ApoE Knockout Mice Research Articles

Infrared laser therapy decreases systemic oxidative stress and inflammation in hypercholesterolemic mice with periodontitis

BackgroundNear-infrared irradiation photobiomodulation (NIR-PBM) has been successfully used in periodontal treatment as an adjuvant tool to locally improve cell function and regeneration. Although the relationship between periodontitis and systemic disease constitutes an important aspect of periodontal clinical research, the systemic effects of NIR-PBM in periodontitis are not well known. In this study, we aimed to investigate the effects of NIR-PBM on systemic oxidative stress and inflammation in an apolipoprotein E (ApoE) knockout mouse model of periodontal disease (PD).MethodsWe evaluated alveolar bone loss by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC), reactive oxygen species (ROS) production in blood cells, inflammatory activity, plasma cholesterol levels, and lipid peroxidation levels in three experimental groups: (1) ApoEC, control group without intervention; (2) ApoEP, first molar ligation-induced periodontitis for 4 weeks; and (3) ApoEP + PBM, exposed to 808 nm continuous wave, ø ~ 3 mm2, 100 mW, 60 s of NIR-PBM for 7 consecutive days after 4 weeks of periodontitis. At the end of the experimental protocols, ApoEP mice presented significantly increased alveolar bone loss, ROS production, inflammatory activity, plasma cholesterol, and lipid peroxidation levels compared to the ApoEC group (P < 0.05). NIR-PBM for 7 days in the ApoEP + PBM mice significantly decreased systemic ROS production, inflammatory response, plasma cholesterol, and lipid peroxidation levels, similar to those found in the ApoEC group (P > 0.05). However, it was not capable of preventing alveolar bone loss (P > 0.05 compared to ApoEP mice).ConclusionA 7-day treatment with NIR-PBM effectively reduces systemic oxidative stress and inflammatory parameters in hypercholesterolemic mice with PD. However, more studies with longer evaluation times are needed to confirm the systemic effects of locally applied NIR-PBM on PD associated with hypercholesterolemia.

Exosomal miR-129 and miR-342 derived from intermittent hypoxia-stimulated vascular smooth muscle cells inhibit the eIF2α/ATF4 axis from preventing calcified aortic valvular disease.

This study aims to elucidate the role of miR-129/miR-342 loaded in exosomes derived from vascular smooth muscle cells (VSMCs) stimulated by intermittent hypoxia in calcified aortic valvular disease (CAVD). Bioinformatics analysis was conducted to identify differentially expressed miRs in VSMCs-derived exosomes and CAVD samples, and their potential target genes were predicted. VSMCs were exposed to intermittent hypoxia to induce stimulation, followed by isolation of exosomes. Valvular interstitial cells (VICs) were cultured in vitro to investigate the impact of miR-129/miR-342 on VICs' osteogenic differentiation and aortic valve calcification with eIF2α. A CAVD mouse model was established using ApoE knockout mice for in vivo validation. In CAVD samples, miR-129 and miR-342 were downregulated, while eIF2α and ATF4 were upregulated. miR-129 and miR-342 exhibited inhibitory effects on eIF2α through targeted regulation. Exosomes released from intermittently hypoxia-stimulated VSMCs contained miR-129 and miR-342. Overexpression of miR-129 and miR-342, or silencing ATF4, suppressed VICs' osteogenic differentiation and aortic valve calcification, which could be rescued by overexpressed eIF2α. Collectively, intermittent hypoxia stimulation of VSMCs leads to the secretion of exosomes that activate the miR-129/miR-342 dual pathway, thereby inhibiting the eIF2α/ATF4 axis and attenuating VICs' osteogenic differentiation and CAVD progression.

The Complementary Effects of Dabigatran Etexilate and Exercise Training on the Development and Stability of the Atherosclerotic Lesions in Diabetic ApoE Knockout Mice.

