Low-density Lipoprotein Receptor-deficient Mice Research Articles

Myeloid GSK3α Deficiency Reduces Lesional Inflammation and Neovascularization during Atherosclerotic Progression.

The molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are poorly understood. We have recently shown that genetic ablation of myeloid glycogen synthase kinase (GSK)-3α attenuates atherosclerotic lesion development in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. However, the precise contributions of GSK3α/β in atherogenesis are not known. The aim of this study is to investigate the effect of GSK3α and/or β deficiency on lesional inflammation and plaque vascularization. Five-week-old female Ldlr-/- mice were fed a high-fat diet for 10 weeks to establish atherosclerotic lesions. Mice were harvested at 15 weeks of age and atherosclerotic lesions were characterized. The results indicate that, in addition to significantly reducing plaque volume, GSK3α-deficiency decreases inflammation, reduces vasa vasorum density at the aortic sinus, and reduces plasma c-reactive protein (CRP) levels. GSK3β-deficiency is associated with decreased plasma CRP levels but does not affect lesional inflammation or vascularization. These results suggest GSK3α may be an applicable target for the development of novel anti-atherogenic therapies.

CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Carnitine O-octanoyltransferase (CROT) deficiency in mice leads to an increase of omega-3 fatty acids.

Introduction: Carnitine O-octanoyltransferase (CROT) is a well-established peroxisomal enzyme involved in liver fatty acid oxidation, but less is known about its recently discovered role in promoting vascular calcification, and whether CROT-dependent liver metabolism contributes to the latter. To date, CROT function in the context of calcification potential has been conducted in the dyslipidemic low-density lipoprotein receptor-deficient (Ldlr-/-) mice. Objectives: To differentiate peroxisome and CROT-dependent lipid biology from that of lipoprotein-mediated lipid biology, we therefore conducted a metabolomic analysis of the liver and plasma of normolipidemic CROT-deficient (Crot-/-) mice. Methods: We performed LC-MS-based metabolomics on liver and plasma derived from Crot-/- and Crot +/- mice and sibling Crot+/+ mice, using a dual-phase metabolite extraction protocol, and multiple LC-MS acquisition strategies. Results: We identified between 79 to 453 annotated metabolites from annotated metabolites from liver samples, and 117 to 424 annotated metabolites from plasma samples. Through differential abundance analysis, we determined that omega-3 fatty acids such as EPA, DPA, and DHA were higher in the liver of Crot-/- and Crot +/- mice than Crot+/+ mice. EPA were higher in plasma of Crot-/- mice than Crot+/+ mice. We also determined that the anti-inflammatory dicarboxylic acids, tetradecanedioic acid and azelaic acid, were higher in the plasma of CROT-deficient mice. Conclusion: Our study associated genetic CROT deletion with increased levels of anti-inflammatory molecules in mouse liver and plasma. These results suggest a potential mechanism for anti-calcification effects of CROT suppression and the potential use of omega-3 fatty acids as biomarkers for future CROT inhibition therapies.

Cathelicidin Antimicrobial Peptide Levels in Atherosclerosis and Myocardial Infarction in Mice and Human.

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.

Finasteride delays atherosclerosis progression in mice and is associated with a reduction in plasma cholesterol in men

Finasteride is commonly prescribed to treat benign prostate hyperplasia and male-pattern baldness in cis men and, more recently, trans individuals. However, the effect of finasteride on cardiovascular disease remains elusive. We evaluated the role of finasteride on atherosclerosis using low-density lipoprotein (LDL) receptor-deficient (Ldlr−/−) mice. Next, we examined the relevance to humans by analyzing the data deposited between 2009 and 2016 in the National Health and Nutrition Examination Survey. We show that finasteride reduces total plasma cholesterol and delays the development of atherosclerosis in Ldlr−/− mice. Finasteride reduced monocytosis, monocyte recruitment to the lesion, macrophage lesion content, and necrotic core area, the latter of which is an indicator of plaque vulnerability in humans. RNA sequencing analysis revealed a downregulation of inflammatory pathways and an upregulation of bile acid metabolism, oxidative phosphorylation, and cholesterol pathways in the liver of mice taking finasteride. Men reporting the use of finasteride showed lower plasma levels of cholesterol and LDL-cholesterol than those not taking the drug. Our data unveil finasteride as a potential treatment to delay cardiovascular disease in people by improving the plasma lipid profile.

