Model Of Atherosclerosis Research Articles

FABP4 Inhibitor Alleviates Lipopolysaccharide-Induced HUVEC Injury by Inactivating NF-κB and Activating PPARγ.

The study aims to examine the effect of FABP4 on inflammatory response and angiogenesis in the cell model of atherosclerosis and to explore its potential mechanism.Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, the mRNA and protein levels of FABP4 in human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS) or oxidized low-density lipoprotein for 6, 12, 24, or 48 hours were measured. To silence FABP4 expression and NF-κB signaling in HUVECs, FABP4 inhibitor and NF-κB inhibitor were utilized. To assess cell survival rate and apoptosis, methyl thiazolyl tetrazolium assays and flow cytometry analysis were conducted. Genes related to cholesterol metabolism, including LOX-1, ABCA1, and ABCG1, were subjected to RT-qPCR and western blotting. Moreover, protein levels of apoptotic markers (Bcl-2 and Bax), PPARγ, the phosphorylated levels of NF-κB p65, and angiogenetic markers (ICAM1, VCAM1, and VEGF) were quantified via western blotting. Using an enzyme-linked immunosorbent assay, concentrations of inflammatory cytokines in HUVECs were measured.FABP4 expression was upregulated in LPS-stimulated HUVECs. The silencing of FABP4 lowered LOX-1 and p65 levels while upregulating ABCA1 and ABCG1 expression in the context of LPS. Furthermore, the inhibition of FABP4 or NF-κB signaling promoted the growth of HUVECs, upregulated Bcl-2 and PPARγ protein levels, and reduced Bax levels, angiogenetic markers, and inflammatory cytokines in the context of LPS. The combination of FABP4 inhibitor and NF-κB inhibitor treatment amplified the above-mentioned effects on cell growth, angiogenesis, and inflammation.Inhibition of FABP4 reduces LPS-induced HUVEC cell damage via the inactivation of NF-κB p65 and activation of PPARγ signaling.

STING deficiency ameliorates mouse models of lupus and atherosclerosis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome characterized by autoreactive responses to nucleic acids, dysregulation of the type I Interferon (IFN-I) pathway, and accelerated atherosclerosis. The stimulator of interferon genes (STING), a cytosolic DNA-sensor, has pathogenic implications in various inflammatory diseases. However, its specific role in SLE pathogenesis, particularly in tissue damage, remains unclear. This study aimed to elucidate the role of STING in murine models of TLR7-driven lupus and atherosclerosis. A TLR7-driven lupus model was induced using imiquimod (IMQ) in wild type (WT) and STING knockout (Sting1-/-) mice on a B6 background. Mice were assessed for organ involvement, serum autoantibodies, and innate and adaptive immune responses. Additionally, Sting1-/- mice were backcrossed to Apolipoprotein E knockout (Apoe-/-) mice, and both Apoe-/- and Apoe-/-Sting1-/- mice were fed a high-fat chow (HFC) diet to induce atherosclerosis. Phenotypic assessments were conducted. Compared to IMQ-treated WT mice, Sting1-/- mice exhibited reduced disease severity in the lupus-like phenotype, characterized by decreased splenomegaly, lower renal immune complex deposition and renal damage, diminished expansion of myeloid cells, and reduced activation of T and B lymphocytes. IMQ-induced DNA release associated with IFN-β production and subsequent IFN-induced responses were attenuated in Sting1-/- mice. DNase I treatment mitigated IMQ-induced pro-inflammatory responses in WT mice but had no effect in Sting1-/- mice. Furthermore, STING deficiency conferred protection against vascular damage and reduced atherosclerosis burden, accompanied by decreased IFN-I production. Human monocyte-derived macrophages treated with IFN-I significantly internalized more acetylated LDL when compared to untreated cells, while an association between oxidized nucleic acids and disease activity and vascular damage was found in human SLE. These findings highlight a pathogenic role of STING and downstream IFN responses in TLR7-driven autoimmunity, vascular damage and atherosclerosis, supporting a therapeutic potential for STING inhibition in SLE treatment. Further research is warranted to elucidate the mechanisms underlying STING's involvement in these processes and to explore the feasibility of targeting STING as a therapeutic strategy in SLE and related autoimmune disorders.

