Development Of Atherosclerosis In Mice Research Articles

Abstract 1036: Mechanisms Of Foamy Monocyte Formation In Atherosclerosis

Diseases linked to atherosclerosis are the leading cause of death in the United States. Transmigration of monocytes across the endothelial layer and their differentiation into macrophages play an important role in the initiation and progression of atherosclerosis. These monocyte-derived macrophages internalize modified LDL in the arterial wall and drive the pathogenesis of atherosclerosis. Recent studies have shown that pharmacological depletion of foamy monocytes inhibits atherosclerosis development in mice, identifying monocyte uptake of plasma LDL as a therapeutic target in atherosclerosis. However, the precise mechanisms by which monocytes internalize plasma LDL remain unknown. The goal of our study was to investigate the role, and relative contribution, of macropinocytosis and scavenger receptors ( Cd36 and Sr-a ) to foamy monocyte formation. The presence of foamy monocytes was confirmed in hypercholesterolemic mice and humans via flow cytometry analysis of Nile Red fluorescence. High resolution scanning electron microscopy and flow cytometry analysis of dextran internalization confirmed macropinocytosis stimulation in THP-1 and primary murine monocytes. Stimulation of macropinocytosis in monocytes induced uptake of both native LDL and oxLDL, leading to foamy monocyte formation in vitro . Pharmacological (EIPA) and genetic inhibition of macropinocytosis ( Lysm - CreER T2+/- Nhe1 -/- ) inhibited foamy monocyte formation (58.9%) in hypercholesterolemic mice in vivo . RT-PCR confirmed expression of Cd36 and Sr-a in monocytes and deletion of Cd36 and Sr-a inhibited foamy monocyte formation in hypercholesterolemic mice (10.3%) compared to wild type controls. FACS and bulk RNA sequencing characterized monocyte subsets including classical, intermediate, and non-classical monocytes in hypercholesterolemic Cd36 -/- / Sr-a -/- and Lysm - CreER T2+/- Nhe1 -/- mice and their respective wild type controls. Mechanistic studies identified NADPH oxidase 2 (Nox2) and downstream redox regulation of actin-cytoskeleton as a major stimulator of monocyte macropinocytosis. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets in the treatment of atherosclerosis.

Cytosolic DNA initiates a vicious circle of aging-related endothelial inflammation and mitochondrial dysfunction via STING: the inhibitory effect of Cilostazol.

Endothelial senescence, aging-related inflammation, and mitochondrial dysfunction are prominent features of vascular aging and contribute to the development of aging-associated vascular disease. Accumulating evidence indicates that DNA damage occurs in aging vascular cells, especially in endothelial cells (ECs). However, the mechanism of EC senescence has not been completely elucidated, and so far, there is no specific drug in the clinic to treat EC senescence and vascular aging. Here we show that various aging stimuli induce nuclear DNA and mitochondrial damage in ECs, thus facilitating the release of cytoplasmic free DNA (cfDNA), which activates the DNA-sensing adapter protein STING. STING activation led to a senescence-associated secretory phenotype (SASP), thereby releasing pro-aging cytokines and cfDNA to further exacerbate mitochondrial damage and EC senescence, thus forming a vicious circle, all of which can be suppressed by STING knockdown or inhibition. Using next-generation RNA sequencing, we demonstrate that STING activation stimulates, whereas STING inhibition disrupts pathways associated with cell senescence and SASP. In vivo studies unravel that endothelial-specific Sting deficiency alleviates aging-related endothelial inflammation and mitochondrial dysfunction and prevents the development of atherosclerosis in mice. By screening FDA-approved vasoprotective drugs, we identified Cilostazol as a new STING inhibitor that attenuates aging-related endothelial inflammation both in vitro and in vivo. We demonstrated that Cilostazol significantly inhibited STING translocation from the ER to the Golgi apparatus during STING activation by targeting S162 and S243 residues of STING. These results disclose the deleterious effects of a cfDNA-STING-SASP-cfDNA vicious circle on EC senescence and atherogenesis and suggest that the STING pathway is a promising therapeutic target for vascular aging-related diseases. A proposed model illustrates the central role of STING in mediating a vicious circle of cfDNA-STING-SASP-cfDNA to aggravate age-related endothelial inflammation and mitochondrial damage.