Aim: To determine the complementary effects of dabigatran etexilate (DE), exercise training (ET), and combination (DE + ET) on the development and stability of the atherosclerotic lesions in diabetic apoE knockout (apoE-/-) mice. Methods: In 48 male apoE-/- diabetic mice, streptozotocin (STZ) was induced for 5 consecutive days. Mice received a high-fat diet (HFD) for 8 weeks and then were randomized into four groups (1. Control/CG, 2. DEG: HFD with DE, 3. ETG: ET on treadmill, 4. DE + ETG: combination DE and ET treatment). At the end of the eighth week, all mice were euthanatized and morphometry of the aortic lesions at the level of aortic valve was obtained. Collagen, elastin, MCP-1, TNF-a, matrix metalloproteinases (MMP-2,-3,-9), and TIMP-1 concentrations within plaques at the aortic valve were determined. Results: All active groups had significantly smaller aorta stenosis (DEG:7.9 ± 2.2%, ETG:17.3 ± 5.3%, DE + ETG:7.1 ± 2.7%) compared to CG (23.3 ± 5.5% p < 0.05), reduced the relative intra-plaque content of MCP-1, macrophages, MMP-3, and MMP-9, and considerably increased collagen, elastin, and TIMP-1 (p < 0.05). Group 4 showed the most pronounced results (p < 0.05). Both DEG and DE + ETG significantly reduced MMP-2 and TNF-a concentrations compared to ETG and CG (p < 0.010). Conclusion: DE and ET treatment of diabetic apoE-/- mice resulted in complementary amelioration of atherosclerotic lesions development and stability, mediated by the anti-inflammatory modulation of both DE and ET.

Alleviating Effects of Ovatodiolide and Antcin K Supplements on High-Fat Diet-Induced Cardiovascular Dysfunction in ApoE-Knockout Mice by Attenuating Oxidative Stress.

A high-fat diet (HFD) is a major risk factor for cardiovascular diseases. Many pure compounds have been demonstrated to be effective in treating cardiovascular diseases. In this study, we investigated the alleviating effects of oral ovatodiolide and antcin K (OAK) supplements on HFD-induced cardiovascular dysfunction in apolipoprotein E (ApoE)-knockout mice. Cardiovascular dysfunction was induced in ApoE-knockout mice by feeding them an HFD for 12 weeks. The degree of cardiovascular dysfunction was assessed through echocardiography, hematological and biochemical analyses, and immunofluorescence and immunohistochemical staining. The HFD-fed mice exhibited cardiovascular dysfunction-abnormal blood biochemical index. The arterial wall tissue exhibited the marked deposition of lipids, upregulated expression of vascular cell adhesion molecule-1 and CD36 receptors, and downregulated expression of the ABCA1 receptor. Macrophages isolated from the peritoneal cavity of the mice exhibited increased levels of lipid accumulation, reactive oxygen species, and CD11b expression but reduced mitochondrial membrane potential. The expression of superoxide dismutase 2 was downregulated and that of tumor necrosis factor-α was upregulated in the myocardial tissue. Oral OAK supplements twice a day for 12 weeks significantly mitigated HFD-induced cardiovascular dysfunction in the experimental mice. Oral OAK supplements appear to be a promising strategy for treating HFD-induced cardiovascular dysfunction. The underlying mechanisms may involve the reduction of lipid accumulation in the artery and oxidative stress and inflammation in the cardiovascular tissue.

