Macrophage-Derived GSDMD Plays an Essential Role in Atherosclerosis and Cross Talk Between Macrophages via the Mitochondria-STING-IRF3/NF-κB Axis.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE-/- and Gsdmd-/- ApoE-/- mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd-/- attenuates the atherosclerotic lesion area in high-fat diet-fed ApoE-/- mice. We performed single-cell RNA sequencing of ApoE-/- and Gsdmd-/- ApoE-/- mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

AIMS: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. METHODS AND RESULTS: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. CONCLUSION: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Hypoxia inducible factor 1α inhibitor PX-478 reduces atherosclerosis in mice.

BACKGROUND AND AIMS: Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis. METHODS: We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE-/- mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline. RESULTS: PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE-/- mice. Russell-Movat's Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE-/- mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE-/- mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE-/- mice treated with PX-478. CONCLUSIONS: These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

LncRNA KCNQ1OT1 depletion inhibits the malignant development of atherosclerosis by miR-145-5p.

BACKGROUND: Atherosclerosis (AS) is a lipid-driven inflammatory disease of the arterial intima. Evidence is growing that dysregulation of lncRNAs is implicated in the pathogenesis of AS. In this research, the role of lncRNA KCNQ1OT1 in AS was investigated. METHODS: ApoE-/- mice were fed on a high fat diet to establish mouse models of AS. Macrophages (THP-1) were treated with oxidized low-density lipoprotein (ox-LDL) to establish cell models of AS. Atherosclerotic lesions of AS mice were determined by performing Oil red O staining. Lipid metabolic disorders and inflammatory were detected using specific assay kits. KCNQ1OT1 and miR-145-5p expression was measured using RT-qPCR. Levels of PPARα and CPT1 were measured using western blot. RESULTS: KCNQ1OT1 expression was upregulated and miR-145-5p was downregulated in atherosclerotic plaques of AS mice and ox-LDL-treated THP-1 cells. Lipid metabolic disorders and inflammation in vivo and in vitro were attenuated by either KCNQ1OT1 knockdown or miR-145-5p overexpression. Additionally, KCNQ1OT1 acted as a molecular sponge of miR-145-5p and downregulated miR-145-5p expression. Furthermore, silencing miR-145-5p abolished the effect of KCNQ1OT1 knockdown. CONCLUSION: Silencing KCNQ1OT1 attenuates AS progression by sponging miR-145-5p.

Macrophage LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Is Required for the Effect of CD47 Blockade on Efferocytosis and Atherogenesis-Brief Report.

OBJECTIVE: Antibody blockade of the "do not eat me" signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE-/-), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE-/- mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls (P<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. CONCLUSIONS: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE-/- model of atherosclerosis.

Calpain inhibitor prevents atherosclerosis in apolipoprotein E knockout mice by regulating mRNA expression of genes related to cholesterol uptake and efflux.

PURPOSE: We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol. METHODS: Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated. RESULTS: CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages. CONCLUSIONS: CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.

Deficiency of inactive rhomboid protein 2 (iRhom2) attenuates diet-induced hyperlipidaemia and early atherogenesis.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall and anti-inflammatory treatment strategies are currently pursued to lower cardiovascular disease burden. Modulation of recently discovered inactive rhomboid protein 2 (iRhom2) attenuates shedding of tumour necrosis factor-alpha (TNF-α) selectively from immune cells. The present study aims at investigating the impact of iRhom2 deficiency on the development of atherosclerosis. METHODS AND RESULTS: Low-density lipoprotein receptor (LDLR)-deficient mice with additional deficiency of iRhom2 (LDLR-/-iRhom2-/-) and control (LDLR-/-) mice were fed a Western-type diet (WD) for 8 or 20 weeks to induce early or advanced atherosclerosis. Deficiency of iRhom2 resulted in a significant decrease in the size of early atherosclerotic plaques as determined in aortic root cross-sections. LDLR-/-iRhom2-/- mice exhibited significantly lower serum levels of TNF-α and lower circulating and hepatic levels of cholesterol and triglycerides compared to LDLR-/- mice at 8 weeks of WD. Analyses of hepatic bile acid concentration and gene expression at 8 weeks of WD revealed that iRhom2 deficiency prevented WD-induced repression of hepatic bile acid synthesis in LDLR-/- mice. In contrast, at 20 weeks of WD, plaque size, plaque composition, and serum levels of TNF-α or cholesterol were not different between genotypes. CONCLUSION: Modulation of inflammation by iRhom2 deficiency attenuated diet-induced hyperlipidaemia and early atherogenesis in LDLR-/- mice. iRhom2 deficiency did not affect diet-induced plaque burden and composition in advanced atherosclerosis in LDLR-/- mice.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

Hydrogen sulfide prevents arterial medial calcification in rats with diabetic nephropathy.