Endothelial Rho kinase controls blood vessel integrity and angiogenesis.

The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. We generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo . Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

Toxicity and efficacy study of a combination of two retinoic acids in an ApoE knockout mouse model of atherosclerosis.

Atherosclerosis is a major contributor to cardiovascular disease, characterized by inflammation and lipid accumulation in arterial walls, leading to plaque formation. Elevated low-density lipoprotein cholesterol is a primary risk factor for atherosclerosis. All-trans retinoic acid (ATRA), a metabolite of vitamin A, has demonstrated anti-inflammatory effects and potential in regulating vascular injury. 9-cisretinoic acid (9cRA) is an active metabolite of vitamin A and activates the retinoid X receptor. This study investigates whether potassium retinoate (PA9RA), a synthetic combination of ATRA and 9cRA, offers superior efficacy in treating atherosclerosis compared to established treatments such as clopidogrel and atorvastatin. Male ApoE-/- mice were fed a Western-type diet and treated with PA9RA, clopidogrel, or atorvastatin for 10 weeks. The body weight, organ weight, serum biochemistry, and histopathology, including atherosclerotic lesion area and liver steatosis were assessed. PA9RA treatment led to a significant reduction in body weight and inguinal fat, with the 45 mg/kg/day dose showing marked efficacy in decreasing atherosclerotic lesion size and ameliorating liver steatosis. Histopathological evaluation revealed decreased foam cell formation and improved liver histology in PA9RA-treated groups compared to controls. Notable side effects included epidermal hyperplasia and gastric hyperplasia at high doses of PA9RA. PA9RA exhibits superior efficacy over clopidogrel and atorvastatin in ameliorating atherosclerosis and fatty liver in ApoE-/- mice. This study highlights PA9RA's potential as a promising therapeutic agent for atherosclerosis. Further research is needed to elucidate its mechanisms of action and assess long-term safety and efficacy.

Abstract 4129709: Aspirin-Nanoparticle for Dual Therapies: Targeted Anti-Inflammatory and Prolonged Anti-Platelet Effects for Atherosclerosis

Background: The current unmet needs for aspirin usage in atherosclerosis lie in its short half-life and narrow indication for anti-platelet effects. Daily aspirin intake is mandatory, and the anti-inflammatory effects of aspirin for atherosclerosis have not successfully translated to clinical practice. Nanoparticles remain in circulation for 2-3 days, with a large portion being cleared by splenic monocytes, which are known to inherently target inflamed sites. Hypothesis: By altering the pharmacokinetics of aspirin through loading into nanoparticles, aspirin-nanoparticles can exert prolonged anti-platelet effects and target atherosclerosis sites via monocyte carriers for anti-inflammatory effects, resulting in dual therapies. Methods: Splenic monocytes were loaded with aspirin-liposomes and co-cultured with endothelial cells or platelets to examine the interactions between them using high-resolution time-series microscopy. Prolonged anti-platelet effects and targeted anti-inflammatory effects of aspirin were validated in intact mice and hindlimb ischemia models, respectively. Furthermore, the dual therapies of aspirin-liposomes were validated in an atherosclerotic mouse model created by partial carotid ligation and a western diet in apoE gene knock-out mice. Results: When splenic monocytes were loaded with aspirin-liposomes, they emitted extracellular vesicles (EVs) loaded with aspirin towards endothelial cells or platelets. As inflamed cells upregulate caveolin expression, they uptake an increased amount of transferred EVs compared to non-inflamed cells. Additionally, aspirin-liposomes showed prolonged circulation time and increased splenic accumulation compared to aspirin itself, resulting in prolonged anti-platelet effects (>7 days) and targeted anti-inflammatory effects at inflamed sites. Compared to the daily oral aspirin group in the atherosclerosis model, the weekly intravenous aspirin-liposome group showed superior therapeutic effects, including attenuated systemic and local inflammation and patent lumen in atherosclerotic sites. Conclusion: Aspirin-nanoparticles can exert prolonged anti-platelet effects combined with targeted anti-inflammatory effects, resulting in superior therapeutic effects on atherosclerosis.