ОЦЕНКА ГЕНОТОКСИЧЕСКОГО ВОЗДЕЙСТВИЯ МИТОМИЦИНА С НА ПРИМЕРЕ APOE НОКАУТНЫХ МЫШЕЙ

BACKGROUND Mitomycin C (MMC) has a wide spectrum of genotoxicity, including inhibition of DNA synthesis, clastogenesis and mutagenesis. As an immediate-action clastogen requiring exclusively intracellular reductive activation, MMC initiates efficient DNA cross-linking. Most studies are aimed at studying single-stage acute effects, which are caused by high doses of mutagen. In turn, there are no or very few studies aimed at studying the chronic effects of MMC. Polychrome red blood cells have been accepted as a suitable target for the evaluation of micronuclei in both acute and cumulative injury. The in vivo micronucleus test is well established as a standard assay for assessing genotoxicity at the chromosomal level of mouse erythrocytes. THE AIM OF THE STUDY To create a chronic genotoxic effect of mitomycin C without lethal outcome in ApoE knockout mice while selecting the optimal dose of MMC. MATERIALS AND METHODS The study design included 6 groups of ApoE-/- mice, two doses of MMC with a concentration of 0.1 and 0.5 mg/kg, one-time and three-time administration. To assess genotoxicity, 1000 polychrome erythrocytes (PCEs) extracted from the bone marrow of a mousefemur were counted on each sample, and PCEs with micronuclei were identified. RESULTS This study aimed to find the optimal dose of MMC that has a clear genotoxic effect and does not lead to death in an ApoE knockout mouse model. With a single injection, PCEs with chromosomal damage were more common (more than 2 times; p &lt; 0.05) in groups of mice with MMC administration (0.1 and 0.5 mg/kg; 0.39 % and 0.26 %, respectively) compared to the control group (0.15 %). We also found that the frequency of occurrence of PCEs with micronuclei in groups of mice with MMC dose of 0.1 and 0.5 mg/kg (0.36 % and 0.47 %, respectively) and three-time administration exceeded this indicator in mice from the control group (0.2 %). CONCLUSION The present study on the determination of the optimal dose of mitomycin C provides further evidence that 0.1 mg/kg is the threshold value for genotoxic effects caused by MMC. An increase in the frequency of micronucleated immature red blood cells in animals exposed to a mutagen is an indicator of induced structural or numerical chromosomal aberrations. Our results further suggest that careful selection of MMC dose is critical. Dose-response studies in rodents can provide useful information on mechanisms and dose selection for long-term toxicity studies.

Apolipoprotein E deficiency attenuated osteogenesis via down-regulating osterix.

Apolipoprotein E (ApoE), a ligand for low-density lipoprotein receptors, is strongly induced during osteogenesis and has a physiologic role in regulating osteoblast function, but the mechanisms of its action are still unclear. The study aims to elucidate the influence and molecular mechanisms of ApoE on bone formation. An ovariectomy-induced osteoporotic model were conducted in ApoE knockout (ApoE-/-) mice to study the effect of ApoE on the bone system. Bone quality were assessed through bone mineral density and histomorphometric analysis. To investigate the underlying role and mechanisms of ApoE during osteogenesis, primary osteoblasts from the calvariums of newborn ApoE-/- or wild-type (WT) mice were cultured in the osteoblastic differentiation medium in vitro for further research. Our animal experiment data showed that ApoE-/- mice exhibited bone loss, exacerbated by estrogen deprivation after ovariectomy. ApoE deficiency attenuated osteoblast activity and inhibited osteoblast osteogenesis, accompanied by decreased osterix expression. ApoE deficiency did not affect primary osteoblast viability and collagen-1 expression. Moreover, osteoprotegerin expression in ApoE-/- osteoblasts was reduced compared to WT controls. Our study demonstrated that ApoE gene deficiency contributed to bone loss and attenuated osteogenesis by down-regulating osterix expression.

NPRC deletion mitigated atherosclerosis by inhibiting oxidative stress, inflammation and apoptosis in ApoE knockout mice

