Tumor necrosis factor receptor-associated factor 5 protects against intimal hyperplasia by regulation of macrophage polarization via directly targeting PPARγ.

OBJECTIVES: Intimal hyperplasia is a serious clinical problem associated with the failure of therapeutic methods in multiple atherosclerosis-related coronary heart diseases, which are initiated and aggravated by the polarization of infiltrating macrophages. The present study aimed to determine the effect and underlying mechanism by which tumor necrosis factor receptor-associated factor 5 (TRAF5) regulates macrophage polarization during intimal hyperplasia. METHODS: TRAF5 expression was detected in mouse carotid arteries subjected to wire injury. Bone marrow-derived macrophages, mouse peritoneal macrophages and human myeloid leukemia mononuclear cells were also used to test the expression of TRAF5 in vitro. Bone marrow-derived macrophages upon to LPS or IL-4 stimulation were performed to examine the effect of TRAF5 on macrophage polarization. TRAF5-knockout mice were used to evaluate the effect of TRAF5 on intimal hyperplasia. RESULTS: TRAF5 expression gradually decreased during neointima formation in carotid arteries in a time-dependent manner. In addition, the results showed that TRAF5 expression was reduced in classically polarized macrophages (M1) subjected to LPS stimulation but was increased in alternatively polarized macrophages (M2) in response to IL-4 administration, and these changes were demonstrated in three different types of macrophages. An in vitro loss-of-function study with TRAF5 knockdown plasmids or TRAF5-knockout mice revealed high expression of markers associated with M1 macrophages and reduced expression of genes related to M2 macrophages. Subsequently, we incubated vascular smooth muscle cells with conditioned medium of polarized macrophages in which TRAF5 expression had been downregulated or ablated, which promoted the proliferation, migration and dedifferentiation of VSMCs. Mechanistically, TRAF5 knockdown inhibited the activation of anti-inflammatory M2 macrophages by directly inhibiting PPARγ expression. More importantly, TRAF5-deficient mice showed significantly aggressive intimal hyperplasia. CONCLUSIONS: Collectively, this evidence reveals an important role of TRAF5 in the development of intimal hyperplasia through the regulation of macrophage polarization, which provides a promising target for arterial restenosis-related disease management.

Inhibition of P21-activated Kinase 1 Promotes Vascular Smooth Muscle Cells Apoptosis Through Reduction of Phosphorylation of Bad.

BACKGROUND: P21-activated kinase 1 (Pak1) has an effect on cell apoptosis and has recently been reported to play an important role in various cardiovascular diseases, in which vascular smooth muscle cell (VSMC) apoptosis is a key process. Thus, we hypothesized that Pak1 may be a novel target to regulate VSMC behaviors. METHODS AND RESULTS: In the present study, we found that the expression of Pak1 was dramatically upregulated in vascular smooth muscle cells (VSMCs) on H2O2 administration and was dependent on stimulation time. Through a loss-of-function approach, Pak1 knockdown increased apoptosis of VSMCs, as tested by TUNEL (TdT-mediated dUTP Nick-End Labeling) immunofluorescence staining, whereas it inhibited the proliferation of VSMCs examined by EdU staining. Moreover, we also noticed that Pak1 silencing promoted the mRNA and protein levels of pro-apoptosis genes but decreased anti-apoptosis marker expression. Importantly, we showed that Pak1 knockdown reduced the phosphorylation of Bad. Moreover, increased Pak1 expression was also noticed in carotid arteries on the wire jury. CONCLUSIONS: Our study identified that Pak1 acted as a novel regulator of apoptosis of VSMCs partially through phosphorylation of Bad.

N-Acetylcysteine Alleviates Phenylephrine-Induced Cardiomyocyte Dysfunction via Engaging PI3K/AKT Signaling Pathway.