Abstract 4114308: Defective in LYPLA1-Mediated Lysophosphatidylcholine Metabolism Contributes to Diabetes-Induced Atherosclerosis and Subsequent Myocardial Dysfunction

Background and Objective: Diabetes mellitus (DM) is a chronic metabolic condition that can cause serious damage to blood vessels and the heart. While current metabolomics research mainly focused on metabolic profiles during disease onset or at advanced stages, there remains a gap in understanding the molecular mechanisms of metabolites in the progression of diabetic cardiovascular disease. This study aims to explore effective strategies for preventing cardiovascular complications in diabetics by excavating metabolic pathways and mechanisms. Methods: Peripheral blood samples were obtained from 21 healthy individuals, 20 patients diagnosed with DM, 20 patients with coronary heart disease (CHD), and 25 patients with both DM and CHD (DC). Cardiac function, biochemical parameters and serum metabolome were detected. A specialized methodology with rigorous screening criteria was employed to identify differential metabolites associated with diabetes-induced CHD and cardiomyopathy. Mice were injected with LPC16:0 to assess cardiovascular function. Models of diabetes and diabetic atherosclerosis were induced by STZ injection and high-fat diet feeding in WT and ApoE -/- mice. Cardiac function and differential metabolites in aortic and cardiac tissues were assessed. The effect and molecular mechanism of LYPLA1 on vascular function, mitochondrial function, and metabolites-related enzymes were examined in human aortic endothelial cells (HAECs) and AC16 cardiomyocytes. Results: Cardiac function was markedly declined in DM and DC patients, as well as in mice with diabetes and diabetic atherosclerosis. However, CHD patients and atherosclerosis alone did not exhibit evident cardiac dysfunction. Key metabolites associated with diabetic cardiomyopathy and diabetes-induced CHD were identified as LPE18:1 and LPC16:0, respectively. Intraperitoneal administration of LPC16:0 in mice led to decreased LYPLA1, severe myocardial anomalies, and the appearance of plaques in the aortic arch. Either LYPLA1 knockdown or the treatment with high glucose and palmitic acid in HAECs resulted in impaired mitochondrial function, reduced intracellular NO and eNOS activity, which were mitigated by LYPLA1 overexpression. Conclusion: The metabolite LPC16:0 and the regulatory enzyme LYPLA1 are pivotal in driving the advancement of cardiovascular complications triggered by diabetes. Targeting LYPLA1 could represent a promising strategy for preventing diabetes-induced CHD and subsequent cardiomyopathy.

Abstract 4113617: Single-cell Analysis Reveals Endothelial Cell CD45 Deficiency Prevents Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE -/- ) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis. Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE -/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters. Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE -/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE -/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.

Association of circulating Robo4 with obesity, hypertension and atherosclerotic plaque burden.