DHCR24 inhibitor SH42 increases desmosterol without preventing atherosclerosis development in mice

The liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol, a final intermediate in cholesterol biosynthesis, has been identified as a selective LXR ligand that suppresses inflammation without inducing lipogenesis. Δ24-Dehydrocholesterol reductase (DHCR24) converts desmosterol into cholesterol, and we previously showed that the DHCR24 inhibitor SH42 increases desmosterol to activate LXR and attenuate experimental peritonitis and metabolic dysfunction-associated steatotic liver disease. Here, we aimed to evaluate the effect of SH42 on atherosclerosis development in APOE∗3-Leiden.CETP mice and low-density lipoproteins (LDL) receptor knockout mice, models for lipid- and inflammation-driven atherosclerosis, respectively. In both models, SH42 increased desmosterol without affecting plasma lipids. While reducing liver lipids in APOE∗3-Leiden.CETP mice, and regulating populations of circulating monocytes in LDL receptor knockout mice, SH42 did not attenuate atherosclerosis in either model.

Gsα Regulates Macrophage Foam Cell Formation During Atherosclerosis.

Many cardiovascular pathologies are induced by signaling through G-protein-coupled receptors via Gsα (G protein stimulatory α subunit) proteins. However, the specific cellular mechanisms that are driven by Gsα and contribute to the development of atherosclerosis remain unclear. High-throughput screening involving data from single-cell and bulk sequencing were used to explore the expression of Gsα in atherosclerosis. The differentially expression and activity of Gsα were analyzed by immunofluorescence and cAMP measurements. Macrophage-specific Gsα knockout (Mac-GsαKO) mice were generated to study the effect on atherosclerosis. The role of Gsα was determined by transplanting bone marrow and performing assays for foam cell formation, Dil-ox-LDL (oxidized low-density lipoprotein) uptake, chromatin immunoprecipitation, and luciferase reporter assays. ScRNA-seq showed elevated Gnas in atherosclerotic mouse aorta's cholesterol metabolism macrophage cluster, while bulk sequencing confirmed increased GNAS expression in human plaque macrophage content. A significant upregulation of Gsα and active Gsα occurred in macrophages from human and mouse plaques. Ox-LDL could translocate Gsα from macrophage lipid rafts in short-term and promote Gnas transcription through ERK1/2 activation and C/EBPβ phosphorylation via oxidative stress in long-term. Atherosclerotic lesions from Mac-GsαKO mice displayed decreased lipid deposition compared with those from control mice. Additionally, Gsα deficiency alleviated lipid uptake and foam cell formation. Mechanistically, Gsα increased the levels of cAMP and transcriptional activity of the cAMP response element binding protein, which resulted in increased expression of CD36 and SR-A1. In the translational experiments, inhibiting Gsα activation with suramin or cpGN13 reduced lipid uptake, foam cell formation, and the progression of atherosclerotic plaques in mice in vivo. Gsα activation is enhanced during atherosclerotic progression and increases lipid uptake and foam cell formation. The genetic or chemical inactivation of Gsα inhibit the development of atherosclerosis in mice, suggesting that drugs targeting Gsα may be useful in the treatment of atherosclerosis.

Integrin α9β1 deficiency does not impact the development of atherosclerosis in mice

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes cardiovascular disease in humans and mice. However, the receptor mediating the effect of SVEP1 on the development of disease remains unclear. We previously demonstrated that depleting either vascular smooth muscle cell (VSMC)- or myeloid cell-derived integrin α9β1, the first receptor that was identified to interact with SVEP1, did not phenocopy the disease-abrogating effect of depleting SVEP1. Due to its wide expression in tissues and cell types, here we extend this line of investigation to definitively determine if integrin α9β1 impacts the development of atherosclerosis. In a mouse model of atherosclerosis, we found that depleting integrin α9β1 in all cells did not alter plaque size or characteristics of plaque complexity when compared to wild type mice. Further, the significant SVEP1-mediated effects on increase in macrophage content and VSMC proliferation within the atherosclerotic plaque were not altered in animals lacking integrin α9β1. Together, these findings strongly suggest that integrin α9β1 is not responsible for mediating the SVEP1-induced promotion of atherosclerosis and support further studies aimed at characterizing other receptors whose interaction with SVEP1 may represent a therapeutically targetable interaction.