Enhancing aortic valve drug delivery with PAR2-targeting magnetic nano-cargoes for calcification alleviation

Calcific aortic valve disease is a prevalent cardiovascular disease with no available drugs capable of effectively preventing its progression. Hence, an efficient drug delivery system could serve as a valuable tool in drug screening and potentially enhance therapeutic efficacy. However, due to the rapid blood flow rate associated with aortic valve stenosis and the lack of specific markers, achieving targeted drug delivery for calcific aortic valve disease has proved to be challenging. Here we find that protease-activated-receptor 2 (PAR2) expression is up-regulated on the plasma membrane of osteogenically differentiated valvular interstitial cells. Accordingly, we develop a magnetic nanocarrier functionalized with PAR2-targeting hexapeptide for dual-active targeting drug delivery. We show that the nanocarriers effectively deliver XCT790—an anti-calcification drug—to the calcified aortic valve under extra magnetic field navigation. We demonstrate that the nano-cargoes consequently inhibit the osteogenic differentiation of valvular interstitial cells, and alleviate aortic valve calcification and stenosis in a high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr−/−) mouse model. This work combining PAR2- and magnetic-targeting presents an effective targeted drug delivery system for treating calcific aortic valve disease in a murine model, promising future clinical translation.

In situ lipid-loading activates peripheral dendritic cell subsets characterized by cellular ROS accumulation but compromises their capacity to prime naïve T cells

Background and aimsDendritic cells (DCs), professional antigen-presenting cells, play an important role in pathologies by controlling adaptive immune responses. However, their adaptation to and functionality in hypercholesterolemia, a driving factor in disease onset and progression of atherosclerosis remains to be established. MethodsIn this study, we addressed the immediate impact of high fat diet-induced hypercholesterolemia in low-density lipoprotein receptor deficient (Ldlr−/−) mice on separate DC subsets, their compartmentalization and functionality. ResultsWhile hypercholesterolemia induced a significant rise in bone marrow myeloid and dendritic cell progenitor (MDP) frequency and proliferation rate after high fat diet feeding, it did not affect DC subset numbers in lymphoid tissue. Hypercholesterolemia led to almost immediate and persistent augmentation in granularity of conventional DCs (cDCs), in particular cDC2, reflecting progressive lipid accumulation by these subsets. Plasmacytoid DCs were only marginally and transiently affected. Lipid loading increased co-stimulatory molecule expression and ROS accumulation by cDC2. Despite this hyperactivation, lipid-laden cDC2 displayed a profoundly reduced capacity to stimulate naïve CD4+ T cells. ConclusionOur data provide evidence that in hypercholesterolemic conditions, peripheral cDC2 subsets engulf lipids in situ, leading to a more activated status characterized by cellular ROS accumulation while, paradoxically, compromising their T cell priming ability. These findings will have repercussions not only for lipid driven cardiometabolic disorders like atherosclerosis, but also for adaptive immune responses to pathogens and/or endogenous (neo) antigens under conditions of hyperlipidemia.

Sortilin-induced lipid accumulation and atherogenesis are suppressed by HNF1b SUMOylation promoted by flavone of Polygonatum odoratum.

This study aims to investigate the impact of hepatocyte nuclear factor 1β (HNF1b) on macrophage sortilin-mediated lipid metabolism and aortic atherosclerosis and explore the role of the flavone of Polygonatum odoratum (PAOA-flavone)-promoted small ubiquitin-related modifier (SUMO) modification in the atheroprotective efficacy of HNF1b. HNF1b was predicted to be a transcriptional regulator of sortilin expression via bioinformatics, dual-luciferase reporter gene assay, and chromatin immunoprecipitation. HNF1b overexpression decreased sortilin expression and cellular lipid contents in THP-1 macrophages, leading to a depression in atherosclerotic plaque formation in low-density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice. Multiple SUMO1-modified sites were identified on the HNF1b protein and co-immunoprecipitation confirmed its SUMO1 modification. The SUMOylation of HNF1b protein enhanced the HNF1b-inhibited effect on sortilin expression and reduced lipid contents in macrophages. PAOA-flavone treatment promoted SUMO-activating enzyme subunit 1 (SAE1) expression and SAE1-catalyzed SUMOylation of the HNF1b protein, which prevented sortilin-mediated lipid accumulation in macrophages and the formation of atherosclerotic plaques in apolipoprotein E-deficient (ApoE-/-) mice. Interference with SAE1 abrogated the improvement in lipid metabolism in macrophage cells and atheroprotective efficacy in vivo upon PAOA-flavone administration. In summary, HNF1b transcriptionally suppressed sortilin expression and macrophage lipid accumulation to inhibit aortic lipid deposition and the development of atherosclerosis. This anti-atherosclerotic effect was enhanced by PAOA-flavone-facilitated, SAE1-catalyzed SUMOylation of the HNF1b protein.

Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

Neovascular age-related macular degeneration AMD (nAMD) is characterized by choroidal neovascularization (CNV) and could lead to irreversible blindness. However, anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy. Therefore, we generated a chimpanzee adenoviral vector (AdC68-PFC) containing three genes, pigment endothelial-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble forms of CD59 (sCD59), to treat nAMD. The results showed that AdC68-PFC mediated a strong onset of PEDF, sFlt-1, and sCD59 expression both invivo and invitro. AdC68-PFC showed preventive and therapeutic effects following intravitreal (IVT) injection in the laser-induced CNV model and very low-density lipoprotein receptor-deficient (Vldlr-/-) mouse model. Invitro assessment indicated that AdC68-PFC had a strong inhibitory effect on endothelial cells. Importantly, the safety test showed no evidence of invivo toxicity of adenovirus in murine eyes. Our findings suggest that AdC68-PFC may be a long-acting and safe gene therapy vector for future nAMD treatments.

Abstract 217: Protease Activated Receptor 2 Is Critical For Vascular Smooth Muscle Cell Transition To A Macrophage-like State

Objective: Recent lineage tracing studies have shown vascular smooth muscle cells (VSMCs) develop macrophage-like characteristics (MLCs) in late-stage atherosclerosis resulting in accelerated inflammation and disease severity. Our objective was to determine if the inflammatory receptor, protease activated receptor 2 (PAR2), accelerates VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Our lab demonstrated Par2 -/- mice have decreased atherosclerotic lesion area versus proficient controls via downregulation of VSMC migration and cytokine release. To examine the role of PAR2 in VSMC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10 ug/mL methyl- β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers (α - actin and myosin heavy chain) and upregulated macrophage markers (CD68 and Mac-2), Par2 -/- VSMCs remained stable. Looking at macrophage characteristics, Par2 -/- VSMCs phagocytized significantly less latex beads than Par2 +/+ VSMCs. Analysis of a hybrid mouse diversity panel (HMDP), conducted on 101 strains of mice with induced atherosclerosis, determined PAR2 was significantly correlated with the MLC super-gene Krüppel-like factor 4 (KLF4), which was found to have lower genetic expression in Par2 deficient VSMCs. Previous studies have demonstrated Par2 mRNA is bound and stabilized by the RNA binding protein human antigen R (HuR). Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression and activity, suggesting a role of HuR in PAR2 mRNA stability. Par2 flox/flox mice bred with transgelin (SM22-alpha) Cre and low-density lipoprotein receptor deficient mice l have been establishing in our lab to investigate the effects of cell-specific deletions of PAR2 in atherosclerosis. VSMC-specific Par2 deficiency blunts the formation of atherosclerosis versus the Cre + littermates. Conclusions: These results suggest that VSMC-specific PAR2 activation mediates the dedifferentiation of VSMCs via upregulation of KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. Future studies will continue to study VSMC dedifferentiation utilizing tagged lineage-tracing mouse models and various in vitro techniques.

Abstract 313: Finasteride Delays Atherosclerosis Progression and Reduces Hepatic Inflammation in Low-density Lipoprotein Receptor-deficient Mice

Finasteride is commonly prescribed to treat benign hyperplasia and male-pattern baldness in cis men and, more recently, trans individuals. However, the effect of finasteride on cardiovascular disease remains elusive. We evaluated the role of finasteride on atherosclerosis using low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice fed a Western diet containing increasing doses of finasteride for 12 weeks. We did not observe changes in food intake, body weight, or liver markers for toxicity for any of our experimental conditions. At the highest dose, finasteride reduced plasma cholesterol and triglycerides to approximately 25% compared to the control group. Plaque content at the aortic arch and aortic root level showed a reduction in lesion burden. Finasteride reduced monocytosis, monocyte recruitment to the lesion, macrophage lesion content, and necrotic area. RNA sequencing analysis revealed a downregulation of inflammatory pathways and an upregulation of bile acid metabolism, oxidative phosphorylation, and cholesterol pathways in the liver of mice taking finasteride. Our data unveil finasteride as a potential treatment to delay cardiovascular disease by its effects on plasma lipid profile.

Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Celastrol Supplementation Ablates Sexual Dimorphism of Abdominal Aortic Aneurysm Formation in Mice.

Abdominal aortic aneurysms (AAAs) are permanent dilations of the abdominal aorta with 4-5 times greater prevalence in males than in females. The aim of this study is to define whether Celastrol, a pentacyclic triterpene from the root extracts of Tripterygium wilfordii, supplementation influences angiotensin II (AngII)-induced AAAs in hypercholesterolemic mice. Age-matched (8-12 weeks old) male and female low-density lipoprotein (Ldl) receptor-deficient mice were fed a fat-enriched diet supplemented with or without Celastrol (10 mg/kg/day) for five weeks. After one week of diet feeding, mice were infused with either saline (n = 5 per group) or AngII (500 or 1000 ng/kg/min, n = 12-15 per group) for 28 days. Dietary supplementation of Celastrol profoundly increased AngII-induced abdominal aortic luminal dilation and external aortic width in male mice as measured by ultrasonography and ex vivo measurement, with a significant increase in incidence compared to the control group. Celastrol supplementation in female mice resulted in significantly increased AngII-induced AAA formation and incidence. In addition, Celastrol supplementation significantly increased AngII-induced aortic medial elastin degradation accompanied by significant aortic MMP9 activation in both male and female mice compared to saline and AngII controls. Celastrol supplementation to Ldl receptor-deficient mice ablates sexual dimorphism and promotes AngII-induced AAA formation, which is associated with increased MMP9 activation and aortic medial destruction.

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.

LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

Long non-coding RNAs (lncRNAs) are emerging as important players in gene regulation and cardiovascular diseases. However, the roles of lncRNAs in atherosclerosis are poorly understood. In the present study, we found that the levels of NIPA1-SO were decreased while those of NIPA1 were increased in human atherosclerotic plaques. Furthermore, NIPA1-SO negatively regulated NIPA1 expression in human umbilical vein endothelial cells (HUVECs). Mechanistically, NIPA1-SO interacted with the transcription factor FUBP1 and the NIPA1 gene. The effect of NIPA1-SO on NIPA1 protein levels was reversed by the knockdown of FUBP1. NIPA1-SO overexpression increased, whilst NIPA1-SO knockdown decreased BMPR2 levels; these effects were enhanced by the knockdown of NIPA1. The overexpression of NIPA1-SO reduced while NIPA1-SO knockdown increased monocyte adhesion to HUVECs; these effects were diminished by the knockdown of BMPR2. The lentivirus-mediated-overexpression of NIPA1-SO or gene-targeted knockout of NIPA1 in low-density lipoprotein receptor-deficient mice reduced monocyte-endothelium adhesion and atherosclerotic lesion formation. Collectively, these findings revealed a novel anti-atherosclerotic role for the lncRNA NIPA1-SO and highlighted its inhibitory effects on vascular inflammation and intracellular cholesterol accumulation by binding to FUBP1 and consequently repressing NIPA1 expression.

LDR-adapted liver-derived cytokines have potential to induce atherosclerosis

Purpose Atherosclerosis is a lipid-driven chronic inflammatory disease that causes cardiovascular diseases (CVD). The association between radiation and atherosclerosis has already been demonstrated; however, the effects of low-dose radiation (LDR) exposure on atherosclerosis have not been reported. Our study aims to propose that LDR may cause atherosclerosis phenotypes by the upregulation of plasminogen activator inhibitor-1 (PAI-1) and downregulation of androgen receptor (AR), which are cytokines secreted from the liver. Methods Low-density lipoprotein (LDL) receptor deficient (Ldlr−/−) mice were irradiated at 50 mGy, 100 mGy, and 1000 mGy. LDR irradiated Ldlr−/− mice serum was analyzed by cytokine array and proteomics with silver staining. Oil Red O staining and BODIPY staining were performed to determine lipid accumulation in Human umbilical vein endothelial cells (HUVECs). Foam cell formation and monocyte recruitment were assessed through co-culture system with HUVECs and THP-1 cells. Results After irradiation with LDR (100 mGy) the mice showed atherosclerotic phenotypes and through analysis results, we selected regulated cytokines, PAI-1 and AR, and found that these were changed in the liver. LDR-regulated cytokines have the potential to be transported to endothelial cells and induce lipid accumulation, inflammation of monocytes, increased oxidized low-density lipoprotein (oxLDL) and foam cells formation, that were series of phenotypes lead to plaque formation in endothelial cells and induces atherosclerosis. As a further aspect of this study, testosterone undecanoate (TU) was found to pharmacologically inhibit a series of atherosclerotic phenotypes exhibited by LDR. This study suggests a role for PAI-1 and AR in regulating the development of atherosclerosis after LDR exposure. Targeting PAI-1 and AR could serve as an attractive strategy for the management of atherosclerosis following LDR exposure.