BACKGROUND: Increased reactive oxygen species (ROS) and oxidative stress response lead to cardiomyocyte hypertrophy and apoptosis, which play crucial roles in the pathogenesis of heart failure. The purpose of current research was to explore the role of antioxidant N-acetylcysteine (NAC) on cardiomyocyte dysfunction and the underlying molecular mechanisms. METHODS AND RESULTS: Compared with control group without NAC treatment, NAC dramatically inhibited the cell size of primary cultured neonatal rat cardiomyocytes (NRCMs) tested by immunofluorescence staining and reduced the expression of representative markers associated with hypertrophic, fibrosis and apoptosis subjected to phenylephrine administration examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Moreover, enhanced ROS expression was attenuated, whereas activities of makers related to oxidative stress response examined by individual assay Kits, including total antioxidation capacity (T-AOC), glutathione peroxidase (GSH-Px), and primary antioxidant enzyme Superoxide dismutase (SOD) were induced by NAC treatment in NRCMs previously treated with phenylephrine. Mechanistically, we noticed that the protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and AKT were increased by NAC stimulation. More importantly, we identified that the negative regulation of NAC in cardiomyocyte dysfunction was contributed by PI3K/AKT signaling pathway through further utilization of PI3K/AKT inhibitor (LY294002) or agonist (SC79). CONCLUSIONS: Collected, NAC could attenuate cardiomyocyte dysfunction subjected to phenylephrine, partially by regulating the ROS-induced PI3K/AKT-dependent signaling pathway.

Nek6 knockdown polarized macrophages into a pro-inflammatory phenotype via inhibiting STAT3 expression.

Recently macrophage polarization has emerged as playing an essential role in the oathogenesis of atherosclerosis, which is the most important underlying process in many types of cardiovascular diseases. Although Nek6 has been reported to be involved in various cellular processes, the effect of Nek6 on macrophage polarization remains unknown. Macrophages exposed to lipopolysaccharide (LPS) or IL-4 were used to establish an in vitro model for the study of regulation of classically (M1) or alternatively (M2) activated macrophage. Bone marrow-derived macrophages (BMDMs) transfected with short hairpin RNA-targeting Nek6 were then in functional studies. We observed that Nek6 expression was decreased in both peritoneal macrophages (PMs) and BMDMs stimulated by LPS. This effect was seen at both mRNA and protein level. The opposite results were obtained after administration of IL-4. Macrophage-specific Nek6 knockdown significantly exacerbated pro-inflammatory M1 polarized macrophage gene expression in response to LPS challenge, but the anti-inflammatory response gene expression that is related to M2 macrophages was attenuated by Nek6 silencing followed by treatment with IL-4. Mechanistic studies exhibited that Nek6 knockdown inhibited the phosphorylated STAT3 expression that mediated the effect on macrophage polarization regulated by AdshNek6. Moreover, decreased Nek6 expression was also observed in atherosclerotic plaques. Collectively, these evidences suggested that Nek6 acts as a crucial site in macrophage polarization, and that this operates in a STAT3-dependent manner.

MicroRNA-183 as a Novel Regulator Protects Against Cardiomyocytes Hypertrophy via Targeting TIAM1.

BACKGROUND: MicroRNAs serve as important regulators of the pathogenesis of cardiac hypertrophy. Among them, miR-183 is well documented as a novel tumor suppressor in previous studies, whereas it exhibits a downregulated expression in cardiac hypertrophy recently. The present study was aimed to examine the effect of miR-183 on cardiomyocytes hypertrophy. METHODS: Angiotensin II (Ang II) was used for establishment of cardiac hypertrophy model in vitro. Neonatal rat ventricular cardiomyocytes transfected with miR-183 mimic or negative control were further utilized for the phenotype analysis. Moreover, the bioinformatics analysis and luciferase reporter assays were used for exploring the potential target of miR-183 in cardiomyocytes. RESULTS: We observed a significant decreased expression of miR-183 in hypertrophic cardiomyocytes. Overexpression of miR-183 significantly attenuated the cardiomyocytes size morphologically and prohypertrophic genes expression. Moreover, we demonstrated that TIAM1 was a direct target gene of miR-183 verified by bioinformatics analysis and luciferase reporter assays, which showed a decreased mRNA and protein expression in the cardiomyocytes transfected with miR-183 upon Ang II stimulation. Additionally, the downregulated TIAM1 expression was required for the attenuated effect of miR-183 on cardiomyocytes hypertrophy. CONCLUSIONS: Taken together, these evidences indicated that miR-183 acted as a cardioprotective regulator for the development of cardiomyocytes hypertrophy via directly regulation of TIAM1.

