apoE-null Mice Research Articles

Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome.

Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear. Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFβ (transforming growth factor β) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFβ1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root. Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFβ1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype. Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFβ1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.

Role of Apolipoprotein E in the Hippocampus and Its Impact following Ionizing Radiation Exposure.

Apolipoprotein E (ApoE) is a lipid carrier in both the peripheral and the central nervous systems (CNSs). Lipid-loaded ApoE lipoprotein particles bind to several cell surface receptors to support membrane homeostasis and brain injury repair. In the brain, ApoE is produced predominantly by astrocytes, but it is also abundantly expressed in most neurons of the CNS. In this study, we addressed the role of ApoE in the hippocampus in mice, focusing on its role in response to radiation injury. To this aim, 8-week-old, wild-type, and ApoE-deficient (ApoE-/-) female mice were acutely whole-body irradiated with 3 Gy of X-rays (0.89 Gy/min), then sacrificed 150 days post-irradiation. In addition, age-matching ApoE-/- females were chronically whole-body irradiated (20 mGy/d, cumulative dose of 3 Gy) for 150 days at the low dose-rate facility at the Institute of Environmental Sciences (IES), Rokkasho, Japan. To seek for ApoE-dependent modification during lineage progression from neural stem cells to neurons, we have evaluated the cellular composition of the dentate gyrus in unexposed and irradiated mice using stage-specific markers of adult neurogenesis. Our findings indicate that ApoE genetic inactivation markedly perturbs adult hippocampal neurogenesis in unexposed and irradiated mice. The effect of ApoE inactivation on the expression of a panel of miRNAs with an established role in hippocampal neurogenesis, as well as its transcriptional consequences in their target genes regulating neurogenic program, have also been analyzed. Our data show that the absence of ApoE-/- also influences synaptic functionality and integration by interfering with the regulation of mir-34a, mir-29b, and mir-128b, leading to the downregulation of synaptic markers PSD95 and synaptophysin mRNA. Finally, compared to acute irradiation, chronic exposure of ApoE null mice yields fewer consequences except for the increased microglia-mediated neuroinflammation. Exploring the function of ApoE in the hippocampus could have implications for developing therapeutic approaches to alleviate radiation-induced brain injury.

Abstract 3055: Cholesterol And Amino Acids Derived From Dietary Sources Drive Atherosclerosis Via Synergistic Activation Of Macrophage Mtorc1 Signaling.

The pathogenic role for lipids, especially cholesterol, in atherosclerosis is well established although relevant mechanisms of action continue to be uncovered. Recent studies linking cholesterol, cholesterol sensing, and the induction of proatherogenic mammalian target of rapamycin (mTOR) signaling is one area with significant therapeutic potential. Our previous studies demonstrated a critical role for amino acids, especially leucine, derived from dietary protein in promoting plaque progression through activation of macrophage mTORC1. We thus aimed to understand the role of cholesterol in macrophage mTOR signaling, its relation to amino acid-mediated effects, and cooperation between dietary lipids and protein in plaque mTOR activation and atherosclerosis. Here, we show LDL cholesterol as well as atherogenic lipids containing modified cholesterol (7-Ketocholesterol and oxLDL) are potent inducers of macrophage mTORC1 especially inconcert with leucine. Importantly, cholesterol and leucine synergistically dampen autophagy/mitophagy, resulting in mitochondrial dysfunction and apoptosis, a process which is completely mTORC1-dependent. In vivo, we find lesional macrophage mTORC1 is stimulated by elevation of plasma cholesterol in ApoE-null mice (n=8-10 per group) fed a Western Diet, while increases in circulating amino acids and cholesterol as a result of diets enriched in protein and lipids lead to mTORC1 hyperactivation and concomitant reduction in autophagy. In conclusion, our study identifies a previously unrecognized signaling role for cholesterol in macrophages and highlights a unique and therapeutically relevant mechanism by which dietary nutrients such as lipids and amino acids can cooperate to drive atherosclerosis.