Previous studies suggested a beneficial effect of natriuretic peptides in animal models of cardiovascular disease, but the role of natriuretic peptide receptor C (NPRC) in the pathogenesis of atherosclerosis (AS) remains unknown. This study was designed to test the hypothesis that NPRC may promote AS lesion formation and instability by enhancing oxidative stress, inflammation, and apoptosis via protein kinase A (PKA) signaling. ApoE−/− mice were fed chow or Western diet for 12 weeks and NPRC expression was significantly increased in the aortic tissues of Western diet-fed mice. Systemic NPRC knockout mice were crossed with ApoE−/− mice to generate ApoE−/−NPRC−/− mice, and NPRC deletion resulted in a significant decrease in the size and instability of aortic atherosclerotic lesions in ApoE−/−NPRC−/− versus ApoE−/− mice. In addition, endothelial cell-specific NPRC knockout attenuated atherosclerotic lesions in mice. In contrast, endothelial cell overexpression of NPRC aggravated the size and instability of atherosclerotic aortic lesions in mice. Experiments in vitro showed that NPRC knockdown in human aortic endothelial cells (HAECs) inhibited ROS production, pro-inflammatory cytokine expression and endothelial cell apoptosis, and increased eNOS expression. Furthermore, NPRC knockdown in HAECs suppressed macrophage migration, cytokine expression, and phagocytosis via its effects on endothelial cells. On the contrary, NPRC overexpression in endothelial cells resulted in opposite effects. Mechanistically, the anti-inflammation and anti-atherosclerosis effects of NPRC deletion involved activation of cAMP/PKA pathway, leading to downstream upregulated AKT1 pathway and downregulated NF-κB pathway. In conclusion, NPRC deletion reduced the size and instability of atherosclerotic lesions in ApoE−/− mice via attenuating inflammation and endothelial cell apoptosis and increasing eNOS expression by modulating cAMP/PKA-AKT1 and NF-κB pathways. Thus, targeting NPRC may provide a promising approach to the prevention and treatment of atherosclerosis.

Endocytosis of red blood cell extracellular vesicles by macrophages leads to cytoplasmic heme release and prevents foam cell formation in atherosclerosis.

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis.

Targeting single inflammation marker IL-1Β does not alter development of advanced atherosclerotic plaques in high-fat diet-fed ApoE knockout mice

Targeting single inflammation marker IL-1Β does not alter development of advanced atherosclerotic plaques in high-fat diet-fed ApoE knockout mice

Intraperitoneal Injection of Human Ferritin Heavy Chain Attenuates the Atherosclerotic Process in APOE-Knockout Mice.

Background: Iron overload can accelerate the accumulation of lipid oxides and contribute to the progression of atherosclerosis. Ferritin heavy chain (FT-H) exhibits oxidase activity, which inhibits the toxicity of ferrous ions and reduces oxidative damage. We investigated the effect of the intraperitoneal injection of FT-H on the progression of atherosclerosis in APOE-knockout mice (Apo-E(-/-) mice). Methods: All mice were fed on a high-fat diet. After 10 weeks, the mice were divided into an injection group (n = 4) and a control group (n = 4). The injection group was injected intraperitoneally with FT-H (50 mg/kg, once a week), and the control group was treated with PBS buffer (at an equal volume to the injection group, once a week). After 10 weeks of intervention, MRI of the aortas was performed. Then, the animals were sacrificed, and tissues were taken. Hematoxylin-eosin (HE) staining was used for histomorphometry, Masson staining was used to quantify the collagen content in the arteries, Prussian blue staining was used to visualize iron deposition in the arteries, and MRI was used to analyze the structure of the aorta in vivo. Immunohistochemistry was performed to detect the expression of MCP-1, MMP-2, MMP-9, FT-H, FT-L, TfR1, NRF-2 and GPX-4. Results: The serological results showed that the injection group had lower levels of glucose (Glu), triacylglycerol (TG), cholesterol (CHO), low-density lipoprotein-C (LDL-C) and malondialdehyde (MDA) (p = 0.0058, p = 0.0098, p = 0.0019, p = 0.0368 and p = 0.0025, respectively), and their serum ferritin (SF) and superoxide dismutase (SOD) levels were higher (p = 0.0004 and p < 0.0001). The Masson staining and MRI results showed that the injection group had less collagen deposition (p = 0.0226), a larger arterial lumen area and arterial volume (p = 0.0006 and p = 0.0005), thinner arterial wall thickness (p = 0.0013) and a more stable arterial plaque structure (p < 0.0001). The immunohistochemical results showed reduced expression of FT-H, FT-L, TfR1, MMP-2, MMP-9, MCP-1 and NRF-2 in the injection group (p = 0.0054, p = 0.0242, p = 0.0221, p = 0.0477, p = 0.0131, p = 0.0435 and p = 0.0179). Prussian blue staining showed that the area of iron-positive areas in the aortic plaques of the control group was larger than that of injected group. The expression of GPX-4 was lower in the control group than in the injection group (p = 0.016). Conclusions: The intraperitoneal administration of FT-H to Apo-E(-/-) mice resulted in lower blood glucose and lipid levels; reduced iron and iron metabolism protein deposition in the aorta; reduced indices of their ferroptosis, oxidation and inflammatory aggregation; and reduced collagen deposition in the aorta, which delayed the process of aortic atherosclerosis in mice.

Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis

BackgroundCXCR3 is a chemokine receptor and is expressed in innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, detection of CXCR3 by positron emission tomography (PET) radiotracer can be a useful tool for detecting the development of atherosclerosis in a noninvasive manner. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, 18F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis.ResultsThe reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured Ki values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [18F]1 was prepared by a two-step radiosynthesis with a decay-corrected radiochemical yield of 13 ± 2%, radiochemical purity > 99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (n = 6). The baseline studies showed that [18F]1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue in Apolipoprotein E (ApoE) knockout (KO) mice fed with a high-fat diet over 12 weeks. The uptake of [18F]1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [18F]1 in the abdominal aorta of C57BL/6 control mice fed with a normal diet were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. Immunohistochemistry studies demonstrated that [18F]1-positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [18F]1, and their CXCR3 expressions were minimal.Conclusion[18F]1 was synthesized with good radiochemical yield and high radiochemical purity. In PET imaging studies, [18F]1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [18F]1 visualized CXCR3 expression in different regions in mice aligned with the tissue histology studies. Taken together, [18F]1 is a potential PET radiotracer for imaging CXCR3 in atherosclerosis.

Protective effects of tanshinone IIA on Porphyromonas gingivalis-induced atherosclerosis via the downregulation of the NOX2/NOX4-ROS mediation of NF-κB signaling pathway

Tanshinone IIA (TSA), an active component isolated from Danshen, possess high medicinal values against atherosclerosis by reducing vascular oxidative stress, inhibiting platelet aggregation, and protecting the endothelium from damage. The periodontal pathogen Porphyromonas gingivalis (P. gingivalis) has been proven to accelerate the development of atherosclerosis. We aim to determine the effects of TSA on P. gingivalis-induced atherosclerosis in ApoE-knockout (ApoE−/−) mice. After feeding with a high-lipid diet and infected with P. gingivalis three times per week for four weeks, TSA-treated (60 mg/kg/d) mice greatly inhibited atherosclerotic lesions both morphologically and biochemically and exhibited significantly reduction ROS, 8-OHdG, and ox-LDL levels in serum compared with P. gingivalis-infected mice. Additionally, TSA-treated mice were observed a marked reduction of ROS, 8-OHdG and ox-LDL in the serum, mRNA levels of COX-2, LOX-1, NOX2 and NOX4 in the aorta, as well as the levels of NOX2, NOX4, and NF-κB. These results suggest that TSA attenuates oxidative stress by decreasing NOX2 and NOX4 and downregulating NF-κB signaling pathway, which might be contributed to the amelioration of atherosclerosis.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

IL-17A induces valvular endothelial inflammation and aggravates calcific aortic valve disease

