Male Low-density Lipoprotein Receptor Research Articles

Dihydromyricetin Alleviates Nonalcoholic Fatty Liver Disease and Its Associated Metabolic Syndrome by Inhibiting Endoplasmic Reticulum Stress in LDLR−/− Mice Fed with a High-Fat and High-Fructose Diet

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease. Previous studies have shown that dihydromyricetin (DHM) is beneficial for NAFLD. However, whether DHM alleviates NAFLD by inhibiting liver endoplasmic reticulum (ER) stress remains unknown. Thus, this study aimed to identify the potential roles and mechanisms of DHM. Twenty-four male low-density lipoprotein receptor (LDLR−/−) knockout mice aged 8 weeks were randomly divided into normal control, control, and DHM groups. Normal control mice were fed a normal diet (ND), and the last two groups of mice were fed a high-fat and high-fructose diet (HFD) for 12 weeks, treated with or without DHM. DHM alleviated diet-induced hyperlipidemia as early as 4 weeks after and until the end of HFD feeding. HFD increased insulin resistance, and the opposite was observed in the DHM group. Compared to the control group, the body weight of the mice and adipocyte size and weight of the retroperitoneal and epididymal fat were remarkably reduced in the DHM group. The expression of genes related to lipid metabolism, such as Acox1 and Cpt1α, was significantly upregulated. Moreover, Mttp was downregulated in the two fat sits in the DHM group. DHM alleviated diet-induced lipid deposition in the liver and decreased liver triglyceride and total cholesterol content. DHM improved liver function by inhibiting ER stress, alleviating atherogenesis, and promoting vascular remodeling. In conclusion, dihydromyricetin improved NAFLD and related insulin resistance, hyperlipidemia, and atherogenesis by inhibiting liver ER stress in HFD-fed LDLR−/− mice.

PRMT5 inhibition induces pro-inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice.

Protein arginine methyltransferase 5 (PRMT5) controls inflammation and metabolism through modulation of histone methylation and gene transcription. Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p< 0.05) and significantly increased hepatic triglyceride levels (+50%; p< 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.

Antisense oligonucleotides targeting hepatic angiotensinogen reduce atherosclerosis and liver steatosis in hypercholesterolemic mice.

Hepatocyte-derived angiotensinogen (AGT) is the precursor of angiotensin II (AngII). We determined the effects of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASO) on AngII-mediated blood pressure (BP) regulation and atherosclerosis and compared its effects with losartan, an AngII type 1 (AT1) receptor blocker, in hypercholesterolemic mice. Eight-week-old male low-density lipoprotein (LDL) receptor deficient mice were administered vehicle or GalNAc AGT ASO (1, 2.5, or 5 mg/kg) subcutaneously beginning 2 weeks before the initiation of Western diet feeding. All mice were fed Western diet for 12 weeks. Their systolic BP was monitored by the tail-cuff technique, and the atherosclerotic lesion area was measured by an en face method. Although the effects of all 3 doses of GalNAc AGT ASO on plasma AGT concentrations were similar, GalNAc AGT ASO reduced BP and atherosclerotic lesion size in a dose-dependent manner. Subsequently, we compared the effects of GalNAc AGT ASO (5 mg/kg) with losartan (15 mg/kg/day). Compared to losartan, GalNAc AGT ASO led to more profound increases in plasma renin and reduction in BP but had similar effects on atherosclerosis. Remarkably, GalNAc AGT ASO also reduced liver steatosis, which was not observed in losartan-treated mice. In conclusion, the BP increase and atherosclerosis development in hypercholesterolemic mice are dependent on AngII generated from hepatic AGT. Deleting hepatic AGT improves diet-induced liver steatosis, and this occurs in an AT1 receptor-independent manner.

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.

(Pro)renin Receptor Inhibition Reduces Plasma Cholesterol and Triglycerides but Does Not Attenuate Atherosclerosis in Atherosclerotic Mice.