PAK1 Silencing Attenuated Proinflammatory Macrophage Activation and Foam Cell Formation by Increasing PPARγ Expression.

Macrophage polarization in response to environmental cues has emerged as an important event in the development of atherosclerosis. Compelling evidences suggest that P21-activated kinases 1 (PAK1) is involved in a wide variety of diseases. However, the potential role and mechanism of PAK1 in regulation of macrophage polarization remains to be elucidated. Here, we observed that PAK1 showed a dramatically increased expression in M1 macrophages but decreased expression in M2 macrophages by using a well-established in vitro model to study heterogeneity of macrophage polarization. Adenovirus-mediated loss-of-function approach demonstrated that PAK1 silencing induced an M2 macrophage phenotype-associated gene profiles but repressed the phenotypic markers related to M1 macrophage polarization. Additionally, dramatically decreased foam cell formation was found in PAK1 silencing-induced M2 macrophage activation which was accompanied with alternation of marker account for cholesterol efflux or influx from macrophage foam cells. Moderate results in lipid metabolism and foam cell formation were found in M1 macrophage activation mediated by AdshPAK1. Importantly, we presented mechanistic evidence that PAK1 knockdown promoted the expression of PPARγ, and the effect of macrophage activation regulated by PAK1 silencing was largely reversed when a PPARγ antagonist was utilized. Collectively, these findings reveal that PAK1 is an independent effector of macrophage polarization at least partially attributed to regulation of PPARγ expression, which suggested PAK1-PPARγ axis as a novel therapeutic strategy in atherosclerosis management.

ALK7 Acts as a Positive Regulator of Macrophage Activation through Down-Regulation of PPARγ Expression.

AIM: Activin receptor-like kinase 7 (ALK7) acts as a key receptor for TGF-ß family members, which play important roles in regulating cardiovascular activity. However, ALK7's potential role, and underlying mechanism, in the macrophage activation involved in atherogenesis remain unexplored. METHODS: ALK7 expression in macrophages was tested by RT-PCR, western blot, and immunofluorescence co-staining. The loss-of-function strategy using AdshALK7 was performed for functional study. Oil Red O staining was used to observe the foam cell formation, while inflammatory mediators and genes related to cholesterol efflux and influx were determined by RT-PCR and western blot. A PPARγ inhibitor (G3335) was used to reveal whether PPARγ was required for ALK7 to affect macrophage activation. RESULTS: The results exhibited upregulated ALK7 expression in oxidized low-density lipoprotein (Ox-LDL) induced bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs), isolated from ApoE-deficient mice, while ALK7's strong immunoreactivity in BMDMs was observed. ALK7 knockdown significantly attenuated pro-inflammatory, but promoted anti-inflammatory, macrophage markers expression. Additionally, ALK7 silencing decreased foam cell formation, accompanied by the up-regulation of ABCA1 and ABCG1 involved in cholesterol efflux but the down-regulation of CD36 and SR-A implicated in cholesterol influx. Mechanistically, ALK7 knockdown upregulated PPARγ expression, which was required for the ameliorated effect of ALK7 silencing macrophage activation. CONCLUSIONS: Our study demonstrated that ALK7 was a positive regulator for macrophage activation, partially through down-regulation of PPARγ expression, which suggested that neutralizing ALK7 might be promising therapeutic strategy for treating atherosclerosis.

Effective Antiviral Activity of the Tyrosine Kinase Inhibitor Sunitinib Malate against Zika Virus / 감염과화학요법