NOS3 prevents MMP-9, and MMP-13 induced extracellular matrix proteolytic degradation through specific microRNA-targeted expression of extracellular matrix metalloproteinase inducer in hypertension-related atherosclerosis.

Endothelial nitric oxide synthase (NOS3) elicits atheroprotection by preventing extracellular matrix (ECM) proteolytic degradation through inhibition of extracellular matrix metalloproteinase inducer (EMMPRIN) and collagenase MMP-13 by still unknown mechanisms. C57BL/6 mice lacking ApoE , NOS3, and/or MMP13 were fed with a high-fat diet for 6 weeks. Entire aortas were extracted and frozen to analyze protein and nucleic acid expression. Atherosclerotic plaques were detected by ultrasound imaging, Oil Red O (ORO) staining, and Western Blot. RNA-seq and RT-qPCR were performed to evaluate EMMPRIN, MMP-9, and EMMPRIN-targeting miRNAs. Mouse aortic endothelial cells (MAEC) were incubated to assess the role of active MMP-13 over MMP-9. One-way ANOVA or Kruskal-Wallis tests were performed to determine statistical differences. Lack of NOS3 in ApoE null mice fed with a high-fat diet increased severe plaque accumulation, vessel wall widening, and high mortality, along with EMMPRIN-induced expression by upregulation of miRNAs 46a-5p and 486-5p. However, knocking out MMP-13 in ApoE/NOS3 -deficient mice was sufficient to prevent mortality (66.6 vs. 26.6%), plaque progression (23.1 vs. 8.8%), and MMP-9 expression, as confirmed in murine aortic endothelial cell (MAEC) cultures, in which MMP-9 was upregulated by incubation with active recombinant MMP-13, suggesting MMP-9 as a new target of MMP-13 in atherosclerosis. We describe a novel mechanism by which the absence of NOS3 may worsen atherosclerosis through EMMPRIN-induced ECM proteolytic degradation by targeting the expression of miRNAs 146a-5p and 485-5p. Focusing on NOS3 regulation of ECM degradation could be a promising approach in the management of atherosclerosis.

Lifetime evaluation of left ventricular structure and function in male ApoE null mice after gamma and space-type radiation exposure.

The space radiation (IR) environment contains high charge and energy (HZE) nuclei emitted from galactic cosmic rays with the ability to overcome current shielding strategies, posing increased IR-induced cardiovascular disease risks for astronauts on prolonged space missions. Little is known about the effect of 5-ion simplified galactic cosmic ray simulation (simGCRsim) exposure on left ventricular (LV) function. Three-month-old, age-matched male Apolipoprotein E (ApoE) null mice were irradiated with 137Cs gamma (γ; 100, 200, and 400cGy) and simGCRsim (50, 100, 150cGy all at 500 MeV/nucleon (n)). LV function was assessed using transthoracic echocardiography at early/acute (14 and 28days) and late/degenerative (365, 440, and 660days) times post-irradiation. As early as 14 and 28-days post IR, LV systolic function was reduced in both IR groups across all doses. At 14days post-IR, 150cGy simGCRsim-IR mice had decreased diastolic wall strain (DWS), suggesting increased myocardial stiffness. This was also observed later in 100cGy γ-IR mice at 28days. At later stages, a significant decrease in LV systolic function was observed in the 400cGy γ-IR mice. Otherwise, there was no difference in the LV systolic function or structure at the remaining time points across the IR groups. We evaluated the expression of genes involved in hemodynamic stress, cardiac remodeling, inflammation, and calcium handling in LVs harvested 28days post-IR. At 28days post-IR, there is increased expression of Bnp and Ncx in both IR groups at the lowest doses, suggesting impaired function contributes to hemodynamic stress and altered calcium handling. The expression of Gals3 and β-Mhc were increased in simGCRsim and γ-IR mice respectively, suggesting there may be IR-specific cardiac remodeling. IR groups were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). No lower threshold was determined using the observed dose-response curves. These findings do not exclude the possibility of the existence of a lower IR threshold or the presence of IR-induced cardiovascular disease (CVD) when combined with additional space travel stressors, e.g., microgravity.