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide. Type 2 diabetes mellitus (T2DM) is a major risk factor for ASCVD development, being associated with endothelial dysfunction and accelerated atherosclerosis. In the current study we aimed to explore the potential value of circulating Robo4 (soluble Robo4, sRobo4), an endothelial-specific receptor that regulates endothelial cell (EC) junctional integrity, vascular permeability, EC-mediated inflammatory responses and angiogenesis, as a marker of vascular disease in individuals with or without T2DM. Thirty-four individuals with T2DM and 15 non-diabetic individuals (control) of similar age and sex were included in the study. sROBO4 was comparable between individuals with T2DM and control (T2DM: 1.54±1.19 vs control: 1.59±0.77 ng/mL, P=0.46). Of interest, sRobo4 positively correlated with body mass index (BMI) (rs=0.384, P=0.006), and negatively correlated with renal function as determined by estimated glomerular filtration rate (rs= -0.429, P=0.003). Moreover, individuals with arterial hypertension had higher sRobo4 compared to normotensive individuals (P=0.03). More importantly, we observed that individuals with 2 or more atherosclerotic plaques (n=28) in their femoral and/or carotid arteries had significantly higher sRobo4 levels compared to individuals with a total of none or 1 atherosclerotic plaque (n=20) (P=0.008). The association of sRobo4 with higher atherosclerotic burden remained significant when separately controlling for the effect of age, sex, presence of T2DM, total cholesterol, or BMI levels. In vivo studies utilizing models of atherosclerosis as well as larger prospective cohort studies are warranted to delineate causality and prognostic value of sRobo4 in atherosclerotic cardiovascular disease.

The human milk oligosaccharide 3'sialyllactose reduces low-grade inflammation and atherosclerosis development in mice.

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of TLR4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of liver X receptor (LXR) and sterol regulatory element binding protein-1 (SREBP1). These acute antiinflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of LPS-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), IFN regulatory factor 2 (IRF2), B cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both s.c. and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

Macrophage foam cell-derived mediator promotes spontaneous fat lipolysis in atherosclerosis models.

Ectopic lipid accumulation in macrophages is responsible for the formation of macrophage foam cells (MFCs) which are involved in the crosstalk with the perivascular adipose tissue (PVAT) of the vascular wall that plays a pivotal role in the progression of atherosclerosis. However, the interrelationship between MFCs and PVAT implementing adipocyte dysfunction during atherosclerosis has not yet been established. We hypothesized that MFC-secreted mediator(s) is causally linked with PVAT dysfunction and the succession of atherosclerosis. To test this hypothesis, MFCs were cocultured with adipocytes, or the conditional media of MFCs (MFC-CM) were exposed to adipocytes and found a significant induction of fat lipolysis in adipocytes. The molecular filtration followed by the high-performance liquid chromatography (HPLC) fractionation and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis of MFC-CM revealed a novel mediator fetuin-A (FetA) that significantly augments toll-like receptor 4 (TLR4)-dependent fat lipolysis in adipocytes. Mechanistically, MFC-derived FetA markedly increased TLR4-dependent c-Jun N-terminal kinases (JNK)/extracellular signal-regulated kinases (ERK) activation that causes spontaneous fat lipolysis implementing adipocyte dysfunction. Thus, the present study provides the first evidence of MFC-derived FetA that induces adipocyte dysfunction by the stimulation of spontaneous fat lipolysis. Therefore, targeting the crosstalk between MFCs and adipocytes could be a newer approach to counter the progression of atherosclerosis.

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis.

Environmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. Single-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. Modulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. Dioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. Exposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis.Dioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaqueAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype.

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

Morin, a matrix metalloproteinase 9 inhibitor, attenuates endothelial-to-mesenchymal transition in atherosclerosis by downregulating Notch-1 signaling

ObjectiveAtherosclerotic cardiovascular disease poses a significant health challenge globally. Recent findings highlight the pivotal role of the endothelial-to-mesenchymal transition (EndMT) in atherosclerosis. Morin is a bioflavonoid mainly extracted from white mulberry, a traditional Chinese herbal medicine with anti-inflammatory and antioxidant properties. This study examines whether morin can alleviate atherosclerosis by suppressing EndMT and seeks to elucidate the underlying mechanism. MethodsWe induced an in vitro EndMT model in human umbilical vein endothelial cells (HUVECs) by stimulating the cells with transforming growth factor β1 (TGF-β1) (10 ng/mL) for 48 h. The in vivo experiments were performed in an atherosclerosis model using apolipoprotein E (ApoE)–/– mice fed with a high-fat diet (HFD). Mice in the intervention group were given morin (50 mg/kg) orally for 4 weeks. Molecular docking and microscale thermophoresis were assayed to understand the interactions between morin and matrix metalloproteinase 9 (MMP-9). ResultsMorin inhibited the expression of EndMT markers in a dose-dependent manner in TGF-β1-treated HUVECs. Administering 50 μmol/L morin suppressed the upregulation of MMP-9 and Notch-1 signaling in TGF-β1-induced EndMT. Moreover, the overexpression of MMP-9 activated Notch-1 signaling, thereby reversing morin’s inhibitory effect on EndMT. In the HFD-induced atherosclerotic ApoE–/– mice, morin notably reduced aortic intimal hyperplasia and plaque formation by suppressing EndMT. Furthermore, morin demonstrated a strong binding affinity for MMP-9. ConclusionMorin acts as an MMP-9 inhibitor to disrupt EndMT in atherosclerosis by limiting the activation of Notch-1 signaling. This study underscores morin’s potential utility in the development of anti-atherosclerotic medication.