Background@#(Introduction): Zika virus (ZIKV), a mosquito-borne flavivirus, causes the outbreaks of Latin America in 2015 - 2016, with the incidence of neurological complications. Sunitinib malate, an orally bioavailable malate salt of the tyrosine kinase inhibitor, is suggested as a broadspectrum antiviral agent against emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. @*Materials and Methods@#This study investigated the antiviral efficacy and antiviral mechanisms of sunitinib malate against ZIKV infection using cytopathic effect reduction, virus yield, and time-of-addition assays. @*Results@#Sunitinib malate concentration-dependently reduced ZIKV-induced cytopathic effect, the expression of viral proteins, and ZIKV yield in supernatant with 50% inhibitory concentration (IC 50 ) value of 0.015 μM, and the selectivity index of greater than 100 against ZIKV infection, respectively. Sunitinib malate had multiple antiviral actions during entry and post-entry stages of ZIKV replication. Sunitinib malate treatment at entry stage significantly reduced the levels of ZIKV RNA replication with the reduction of (+) RNA to (-) RNA ratio and the production of new intracellular infectious particles in infected cells. The treatment at post-entry stage caused a concentration-dependent increase in the levels of ZIKV (+) RNA and (-) RNA in infected cells, along with enlarging the ratio of (+) RNA to (-) RNA, but caused a pointed increase in the titer of intracellular infectious particles by 0.01 and 0.1 μM, and a substantial decrease in the titer of intracellular infectious particles by 1 μM. @*Conclusion@#The study discovered the antiviral actions of sunitinib malate against ZIKV infection, demonstrating a repurposed, host-targeted approach to identify potential antiviral drugs for treating emerging and global viral diseases.

ALK7 Promotes Vascular Smooth Muscle Cells Phenotypic Modulation by Negative Regulating PPARγ Expression.

As a receptor for transforming growth factor-ß, nodal and activin, activin receptor-like kinase 7 (ALK7) previously acts as a suppressor of tumorigenesis and metastasis, which has emerged to play a key role in cardiovascular diseases. However, the potential effect and molecular mechanism of ALK7 on vascular smooth muscle cells' (VSMCs) phenotypic modulation have not been investigated. Using cultured mouse VSMCs with platelet-derived growth factor-BB administration, we observed that ALK7 showed a significantly increased expression in VSMCs accompanied by decreased VSMCs differentiation marker genes. Loss-of-function study demonstrated that ALK7 knockdown inhibited platelet-derived growth factor-BB-induced VSMCs phenotypic modulation characterized by increased VSMCs differentiation markers, reduced proliferation, and migration of VSMCs. Such above effects were reversed by ALK7 overexpression. Notably, we noticed that ALK7 silencing dramatically enhanced PPARγ expression, which was required for the attenuated effect of ALK7 knockdown on VSMCs phenotypic modulation. Collected, we identified that ALK7 acted as a novel and positive regulator for VSMCs phenotypic modulation partially through inactivation of PPARγ, which suggested that neutralization of ALK7 might act as a promising therapeutic strategy of intimal hyperplasia.

Cis-3-O-p-hydroxycinnamoyl Ursolic Acid Induced ROS-Dependent p53-Mediated Mitochondrial Apoptosis in Oral Cancer Cells

Cis-3-O-p-hydroxycinnamoyl ursolic acid (HCUA), a triterpenoid compound, was purified from Elaeagnus oldhamii Maxim. This traditional medicinal plant has been used for treating rheumatoid arthritis and lung disorders as well as for its anti-inflammation and anticancer activities. This study aimed to investigate the anti-proliferative and apoptotic-inducing activities of HCUA in oral cancer cells. HCUA exhibited anti-proliferative activity in oral cancer cell lines (Ca9-22 and SAS cells), but not in normal oral fibroblasts. The inhibitory concentration of HCUA that resulted in 50% viability was 24.0 µM and 17.8 µM for Ca9-22 and SAS cells, respectively. Moreover, HCUA increased the number of cells in the sub-G1 arrest phase and apoptosis in a concentration-dependent manner in both oral cancer cell lines, but not in normal oral fibroblasts. Importantly, HCUA induced p53-mediated transcriptional regulation of pro-apoptotic proteins (Bax, Bak, Bim, Noxa, and PUMA), which are associated with mitochondrial apoptosis in oral cancer cells via the loss of mitochondrial membrane potential. HCUA triggered the production of intracellular reactive oxygen species (ROS) that was ascertained to be involved in HCUA-induced apoptosis by the ROS inhibitors YCG063 and N-acetyl-L-cysteine. As a result, HCUA had potential antitumor activity to oral cancer cells through eliciting ROS-dependent and p53-mediated mitochondrial apoptosis. Overall, HCUA could be applicable for the development of anticancer agents against human oral cancer.