Abstract 13977: Insulin-Like Growth Factor Binding Protein 7 is Associated With Development of Cell Senescence and Progression of Coronary Atherosclerosis

Introduction: Cellular senescence, an irreversible cell cycle arrest, has been associated with atherosclerosis. Senescent cells (SC) secrete proinflammatory molecules contributing to disease progression. Insulin-like growth factor binding protein 7 (IGFBP7) binds IGF-1 and also interacts with IGF-1 receptor, suppressing intracellular IGF-1 signaling. IGFBP7 has been linked to heart failure-associated cell senescence. Hypothesis: We hypothesize that SC secrete IGFBP7, and IGFBP7 contributes to atherogenesis. Methods: Premature cell senescence was induced by treatment of smooth muscle cells (SMC) with H2O2. IGFBP7 levels were measured by immunoblotting of cell lysates and conditioned media (CM) and by immunohistochemistry in Apoe-null mice and in Rapacz hypercholesterolemic pigs. Binding of IGFBP7 with IGF-1 receptor was quantified by proximity ligation assay. Results: H2O2-treated SMC have marked increase in expression of the senescence markers beta-galactosidase (SA-βGal) and γH2A.X, and 3-fold (P<0.05) increase in IGFBP7 in cell CM. Abundant senescent SMC (αSMA + /SA-βGal + ) were detected in fibrous cap’s plaque in murine innominate artery and these cells had increased IGFBP7 compared to SMC in the vascular media. Increased IGFBP7 levels (1.8-fold increase, P<0.05) were also detected in porcine coronary plaque and IGFBP7 upregulation correlated with elevated IGFBP7/IGF-1 receptor binding. These data suggest a mechanistic link between senescence, IGFBP7 upregulation and atherogenesis. SMC were incubated with CM from senescent SMC (or non-senescent controls) to determine if SC-secreted IGFBP7 inhibits IGF-1 signaling. Preincubation with CM from SC inhibited IGF-1-induced Akt phosphorylation, however co-incubation with anti-IGFBP7 antibody reversed this effect, indicating that IGFBP7 in the CM mediates the inhibitory effect on IGF-1 signaling. Conclusions: We have shown that SMC senescence induced IGFBP7 and that high IGFBP7 levels were associated with senescent SMC in the atherosclerotic plaque. IGFBP7 inhibits IGF-1 signaling in vitro and binds IGF-1 receptor in plaque SMC. Our results suggest a novel role of IGFBP7 in senescence and in atherogenesis and identify IGFBP7 as a potential target for anti-atherogenic therapy.