18F-fluorodeoxyglucose PET-MR characterization of aortic inflammation in ApoE-/- mouse models of accelerated atherosclerosis: comparison of Western diet vs. uremia.

ApoE-/- mice are a widely used preclinical model of atherosclerosis, potentially accelerated by a Western diet (WD) or uremia. We aimed to compare hybrid 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography-magnetic resonance (PET-MR) and immunostaining in ApoE-/- models of accelerated atherosclerosis.Five groups were studied: standard diet-fed ApoE-/- (n = 7), standard diet-fed and uremic ApoE-/- (n = 7), WD ApoE-/- (n = 7), WD and uremic ApoE-/- (n = 6), and control C57BL/6J mice (n = 6). Uremia was induced by electrocoagulation of the right kidney at 8 weeks old, followed 2 weeks later by a contralateral nephrectomy. 18F-FDG PET-MR imaging and histological staining (anti-CD4, -CD8, -CD11c, -CD20, -CD31, -CD68, -CD163, -interferon-γ, interleukin-1α, -1β, -6, -17A antibodies) were performed in 18-week-old mice, i.e., 8 weeks after 5/6 nephrectomy and/or WD.18F-FDG uptake was similar in all groups. In contrast, histological staining highlighted higher percentages of CD8-, CD68-, or CD11c-positive cells in ApoE-/- aortic samples than in wild-type aortic samples. In addition, immunostaining revealed some differences between ApoE-/- mouse groups. Only the WD seemed to contribute to these differences.Using immunostaining, WD appeared to be a stronger accelerator of atherosclerosis than uremia. However, 18F-FDG PET-MR imaging failed to demonstrate in vivo increased aortic glucose uptake in these models.

Effect of Oral Administration of Collagen Peptide OG-5 on Advanced Atherosclerosis Development in ApoE-/- Mice.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, which involves multiple cell types. Peptide OG-5 is identified from collagen hydrolysates derived from Salmo salar and exhibits an inhibitory effect on early atherosclerosis. The primary objective of this study was to investigate the impact of OG-5 on advanced atherosclerotic lesions as well as its stability during absorption. In this study, the ApoE-/- mice were employed to establish advanced atherosclerosis model to investigate the treatment effect of peptide OG-5. The results showed that oral administration of OG-5 at a dosage of 150 mg/kg bw resulted in a 30% reduction in the aortic plaque formation area in ApoE-/- mice with few bleeding risks. Specifically, intervention with a low dose of OG-5 (50 mg/kg bw), initiated in the early stage of atherosclerosis, continues to provide benefits into the middle and late stages without bleeding risks. Furthermore, treatment of OG-5 increased expression levels of contractile phenotype markers and reduced the accumulation of lipoprotein in VSMCs induced by ox-LDL. Peptide OG-5 could ensure transport across Caco-2 cell monolayers, exhibiting a Papp value of 1.80 × 10-5 cm/s, and exhibited a robust stability in plasma with remaining content >70% after 8 h incubation. In vivo studies revealed that OG-5 reached maximum concentration in blood after 120 min. The present results demonstrate the potential efficacy of peptide OG-5 as a promising agent for intervention in anti-atherogenesis strategies.