BACKGROUND: Arterial medial calcification (AMC) is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes and chronic kidney disease. Here, we tested whether hydrogen sulfide (H2S) can prevent AMC in rats with diabetic nephropathy (DN). METHODS: DN was induced by a single injection of streptozotocin and high-fat diet (45% kcal as fat) containing 0.75% adenine in Sprague-Dawley rats for 8 weeks. RESULTS: Rats with DN displayed obvious calcification in aorta, and this was significantly alleviated by Sodium Hydrosulfide (NaHS, a H2S donor, 50 µmol/kg/day for 8 weeks) treatment through decreasing calcium and phosphorus content, ALP activity and calcium deposition in aorta. Interestingly, the main endogenous H2S generating enzyme activity and protein expression of cystathionine-γ-lyase (CSE) were largely reduced in the arterial wall of DN rats. Exogenous NaHS treatment restored CSE activity and its expression, inhibited aortic osteogenic transformation by upregulating phenotypic markers of smooth muscle cells SMα-actin and SM22α, and downregulating core binding factor α-1 (Cbfα-1, a key factor for bone formation), protein expressions in rats with DN when compared to the control group. NaHS administration also significantly reduced Stat3 activation, cathepsin S (CAS) activity and TGF-ß1 protein level, and improved aortic elastin expression. CONCLUSIONS: H2S may have a clinical significance for treating AMC in people with DN by reducing Stat3 activation, CAS activity, TGF-ß1 level and increasing local elastin level.

Allisartan isoproxil reduces mortality of stroke-prone rats and protects against cerebrovascular, cardiac, and aortic damage.

Stroke is a common cause of death and disability. Allisartan isoproxil (ALL) is a new angiotensin II receptor blocker and a new antihypertensive drug discovered and developed in China. In the present study we investigated the therapeutic effects of ALL in stroke-prone renovascular hypertensive rats (RHR-SP) and the underlying mechanisms. The model rats were generated via two-kidney two-clip (2K2C) surgery, which led to 100% of hypertension, 100% of cerebrovascular damage as well as 100% of mortality 1 year after the surgery. Administration of ALL (30 mg · kg-1 · d-1 in diet, for 55 weeks) significantly decreased stroke-related death and prolonged lifespan in RHR-SP, but the survival ALL-treated RHR-SP remained of hypertension and cardiovascular hypertrophy compared with sham-operated normal controls. In addition to cardiac, and aortic protection, ALL treatment for 10 or 12 weeks significantly reduced cerebrovascular damage incidence and scoring, along with a steady reduction of blood pressure (BP) in RHR-SP. Meanwhile, it significantly decreased serum aldosterone and malondialdehyde levels and cerebral NAD(P)H oxidase expressions in RHR-SP. We conducted 24 h continuous BP recording in conscious freely moving RHR-SP, and found that a single intragastric administration of ALL produced a long hypotensive effect lasting for at least 12 h on systolic BP. Taken together, our results in RHR-SP demonstrate that ALL can be used for stroke prevention via BP reduction and organ protection, with the molecular mechanisms related to inhibition of angiotensin-aldosterone system and oxidative stress. This study also provides a valuable scoring for evaluation of cerebrovascular damage and drug efficacy.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway.

C1q tumor necrosis factor-related protein 12 (CTRP12), a conserved paralog of adiponectin, is closely associated with cardiovascular disease. However, little is known about its role in atherogenesis. The aim of this study was to examine the influence of CTRP12 on atherosclerosis and explore the underlying mechanisms. Our results showed that lentivirus-mediated CTRP12 overexpression inhibited lipid accumulation and inflammatory response in lipid-laden macrophages. Mechanistically, CTRP12 decreased miR-155-5p levels and then increased its target gene liver X receptor α (LXRα) expression, which increased ATP binding cassette transporter A1 (ABCA1)- and ABCG1-dependent cholesterol efflux and promoted macrophage polarization to the M2 phenotype. Injection of lentiviral vector expressing CTRP12 decreased atherosclerotic lesion area, elevated plasma high-density lipoprotein cholesterol levels, promoted reverse cholesterol transport (RCT), and alleviated inflammatory response in apolipoprotein E-deficient (apoE-/-) mice fed a Western diet. Similar to the findings of in vitro experiments, CTRP12 overexpression diminished miR-155-5p levels but increased LXRα, ABCA1, and ABCG1 expression in the aortas of apoE-/- mice. Taken together, these results suggest that CTRP12 protects against atherosclerosis by enhancing RCT efficiency and mitigating vascular inflammation via the miR-155-5p/LXRα pathway. Stimulating CTRP12 production could be a novel approach for reducing atherosclerosis.