PS-BPB04-1: DNA-APTAMER RAISED AGAINST RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS INHIBITS ATHEROSCLEROSIS

Background and Aims: Interaction of advanced glycation end products (AGEs) with the receptor RAGE could accelerate atherosclerotic plaque formation. However, effect of DNA-aptamer raised against RAGE on atherosclerosis remains unclear. In this study, we aimed to elucidate whether and how RAGE-aptamer could inhibit atherosclrotic lesions and oxidized-LDL uptake of macrophages derived from apolipoprotein E (ApoE)-null mice, a representative animal model of atherosclerosis. We also examined whether RAGE-aptamer could inhibit oxidized-LDL uptake in AGE-exposed mouse peripheral macrophages and human U937 cells. Materials and Methods: Seventeen-week-old male ApoE-null mice were subcutaneously infused with RAGE-aptamer (10 pmoL/g/day) or control-aptamer by osmotic pump for 4 weeks. Peritoneal macrophages were extracted from mice at 21 weeks of age just after intraperitoneal injection of thioglycollate broth. The entire aortae and cross-sections of the aortic roots were stained with Oil Red O or MOMA-2 for the assessment of atherosclerotic lesions or macrophage infiltration. Mouse macrophages and human U937 cells were incubated with 100 μg/mL AGE-bovine serum albumin (AGE-BSA) or non-glycated BSA for 24 hours. Then the cells were treated with RAGE-aptamer or control-aptamer for 18 hours. Oxidized-LDL uptake was determined by Dil-oxidized-LDL fluorescent intensity using immunohistochemistry. Quantitative RT-PCR analysis was performed using TaqMan-based probes and SYBR-based primers. Results: Atherosclerotic lesions and infiltration of macrophages in the aortae isolated from ApoE-null mice infused subcutaneously with RAGE-aptamer were significantly suppressed compared with control-aptamer-treated mice. Oxidized-LDL uptake, and cyclin-dependent kinase 5 (Cdk5) and CD36 gene expression in macrophages extracted from ApoE-null mice infused with RAGE-aptamer were attenuated in comparison to those from mice infused with control-aptamer. Furthermore, RAGE-aptamer significantly inhibited oxidized-LDL uptake, and Cdk5 and CD36 gene expression in mouse macrophages and human U937 cells compared with those exposed with control-aptamer. Gene expression level of Cdk5 in mouse or human U937 cells was correlated with that of CD36. Conclusion: Our present findings suggest that RAGE-aptamer could play a protective role against atherosclerosis through the inhibition of macrophage foam cell formation via Cdk5-CD36 pathway in mice and human, which may be caused by attenuating the harmful effects of AGEs.

Combination of folic acid with nifedipine is completely effective in attenuating aortic aneurysm formation as a novel oral medication

Aortic aneurysms are prevalent and severe vascular diseases with high mortality from unpredicted ruptures, while the only treatment option is surgical correction of large aneurysms with considerable risk. We have shown that folic acid (FA) is highly effective in alleviating development of aneurysms although not sufficient to completely attenuate aneurysm formation. Here, we examined therapeutic effects on aneurysms of combining FA with Nifedipine as novel and potentially more effective oral medication. Oral administration with FA (15 mg/kg/day) significantly reduced incidence of AAA from 85.71% to 18.75% in Ang II-infused apolipoprotein E (apoE) null mice, while combination of FA with Nifedipine (1.5, 5.0 or 20 mg/kg/day) substantially and completely further reduced incidence of AAA to 12.5%, 11.76% and 0.00% respectively in a dose-dependent manner. The combinatory therapy substantially and completely further alleviated enlargement of abdominal aortas defined by ultrasound, vascular remodeling characterized by elastin degradation and adventitial hypertrophy, as well as aortic superoxide production and eNOS uncoupling activity also in a dose-dependent manner, with combination of FA with 20 mg/kg/day Nifedipine attenuating all of these features by 100% to control levels. Aortic NO and H4B bioavailabilities were also dose-dependently further improved by combining FA with Nifedipine. These data establish entirely innovative and robust therapeutic regime of FA combined with Nifedipine for the treatment of aortic aneurysms. The comminatory therapy can serve as a first-in-class and most effective oral medication for aortic aneurysms, which can be rapidly translated into clinical practice to revolutionize management of the devastating vascular diseases of aortic aneurysms known as silent killers.

