophagy. Macrophage forms a tightly sealed, acidified extracellular compartment around agLDL that is stabilized by F-actin. Lysosomes are secreted into this extracellular compartment, allowing lysosomal acid lipase to catabolize cholesteryl esters in the core of agLDL to free cholesterol. However, it remains unclear how the extracellular free cholesterol is transported into macrophages for lipid droplet formation. Here, we identify the cholesterol transport protein StarD4 as a major player in the intracellular movement of free cholesterol released by digestive exophagy. We show that StarD4 knockout bone marrow-derived macrophages have delayed onset in foam cell formation when encountering agLDL. However, no difference is observed when StarD4 knockout macrophages are treated with monomeric acetylated LDL, suggesting the effect of StarD4 is specific to cholesterol delivered to the plasma membrane. Moreover, loss of StarD4 does not alter the rate of digestive exophagy, and no difference in lysosome exocytosis is observed between StarD4 knockout and wildtype macrophages. Therefore, our results indicate that StarD4 mediated plasma membrane to ER cholesterol transport is a critical step in digestive exophagy of agLDL. Loss of StarD4 can delay the onset of foam cell formation, which may provide insight into novel therapeutic strategies for atherosclerosis.

The calpain/ABCA1 pathway drives mast cell- initiated vascular leakage and inflammation in the skin.

Abstract Cutaneous mast cells (MCs) initiate local and systemic immune responses. To reach this end, activated MCs immediately release an arsenal of pro-inflammatory proteases. In this study, we evaluated the role of the cysteine protease, calpain in MC function. Calpain activity and protein were confirmed in the supernatants of MCs activated via either the FceR or the MrgprB2. In vivo, extracellular calpain induces vascular leakage in a MC- dependent manner. We then investigated the mechanism of calpain release. In vitro and in vivo studies suggest that the cholesterol transporter, the ATP- binding cassette A1 transporter (ABCA1) externalizes calpain. To investigate the role of the ABCA1 in MC biology, we generated mice lacking the Abca1 specifically on connective tissue MCs (Mcpt5-Cre+Abca1fl/fl mice). In vitro, Abca1-deficient MCs accumulate intracellular cholesterol, have reduced calpain release and reduced pro-inflammatory cytokines levels. In vivo, in the MC903-induced atopic dermatitis model, Mcpt5-Cre+Abca1fl/fl mice have reduced inflammation and a fewer MCs in affected skin compared to littermate controls. Corroborating this finding, global ABCA1 blockade with FDA-approved glyburide similarly reduced inflammation induced by MC903. Collectively, these data suggest that targeting the calpain/ABCA1 pathway may open new therapeutic avenues for MC -mediated inflammation in the skin.

Abstract 3039: TOMM40 Knockdown In Macrophages Inhibits Oxidized LDL-induced NLRP3 Activation And Promotes Reverse Cholesterol Transport

Atherogenesis has been shown to be promoted by activation of the NLRP3 inflammasome, a cytosolic innate immune sensor activated by a broad range of factors, including oxidized LDL (oxLDL). Our group has shown that hepatocyte knockdown of Translocase of Outer Mitochondrial Membrane 40 ( TOMM40), which encodes a key component of the mitochondrial importer TOM complex, results in increased levels of oxysterols that activate the transcription factors LXRα and β. As LXR agonists have been shown to inhibit NLRP3 activation, we tested whether TOMM40 knockdown reduces NLRP3 activation in THP-1 monocyte-derived macrophages. On stimulating these cells with both the NLRP3 activator Nigericin and oxLDL following TOMM40 knockdown by siRNA, we found that pro-inflammatory cytokine release and NLRP3 activation were significantly inhibited (Two-way ANOVA, p<0.0001, n=6). We further showed that TOMM40 knockdown resulted in significant LXRβ upregulation (unpaired t-test, p=0.007, n=3), and reduced oxLDL-induced upregulation of both NLRP3 and CASP1 , which encodes a protein recruited in NLRP3 activation (One-way ANOVA, p=0.0007, n=3). Moreover, pre-treatment with an LXR antagonist, GSK2033, abrogated TOMM40 knockdown-induced NLRP3 inhibition, supporting our hypothesis that this inhibition is likely LXR dependent. Since LXRs are known to promote cholesterol efflux from macrophages, we also examined the effect of TOMM40 knockdown on expression of the cholesterol transporter genes, ABCA1, ABCG1, and SR-B1 , and found that ABCG1 and SR-B1 were significantly upregulated (unpaired t-test, p=0.02 and p=0.003 respectively, n=3). Taken together, these findings identify mechanisms by which TOMM40 knockdown exerts anti-inflammatory effects and promotes reverse cholesterol transport. While TOMM40 has been studied largely in relation to APOE and neurodegenerative disease due to its genomic location, we identify a novel potential impact of TOMM40 on atherogenesis.