Apolipoprotein A1 Protects Against Necrotic Core Development in Atherosclerotic Plaques: PDZK1-Dependent High-Density Lipoprotein Suppression of Necroptosis in Macrophages.

Atherosclerosis is a chronic disease affecting artery wall and a major contributor to cardiovascular diseases. Large necrotic cores increase risk of plaque rupture leading to thrombus formation. Necrotic cores are rich in debris from dead macrophages. Programmed necrosis (necroptosis) contributes to necrotic core formation. HDL (high-density lipoprotein) exerts direct atheroprotective effects on different cells within atherosclerotic plaques. Some of these depend on the SR-B1 (scavenger receptor class B type I) and the adapter protein PDZK1 (postsynaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1). However, a role for HDL in protecting against necroptosis and necrotic core formation in atherosclerosis is not completely understood. Low-density lipoprotein receptor-deficient mice engineered to express different amounts of ApoA1 (apolipoprotein A1), or to lack PDZK1 were fed a high fat diet for 10 weeks. Atherosclerotic plaque areas, necrotic cores, and key necroptosis mediators, RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like protein) were characterized. Cultured macrophages were treated with HDL to determine its effects, as well as the roles of SR-B1, PDZK1, and the PI3K (phosphoinositide 3-kinase) signaling pathway on necroptotic cell death. Genetic overexpression reduced, and ApoA1 knockout increased necrotic core formation and RIPK3 and MLKL within atherosclerotic plaques. Macrophages were protected against necroptosis by HDL and this protection required SR-B1, PDZK1, and PI3K/Akt pathway. PDZK1 knockout increased atherosclerosis in LDLRKO mice, increasing necrotic cores and phospho-MLKL; both of which were reversed by restoring PDZK1 in BM-derived cells. Our findings demonstrate that HDL in vitro and ApoA1, in vivo, protect against necroptosis in macrophages and necrotic core formation in atherosclerosis, suggesting a pathway that could be a target for the treatment of atherosclerosis.

Identification of diagnostic biomarkers and therapeutic targets in peripheral immune landscape from coronary artery disease

BackgroundPeripheral biomarkers are increasingly vital non-invasive methods for monitoring coronary artery disease (CAD) progression. Their superiority in early detection, prognosis evaluation and classified diagnosis is becoming irreplaceable. Nevertheless, they are still less explored. This study aimed to determine and validate the diagnostic and therapeutic values of differentially expressed immune-related genes (DE-IRGs) in CAD.MethodsWe downloaded clinical information and RNA sequence data from the GEO database. We used R software, GO, KEGG and Cytoscape to analyze and visualize the data. A LASSO method was conducted to identify key genes for diagnostic model construction. The ssGSEA analysis was used to investigate the differential immune cell infiltration. Besides, we constructed CAD mouse model (low-density lipoprotein receptor deficient mice with high fat diet) to discover the correlation between the screened genes and severe CAD progress. We further uncovered the role of IL13RA1 might play in atherosclerosis.ResultsA total of 762 differential genes were identified between the peripheral blood of 218 controls and 199 CAD patients, which were significantly associated with infection, immune response and neural activity. 58 DE-IRGs were obtained by overlapping the differentially expressed genes(DEGs) and immune-related genes downloaded from ImmpDb database. Through LASSO regression, CCR9, CER1, CSF2, IL13RA1, INSL5, MBL2, MMP9, MSR1, NTS, TNFRSF19, CXCL2, HTR3C, IL1A, and NR4A2 were distinguished as peripheral biomarkers of CAD with eligible diagnostic capabilities in the training set (AUC = 0.968) and test set (AUC = 0.859). The ssGSEA analysis showed that the peripheral immune cells had characteristic distribution in CAD and also close relationship with specific DE-IRGs. RT-qPCR test showed that CCR9, CSF2, IL13RA1, and NTS had a significant correlation with LDLR−/− mice. IL13RA1 knocked down in RAW264.7 cell lines decreased SCARB1 and ox-LDL-stimulated CD36 mRNA expression, TGF-β, VEGF-C and α-SMA protein levels and increased the production of IL-6, with downregulation of JAK1/STAT3 signal pathway.ConclusionsWe constructed a diagnostic model of advanced-stage CAD based on the screened 14 DE-IRGs. We verified 4 genes of them to have a strong correlation with CAD, and IL13RA1 might participate in the inflammation, fibrosis, and cholesterol efflux process of atherosclerosis by regulating JAK1/STAT3 pathway.