Calcific aortic valve disease (CAVD) is an aging related disease characterized by inflammation and fibrocalcific remodeling. IL-17A is a key cytokine associated with pathophysiology of inflammatory and fibrotic disease. Previous studies showed accumulation of IL-17A-producing T helper lymphocytes in human calcified aortic valves and significantly elevated IL-17RA expression in calcified valves. However, the role of IL-17A signaling in the initiation and development of CAVD is still unclear. In this study, by analyzing public transcriptome databases, we found that IL-17A-IL-17RA signaling is activated in calcified valves. Gene expression analysis revealed significantly increased IL-17A, IL-17RA, and RUNX2 expression in calcified human aortic valves compared to in non-calcified valves, and the expression of IL-17A and IL-17RA were positively correlated with RUNX2 expression. A 5/6 nephrectomy was performed in Apoe−/− (Apoe knockout) mice to establish a CAVD mouse model. IL-17A-neutralizing antibodies significantly reduced valve calcium deposition and decreased expression of RUNX2 in aortic valves. Immunofluorescence staining of human aortic valves and qRT-PCR analysis of primary aortic valve cells revealed abundant expression of IL-17RA in valvular endothelial cells (VECs). RNA sequencing indicated that IL-17A promoted the activation of inflammatory signaling pathways in VECs. Furthermore, qRT-PCR and cytometric bead array analysis confirmed that IL-17A promoted the expression or secretion of inflammatory cytokines IL-6 and IL-1β, chemokines CXCL2 and CXCL8, and fibrosis-related gene COL16A1. Our findings indicate that elevated IL-17A in CAVD may promote valve inflammation, fibrosis, and calcification by inducing endothelial activation and inflammation. Targeting IL-17A-IL-17RA signaling may be a potential therapeutic strategy for CAVD.

Gene deletion of Interleukin-1α reduces ER stress-induced CHOP expression in macrophages and attenuates the progression of atherosclerosis in apoE-deficient mice

The pathophysiology of atherosclerosis initiation and progression involves many inflammatory cytokines, one of them is interleukin (IL)-1α that has been shown to be secreted by activated macrophages. We have previously shown that IL-1α from bone marrow-derived cells is critical for early atherosclerosis development in mice. It is known that endoplasmic reticulum (ER) stress in macrophages is involved in progression to more advanced atherosclerosis, but it is still unknown whether this effect is mediated through cytokine activation or secretion. We previously demonstrated that IL-1α is required in ER stress-induced activation of inflammatory cytokines in hepatocytes and in the associated induction of steatohepatitis. In the current study, we aimed to examine the potential role of IL-1α in ER stress-induced activation of macrophages, which is relevant to progression of atherosclerosis. First, we demonstrated that IL-1α is required for atherosclerosis development and progression in the apoE knockout (KO) mouse model of atherosclerosis. Next, we showed that ER stress in mouse macrophages results in the protein production and secretion of IL-1α in a dose-dependent manner, and that IL-1α is required in ER stress-induced production of the C/EBP homologous protein (CHOP), a critical step in ER stress-mediated apoptosis. We further demonstrated that IL-1α-dependent CHOP production in macrophages is specifically mediated through the PERK-ATF4 signaling pathway. Altogether, these findings highlight IL-1α as a potential target for prevention and treatment of atherosclerotic cardiovascular disease.

TREM2 promotes cholesterol uptake and foam cell formation in atherosclerosis.

Disordered lipid accumulation in the arterial wall is a hallmark of atherosclerosis. Previous studies found that the expression of triggering receptor expressed on myeloid cells 2 (TREM2), a transmembrane receptor of the immunoglobulin family, is increased in mouse atherosclerotic aortic plaques. However, it remains unknown whether TREM2 plays a role in atherosclerosis. Here we investigated the role of TREM2 in atherosclerosis using ApoE knockout (ApoE-/-) mouse models, primary vascular smooth muscle cells (SMCs), and bone marrow-derived macrophages (BMDMs). In ApoE-/- mice, the density of TREM2-positive foam cells in aortic plaques increased in a time-dependent manner after the mice were fed a high-fat diet (HFD). Compared with ApoE-/- mice, the Trem2-/-/ApoE-/- double-knockout mice showed significantly reduced atherosclerotic lesion size, foam cell number, and lipid burden degree in plaques after HFD feeding. Overexpression of TREM2 in cultured vascular SMCs and macrophages exacerbates lipid influx and foam cell formation by upregulating the expression of the scavenger receptor CD36. Mechanistically, TREM2 inhibits the phosphorylation of p38 mitogen-activated protein kinase and peroxisome proliferator activated-receptor gamma (PPARγ), thereby increasing PPARγ nuclear transcriptional activity and subsequently promoting the transcription of CD36. Our results indicate that TREM2 exacerbates atherosclerosis development by promoting SMC- and macrophage-derived foam cell formation by regulating scavenger receptor CD36 expression. Thus, TREM2 may act as a novel therapeutic target for the treatment of atherosclerosis.