Vaccination targeting senescence-associated transmembrane protein alleviated cardiovascular pathology in the mice

Abstract Introduction Accumulation of senescent cells is observed in various organs including vasculature, heart and white adipose tissue with age, and known to become a substrate of pathophysiology of various cardiovascular-metabolic diseases. We have previously developed a vaccine to eliminate senescent cells, so-called "senolytic vaccine" by targeting Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) as a cellular-senescence-associated cell surface protein. We have demonstrated that GPNMB senolytic vaccine could remove senescent cells and alleviate the pathology cardiovascular-metabolic diseases. Senescence-associated adhesion molecule 1 (SAAM-1) is also known as a cell-surface membrane to increase its expression during cellular senescence in endothelial cells as well as the development of cardiovascular diseases. Here, we identified SAAM-1 as another senescence antigen and created a vaccine to eliminate senescent cells by targeting SAAM-1. Purpose This study was purposed to evaluate the efficacy of SAAM-1 vaccine for the treatment of cardiovascular diseases in mice models. Methods As a pressure overload-induced heart failure (HF) model, C57BL/6N wild type (WT) mice were underwent transverse aortic constriction (TAC) or sham opretaion at 13 weeks of age. SAAM-1 or control vaccination were given at 8 and 10 weeks of age before operation. Echocardiography examination was conducted two weeks after TAC operation, followed by tissue sampling 3 weeks after the examination. As an atherosclerosis model, ApoE KO mice were subjected to a high-fat diet (HFD) starting at 4 weeks of age. Vaccination of ApoE KO mice was administered at 8 weeks of age with SAAM-1-vaccine or control-vaccine, followed by sacrifice 4 weeks later for further analysis. ApoE-KO mice were also crossed with p16-mediated Luciferase expressing mice (p16-Luc mice) and subjected to generation of atherosclerosis model. Results SAAM-1 expression was significantly increased in left ventricle (LV) in TAC mice but its increase was suppressed by SAAM-1 vaccination. Interestingly, both worsening systolic function and dilatation of LV by pressure overload were significantly alleviated by SAAM-1 vaccination. Furthermore, expression level of senescent markers including p16 and p21, as well as several inflammation markers were increased in failing heart along with development of cardiac fibrosis, but SAAM-1 vaccine could significantly attenuate this pathological change. In ApoE KO mice, SAAM-1 vaccine decreased expression level of several inflammation markers in the mice, along with a decreasing p16 signal in the aorta. Conclusions Targeting SAAM-1 for vaccination would be a novel and promising approach to alleviating age-related cardiovascular disease.

Therapeutic effects of FGF21 mimetic bFKB1 on MASH and atherosclerosis in Ldlr-/-.Leiden mice.

Fibroblast growth factor 21 (FGF21) is a promising target for treatment of obesity-associated diseases including metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis. We evaluated the effects of the bispecific anti-FGF21-β klotho (KLB) agonist antibody bFKB1 in a preclinical model of MASH and atherosclerosis. Low-density lipoprotein receptor knockout (Ldlr-/-).Leiden mice received a high-fat diet for 20 weeks, followed by treatment with an isotype control antibody or bFKB1 for 12 weeks. Effects on plasma risk markers and (histo)pathology of liver, adipose tissue, and heart were evaluated alongside hepatic transcriptomics analysis. bFKB1 lowered body weight (-21%) and adipose tissue mass (-22%) without reducing food intake. The treatment also improved plasma insulin (-80%), cholesterol (-48%), triglycerides (-76%), alanine transaminase (ALT: -79%), and liver weight (-43%). Hepatic steatosis and inflammation were strongly reduced (macrovesicular steatosis -34%; microvesicular steatosis -100%; inflammation -74%) and while the total amount of fibrosis was not affected, bFKB1 did decrease new collagen formation (-49%). Correspondingly, hepatic transcriptomics and pathway analysis revealed the mechanistic background underlying these histological improvements, demonstrating broad inactivation of inflammatory and profibrotic transcriptional programs by bFKB1. In epididymal white adipose tissue, bFKB1 reduced adipocyte size (-16%) and inflammation (-52%) and induced browning, signified by increased uncoupling protein-1 (UCP1) protein expression (8.5-fold increase). In the vasculature, bFKB1 had anti-atherogenic effects, lowering total atherosclerotic lesion area (-38%). bFKB1 has strong beneficial metabolic effects associated with a reduction in hepatic steatosis, inflammation, and atherosclerosis. Analysis of new collagen formation and profibrotic transcriptional programs indicate that bFKB1 treatment may have antifibrotic potential in a longer treatment duration as well.

