Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

Loss of CD226 protects apolipoprotein E-deficient mice from diet-induced atherosclerosis

CD226 is a costimulatory molecule that regulates immune cell functions in T cells, natural killer cells, and macrophages. Because macrophage-derived foam cell formation is a crucial factor contributing to the development of atherosclerosis, we aimed to evaluate the potential roles of CD226 in the pathogenesis of atherosclerosis. The effects of CD226 on atherosclerosis were investigated in CD226 and apolipoprotein E double-knockout (CD226^?/? ApoE^?/?) mice fed with a high-cholesterol atherogenic diet. CD226 expression in macrophages was evaluated using flow cytometry. Histopathological analysis was performed to evaluate the atherosclerotic lesions. Inflammatory cell infiltration was detected using immunofluorescence staining. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PEMs) were isolated from the mice and used to explore the mechanism in vitro. The in vivo results indicated that CD226 knockdown protected against atherosclerosis in ApoE^?/? mice, evidenced by reduced plaque accumulation in the brachiocephalic artery, aortic roots, and main aortic tree. CD226 gene-deficient macrophages showed reduced foam cell formation under ox-low density lipoprotein stimulation compared with wild-type (WT) cells. CD226 deficiency also decreased the expression of CD36 and scavenger receptor (SR)-A (responsible for lipoprotein uptake) but increased the expression of ATP-binding cassette transporter A1 and G1 (two transporters for cholesterol efflux). Therefore, loss of CD226 hinders foam cell formation and atherosclerosis progression, suggesting that CD226 is a promising new therapeutic target for atherosclerosis.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Aldehyde dehydrogenase 2 deficiency promotes atherosclerotic plaque instability through accelerating mitochondrial ROS-mediated vascular smooth muscle cell senescence

Previous evidence has indicated a beneficial role for aldehyde dehydrogenase 2 (ALDH2) in suppressing atherosclerotic plaque progression and instability. However, the underlying mechanism remains somewhat elusive. This study was designed to examine the effect of ALDH2 deficiency on high-cholesterol diet-induced atherosclerotic plaque progression and plaque vulnerability in atherosclerosis-prone ApoE knockout (ApoE^?/?) mice with a focus on foam cell formation in macrophages and senescence of vascular smooth muscle cells (VSMCs). Serum lipid profile, plaque progression, and plaque vulnerability were examined in ApoE^?/? and ALDH2/ApoE double knockout (ALDH2^?/?ApoE^?/?) mice after high-cholesterol diet intake for 8 weeks. ALDH2 deficiency increased the serum levels of triglycerides while it decreased levels of total cholesterol and high-density lipoprotein cholesterol. Unexpectedly, ALDH2 deficiency reduced the plaque area by 58.9% and 37.5% in aorta and aortic sinus, respectively. Plaque instability was aggravated by ALDH2 deficiency along with the increased necrotic core size, decreased collagen content, thinner fibrous cap area, decreased VSMC content, and increased macrophage content. In atherosclerotic lesions, ALDH2 protein was located in both macrophages and VSMCs. Further results revealed downregulated ALDH2 expression in aorta of aged ApoE^?/? mice compared with young mice. However, in vitro study suggested that ALDH2 expression was upregulated in bone marrow-derived macrophages (BMDMs) with an opposite effect in VSMCs following 80 μg/ml oxidized low-density lipoprotein (oxLDL) treatment. Interestingly, ALDH2 deficiency displayed little effect in oxLDL-induced foam cell formation from BMDMs, while ALDH2 knockdown by siRNA and ALDH2 overexpression by lentivirus infection promoted and retarded oxLDL-induced VSMC senescence, respectively. Mechanistically, ALDH2 mitigated oxLDL-induced overproduction of mitochondrial reactive oxygen species (mROS) and activation of downstream p53/p21/p16 pathway. Clearance of mROS by mitoTEMPO significantly reversed the promotive effect of ALDH2 knockdown on VSMC senescence. Taken together, our data revealed that ALDH2 deficiency suppressed atherosclerotic plaque area while facilitating plaque instability possibly through accelerating mROS-mediated VSMC senescence.This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Intact endothelial autophagy is required to maintain vascular lipid homeostasis

The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular ¹²⁵I‐LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE^?/?mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age‐dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

Undaria pinnatifida soluble fiber regulates Angptl3-LPL pathway to lessen hyperlipidemia in mice

Angiopoietin-like protein 3 (Angptl3)–lipoprotein lipase (LPL) pathway may be a useful pharmacologic target for hyperlipidemia. The present study was conducted to test the effect of soluble fiber extracted from Undaria pinnatifida (UP), on hyperlipidemia in apolipoprotein E-deficient (ApoE^?/?) mice. Forty mice were divided into four groups (n?=?10): control group (C57BL/6J mice), ApoE^?/? mice group, and two groups of ApoE^?/? mice treated with UP fiber (5 or 10 % per day). UP soluble fiber treatment significantly decreased plasma and hepatic total cholesterol, triglycerides levels, plasma low-density lipoprotein cholesterol, and malondialdehyde concentrations and increased plasma high-density lipoprotein cholesterol level and downregulated protein expression of Angptl3 concomitantly with upregulated protein expression of LPL. In addition, T0901317 caused elevated expression of hepatic Angptl3 protein, and the effect of T0901317 was also abrogated by UP soluble fiber in C57BL/6J mice. The present results suggest that the UP soluble fiber regulates Angptl3-LPL pathway to lessen hyperlipidemia in mice.     