Non-canonical NF-κB contributes to endothelial pyroptosis and atherogenesis dependent on IRF-1

Cell inflammation and death are closely linked processes contributing to endothelial dysfunction, which plays a critical role in atherogenesis. Activation of the NLRP3 inflammasome causes pyroptosis, the Gasdermin D (GSDMD)-mediated inflammatory cell death. The non-canonical NF-κB pathway has been implicated in inflammation; however, its role in NLRP3 inflammasome-mediated endothelial dysfunction has not been investigated. This study investigated a role for the non-canonical NF-κB pathway in regulating endothelial pyroptosis as it relates to atherogenesis. Immunohistochemistry indicated inflammasome activation in the endothelial cells (EC) of human atherosclerotic arteries. Flow cytometry and Western blot analysis revealed that oxidized low-density lipoprotein (oxLDL) activated the NLRP3 inflammasome, concomitant with the activation of non-canonical NF-κB in primary human aortic EC. Interference of NF-κB inducing kinase (NIK), the key regulator of the non-canonical pathway, significantly attenuated oxLDL- or LPS/ATP-induced NLRP3 inflammasome activation, pyroptosis, IL-1β, and IL-18 secretion. In contrast, overexpression of NIK exacerbated these responses. Chromatin immunoprecipitation revealed that activation of the non-canonical NF-κB pathway upregulated the transcription factor IRF-1 through RelB/p52 binding to its promoter region at -782/-770. In addition to the known target CASP1, RNA sequencing further identified GSDMD as a target gene of IRF-1. IRF-1 but not RelB/p52 interacted with the GSDMD promoter at -526/-515 and the CASP1 promoter at -11/10 to promote the expression and CASP1-mediated activation of GSDMD. Consistent with the observations in cultured endothelium, endothelial-specific deficiency of NIK or IRF-1 attenuated atherosclerosis in high-fat diet-fed Apoe-null mice. These data demonstrate that the non-canonical NF-κB pathway contributes to NLRP3 inflammasome-mediated endothelial pyroptosis and the development of atherosclerosis through GSDMD activation in a manner dependent on IRF-1. Further investigation may facilitate the identification of specific therapeutic targets for atherosclerotic heart diseases.

Conjugated linoleic acid isomers induced dyslipidemia and lipoatrophy are exacerbated by rosiglitazone in ApoE null mice fed a Western diet

BACKGROUND: Insulin sensitizers have been used to treat Type 2 diabetes. However, their non-negligible side effects have led to cardiovascular concerns and the withdrawal of a member, rosiglitazone. OBJECTIVE: We combined conjugated linoleic acid (CLA) with rosiglitazone to test for amelioration of side effects posed by rosiglitazone in vivo. METHODS: We utilized ApoE null mice fed with Western diet (WD) to test our hypothesis. Mice were fed WD, with or without CLA administration, for 12 weeks. CLA utilized in our study consisted of a 1:1 ratio of 95% pure c9,t11, and t10,c12 isomers at a concentration of 0.1% w/v in fat-free milk. Starting from Week 12, select mice received rosiglitazone. RESULTS: It was found that mice receiving CLA from Week 0 and rosiglitazone from Week 12 had the lowest body weight and exacerbated hepatomegaly. Although these mice had attenuated insulin resistance compared to mice receiving only Western diet, they display a marked increase in total plasma cholesterol and low-density lipoprotein (LDL) cholesterol. Mice receiving early CLA administration developed hyperleptinemia, which was not restored by rosiglitazone. CONCLUSION: Taken together, against the background of ApoE null genotype and WD feeding, simultaneous administration of 1:1 CLA and rosiglitazone led to dyslipidemic lipoatrophy.

Repurposing the anti-cancer drug docetaxel for the prevention and treatment of atherosclerosis

Background and Aims : We have identified that a negative regulator of high-density lipoprotein (HDL) biogenesis, desmocollin 1 (DSC1) contributes to cholesterol accumulation in the atherosclerotic plaque. With screening of 10 million small molecules, the FDA-approved chemotherapy drug docetaxel was identified to inhibit the DSC1 activity and promote HDL biogenesis. To test whether docetaxel reduces atherosclerosis in an animal model, apoE-null mice were fed a high-fat diet to promote dyslipidemia.