Objective: Elevated plasma cholesterol concentrations contributes to ischemic cardiovascular diseases. Recently, we showed that inhibiting hepatic (pro)renin receptor [(P)RR] attenuated diet-induced hypercholesterolemia and hypertriglyceridemia in low-density lipoprotein receptor (LDLR) deficient mice. The purpose of this study was to determine whether inhibiting hepatic (P)RR could attenuate atherosclerosis.Approach and Results: Eight-week-old male LDLR−/− mice were injected with either saline or N-acetylgalactosamine-modified antisense oligonucleotides (G-ASOs) primarily targeting hepatic (P)RR and were fed a western-type diet (WTD) for 16 weeks. (P)RR G-ASOs markedly reduced plasma cholesterol concentrations from 2,211 ± 146 to 1,128 ± 121 mg/dL. Fast protein liquid chromatography (FPLC) analyses revealed that cholesterol in very low-density lipoprotein (VLDL) and intermediate density lipoprotein (IDL)/LDL fraction were potently reduced by (P)RR G-ASOs. Moreover, (P)RR G-ASOs reduced plasma triglyceride concentrations by more than 80%. Strikingly, despite marked reduction in plasma lipid concentrations, atherosclerosis was not reduced but rather increased in these mice. Further testing in ApoE−/− mice confirmed that (P)RR G-ASOs reduced plasma lipid concentrations but not atherosclerosis. Transcriptomic analysis of the aortas revealed that (P)RR G-ASOs induced the expression of the genes involved in immune responses and inflammation. Further investigation revealed that (P)RR G-ASOs also inhibited (P)RR in macrophages and in enhanced inflammatory responses to exogenous stimuli. Moreover, deleting the (P)RR in macrophages resulted in accelerated atherosclerosis in WTD fed ApoE−/− mice.Conclusion: (P)RR G-ASOs reduced the plasma lipids in atherosclerotic mice due to hepatic (P)RR deficiency. However, augmented pro-inflammatory responses in macrophages due to (P)RR downregulation counteracted the beneficial effects of lowered plasma lipid concentrations on atherosclerosis. Our study demonstrated that hepatic (P)RR and macrophage (P)RR played a counteracting role in atherosclerosis.

Nobiletin Prevents High-Fat Diet-Induced Dysregulation of Intestinal Lipid Metabolism and Attenuates Postprandial Lipemia.

Nobiletin is a dietary flavonoid that improves insulin resistance and atherosclerosis in mice with metabolic dysfunction. Dysregulation of intestinal lipoprotein metabolism contributes to atherogenesis. The objective of the study was to determine if nobiletin targets the intestine to improve metabolic dysregulation in both male and female mice. Approach and Results: Triglyceride-rich lipoprotein (TRL) secretion, intracellular triglyceride kinetics, and intestinal morphology were determined in male and female LDL (low-density lipoprotein) receptor knockout (Ldlr-/-), and male wild-type mice fed a standard laboratory diet or high-fat, high-cholesterol (HFHC) diet ± nobiletin using an olive oil gavage, radiotracers, and electron microscopy. Nobiletin attenuated postprandial TRL levels in plasma and enhanced TRL clearance. Nobiletin reduced fasting jejunal triglyceride accumulation through accelerated TRL secretion and lower jejunal fatty acid synthesis with no impact on fatty acid oxidation. Fasting-refeeding experiments revealed that nobiletin led to higher levels of phosphorylated AKT (protein kinase B) and FoxO1 (forkhead box O1) and normal Srebf1c expression indicating increased insulin sensitivity. Intestinal length and weight were diminished by HFHC feeding and restored by nobiletin. Both fasting and postprandial plasma GLP-1 (glucagon-like peptide-1; and likely GLP-2) were elevated in response to nobiletin. Treatment with a GLP-2 receptor antagonist, GLP-2(3-33), reduced villus length in HFHC-fed mice but did not impact TRL secretion in any diet group. In contrast to males, nobiletin did not improve postprandial lipid parameters in female mice. Nobiletin opposed the effects of the HFHC diet by normalizing intestinal de novo lipogenesis through improved insulin sensitivity. Nobiletin prevents postprandial lipemia because the enhanced TRL clearance more than compensates for increased TRL secretion.

Preliminary adipose removal did not prevent diet-induced metabolic disorders in mice.