Asiaticoside might attenuate bleomycin‐induced pulmonary fibrosis by activating cAMP and Rap1 signalling pathway assisted by A2AR

Asiaticoside (AS) has been reported to have protective effect on pulmonary fibrosis (PF). In this study, we aimed to explore the potential mechanism of the therapeutic role of AS and its relationship with A2AR in PF. Adenosine 2A receptor gene knockout (A2AR^?/?) mice and wild‐type (WT) mice were used to establish bleomycin (BLM)‐induced PF models and were then treated with AS (50 mg/kg/d). Pulmonary inflammation and fibrosis were observed in the PF model with much higher severity in A2AR^?/?mice than that in WT mice and AS significantly alleviated lung inflammation and fibrosis; however, it was less effective in A2AR^?/? mice than in WT mice via histopathological analysis. Using RNA sequencing analysis, we found up‐regulated differentially expressed genes (DEGs) in BLM group were enriched in immune and inflammation‐associated pathways compared with control group. There were 242 common DEGs between down‐regulated in BLM vs control group and up‐regulated in BLM + AS vs BLM group, which were enriched in cAMP and Rap1 signalling pathways. Furthermore, the expression of five key factors of these two pathways including adenylate cyclase (ADCY1, ADCY5, ADCY8, cAMP and Rap1) were confirmed up‐regulated by AS with the presence of A2AR. Therefore, AS might attenuate BLM‐induced PF by activating cAMP and Rap1 signalling pathways which is assisted by A2AR, making it a promising therapeutic optional for PF.     

Exosome derived from CD137‐modified endothelial cells regulates the Th17 responses in atherosclerosis

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137‐modified ECs (CD137‐Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti‐CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137‐Exo efficiently induced the progression of AS in ApoE^?/? mice. CD137‐Exo increased the proportion of Th17 cells both in vitro and vivo. The IL‐6 contained in CD137‐Exo which is regulated by Akt and NF‐КB pathway was verified to activate Th17 cell differentiation. IL‐17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM‐1), monocyte chemoattractant protein‐1 (MCP‐1) and E‐selectin in the supernatants of ECs after IL‐17 treatment was dramatically increased. CD137‐Exo promoted the progression of AS and Th17 cell differentiation via NF‐КB pathway mediated IL‐6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.     

Prdx1 (peroxiredoxin 1) deficiency reduces cholesterol efflux via impaired macrophage lipophagic flux

Oxidative stress activates macroautophagy/autophagy and contributes to atherogenesis via lipophagic flux, a form of lipid removal by autophagy. However, it is not known exactly how endogenous antioxidant enzymes are involved in lipophagic flux. Here, we demonstrate that the antioxidant PRDX1 (peroxiredoxin 1) has a crucial role in the maintenance of lipophagic flux in macrophages. PRDX1 is more highly expressed than other antioxidant enzymes in monocytes and macrophages. We determined that Prdx1 deficiency induced excessive oxidative stress and impaired maintenance of autophagic flux in macrophages. Prdx1-deficient macrophages had higher intracellular cholesterol mass and lower cholesterol efflux compared with wild type. This perturbation in cholesterol homeostasis was due to impaired lipophagic cholesterol hydrolysis caused by excessive oxidative stress, resulting in the inhibition of free cholesterol formation and the reduction of NR1H3 (nuclear receptor subfamily 1, group H, member 3) activity. Notably, impairment of both lipophagic flux and cholesterol efflux was restored by the 2-Cys PRDX-mimics ebselen and gliotoxin. Consistent with this observation, apoe ^?/? mice transplanted with bone marrow from prdx1^?/?apoe^?/? mice had increased plaque formation compared with apoe^?/? BM-transplanted recipients. This study reveals that PRDX1 is crucial to regulating lipophagic flux and maintaining macrophage cholesterol homeostasis against oxidative stress. We suggest that PRDX1-dependent control of oxidative stress may provide a strategy for treating atherosclerosis and autophagy-related human diseases.     