Effect of Thymoquinone on Renal Damage Induced by Hyperlipidemia in LDL Receptor-Deficient (LDL-R-/-) Mice

Hyperlipidemia is a well-established risk factor for kidney injury, which can lead to chronic kidney disease (CKD). Thymoquinone (TQ) is one of the most active ingredients in Nigella sativa seeds. It has various beneficial properties, including antioxidant and anti-inflammatory activities. TQ also exerts positive effects on doxorubicin- (DOX-) induced nephropathy and ischemia-reperfusion-induced kidney injury in rats. Therefore, in this study, we investigated the possible protective effects of TQ against kidney injury in low-density lipoprotein receptor-deficient (LDL-R−/−) mice. Eight-week-old male LDL-R−/− mice were randomly divided into the following three groups: normal diet (ND group), high-fat diet (HFD group), and HFD combined with TQ (HFD+TQ group). The mice were fed the same diet for eight weeks. After eight weeks, we performed serological analysis of the mice in all three groups. We histologically analyzed the kidney tissue and also investigated the expression of proinflammatory cytokines in the kidney tissue. Metabolic characteristics, including total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and creatinine (CRE) levels, were lower in the LDL-R−/− HFD+TQ mice than in the HFD mice. Periodic acid-Schiff (PAS) and Masson's trichrome staining revealed excessive lipid deposition and collagen accumulation in the kidneys of the LDL-R−/− HFD mice, which were significantly reduced in the LDL-R−/− HFD+TQ mice. Furthermore, macrophages and levels of proinflammatory cytokines were lower in the kidney tissues of the LDL-R−/− HFD+TQ mice than in those of the LDL-R−/− HFD mice. Moreover, profibrosis- and oxidative stress-related protein expression was lower in the kidney tissues of the LDL-R−/− HFD+TQ mice than in those of the LDL-R−/− HFD mice. These results indicate that TQ may be a potential therapeutic agent for kidney damage caused by hyperlipidemia.

Expression of a PCSK9 Gain-of-Function Mutation in C57BL/6J Mice to Facilitate Angiotensin II-Induced AAAs.

Angiotensin II (AngII) infusion in mice has been used widely to investigate mechanisms of abdominal aortic aneurysms (AAAs). To achieve a high incidence of AngII-induced AAAs, mice should be hypercholesterolemic. Therefore, either low-density lipoprotein receptor (LDLR) or apolipoprotein E deficiency have been used as a hypercholesterolemic background. However, it is a time-consuming and expensive process to generate compound deficient strains that have either an LDLR or apolipoprotein E deficient background. Proprotein convertase subtilisin/kexin type 9 (PCSK9) facilitates the degradation of LDL receptors. Previous studies demonstrated profound increases of plasma cholesterol concentrations after a single intraperitoneal injection of adeno-associated viruses (AAV) expressing a gain-of-function mutation of mouse PCSK9 (AAV.mPCSK9D377Y) in C57BL/6J mice fed a Western diet. Of note, injection of AAV.mPCSK9D377Y augmented AngII-induced AAA formation in C57BL/6J mice that had comparable severity of AAAs to LDLR deficient mice. Thus, AAV.mPCSK9D377Y infection greatly expedites studies on a gene of interest using AngII-induced AAAs. This commentary provides a brief technical guide of this approach and discusses the pros and cons of its use in AAA research.