Background:Obesity is a fundamental factor in metabolic disorders such as hyperlipidemia, insulin resistance, fatty liver, and atherosclerosis. However, effective preventive measures are still lacking. This study aimed to investigate different surgical protocols for removing partial adipose tissue before the onset of obesity and determine whether, and by which protocol, preliminary adipose removal could exert potent preventive effects against diet-induced metabolic disorders.Methods:Male low-density lipoprotein receptor (LDL-R) knockout (KO) mice were randomly divided into four groups and subjected to epididymal fat removal (Epi-FR) surgery, subcutaneous fat removal (suQ-FR) surgery, both subcutaneous and epididymal fat removal (Epi + suQ-FR) surgery, or sham-operation. After 1 week of recovery, all mice were given a high-fat diet (HFD) for 10 weeks to induce metabolic disorders.Results:In the Epi-FR group and the sham-operated group, the mean numbers of the residual subcutaneous fat were 28.59 mg/g and 18.56 mg/g, respectively. The expression of relative genes such as Pparg, Cebpa, Dgat2, Fabp4 and Cd36 in the residual subcutaneous fat increased 2.62, 3.90, 3.11, 2.06, 1.78 times in the Epi-FR group compared with that in the sham-operated group. Whereas in the other fat-removal groups, the residual fat depots had no significant change in either size or gene expression, as compared with those of the sham-operated group. Plasma lipid and glucose levels and insulin sensitivity, as detected by the glucose tolerance test, were not significantly alleviated in the three fat removal groups. Liver mass or lipid content was not attenuated in any of the three fat removal groups. The atherosclerosis burdens in the entire inner aorta and aortic root did not decrease in any of the three fat removal groups.Conclusions:Our data suggest that removal of epididymal adipose or subcutaneous adipose alone or in combination before the onset of obesity did not protect against hyperlipidemia, insulin resistance, fatty liver, or atherosclerosis in LDL-R KO mice fed with a HFD. Hence, adipose removal possibly does not represent a potential approach in preventing obesity-related metabolic disorders in the obesity-susceptible population.

Therapeutic Assessment of Combination Therapy with a Neprilysin Inhibitor and Angiotensin Type 1 Receptor Antagonist on Angiotensin II-Induced Atherosclerosis, Abdominal Aortic Aneurysms, and Hypertension.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor–deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs.SIGNIFICANCE STATEMENTThe combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor–deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Effects of Oat Fiber Intervention on Cognitive Behavior in LDLR-/- Mice Modeling Atherosclerosis by Targeting the Microbiome-Gut-Brain Axis.

It is known that cardiovascular disease can result in cognitive impairment. However, whether oat fiber improves cognitive behavior through a cardiovascular-related mechanism remains unclear. The present work was aimed to elucidate the potential of oat fiber on cognitive behavior by targeting the neuroinflammation signal and microbiome-gut-brain axis in a mouse model of atherosclerosis. Male low-density lipoprotein receptor knock-out (LDLR-/-) mice were treated with a high fat/cholesterol diet without or with 0.8% oat fiber for 14 weeks. Behavioral tests indicated that LDLR-/- mice exhibited a significant cognitive impairment; however, oat fiber can improve cognitive behavior by reducing latency to the platform and increasing the number of crossing and swimming distance in the target quadrant. Oat fiber can inhibit Aβ plaque processing in both the cortex and hippocampus via decreasing the relative protein expression of GFAP and IBα1. Notably, oat fiber inhibited the nod-like receptor family pyrin domain-containing 3 inflammasome activation and blocked the toll-like receptor 4 signal pathway in both the cortex and hippocampus, accompanied by a reduction of circulating serum lipopolysaccharide. In addition, oat fiber raised the expressions of short-chain fatty acid (SCFA) receptors and tight junction proteins (zonula occludens-1 and occludin) and improved intestinal microbiota diversity via increasing the contents of gut metabolites SCFAs. In summary, the present study provided experimental evidence that dietary oat fiber retarded the progression of cognitive impairment in a mouse model of atherosclerosis. Mechanistically, the neuroprotective potential was related to oat fiber and its metabolites SCFAs on the diversity and abundance of gut microbiota that produced anti-inflammatory metabolites, leading to repressed neuroinflammation and reduced gut permeability through the microbiome-gut-brain axis.

Prodigiosin Modulates the Immune Response and Could Promote a Stable Atherosclerotic Lession in C57bl/6 Ldlr-/- Mice.