Colchicine prevents accelerated atherosclerosis development in TET2-mutant clonal hematopoiesis

Abstract Background Somatic mutations in the TET2 gene that lead to clonal hematopoiesis are associated with accelerated atherosclerosis development in mice and a higher risk of atherosclerotic cardiovascular disease events in humans. Mechanistically, these observations have been linked to exacerbated vascular inflammation. Purpose Here, we sought to evaluate whether colchicine, a widely available and inexpensive anti-inflammatory drug, prevents the accelerated atherosclerosis associated with TET2-mutant clonal hematopoiesis. Methods In mice, we mimicked TET2-mutant clonal hematopoiesis using a competitive bone marrow transplantation approach in atherosclerosis prone Ldlr/- mice. We generated hematopoietic chimeras carrying initially 10% Tet2-/- hematopoietic cells, which were fed a high-cholesterol diet and treated with colchicine or placebo. Atherosclerosis development was evaluated histologically. In humans, we analyzed whole exome sequencing data and electronic medical records from 50,291 participants in a hospital-based cohort to examine the potential modifying effect of colchicine prescription on the association between clonal hematopoiesis and myocardial infarction. Results Colchicine prevented the development of accelerated atherosclerosis in the mouse model of TET2-mutant clonal hematopoiesis, without affecting serum cholesterol levels or the expansion of TET2-deficient hematopoietic cells. In humans, consistent with experimental findings, patients who were prescribed colchicine had attenuated associations between TET2-mutant clonal hematopoiesis and myocardial infarction (ORno colchicine [95% CI]: 1.18 [0.93-1.48]; ORcolchicine [95% CI]: 0.78 [0.45-1.35]; Pinteraction=0.03). This interaction was not observed for carriers of other clonal hematopoiesis-related mutations, such as those affecting DNMT3A or ASXL1. Conclusions Our results highlight the potential value of colchicine as a targeted preventive care strategy to mitigate the higher cardiovascular risk of carriers of somatic TET2 mutations in blood cells. These observations set the basis for the development of clinical trials that evaluate the efficacy of precision medicine approaches tailored to the effects of specific somatic mutations linked to clonal hematopoiesis.

Genetic inhibition of CARD9 accelerates the development of atherosclerosis in mice through CD36 dependent-defective autophagy

Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1β production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.

Effect of LDL desialylation on the development of atherosclerosis in mice

Effect of LDL desialylation on the development of atherosclerosis in mice

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.

Impact of dietary choline on lipid metabolism and atherosclerosis development in apoEKO mice deficient or overexpressing apolipoprotein A-I: Selected Abstract - XVI SITeCS Congress

TMAO, a metabolite of dietary choline, is considered a pro-atherogenic molecule for its ability to interfere with the reverse cholesterol transport, in which apolipoprotein A-I and HDL play a key role. In the present work it was evaluated how TMAO impacts on the development of atherosclerosis in mice with different levels of apoA-I/HDL. Mice deficient in both murine apoA-I and apoE (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I), characterized by extremely low or high plasma HDL levels respectively, were fed for 16 weeks two standard rodent diets, differing only in their choline content (0.09% or 1.2%). At the end of the dietary treatment, atherosclerosis development was quantified at the aortic sinus, targeted plasma metabolomics was performed, and gene expression was evaluated in liver, duodenum, jejunum and ileum. With both diets, DKO mice developed much larger plaques than DKO/hA-I mice. High-choline diet increased plasma TMAO levels in both genotypes. Interestingly, a worsening of plaque development by high choline diet occurred in DKO/hA-I mice only (0.057±0.048 mm2 vs 0.0988±0.064 mm2, p<0.01). Plasma metabolomics indicated that choline supplementation, only in the presence of HDL, significantly increased the concentration of some ceramide species in addition to several markers of impaired kidney function. High-choline diet increased the hepatic gene expression of Fmo1 and Fmo2 in DKO/hA-I, whereas the expression of Scarb1 was lower in DKO/hA-I compared to DKO mice, regardless of the dietary treatment. Intestinal expression of genes involved in inflammatory response and in lipid metabolism was comparable between genotypes and was not modified by choline supplementation. In conclusion, high choline diet increased plasma TMAO concentration in both genotypes, but affected atherosclerosis development, plasma metabolome and hepatic gene expression only in high HDL mice. Intestinal gene expression was not affected neither by genotype nor by dietary choline content.