Administration of the KCa channel activator SKA-31 improves endothelial function in the aorta of atherosclerosis-prone mice

Atherosclerosis is a major risk factor for cardiovascular disease and is induced by hyperlipidemia and endothelial dysfunction (ED), leading to fatty plaque formation and impaired vascular function. Pharmacological activation of endothelial Ca 2+ -activated K + channels (KCa2.3 and KCa3.1) can oppose ED by enhancing endothelium-dependent vasodilation.We hypothesized that improving endothelial function will mitigate the development and/or severity of atherosclerosis in Apoe knockout (Apoe-/-) mice. Administration of the KCa channel activator SKA-31 was utilized to improve endothelial function in vivo.Experimentally, 8-week-old male Apoe-/- mice on a high fat diet (HFD) were administered one of three daily oral treatments for 12 weeks: the KCa channel activator SKA-31 (10 mg/kg), the KCa3.1 channel blocker senicapoc (40 mg/kg), or drug vehicle alone. Pharmacological inhibition of KCa3.1 channels is reported to reduce atherosclerosis in Apoe-/- mice and was utilized as a benchmark. Cardiac function was assessed by echocardiography under isoflurane anesthesia. Atherosclerotic lesions in the thoracic aorta were visualized by Oil-Red-O staining, and abdominal aortic contractility and relaxation were measured by wire myography in Apoe-/- mice and age/sex-matched wild-type C57BL/6 mice.At the end of drug treatment, left ventricular (LV) ejection fraction, fractional shortening, and LV posterior wall thickness were not different in Apoe-/- mice treated with either SKA-31 or senicapoc compared with vehicle. Oil-Red-O staining of the thoracic aorta and aortic arch revealed fatty plaque formation in Apoe-/- mice (~15% of area) compared with WT controls (&lt;1% of area), but neither SKA-31 nor senicapoc treatments reduced plaque formation vs. vehicle. Phenylephrine (PE)-evoked contraction of abdominal aortic rings was similar in WT and vehicle/drug treated Apoe-/- mice. Conversely, endothelium-dependent, acetylcholine-induced relaxation was significantly enhanced in PE-constricted aortic rings from SKA-31-treated mice (mean ± SD, 75.6 ± 21.1 %) vs. vehicle (51.2 ± 11.9 %, p=0.0024). Acetylcholine-induced relaxation was not improved by senicapoc administration. Relaxation induced by the smooth muscle vasodilator sodium nitroprusside was similar in WT and vehicle/drug treated Apoe-/- aortic rings. Summary: SKA-31 administration improved aortic endothelial function without reducing lesion density. Future studies will examine how SKA-31 improves endothelium-dependent relaxation and whether enhanced KCa channel activity (with SKA-31) in combination with lipid-lowering drugs (e.g., statins) can mitigate atherosclerosis more effectively. Enhancement of endothelial KCa channel activity may thus represent a viable stand-alone or “add-on” strategy to reduce morbidity and mortality associated with ED and atherosclerosis. Funding provided by the CIHR and NSERC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Apoe-knockout induces strong vascular oxidative stress and significant changes in the gene expression profile related to the pathways implicated in redox, inflammation, and endothelial function