Abstract 1053: The Role Of Bile Acid-initiated Signaling In Vascular Smooth Muscle Cell Phenotypic Switching And Atherosclerosis

Atherosclerosis is a chronic inflammatory disease in which foam cells (lipid-laden macrophage-like cells) accumulate in the arterial wall. Vascular smooth muscle cells (VSMCs) are not terminally differentiated; they switch to a foam cell-like phenotype in response to various stimuli, such as cholesterol (CHOL), increasing susceptibility to vascular diseases. Modulated SMCs are the main contributors to atherosclerotic plaque formation. The intracellular metabolism of CHOL via peroxisomes in hepatocytes produces bile acids like cholic or deoxycholic acid, which agonize liver X receptor (LXR). LXR facilitates the first step of reverse CHOL transport. It is unknown if VSMCs synthesize bile acids and whether peroxisomes and the bile acids they produce play a role in VSMC-derived foam cell formation. Using human aortic SMCs (HASMCs), we found that CHOL treatment decreased expression of several peroxisomal proteins. Peroxisomal loss-of-function experiments demonstrated increased intracellular lipid accumulation, as visualized with Oil Red O (ORO) staining. We have shown that HASMCs synthesize bile acids after exposure to CHOL, which led us to hypothesize that bile acid-initiated signaling participates in the phenotypic modulation initiated by CHOL in VSMCs. Treatment of HASMCs with LXR agonists or bile acids increased expression of the efflux CHOL transporter ABCA1, bile acid-related peroxisomal enzymes, and differentiation markers; they decreased expression of macrophage and foam cell markers and ORO staining. These data show that VSMCs have the metabolic capacity to synthesize bile acids, which is impaired if peroxisomes are non-functional or absent. Our data indicates that activation of bile acid receptors upregulates ABCA1, favoring a differentiated VSMC phenotype. Together, these data suggest that activation of LXR plays a role in CHOL-induced VSMC to foam cell transition and that LXR is a potential therapeutic target which promotes VSMC differentiation and decreases atherosclerotic plaque formation.

Extract of Gualou-Xiebai Herb Pair Improves Lipid Metabolism Disorders by Enhancing the Reverse Cholesterol Transport in Atherosclerosis Mice.

Gualou is derived from the fruit of Trichosanthes kirilowii Maxim, while Xiebai from the bulbs of Allium macrostemon Bunge. Gualou and Xiebai herb pair (2:1) is widely used in clinical practice to treat atherosclerotic cardiovascular diseases. However, the mechanism underlying its potential activity on atherosclerosis (AS) has not been fully elucidated. The extract of Gualou-Xiebai herb pair (GXE) was prepared from Gualou (80 g) and Xiebai (40 g) by continuous refluxing with 50% ethanol for 2 h at 80°C. In vivo, ApoE-/- mice were fed a high-fat diet (HFD) for 10 weeks to induce an AS model, and then the mice were treated with GXE (3, 6, 12 g/kg) or atorvastatin (10 mg/kg) via oral gavage. Besides, RAW264.7 macrophages were stimulated by ox-LDL to establish a foam cell model in vitro. GXE suppressed plaque formation, regulated plasma lipids, and promoted liver lipid clearance in AS mice. In addition, 0.5, 1, and 2 mg/mL GXE significantly reduced the TC and FC levels in ox-LDL (50 μg/mL)-stimulated foam cells. GXE increased cholesterol efflux from the foam cells to ApoA-1 and HDL, and enhanced the protein expressions of ABCA1, ABCG1, and SR-BI, which were reversed by the PPARγ inhibitor. Meanwhile, GXE increased the LCAT levels, decreased the lipid levels and increased the TBA levels in the liver of AS mice. Molecular docking indicated that some compounds in GXE showed favorable binding energy with PPARγ, LCAT and CYP7A1 proteins, especially apigenin-7-O-β-D-glucoside and quercetin. In summary, our results suggested that GXE improved lipid metabolism disorders by enhancing RCT, providing a scientific basis for the clinical use of GXE in AS treatment.