Vascular smooth muscle- and myeloid cell-derived integrin α9β1 does not directly mediate the development of atherosclerosis in mice

Background and aimsSushi, von Willebrand factor type A, EGF pentraxin domain-containing 1 (SVEP1), an extracellular matrix protein, is a human coronary artery disease locus that promotes atherosclerosis. We previously demonstrated that SVEP1 induces vascular smooth muscle cell (VSMC) proliferation and an inflammatory phenotype in the arterial wall to enhance the development of atherosclerotic plaque. The only receptor known to interact with SVEP1 is integrin α9β1, a cell surface receptor that is expressed by VSMCs and myeloid lineage-derived monocytes and macrophages. Our previous in vitro studies suggested that integrin α9β1 was necessary for SVEP1-induced VSMC proliferation and inflammation; however, the underlying mechanisms mediated by integrin α9β1 in these cell types during the development of atherosclerosis remain poorly understood. Methods and ResultsHere, using cell-specific gene targeting, we investigated the effects of the integrin α9β1 receptor on VSMCs and myeloid cells in mouse models of atherosclerosis. Interestingly, we found that depleting integrin α9β1 in either VSMCs or myeloid cells did not affect the formation or complexity of atherosclerotic plaque in vessels after either 8 or 16 weeks of high fat diet feeding. ConclusionsOur results indicate that integrin α9β1 in these two cell types does not mediate the in vivo effect of SVEP1 in the development of atherosclerosis. Instead, our results suggest either the presence of other potential receptor(s) or alternative integrin α9β1-expressing cell types responsible for SVEP1 induced signaling in the development of atherosclerosis.

ILRUN Promotes Atherosclerosis Through Lipid-Dependent and Lipid-Independent Factors.

Common genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domains (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice. For our study, we generated global Ilrun-deficient (IlrunKO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout (LdlrKO) and apolipoprotein E knockout (ApoeKO; double knockout [DKO]). Compared with littermate control mice (single LdlrKO or ApoeKO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the ApoeKO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions. Our results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.

Platelets inhibit development of atherosclerosis in atherosclerotic mice

ABSTRACT Platelets can protect from lipopolysaccharide-induced septic shock by inhibiting inflammation, but it is unknown whether platelets have an anti-atherosclerotic effect. The aim of this study was to investigate the effect of platelet transfusion on atherosclerosis (AS) in a mouse model of AS. Apolipoprotein E deficiency (ApoE−/−) mice were fed with a high-fat diet (HFD) for 8 weeks to establish a mouse model of AS. Mice weekly underwent bi-weekly injection with or without platelets during AS induction (HFD+platelet). Hematoxylin–eosin (H&E), Oil Red O, and Sudan IV stainings were used to assess pathological and morphological changes in the aortic tissue. Lipid levels, and liver and kidney function were examined using an automatic biochemical analyzer. Immune histochemical assays were used to detect the infiltration and distribution of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, and monocyte chemotactic protein-1 (MCP-1) in the aortic arch. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to examine the expression levels of TNF-α, IL-1β, IL-6, and MCP-1 in the aorta or the peripheral blood, respectively. Compared with the HFD group, AS pathological lesions from the aortic arch in the HFD+platelet group were significantly smaller and alterations in the lipid metabolism were also less pronounced. Furthermore, TNF-α, IL-1β, IL-6, and MCP-1 levels were all significantly reduced in mice that received platelet injection. Platelets transfusion can effectively ameliorate lipid metabolism, suppress the inflammatory response in the vascular wall, and inhibit the development of AS in mice.