Aspirin inhibits atherosclerosis through macrophage pyroptosis by interacting with Lactobacillus johnsonii and its metabolite butyrate

Abstract Introduction Atherosclerotic cardiovascular disease (ASCVD) is a chronic progressive disease closely related to metabolism and inflammation. Aspirin inhibits cyclooxygenase (COX) and has antiplatelet aggregation effects, considered as the cornerstone of ASCVD treatment. Numerous studies have shown that gut microbiota (GM) and its metabolites affects host metabolism. Studies have also shown that people taking aspirin exhibit specific changes in the microbiota profile. However, whether the interaction of aspirin with GM and its metabolites affects the therapeutic efficacy of aspirin in treating atherosclerosis (AS) have not been reported. Purpose This study aimed to explore the interaction of aspirin with the GM and its metabolites in the treatment of AS, to reveal the specific mechanism of the GM-metabolite axis and provide potential new targets for the combined treatment of AS. Methods To establish the mouse model of atherosclerosis by high-fat diet, and the atherosclerotic plaque burden and composition were analyzed. The role of GM in aspirin anti-atherosclerosis was explored by antibiotics and fecal microbiota transplantation (FMT). The third generation 16S RNA sequencing was used to explore the effects of aspirin on species diversity and abundance of GM, and to screen differential microorganisms for intervention of AS mice. UHPLC-MS and GC-MS were used to explore the changes of intestinal metabolites induced by aspirin. The correlation between gut microbiota and metabolites was also investigated. Finally, in vitro and in vivo experiments were performed to confirm the effects and mechanisms of metabolites on macrophage pyroptosis and atherosclerosis. Results Aspirin has anti-atherosclerosis effect. After removing gut microbiota, the therapeutic effect of aspirin on atherosclerosis was decreased. Three generations of 16S rRNA gene sequencing results showed that aspirin supplementation alters the diversity of gut microbiota, and increased the relative abundance of Lactobacillus johnsonii(L.johnsonii) and Ligilactobacillus murinus(L.murinus). After gavage of L.johnsonii and L.murinus, the plaque burden and macrophage pyroptosis were significantly reduced in AS mice. Colonization with L.johnsonii and L.murinus significantly increased the abundance of probiotics and butyrate level in the feces of AS mice and L.johnsonii and L.murinus were positively correlated with butyrate. Supplementation of butyrate inhibited macrophage pyroptosis and atherosclerosis in vivo. In vitro, butyrate could inhibit the expression of NLRP3/Caspase-1/IL-1β and pyroptosome production, and this process mainly by activating butyrate receptors GPR41, GPR43 and GPR109A. Conclusions The gut microbiota may mediate the anti-AS effect of aspirin by promoting an increase in the abundance of L.johnsonii and L.murinus. Butyrate produced by Lactobacillus may mediate the anti-AS effect of aspirin, and inhibiting macrophage pyroptosis and the progression of AS.