Atherosclerosis is a chronic inflammatory disease, whose progression and stability are modulated, among other factors, by an innate and adaptive immune response. Prodiginines are bacterial secondary metabolites with antiproliferative and immunomodulatory activities; however, their effect on the progression or vulnerability of atheromatous plaque has not been evaluated. This study assessed the therapeutic potential of prodigiosin and undecylprodigiosin on inflammatory marker expression and atherosclerosis. An in vitro and in vivo study was carried out. Migration, low-density lipoprotein (LDL) uptake and angiogenesis assays were performed on cell types involved in the pathophysiology of atherosclerosis. In addition, male LDL receptor null (Ldlr-/-) C57BL/6J mice were treated with prodigiosin or undecylprodigiosin for 28 days. Morphometric analysis of atherosclerotic plaques, gene expression of atherogenic factors in the aortic sinus and serum cytokine quantification were performed. The treatments applied had slight effects on the in vitro tests performed, highlighting the inhibitory effect on the migration of SMCs (smooth muscle cells). On the other hand, although no significant difference in atherosclerotic plaque progression was observed, gene expression of IL-4 and chemokine (C-C motif) ligand 2 (Ccl2) was downregulated. In addition, 50 µg/Kg/day of both treatments was sufficient to inhibit circulating tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in serum. These results suggested that prodigiosin and undecylprodigiosin modulated inflammatory markers and could have an impact in reducing atherosclerotic plaque vulnerability.

Anti-atherogenic properties of Kgengwe (Citrullus lanatus) seed powder in low-density lipoprotein receptor knockout mice are mediated through beneficial alterations in inflammatory pathways.

Kgengwe fruits are commonly consumed in sub-Saharan countries. Recent reports indicated low coronary artery disease rates in those regions. To investigate anti-atherogenic properties and potential mechanisms of action of Kgengwe seed powder (KSP), male low-density lipoprotein receptor knockout (LDL-r-KO) mice were fed with an atherogenic diet supplemented with (treated, n = 10) or without (controls, n = 10) 10% (w/w) KSP for 20weeks. Proximate analysis revealed that KSP contained 38% fibre and 15% lipids. KSP supplementation was not associated with significant changes in body weight gain rate, food intake, and plasma lipid levels. However, the average atherosclerotic lesion size in the aortic roots in the KSP-treated group was 58% smaller than that in the control group (0.26 vs 0.11 mm2, p< 0.05). This strong anti-atherogenic effect was associated with significant increases in the average plasma levels of certain cytokines such as IL-10 (6 vs 13 pg/mL, p< 0.05), GM-CSF (0.1 vs 0.2 pg/mL, p< 0.05), and EPO (7 vs 16 pg/mL, p< 0.05) along with reductions in the average levels of plasma MCP-1 (19 vs 14 pg/mL, p< 0.05) and MIP-2 (28 vs 13 pg/mL, p< 0.05). Except for relatively high levels of saturated fatty acids, KSP possesses balanced nutrient compositions with strong anti-atherogenic properties, which may be mediated through alterations in inflammatory pathways. Additional studies warrant confirmation and mechanism(s) of action of such effects. Novelty: Kgengwe seeds prevent atherogenesis in LDL-r-KO mice. Kgengwe seeds increase circulating levels of IL-10 and EPO. No reduction in plasma total cholesterol levels.

One amino acid change of Angiotensin II diminishes its effects on abdominal aortic aneurysm

Angiotensin (Ang) A is formed by the decarboxylation of the N terminal residue of AngII. The present study determined whether this one amino acid change impacted effects of AngII on abdominal aortic aneurysm (AAA) formation in mice. Computational analyses implicated that AngA had comparable binding affinity to both AngII type 1 and 2 receptors as AngII. To compare effects of these two octapeptides in vivo, male low-density lipoprotein receptor (Ldlr) or apolipoprotein E (Apoe) deficient mice were infused with either AngII or AngA (1 μg/kg/min) for 4 weeks. While AngII infusion induced AAA consistently in both mouse strains, the equivalent infusion rate of AngA did not lead to AAA formation. We also determined whether co-infusion of AngA would influence AngII-induced aortic aneurysm formation in male Apoe−/− mice. Co-infusion of the same infusion rate of AngII and AngA did not change AngII-induced AAA formation. Since it was reported that a 10-fold higher concentration of AngA elicited comparable vasoconstrictive responses as AngII, we compared a 10-fold higher rate (10 μg/kg/min) of AngA infusion into male Apoe−/− mice with AngII (1 μg/kg/min). This rate of AngA led to abdominal aortic dilation in three of ten mice, but no aortic rupture, whereas the 10-fold lower rate of AngII infusion led to abdominal aortic dilation or rupture in eight of ten mice. In conclusion, AngA, despite only being one amino acid different from AngII, has diminished effects on aortic aneurysmal formation, implicating that the first amino acid of AngII has important pathophysiological functions.