Lipopolysaccharide and tyloxapol accelerate the development of atherosclerosis in mice.

The occurrence of atherosclerosis is closely related to inflammation and lipid metabolism disorder. It has been found that lipopolysaccharide (LPS) could induce inflammation, and tyloxapol (Ty) could induce hyperlipidemia. However, the effects of LPS and Ty on the development and mechanism of atherosclerosis have not been investigated thoroughly. To answer this question, we used assay kits to detect total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) content to evaluate dyslipidemia. We used hematoxylin and eosin staining to evaluate the pathological structure of the aorta and liver, and then used Oil Red O staining to access lipid accumulation in the aortic wall. Subsequently, we used the alanine transaminase (ALT) kit to examine the liver injury. Finally, we used the Western blot experiment to measure proteins that regulate lipid metabolism. We found that the LPS + Ty group could increase the levels of TC, TG, and LDL in the serum and promote lipid accumulation in the aortic wall in mice. Moreover, our study showed that the LPS + Ty group induced pathological changes in hepatocytes and increased ALT content in mice. Significantly, we found that the LPS + Ty group could activate acetyl-CoA carboxylase, sterol regulatory element-binding protein-1c, and inhibit peroxisome proliferator-activated receptors α in mice. Therefore, we suppose that LPS and Ty aggravated the development of atherosclerosis by promoting hyperlipidemia and the disorder of lipid metabolism in mice. These findings are significant for the study of the pathogenesis of atherosclerosis and the selection of animal models.

GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression.

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE−/− mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides −750 and −503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo−/− mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

Influence of atherosclerosis on the molecular expression of the TRPC1/BK signal complex in the aortic smooth muscles of mice.

Atherosclerosis (AS) is one a disease that seriously endangers human health. Previous studies have demonstrated that transient receptor potential channel-1 (TRPC1)/large conductance Ca2+ activated K+ channel (BK) signal complex is widely distributed in arteries. Therefore, it was hypothesized that TRPC1-BK signal complex may be a new target for the treatment of AS-related diseases. Apolipoprotein E-/- (ApoE-/-) mice were used to establish an atherosclerotic animal model in the present study, and the association between AS and the TRPC1-BK signal complex was examined. The present study aimed to compare the differences in the expression levels of mRNAs and proteins of the TRPC1-BK signal complex expressed in the aortic vascular smooth muscle tissue, between mice with AS and control mice. There were 10 mice in each group. Reverse transcription PCR, western blotting and immunohistochemistry were used to detect the differences in the mRNA and protein expression levels of TRPC1, BKα (the α subunit of BK) and BKβ1 (the β1 subunit of BK). The mRNA expression level of TRPC1 in AS model mice was significantly higher compared with that in the control group (P<0.05). However, the mRNA expression levels of BKα and BKβ1 were lower compared with those in the controls (both P<0.01). The mice in the ApoE-/- group successfully developed AS. In this group, the protein expression level of TRPC1 was significantly higher than that in the control group (P<0.01), while the protein expression levels of BKα and BKβ1 were lower compared with those in the control group (P<0.01 and P<0.05, respectively). Collectively, it was identified that the protein and mRNA expression levels of the TRPC1/BK signal complex in the aortic vascular smooth muscle tissue could be influenced by the development of AS in mice. Hence, the TRPC1/BK signal complex may be a potential therapeutic target for the prevention and treatment of AS-related complications in the future.