The essential function of CARD9 in diet‐induced inflammation and metabolic disorders in mice

Inflammation and metabolic disorder are common pathophysiological conditions, which play a vital role in the development of obesity and type 2 diabetes. The purpose of this study was to explore the effects of caspase recruitment domain (CARD) 9 in the high fat diet (HFD)‐treated mice and attempt to find a molecular therapeutic target for obesity development and treatment. Sixteen male CARD9^?/? and corresponding male WT mice were fed with normal diet or high fat diet, respectively, for 12 weeks. Glucose tolerance, insulin resistance, oxygen consumption and heat production of the mice were detected. The CARD9/MAPK pathway‐related gene and protein were determined in insulin‐responsive organs using Western blotting and quantitative PCR. The results showed that HFD‐induced insulin resistance and impairment of glucose tolerance were more severe in WT mice than that in the CARD9^?/? mice. CARD9 absence significantly modified O₂ consumption, CO₂ production and heat production. CARD9^?/? mice displayed the lower expression of p38 MAPK, JNK and ERK when compared to the WT mice in both HFD‐ and ND‐treated groups. HFD induced the increase of p38 MAPK, JNK and ERK in WT mice but not in the CARD9^?/? mice. The results indicated that CARD9 absence could be a vital protective factor in diet‐induced obesity via the CARD9/MAPK pathway, which may provide new insights into the development of gene knockout to improving diet‐induced obesity and metabolism disorder.     

Silencing of NONO inhibits abdominal aortic aneurysm in apolipoprotein E‐knockout mice via collagen deposition and inflammatory inhibition

The role of Non‐POU‐domain‐containing octamer‐binding protein (NONO) in the formation and development of angiotensin II (Ang II)‐induced abdominal aortic aneurysm (AAA) in apolipoprotein E‐knockout (ApoE^?/?) mice is still unknown. In Part I, the protein level of NONO was suggestively greater in the AAA tissues compare to that in the normal abdominal aortas. In Part II, 20 ApoE^?/? male mice were used to examine the transfection efficiency of lentivirus by detecting GFP fluorescence. In Part III, mice were arbitrarily separated into two groups: one was the control group without Ang II infusion, and another was the Ang II group. Mice treated with Ang II were further randomly divided into three groups to receive the same volume of physiological saline (NT group), sh‐negative control lentivirus (sh‐NC group) and si‐NONO lentivirus (sh‐NONO group). NONO silencing suggestively reduced the occurrence of AAA and abdominal aortic diameter. Compare to the NT group, NONO silencing markedly augmented the content of collagen and vascular smooth muscle cells but reduced macrophage infiltration in AAA. In addition, knockdown of NONO also increased the expression of prolyl‐4‐hydroxylase α1, whereas also decreased the levels of collagen degradation and pro‐inflammatory cytokines in AAA. We detected the interface of NONO and NF‐κB p65, and found that NONO silencing inhibited both the nuclear translocation and the phosphorylation levels of NF‐κB p65. Silencing of NONO prevented Ang II‐influenced AAA in ApoE^?/? mice through increasing collagen deposition and inhibiting inflammation. The mechanism may be that silencing of NONO decreases the nuclear translocation and phosphorylation of NF‐κB.     

Signalling through Src family kinase isoforms is not redundant in models of thrombo‐inflammatory vascular disease

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad‐spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non‐redundant fashion in the thrombo‐inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE^?/? model of atherosclerosis, we find that SFK signalling regulates platelet‐dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet‐mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr^?/?/ApoE^?/? or Lyn^?/?/ApoE^?/? animals. SFK signalling is not redundant in thrombo‐inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.     

Lysophosphatidylcholine promotes cholesterol efflux from mouse macrophage foam cells via PPARgamma-LXRalpha-ABCA1-dependent pathway associated with apoE

Formation of macrophage-derived foam cells is a hallmark in earlier stages of atherosclerosis (AS). Increased cholesterol efflux from macrophage foam cells promote atherosclerotic regression. In the present study, lysophosphatidylcholine (LPC) promoting cholesterol efflux from macrophage foam cells was observed, and the mechanism underlying the action was investigated. Macrophage foam cells from mice were incubated with different concentrations of LPC (10, 20, 40, 80 microM), and the free cholesterol in medium increased but total intracellular cholesterol decreased. At the same time, the expression of PPARgamma, LXRalpha, ABCA1 was enhanced in a dose-dependent manner. The treatment of macrophage foam cells with 40 microM LPC for 12, 24 and 48 h promoted cellular cholesterol efflux in a time-dependent manner, meanwhile expression of PPARgamma, LXRalpha, ABCA1 was also raised respectively. Addition of different specific inhibitors of PPARgamma (GW9662), LXRalpha (GGPP), ABCA1 (DIDS) to the foam cells significantly suppressed LPC-induced cholesterol efflux. Also treatment with specific inhibitors of PPARgamma or LXRalpha decreased ABCA1 mRNA and protein expressions. LPC (40 microM)-induced cholesterol efflux was significantly lower in macrophage foam cells from apoE deficient mice than from normal C57BL/6J mice. In contrast, 10 microg apoAI-induced cholesterol efflux from foam cells remained in apoE deficient mice. The present results indicate that LPC promotes cholesterol efflux from macrophage foam cells via a PPARgamma-LXRalpha-ABCA1-dependent pathway. Furthermore, apoE may be involved in this process.