Dual role played by NKT cells in the development of atherosclerosis: Evidence from meta-analysis of pre-clinical studies

Background and Aims : NKT cells play crucial role in regulating early inflammation in atherosclerosis. Broadly, there are two subsets, invariant (iNKT) and variant (vNKT), where iNKTs are thought to be pro-atherosclerotic, yet uncertainty remains.Methods: We performed a pre-clinical meta-analysis to understand the true role of NKT cells in atherosclerosis. Twenty studies comprising 550 mice were selected for the meta-analysis. A random-effect model was employed to estimate the effect size. Additionally, we also fed high-fat-diet (HFD) to ApoE-null mice for 5 weeks, followed by identification of NKT lymphocytes from liver, spleen and blood with antibodies and α-GalCer loaded CD1d tetramer and flow cytometric analysis.Conclusions: This is the first meta-analysis on NKT cells and its subsets in preclinical atherosclerotic models. Based on this analysis and our data, we conclude that both NKT and iNKT cells are pro-atherosclerotic in nature, however, a gap still remains in the understanding of interplay of both subsets of NKT cells in atherosclerosis. Background and Aims : NKT cells play crucial role in regulating early inflammation in atherosclerosis. Broadly, there are two subsets, invariant (iNKT) and variant (vNKT), where iNKTs are thought to be pro-atherosclerotic, yet uncertainty remains. Methods: We performed a pre-clinical meta-analysis to understand the true role of NKT cells in atherosclerosis. Twenty studies comprising 550 mice were selected for the meta-analysis. A random-effect model was employed to estimate the effect size. Additionally, we also fed high-fat-diet (HFD) to ApoE-null mice for 5 weeks, followed by identification of NKT lymphocytes from liver, spleen and blood with antibodies and α-GalCer loaded CD1d tetramer and flow cytometric analysis. Conclusions: This is the first meta-analysis on NKT cells and its subsets in preclinical atherosclerotic models. Based on this analysis and our data, we conclude that both NKT and iNKT cells are pro-atherosclerotic in nature, however, a gap still remains in the understanding of interplay of both subsets of NKT cells in atherosclerosis.

Genetic Evidence for a Causal Relationship between Hyperlipidemia and Type 2 Diabetes in Mice.

Dyslipidemia is considered a risk factor for type 2 diabetes (T2D), yet studies with statins and candidate genes suggest that circulating lipids may protect against T2D development. Apoe-null (Apoe-/-) mouse strains develop spontaneous dyslipidemia and exhibit a wide variation in susceptibility to diet-induced T2D. We thus used Apoe-/- mice to elucidate phenotypic and genetic relationships of circulating lipids with T2D. A male F2 cohort was generated from an intercross between LP/J and BALB/cJ Apoe-/- mice and fed 12 weeks of a Western diet. Fasting, non-fasting plasma glucose, and lipid levels were measured and genotyping was performed using miniMUGA arrays. We uncovered a major QTL near 60 Mb on chromosome 15, Nhdlq18, which affected non-HDL cholesterol and triglyceride levels under both fasting and non-fasting states. This QTL was coincident with Bglu20, a QTL that modulates fasting and non-fasting glucose levels. The plasma levels of non-HDL cholesterol and triglycerides were closely correlated with the plasma glucose levels in F2 mice. Bglu20 disappeared after adjustment for non-HDL cholesterol or triglycerides. These results demonstrate a causative role for dyslipidemia in T2D development in mice.