Smooth muscle 22 alpha protein inhibits VSMC foam cell formation by supporting normal LXR\u03b1 signaling, ameliorating atherosclerosis

Vascular smooth muscle cells (VSMCs) are indispensable components in foam cell formation in atherosclerosis. However, the mechanism behind foam cell formation of VSMCs has not been addressed. We found a potential association between deletion of smooth muscle (SM) 22α and deregulated nuclear receptors liver X receptors (LXRs)/retinoid X receptor (RXR) signaling in mice. Here, we investigated the roles of SM22α in LXRα-modulated cholesterol homeostasis, and explore possible mechanisms underlying this process. We identified that the depletion of SM22α was a primary event driving VSMC cholesterol accumulation and the development of atherosclerosis in mice. Proteomic and lipidomic analysis validated that downregulation of SM22α was correlated with reduced expression of LXRα and ATP-binding cassette transporter (ABCA) 1 and increased cholesteryl ester in phenotypically modulated VSMCs induced by platelets-derived growth factor (PDGF)-BB. Notably, LXRα was mainly distributed in the cytoplasm rather than the nucleus in the neointimal and Sm22α−/− VSMCs. Loss of SM22α inhibited the nuclear import of LXRα and reduced ABCA1-mediated cholesterol efflux via promoting depolymerization of actin stress fibers. Affinity purification and mass spectrometry (AP-MS) analysis, co-immunoprecipitation and GST pull-down assays, confocal microscopy, and stochastic optical reconstruction microscopy (STORM) revealed that globular-actin (G-actin), monomeric actin, interacted with and retained LXRα in the cytoplasm in PDGF-BB-treated and Sm22α−/− VSMCs. This interaction blocked LXRα binding to Importin α, a karyopherin that mediates the trafficking of macromolecules across the nuclear envelope, and the resulting reduction of LXRα transcriptional activity. Increasing SM22α expression restored nuclear localization of LXRα and removed cholesterol accumulation via inducing actin polymerization, ameliorating atherosclerosis. Our findings highlight that LXRα is a mechanosensitive nuclear receptor and that the nuclear import of LXRα maintained by the SM22α-actin axis is a potential target for blockade of VSMC foam cell formation and development of anti-atherosclerosis.

Inhibition of Rac1 GTPase Decreases Vascular Oxidative Stress, Improves Endothelial Function, and Attenuates Atherosclerosis Development in Mice.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice.Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration.Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

The human milk oligosaccharide 3’sialyllactose promotes inflammation resolution and reduces atherosclerosis development.

Background and Aims: Macrophages contribute to the induction and resolution of inflammation and play a central role in the 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. In this study, we tested the hypothesis that a specific HMO attenuates atherosclerosis low-grade macrophage inflammation and atherosclerosis development in mice.

Transient Receptor Potential Melastatin 2 Enhances Vascular Reactivity During Development of Atherosclerosis in Mice.

To investigate the function of transient receptor potential melastatin 2 (TRPM2) in vascular reactivity induced by 5-hydroxytryptamine (5-HT) in the aorta during development of atherosclerosis in mice. Forty mice were randomly divided into 4 groups: C57BL/6J on normal diet (C57 + ND), C57BL/6J on high-fat diet (C57 + HFD), apolipoprotein E gene knockout mice (ApoE-/-) on ND (ApoE-/- + ND), and ApoE-/- on HFD (ApoE-/- + HFD). They were fed with a ND or HFD for 16 weeks. Aortic TRPM2 expression and isometric contractions were analyzed. In the ApoE-/- + HFD group, body weight, blood glucose, and blood lipid concentrations were increased, and aortic plaques were developed. Compared with the other 3 groups, aortic TRPM2 mRNA and protein levels were significantly increased in the ApoE-/- + HFD group (P < 0.01). Aortic reactivity to 5-HT was enhanced in ApoE-/- + HFD mice with lower EC50 values. The enhanced reactivity to 5-HT was significantly inhibited by TRPM2 inhibitors, N-p-amylcinnamoyl anthranilic acid (1 µmol/l) and 2-aminoethyl diphenylborinate (10 µmol/l). Aortic TRPM2 expression is upregulated in ApoE knockout mice fed with a HFD. Upregulation of TRPM2 enhances 5-HT vascular reactivity during development of atherosclerosis.