Synthesis and In-Silico Studies of Ortho-Fluorinated Benzenesulfonamides as Putative Anti-CETP Agents

Abstract: Cardiovascular disease is one of the primary causes of death. Atherosclerosis produces artery constriction or obstruction, which can lead to a heart attack or stroke. Cholesteryl Ester Transfer Protein (CETP) is a protein that aids in reverse cholesterol transport. It promotes cholesteryl ester transfer from HDL to LDL and VLDL. So, inhibition of CETP by drugs limits cardiovascular disease by decreasing LDL and increasing HDL cholesterol. In this study, ten ortho-fluoro substituted benzenesulfonamides 6a-6j were prepared, and their structure was fully determined using 1H NMR, 13C NMR, HR-MS, and IR. In vitro biological evaluation showed that compound 6d has the highest inhibitory activity with 100% inhibition, while compounds 6a-6c and 6e-6j had activities ranged from 29% - 83% at 10 μM concentration. Interestingly, para-substituted derivatives (6d, 6g, and 6j) were observed to have greater CETP inhibitory activities than their ortho- and meta- analogues irrespective to the nature of substituent, i.e., CH3, Cl, or NO2. Ligandfit docking experiment revealed the difference in the binding mode among the synthesized compounds, which is reflected in their CETP inhibitory activity.

Abstract 2071: Identification Of Genetic Loci Influencing Lethal Atherothrombosis In Mice

Introduction: Atherothrombosis leading to myocardial infarction and stroke is a major cause of mortality. Ruptured atherosclerotic plaques release prothrombotic molecules such as tissue factor, which initiates thrombosis. Both genetic and environmental factors contribute to atherothrombosis. Combined complete deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes (dKO) on a mixed C57BL/6J (B6) and 129S1/SvImJ (129S1) background leads to an atherothrombotic phenotype and early death before 8 weeks of age. However, the dKO phenotype is dramatically different if the Scarb1 and ApoE deficient mice are backcrossed extensively to the B6 mouse strain, with no dKOs surviving to the weaning age. Hypothesis/Goals: We hypothesized that 129S1 genetic variants reduce atherothrombosis severity in dKO mice and used mouse genetics to identify these loci. Methods: To identify potential genetic factors, we produced and analyzed Scarb1 and ApoE dKO mice from 6 backcross/intercross generations with progressively more B6 genomic contribution. Weights were taken for 38 dKO mice and littermates from 3 weeks old until death of the dKO mice at 8 weeks of age. dKO mice from F1-F3 generations were divided into 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse DNA was subjected to genome wide marker genotyping using the GigaMUGA platform. Quantitative trait loci analysis (QTL) was performed to identify loci responsible for the phenotypic weight differences. Results: dKO weights fell into 3 categories: indistinguishable from littermates, 14%-39% less than littermates, and greater than 39% less than littermates. QTL analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. Niemann Pick type C2 (Npc2), an intracellular cholesterol transporter, is within the interaction region on Chr 12 and is a prime candidate dKO modifier. Conclusion: We used weight data as a representative of overall health to categorize dKO phenotypes relative to non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate modifier gene for the Chromosome 12 locus. Identification of atherothrombosis modifier genes will provide novel therapeutic targets for this complex disease.

Abstract 160: Macrophage-specific Ablation Of Bmal1 Regulates Cholesterol Transport And Enhances Atherosclerosis

We have previously shown that global and liver-specific Bmal1 deficient mice develop enhanced atherosclerosis. Macrophages play a critical role in atherosclerosis, yet the role of macrophage Bmal1 in cholesterol uptake, intracellular transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific Bmal1 deficient mice to clarify the role of macrophage Bmal1 in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Mechanistically, Bmal1 deficient macrophages exhibited: (1) increased expression of Lox-1 and Cd36 and uptake of oxLDL; (2) decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux, and reverse cholesterol transport; and (3) diminished export of free cholesterol from lysosomes due to reduced expression of Npc1 and Npc2. Molecular studies showed that Bmal1 directly regulates Npc1 and Npc2 by binding to E-boxes in their promoters, whereas it indirectly regulates Abca1 and Abcg1 by regulating ZNF202. Furthermore, we provide evidence that Bmal1 interacts with both Clock and Npas2 to regulate ZNF202, Npc1 and Npc2. Our data demonstrate that macrophage Bmal1 is a key regulator of oxLDL uptake, lysosomal cholesterol export, and cholesterol efflux, and, therefore, plays a crucial role in the prevention of atherosclerosis.