MP05-12 DEPENDENT ANTIBIOTIC AND OXALATE INTERACTIONS IMPACT ATHEROSCLEROSIS AND CALCIUM OXALATE STONES IN THE KIDNEYS

You have accessJournal of UrologyCME1 May 2022MP05-12 DEPENDENT ANTIBIOTIC AND OXALATE INTERACTIONS IMPACT ATHEROSCLEROSIS AND CALCIUM OXALATE STONES IN THE KIDNEYS Jose Agudelo, Lucas Osborn, Beckey DeLucia, Mark Brown, Ava Adler, Jan Claesen, Jane Nguyen, and Aaron Miller Jose AgudeloJose Agudelo More articles by this author , Lucas OsbornLucas Osborn More articles by this author , Beckey DeLuciaBeckey DeLucia More articles by this author , Mark BrownMark Brown More articles by this author , Ava AdlerAva Adler More articles by this author , Jan ClaesenJan Claesen More articles by this author , Jane NguyenJane Nguyen More articles by this author , and Aaron MillerAaron Miller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002522.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies have shown a positive relationship between cardiometabolic syndrome and increased risk of urinary stone disease (USD). However, the underlying mechanisms that leads to these co-morbidities are unknown. In the current study, we aimed to determine microbiome and diet dependent effects on an atherosclerosis and calcium oxalate stone formation in a mouse model. METHODS: Sixty-six female ApoE null mice were randomly assigned to either a basal diet or 3% oxalate diet. Half of the animals received antibiotic water and the other half sterile water. Mice consumed diets ad libitum for 12-weeks. At the end of the trial, fecal pellets were collected for 16S rRNA quantification. Mice were necropsied, and kidneys, aortas and liver were collected. Kidneys and aortas underwent polarized light quantification of oxalate content. Kidneys were also assessed for oxalate nephropathy after Von Kossa and Alizarin red staining and aortas were assessed for atherosclerosis plaque quantification. Livers were used for bile acid content assessment. Results were compared on a one-way and two-way ANOVA analyses. RESULTS: Bile acid production was significantly impacted by both oxalate and antibiotics. Renal histopathological analysis revealed that oxalate led to an increase in dilated tubules, oxalate nephropathy and tubular injury metrics, which was exacerbated with antibiotic use. Total atherosclerotic plaque lesions in the aortas and the presence of calcium oxalate in the plaque was significantly higher in the group that received dietary oxalate, which again was exacerbated by antibiotic use. CONCLUSIONS: Our results support the hypothesis that exogenous sources of oxalate may lead to both kidney stones and atherosclerosis through the formation of calcium oxalate plaques, which is exacerbated by a loss of function dysbiosis driven by long-term antibiotic use. Importantly, given the clear and significant formation of kidney stones, the APOE mice may serve as a more appropriate mouse model for kidney stones that what has previously been used. Source of Funding: Seed Funding Lerner Research Institute. Urology Care Foundation Research Scholar Award © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e72 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jose Agudelo More articles by this author Lucas Osborn More articles by this author Beckey DeLucia More articles by this author Mark Brown More articles by this author Ava Adler More articles by this author Jan Claesen More articles by this author Jane Nguyen More articles by this author Aaron Miller More articles by this author Expand All Advertisement PDF DownloadLoading ...

Genetic connection of carotid atherosclerosis with coat color and body weight in an intercross between hyperlipidemic mouse strains.

Atherosclerosis in the carotid artery is a major cause of ischemic stroke and has a strong genetic component. The aim of this study was to identify genetic factors contributing to carotid atherosclerosis. One hundred fifty-four female F2 mice were generated from an intercross between LP/J and BALB/cJ Apoe-null (Apoe-/-) mice and fed 12 wk of Western diet. Atherosclerotic lesions, body weight, and coat color were measured and genotyping was performed using miniMUGA genotyping arrays. A significant quantitative trait locus (QTL) on chromosome (Chr) 7, named Cath20, and five suggestive QTL on Chr 6, 12, 13, 15, and X were identified for carotid lesions. Three significant QTL, Bwfq2, Bw1n, Bwtq6, on Chr 2, 7, and 15 were identified for body weight. Two significant QTL, Chop2 and Albc2, on Chr 4 and 7 were identified for coat color, with Tyr, encoding tyrosinase, being the causal gene of Albc2. Cath20 overlapped with or was close to QTL Bw1n and Albc2 on Chr7. Carotid lesion sizes were significantly correlated with body weight and graded coat color in F2 mice. Cath20 on Chr7 disappeared after adjustment for coat color but remained after adjustment for body weight. Tyr was abundantly expressed in atherosclerotic lesions. These results demonstrate genetic connections of carotid atherosclerosis with body weight and coat color in hyperlipidemic mice and suggest a potential role for Tyr in carotid atherosclerosis.