Tollip Negatively Regulates Vascular Smooth Muscle Cell-Mediated Neointima Formation by Suppressing Akt-Dependent Signaling.

BACKGROUND: Tollip, a well-established endogenous modulator of Toll-like receptor signaling, is involved in cardiovascular diseases. The aim of this study was to investigate the role of Tollip in neointima formation and its associated mechanisms. METHODS AND RESULTS: In this study, transient increases in Tollip expression were observed in platelet-derived growth factor-BB-treated vascular smooth muscle cells and following vascular injury in mice. We then applied loss-of-function and gain-of-function approaches to elucidate the effects of Tollip on neointima formation. While exaggerated neointima formation was observed in Tollip-deficient murine neointima formation models, Tollip overexpression alleviated vascular injury-induced neointima formation by preventing vascular smooth muscle cell proliferation, dedifferentiation, and migration. Mechanistically, we demonstrated that Tollip overexpression may exert a protective role in the vasculature by suppressing Akt-dependent signaling, which was further confirmed in rescue experiments using the Akt-specific inhibitor (AKTI). CONCLUSIONS: Our findings indicate that Tollip protects against neointima formation by negatively regulating vascular smooth muscle cell proliferation, dedifferentiation, and migration in an Akt-dependent manner. Upregulation of Tollip may be a promising strategy for treating vascular remodeling-related diseases.

Mindin deficiency in macrophages protects against foam cell formation and atherosclerosis by targeting LXR-ß.

Mindin, which is a highly conserved extracellular matrix protein, has been documented to play pivotal roles in regulating angiogenesis, inflammatory processes, and immune responses. The aim of the present study was to assess whether mindin contributes to the development of atherosclerosis. A significant up-regulation of Mindin expression was observed in the serum, arteries and atheromatous plaques of ApoE-/- mice after high-fat diet treatment. Mindin-/-ApoE-/- mice and macrophage-specific mindin overexpression in ApoE-/- mice (Lyz2-mindin-TG) were generated to evaluate the effect of mindin on the development of atherosclerosis. The Mindin-/-ApoE-/- mice exhibited significantly ameliorated atherosclerotic burdens in the entire aorta and aortic root and increased atherosclerotic plaque stability. Moreover, bone marrow transplantation further demonstrated that mindin deficiency in macrophages was largely responsible for the alleviated atherogenesis. The Lyz2-mindin-TG mice exhibited the opposite phenotype. Mindin deficiency enhanced foam cell formation by increasing the expression of cholesterol effectors, including ABCA1 and ABCG1. The mechanistic study indicated that mindin ablation promoted LXR-ß expression via a direct interaction. Importantly, LXR-ß inhibition largely reversed the ameliorating effect of mindin deficiency on foam cell formation and ABCA1 and ABCG1 expression. The present study demonstrated that mindin deficiency serves as a novel mediator that protects against foam cell formation and atherosclerosis by directly interacting with LXR-ß.

Reduced atherosclerosis lesion size, inflammatory response in miR-150 knockout mice via macrophage effects.

Atherosclerosis is considered to be a chronic inflammatory disease that can lead to severe clinically important cardiovascular events. miR-150 is a small noncoding RNA that significantly enhances inflammatory responses by upregulating endothelial cell proliferation and migration, as well as intravascular environmental homeostasis. However, the exact role of miR-150 in atherosclerosis remains unknown. Here, we investigated the effect of miR-150 deficiency on atherosclerosis development. Using double-knockout (miR-150-/- and ApoE-/-) mice, we measured atherosclerotic lesion size and stability. Meanwhile, we conducted in vivo bone marrow transplantation to identify cellular-level components of the inflammatory response. Compared with mice deficient only in ApoE, the double-knockout mice had significantly smaller atherosclerotic lesions and displayed an attenuated inflammatory response. Moreover, miR-150 ablation promoted plaque stabilization via increases in smooth muscle cell and collagen content and decreased macrophage infiltration and lipid accumulation. The in vitro experiments indicated that an inflammatory response with miR-150 deficiency in atherosclerosis results directly from upregulated expression of the cytoskeletal protein, PDZ and LIM domain 1 (PDLIM1), in macrophages. More importantly, the decreases in phosphorylated p65 expression and inflammatory cytokine secretion induced by miR-150 ablation were reversed by PDLIM1 knockdown. These findings suggest that miR-150 is a promising target for the management of atherosclerosis.