10,12 Conjugated Linoleic Acid-Driven Weight Loss Is Protective against Atherosclerosis in Mice and Is Associated with Alternative Macrophage Enrichment in Perivascular Adipose Tissue.

The dietary fatty acid 10,12 conjugated linoleic acid (10,12 CLA) promotes weight loss by increasing fat oxidation, but its effects on atherosclerosis are less clear. We recently showed that weight loss induced by 10,12 CLA in an atherosclerosis-susceptible mouse model with characteristics similar to human metabolic syndrome is accompanied by accumulation of alternatively activated macrophages within subcutaneous adipose tissue. The objective of this study was to evaluate whether 10,12 CLA-mediated weight loss was associated with an atheroprotective phenotype. Male low-density lipoprotein receptor deficient (Ldlr−/−) mice were made obese with 12 weeks of a high-fat, high-sucrose diet feeding (HFHS: 36% fat, 36% sucrose, 0.15% added cholesterol), then either continued on the HFHS diet with or without caloric restriction (CR), or switched to a diet with 1% of the lard replaced by either 9,11 CLA or 10,12 CLA for 8 weeks. Atherosclerosis and lipid levels were quantified at sacrifice. Weight loss in mice following 10,12 CLA supplementation or CR as a weight-matched control group had improved cholesterol and triglyceride levels, yet only the 10,12 CLA-treated mice had improved en face and aortic sinus atherosclerosis. 10,12 CLA-supplemented mice had increased lesion macrophage content, with enrichment of surrounding perivascular adipose tissue (PVAT) alternative macrophages, which may contribute to the anti-atherosclerotic effect of 10,12 CLA.

Reversal of adipose tissue loss by probucol in mice with deficiency of both scavenger receptor class B type 1 and LDL receptor on high fat diet

Scavenger receptor class B type 1(SR-B1) and low density lipoprotein receptor (LDLR) play vital roles in cholesterol homeostasis. Previous studies indicated a strong link between cholesterol and adipose tissue (AT). In this study, adult male SR-B1 and LDLR double knockout (DKO) mice were fed with high fat diet (HFD) for 3 months. Interestingly, we found severe loss of AT in DKO mice fed with HFD. To reverse the AT loss in DKO mice, 1% probucol was added in HFD. In DKO mice on HFD, plasma total cholesterol (TC) and free cholesterol (FC) levels were increased 6 and 15 folds respectively compared with wild type (WT) mice. We found severe loss of AT in whole body of DKO mice compared with WT or single KO mice. In AT of DKO mice, histology showed the very small size of adipocytes and infiltration of inflammatory cells; Genes expressions related to fatty acid uptake, lipogenesis and adipogenesis were decreased; TUNEL analysis and related genes expressions of endoplasmic reticulum (ER) stress and inflammation were significantly higher than those of WT or single KO mice. Probucol could reduce increased TC and FC levels, and reverse the loss of fat and apoptosis of AT in DKO mice. AT loss in DKO mice with HFD was probably due to high levels of FC which led to apoptosis induced by ER stress and inflammation of AT. This study provided a novel utility of probucol in rescue of fat loss in DKO mice.

Abstract 422: Protease-Activated Receptor 2 is Critical for the Formation and Progression of Atherosclerosis