Genetic Connection between Hyperglycemia and Carotid Atherosclerosis in Hyperlipidemic Mice.

Type 2 diabetes (T2D) is a major risk for atherosclerosis and its complications. Apoe-null (Apoe−/−) mouse strains exhibit a wide range of variations in susceptibility to T2D and carotid atherosclerosis, with the latter being a major cause of ischemic stroke. To identify genetic connections between T2D and carotid atherosclerosis, 145 male F2 mice were generated from LP/J and BALB/cJ Apoe−/− mice and fed 12 weeks of a Western diet. Atherosclerotic lesions in the carotid arteries, fasting, and non-fasting plasma glucose levels were measured, and genotyping was performed using miniMUGA arrays. Two significant QTL (quantitative trait loci) on chromosomes (Chr) 6 and 15 were identified for carotid lesions. The Chr15 QTL coincided precisely with QTL Bglu20 for fasting and non-fasting glucose levels. Carotid lesion sizes showed a trend toward correlation with fasting and non-fasting glucose levels in F2 mice. The Chr15 QTL for carotid lesions was suppressed after excluding the influence from fasting or non-fasting glucose. Likely candidate genes for the causal association were Tnfrsf11b, Deptor, and Gsdmc2. These results demonstrate a causative role for hyperglycemia in the development of carotid atherosclerosis in hyperlipidemic mice.

Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux

Atherosclerosis is a chronic inflammatory condition in which macrophages play a major role. Janus kinase 2 (JAK2) is a pivotal molecule in inflammatory and metabolic signaling, and Jak2V617F activating mutation has recently been implicated with enhancing clonal hematopoiesis and atherosclerosis. To determine the essential in vivo role of macrophage (M)-Jak2 in atherosclerosis, we generate atherosclerosis-prone ApoE-null mice deficient in M-Jak2. Contrary to our expectation, these mice exhibit increased plaque burden with no differences in macrophage proliferation, recruitment or bone marrow clonal expansion. Notably, M-Jak2-deficient bone marrow derived macrophages show a significant defect in cholesterol efflux. Pharmacologic JAK2 inhibition with ruxolitinib also leads to defects in cholesterol efflux and accelerates atherosclerosis. Liver X receptor agonist abolishes the efflux defect and attenuates the accelerated atherosclerosis that occurs with M-Jak2 deficiency. Macrophages of individuals with the Jak2V617F mutation show increased efflux which is normalized when treated with a JAK2 inhibitor. Together, M-Jak2-deficiency leads to accelerated atherosclerosis primarily through defects in cholesterol efflux from macrophages.

Smooth Muscle Cell Notch2 Is Not Required for Atherosclerotic Plaque Formation in ApoE Null Mice.

We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.

Analysis of differential gene expression and transcript usage in hippocampus of Apoe null mutant mice: Implications for Alzheimer’s disease

A dataset of single-nucleus RNA sequencing (snRNAseq) data was analyzed using Seurat, Sierra, and Ingenuity Pathway Analysis (IPA) programs to assess differentially expressed genes (DEGs) and differential transcript usage (DTU) in mouse hippocampal cell types. Seurat identified DEGs between the wild type (WT) and Apoe knockout (EKO) mice. IPA identified 11 statistically significant canonical pathways in >1 cell type. Sierra identified Sipa1l1 with DTU between WT and EKO samples. Analysis of the Sipa1l1 peak region identified an alternative non-canonical polyadenylation signal and a putative cytoplasmic polyadenylation element. APOE regulation of gene transcription and co-transcriptional RNA processing may underlie Alzheimer’s disease.

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.