Trimethylamine N-oxide ameliorates hepatic damage including reduction of hepatic bile acids and cholesterol in Fxr-null mice

There are conflicting animal experiments on the effect of trimethylamine N-oxide (TMAO), the dietary metabolite, on non-alcoholic fatty liver disease (NAFLD). This study aims to determine the effect of TMAO on NAFLD. A diet containing 0.3% TMAO was fed to farnesoid X receptor (Fxr)-null mice, a model of NAFLD, for 13 weeks. Fxr-null mice fed TMAO showed significant reductions in liver damage markers but not wild-type mice. Hepatic bile acid and cholesterol levels were significantly decreased, and triacylglycerol levels tended to decrease in TMAO-fed Fxr-null mice. Changes in mRNA levels of hepatic bile acid and cholesterol transporters and synthetic enzymes were observed, which could explain the decreased hepatic bile acid and cholesterol levels in Fxr-null mice given the TMAO diet but not in the wild-type mice. These results suggest that TMAO intake ameliorates liver damage in Fxr-null mice, further altering bile acid/cholesterol metabolism in an FXR-independent manner.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Cholesterol transport and beyond: Illuminating the versatile functions of HDL apolipoproteins through structural insights and functional implications.

High-density lipoproteins (HDLs) play a vital role in lipid metabolism and cardiovascular health, as they are intricately involved in cholesterol transport and inflammation modulation. The proteome of HDL particles is indeed complex and distinct from other components in the bloodstream. Proteomics studies have identified nearly 285 different proteins associated with HDL; however, this review focuses more on the 15 or so traditionally named "apo" lipoproteins. Important lipid metabolizing enzymes closely working with the apolipoproteins are also discussed. Apolipoproteins stand out for their integral role in HDL stability, structure, function, and metabolism. The unique structure and functions of each apolipoprotein influence important processes such as inflammation regulation and lipid metabolism. These interactions also shape the stability and performance of HDL particles. HDLs apolipoproteins have multifaceted roles beyond cardiovascular diseases (CVDs) and are involved in various physiological processes and disease states. Therefore, a detailed exploration of these apolipoproteins can offer valuable insights into potential diagnostic markers and therapeutic targets. This comprehensive review article aims to provide an in-depth understanding of HDL apolipoproteins, highlighting their distinct structures, functions, and contributions to various physiological processes. Exploiting this knowledge holds great potential for improving HDL function, enhancing cholesterol efflux, and modulating inflammatory processes, ultimately benefiting individuals by limiting the risks associated with CVDs and other inflammation-based pathologies. Understanding the nature of all 15 apolipoproteins expands our knowledge of HDL metabolism, sheds light on their pathological implications, and paves the way for advancements in the diagnosis, prevention, and treatment of lipid and inflammatory-related disorders.

The Impact of the Apolipoprotein E Genotype on Cardiovascular Disease and Cognitive Disorders.

Apolipoprotein E (ApoE) plays a critical role in cholesterol transport and protection against the development of atherosclerotic cardiovascular disease (ASCVD). Humans have 3 prevalent isoforms of ApoE: apolipoprotein E2 (ApoE2), apolipoprotein E3 (ApoE3), and apolipoprotein E4 (ApoE4). The E4 allele has been associated with higher ASCVD risk. While E4 patients do have higher cholesterol levels, they do not have enough to account for the substantially elevated ASCVD risk relative to E2 and E3 patients. ASCVD risk calculators would underestimate the true effect of E4 if the difference was caused entirely by a difference in cholesterol level. This article reviews the function of ApoE in atherosclerosis, and how each isoform functions differently. We review what is known about the molecular mechanisms through which ApoE prevents endothelial dysfunction and damage, how ApoE stimulates macrophage efflux of cholesterol from atherogenic lesions, and the ways in which ApoE decreases inflammation throughout atherosclerosis. The impact of ApoE on Alzheimer's disease and a discussion of why it is possibly unrelated to ASCVD prevention are included. Clinical applications to hyperlipidemia management and ASCVD prevention in specific patient populations are discussed.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1β (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.