Apolipoprotein E (APOE) was recognized as a key regulator of lipid metabolism, which prompted the Apoe-knockout (Apoe−/−) mouse to be the most widely used atherosclerotic model. However, with more and more important physiological roles of APOE being revealed, it is necessary to reacquaint its comprehensive function in the aorta. In this study, we aimed to reveal how Apoe-knockout impacts the gene pathways and phenotypes in the aorta of mice. We performed transcriptome sequencing to acquire the gene expression profile (GEP) for C57BL/6J and Apoe−/− mouse aorta, and used enrichment analysis to reveal the signal pathways enriched for differentially expressed genes (DEGs). In addition, we used immunofluorescence and ELISA to detect the phenotypic differences of vascular tissues and plasma in the two-group mice. Apoe-knockout resulted in significant changes in the expression of 538 genes, among which about 75% were up-regulated and 134 genes were altered more than twice. In addition to the lipid metabolism pathways, DEGs were also mainly enriched in the pathways implicated in endothelial cell proliferation, migration of epithelial cells, immune regulatory, and redox. GSEA shows that the up-regulated genes are mainly enriched in ‘immune regulation pathways’ and ‘signal regulation’ pathways, while the down-regulated genes are enriched in lipid metabolism pathways, ‘regulation_of_nitric_oxide_synthase_activity’ and the pathways involved in redox homeostasis, including ‘monooxygenase regulation’, ‘peroxisomes’ and ‘oxygen binding’. A significant increase of reactive oxygen species and a remarkable reduction of GSH/GSSG ratio were respectively observed in the vascular tissues and plasma of Apoe−/− mice. In addition, endothelin-1 significantly increased in the vascular tissue and the plasma of Apoe−/− mice. Taken together, our results suggest that besides functioning in lipid metabolism, APOE may be an important signal regulator that mediates the expression of the genes related to the pathways involved in redox, inflammation, and endothelial function. Apoe-knockout-induced strong vascular oxidative stress is also the key factor contributing to atherosclerosis.

SFRP4 Reduces Atherosclerosis Plaque Formation in ApoE Deficient Mice.

Secreted frizzled related protein 4 (SFRP4), a member of the SFRPs family, contributes to a significant function in metabolic and cardiovascular diseases. However, there is not enough evidence to prove the antiatherosclerosis effect of SFRP4 in ApoE knock-out (KO) mice. ApoE KO mice were fed a western diet and injected adenovirus (Ad)-SFRP4 through the tail vein for 12 weeks. Contrasted with the control cohort, the area of atherosclerotic plaque in ApoE KO mice overexpressing SFRP4 was reduced significantly. Plasma high-density lipoprotein cholesterol was elevated in the Ad-SFRP4 group. RNA sequence analysis indicated that there were 96 differentially expressed genes enriched in 10 signaling pathways in the mRNA profile of aortic atherosclerosis lesions. The analysis data also revealed the expression of a number of genes linked to metabolism, organism system, and human disease. In summary, our data demonstrates that SFRP4 could play an important role in improving atherosclerotic plaque formation in the aorta.

Cyclic di-AMP Rescues Porphyromonas gingivalis–Aggravated Atherosclerosis

Growing evidence demonstrates the relationship between periodontitis and atherosclerotic cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis (Pg) has been shown to contribute to the progression of atherosclerosis. Cyclic diadenylate monophosphate (c-di-AMP) has been widely studied as an immune adjuvant for tumor immunotherapy, given its ability to activate the stimulator of interferon genes (STING) and regulate trained immunity. This study sought to elucidate the role of c-di-AMP in Pg-associated atherosclerosis. Periodontitis and atherosclerosis mouse models were established by ligature application around maxillary second molars and feeding ApoE knockout mice with a high-fat diet. We found that periodontitis and atherosclerosis were more severe in mice exposed to Pg than mice that underwent ligature placement only, while prophylactic treatment with c-di-AMP activated trained immunity and elicited significant alleviation of alveolar bone resorption, as well as reduced blood lipid levels and atherosclerotic plaque accumulation. After 3 mo of intervention, c-di-AMP limited the elevation of cytokines interleukin (IL)–6, IL-1β, tumor necrosis factor α, and interferon β; extracellular matrix remodeling enzymes MMP-2 and MMP-9; and adhesion molecules ICAM-1 and VCAM-1 gene expression. The mechanism underlying Pg-aggravated atherosclerosis may be attributed to changes in microbiota composition in oral and aortic plaques and excess inflammatory response, whereas c-di-AMP could prevent the effects of Pg infection due to its potential ability to activate trained immunity and regulate microecological balance. Our findings suggest a positive role of c-di-AMP in alleviating Pg-aggravated atherosclerosis by regulating the immune response and influencing the local microenvironment.