Objective: Protease-activated receptor 2 (PAR-2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. While PAR-2 protein is present in coronary atherosclerotic lesions, the relevance of this finding has not been investigated in experimental models. The objective of this study was to determine the effects of PAR-2 on the development of atherosclerosis. Methods and Results: Relative expression of PAR-2 is increased in human coronary artery (21 fold) and mouse aortic arch (16 fold) atheromas versus control coronary and aortic arch arteries, respectively (P = 0.001). To determine the effect of PAR-2 deficiency on atherosclerosis, male low density lipoprotein receptor deficient ( Ldlr -/- ) mice (8-12 weeks old) that were Par-2 +/+ or Par-2 -/- were fed a fat and cholesterol-enriched diet for 12 (n = 10 each group) or 24 weeks (n = 5 each group). PAR-2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root with no effects on total plasma cholesterol concentrations or lipoprotein distributions after 12 (P = 0.000433) and 24 (P = 0.037) weeks. These reductions were attributable to both hematopoietic and non-hematopoietic-derived PAR-2 from analysis of bone marrow experiments (n = 15 for each of 4 chimeric groups; P &lt; 0.05). Mechanistic studies using ex vivo macrophages show that activation of PAR-2 results in augmented foam cell formation and apoptosis with treatment of oxidized low-density lipoprotein in conjunction with decreased expression of the nuclear receptor LXR-alpha and several cholesterol transporters. In addition, PAR-2 activation of ex-vivo cultured vascular smooth muscle cells (VSMCs) augments their transition to a macrophage-like state (after cholesterol treatment) via upregulation of human antigen R (HuR) and resultant stabilization of the transcription factor Krüppel-like factor 4 (KLF4). Conclusion: Our results indicate PAR-2 deficiency significantly attenuates the initiation (12 weeks) and reduces the progression (24 weeks) of atherosclerosis potentially via regulation of both lipid efflux from macrophages and the phenotypic modulation of VSMCs.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Macrophage IKKα Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis.

The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells. Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Vascular smooth muscle LRP6 limits arteriosclerotic calcification in diabetic LDLR-/- mice by restraining noncanonical Wnt signals.

Wnt signaling regulates key aspects of diabetic vascular disease. We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis. As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation. LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.

Metabolomic Profiling of Arginine Metabolome Links Altered Methylation to Chronic Kidney Disease Accelerated Atherosclerosis.

Atherosclerotic cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD), but the mechanisms underlying vascular disease has not been fully understood. As the nitrogen donor in nitric oxide (NO·) synthesis, arginine and its metabolic products are integrally linked to vascular health and information. We hypothesized that derangements in this pathway could explain, in part, increased atherosclerotic risk in CKD. We developed a targeted metabolomic platform to profile quantitatively arginine metabolites in plasma by liquid chromatography tandem mass spectrometry (LC/MS). Male low-density lipoprotein receptor defcient (LDLr−/−) mice at age 6 weeks were subjected to sham or 5/6 nephrectomy surgery to induce CKD. Subsequently, the animals were maintained on high fat diet for 24 weeks. Targeted metabolomic analysis of arginine metabolites in plasma was performed by isotope dilution LC/MS including asymmetric dimethyl arginine (ADMA), symmetric dimethyl arginine (SDMA), N-mono-methylarginine (NMMA), arginine and citrulline. Although elevated plasma levels of ADMA and SDMA were found in the CKD mice, only higher ADMA level correlated with degree of atherosclerosis. No significant differences were noted in levels of NMMA between the groups. CKD mice had high levels of citrulline and arginine, but ADMA levels had no correlation with either of these metabolites. These fndings strongly implicate altered arginine methylation and accumulation of ADMA, may in part contribute to CKD accelerated atherosclerosis. It raises the possibility that interrupting pathways that generate ADMA or enhance its metabolism may have therapeutic potential in mitigating atherosclerosis.

Noninvasive imaging of aortic atherosclerosis by ultrasound biomicroscopy in a mouse model.

The noninvasive and accurate evaluation of vessel characteristics in mouse models has become an intensive focus of vascular medicine. This study aimed to apply ultrasound biomicroscopy to evaluate aortic atherosclerotic progression in a low-density lipoprotein receptor (LDL-R) knockout mouse model of atherosclerosis. Ten male LDL-R(-/-)C57BL/6 mice aged 16 and 24 weeks and 8 male wild-type C57BL/6 mice aged 16 and 24 weeks were used as experimental and control groups, respectively. Ultrasound biomicroscopy was applied to detect the morphologic characteristics of the aortic root, ascending aorta, aortic arch, and carotid artery and to measure the aortic root intima-media thickness and carotid artery bifurcation. Ultrasound biomicroscopy showed a significant increase in the aortic root intima-media thickness from 0.10 ± 0.03 mm in 16-week-old mice to 0.16 ± 0.04 mm in 24-week-old mice (P < .01). The ultrasound biomicroscopically measured intima-media thickness was highly correlated with the histologic measurement (r = 0.81). Ultrasound biomicroscopy could be used for a noninvasive, accurate, and dynamic analysis of aortic atherosclerosis in LDL-R knockout mice.