Antibodies to HIV-1 gp120 in an experimental model of atherosclerosis caused by immunization with native high-density lipoproteins

Antibodies to bacterial and viral antigens are detected in some systematic autoimmune diseases in the absence of infection. Autoimmune MRL mice and patients with mixed connective tissue disease produce antibodies to gp120 HIV-1, despite the fact that they had never been exposed to HIV-1. Conversely, viral infections may be accompanied by pathological autoimmune reactions. Reactivity to viral antigens in autoimmune diseases and autoimmune reactions in viral diseases are caused by the induction of antibodies via idiotype-anti-idiotype interactions between autoclones and lymphocyte clones against foreign antigens or homology of foreign antigens and autoantigens. Cardiovascular diseases caused by atherosclerosis are currently one of the main causes of mortality in HIV infected patients. It is assumed that HIV infection accelerates atherogenesis. The mechanisms of the association between HIV infection and atherosclerosis are not completely clear. Chronic activation of the immune system, disturbance of cytokine regulation caused by HIV infection, induction of autoantibodies against oxidized low-density lipoproteins (LDL) and antibodies to ApoB-D are considered as possible factors of atherogenesis in HIV infection. The aim of this research was to determine whether an atherogenic immune response against native human high-density lipoproteins (nHDL) could induce antibodies to gp120 HIV-1. Studies were conducted on model of autoimmune atherosclerosis caused by immunization with native human HDL in rabbits. Rabbits (n = 6) were one-time intradermally immunized with human nHDL at a dose of 200 μg of protein per animal in incomplete Freund’s adjuvant. Antibodies to gp120 HIV-1 were measured before immunization of rabbits and on the 28th and 42th day after immunization with HDL, and antibodies to HDL were weekly measured within 42 days after immunization. Histological analysis of the aorta was conducted after eight months after immunization. Transient anti-gp120 HIV-1 antibody production was detected in rabbits immunized with native HDL. The appearance of antibodies to gp120 HIV-1 in response to immunization with native HDL which causes atherosclerosis suggests that the immune response to gp120 HIV-1 during HIV infection may be accompanied by the production of atherogenic antibodies to HDL. Consequently, the antibodies to native lipoproteins caused by an immune response against gp120 HIV-1 may be a factor in atherogenesis during HIV-1 infection.

Alisol A inhibits and stabilizes atherosclerotic plaques by protecting vascular endothelial cells.

Dysfunction of endothelial cells represents a crucial aspect in the pathogenesis of atherosclerosis. The aim of this study was to explore the protective effects of alisol A on vascular endothelial cells and its possible mechanisms. An atherosclerosis model was established by feeding ApoE-/- mice with high-fat chow. Alisol A (150mg/kg/d) or atorvastatin (15mg/kg/d) was administered, and the levels of blood lipids were evaluated. The effect of the drugs on atherosclerotic plaques was observed by staining the aorta with Sudan IV. In vitro experiments were conducted using human aortic endothelial cells (HAECs) to assess the effects of alisol A on cell proliferation, migration, tubulation, secretion, and cellular integrity by CCK-8 assay, wound healing assay, angiogenesis assay, NO secretion, and release of LDH. Transcriptomics and molecular docking were used to explore the mechanism of plaque inhibition and stabilization by alisol A. Alisol A significantly reduced the aortic plaque area in ApoE-/- mice fed with high-fat chow. In vitro, alisol A had a protective effect on HAECs, which was reflected in the inhibition of vascular endothelial cell proliferation, promotion of NO secretion by vascular endothelial cells, inhibition of vascular endothelial cell migration and angiogenesis, and the maintenance of cell membrane integrity. Therefore, alisol A inhibited and stabilized atherosclerotic plaques and slowed down the process of atherosclerosis. Transcriptomics studies showed 4,086 differentially expressed genes (DEGs) in vascular endothelial cells after alisol A treatment. Enrichment analysis indicated that many genes involved in TNF signaling pathway were differentially expressed, and inflammatory genes were suppressed. The molecular docking results verified the hypothesis that alisol A has a low binding energy after docking with TNF target, and TNF could be a potential target of alisol A. Alisol A produced protection on vascular endothelial cells, achieving inhibition and stabilization of atherosclerotic plaques.