E17241 as a Novel ABCA1 (ATP-Binding Cassette Transporter A1) Upregulator Ameliorates Atherosclerosis in Mice.

[Figure: see text].

Metformin directly suppresses atherosclerosis in normoglycaemic mice via haematopoietic adenosine monophosphate-activated protein kinase.

AIMS: Atherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis. METHODS AND RESULTS: Normoglycaemic Ldlr-/- hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10-11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 µM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects. CONCLUSION: Metformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK.

Adoptive transfer of CX3CR1 transduced-T regulatory cells improves homing to the atherosclerotic plaques and dampens atherosclerosis progression.

AIM: Loss of immunosuppressive response supports inflammation during atherosclerosis. We tested whether adoptive cell therapy (ACT) with Tregulatory cells (Tregs), engineered to selectively migrate in the atherosclerotic plaque, would dampen the immune-inflammatory response in the arterial wall in animal models of familial hypercholesterolaemia (FH). METHODS AND RESULTS: FH patients presented a decreased Treg suppressive function associated to an increased inflammatory burden. A similar phenotype was observed in Ldlr -/- mice accompanied by a selective increased expression of the chemokine CX3CL1 in the aorta but not in other districts (lymph nodes, spleen, and liver). Treg overexpressing CX3CR1 were thus generated (CX3CR1+-Tregs) to drive Tregs selectively to the plaque. CX3CR1+-Tregs were injected (i.v.) in Ldlr -/- fed high-cholesterol diet (western type diet, WTD) for 8 weeks. CX3CR1+-Tregs were detected in the aorta, but not in other tissues, of Ldlr -/- mice 24 h after ACT, corroborating the efficacy of this approach. After 4 additional weeks of WTD, ACT with CX3CR1+-Tregs resulted in reduced plaque progression and lipid deposition, ameliorated plaque stability by increasing collagen and smooth muscle cells content, while decreasing the number of pro-inflammatory macrophages. Shotgun proteomics of the aorta showed a metabolic rewiring in CX3CR1+-Tregs treated Ldlr -/- mice compared to controls that was associated with the improvement of inflammation-resolving pathways and disease progression. CONCLUSION: ACT with vasculotropic Tregs appears as a promising strategy to selectively target immune activation in the atherosclerotic plaque.

Deletion of fibroblast activation protein provides atheroprotection.

AIMS: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. METHODS AND RESULTS: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. CONCLUSIONS: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.

Endothelial Rap1 (Ras-Association Proximate 1) Restricts Inflammatory Signaling to Protect From the Progression of Atherosclerosis.

OBJECTIVE: Small GTPase Rap1 (Ras-association proximate 1) is a novel, positive regulator of NO release and endothelial function with a potentially key role in mechanosensing of atheroprotective, laminar flow. Our objective was to delineate the role of Rap1 in the progression of atherosclerosis and its specific functions in the presence and absence of laminar flow, to better define its role in endothelial mechanisms contributing to plaque formation and atherogenesis. Approach and Results: In a mouse atherosclerosis model, endothelial Rap1B deletion exacerbates atherosclerotic plaque formation. In the thoracic aorta, where laminar shear stress-induced NO is otherwise atheroprotective, plaque area is increased in Athero-Rap1BiΔEC (atherogenic endothelial cell-specific, tamoxifen-inducible Rap1A+Rap1B knockout) mice. Endothelial Rap1 deficiency also leads to increased plaque size, leukocyte accumulation, and increased CAM (cell adhesion molecule) expression in atheroprone areas, whereas vascular permeability is unchanged. In endothelial cells, in the absence of protective laminar flow, Rap1 deficiency leads to an increased proinflammatory TNF-α (tumor necrosis factor alpha) signaling and increased NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and elevated inflammatory receptor expression. Interestingly, this increased signaling to NF-κB activation is corrected by AKTVIII-an inhibitor of Akt (protein kinase B) translocation to the membrane. Together, these data implicate Rap1 in restricting Akt-dependent signaling, preventing excessive cytokine receptor signaling and proinflammatory NF-κB activation. CONCLUSIONS: Via 2 distinct mechanisms, endothelial Rap1 protects from the atherosclerosis progression in the presence and absence of laminar flow; Rap1-stimulated NO release predominates in laminar flow, and restriction of proinflammatory signaling predominates in the absence of laminar flow. Our studies provide novel insights into the mechanisms underlying endothelial homeostasis and reveal the importance of Rap1 signaling in cardiovascular disease.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.