The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1.

Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury.SIGNIFICANCE STATEMENT Stroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting the TRAF6-Rac1 axis may provide a novel therapeutic strategy for stroke recovery.

Interferon Regulatory Factor 4 Inhibits Neointima Formation by Engaging Krüppel-Like Factor 4 Signaling.

BACKGROUND: The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown. METHODS: IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)-specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)-TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated. RESULTS: IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation. CONCLUSIONS: IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.

Restoration of Circulating MFGE8 (Milk Fat Globule-EGF Factor 8) Attenuates Cardiac Hypertrophy Through Inhibition of Akt Pathway.

Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting Mfge8 in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific Mfge8 overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either Mfge8-knockout or Mfge8-overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3ß (glycogen synthase kinase-3ß)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by Mfge8 deficiency but suppressed by Mfge8 overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of Mfge8 deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy.

LILRB4 deficiency aggravates the development of atherosclerosis and plaque instability by increasing the macrophage inflammatory response via NF-κB signaling.

Atherosclerosis is a chronic inflammatory disease. LILRB4 is associated with the pathological processes of various inflammatory diseases. However, the potential function and underlying mechanisms of LILRB4 in atherogenesis remain to be investigated. In this study, LILRB4 expression was examined in both human and mouse atherosclerotic plaques. The effects and possible mechanisms of LILRB4 in atherogenesis and plaque instability were evaluated in LILRB4-/-ApoE-/- and ApoE-/- mice fed a high-fat diet. We found that LILRB4 was located primarily in macrophages, and its expression was up-regulated in atherosclerotic lesions from human coronary arteries and mouse aortic roots. LILRB4 deficiency significantly accelerated the development of atherosclerotic lesions and increased the instability of plaques, as evidenced by the increased infiltration of lipids, decreased amount of collagen components and smooth muscle cells. Moreover, LILRB4 deficiency in bone marrow-derived cells promoted the development of atherosclerosis. In vivo and in vitro analyses revealed that the pro-inflammatory effects of LILRB4 deficiency were mediated by the increased activation of NF-κB signaling due to decreased Shp1 phosphorylation. In conclusion, the present study indicates that LILRB4 deficiency promotes atherogenesis, at least partly, through reduced Shp1 phosphorylation, which subsequently enhances the NF-κB-mediated inflammatory response. Thus, targeting the "LILRB4-Shp1" axis may be a novel therapeutic approach for atherosclerosis.

Oncostatin M receptor ß deficiency attenuates atherogenesis by inhibiting JAK2/STAT3 signaling in macrophages.

Oncostatin M (OSM) is a secreted cytokine mainly involved in chronic inflammatory and cardiovascular diseases through binding to OSM receptor ß (OSMR-ß). Recent studies demonstrated that the presence of OSM contributed to the destabilization of atherosclerotic plaque. To investigate whether OSMR-ß deficiency affects atherosclerosis, male OSMR-ß-/-ApoE-/- mice were generated and utilized. Here we observed that OSMR-ß expression was remarkably upregulated in both human and mouse atherosclerotic lesions, which were mainly located in macrophages. We found that OSMR-ß deficiency significantly ameliorated atherosclerotic burden in aorta and aortic root relative to ApoE-deficient littermates and enhanced the stability of atherosclerotic plaques by increasing collagen and smooth muscle cell content, while decreasing macrophage infiltration and lipid accumulation. Moreover, bone marrow transplantation of OSMR-ß-/- hematopoietic cells to atherosclerosis-prone mice displayed a consistent phenotype. Additionally, we observed a relatively reduced level of JAK2 and signal transducer and activator of transcription (STAT)3 in vivo and under Ox-LDL stimulation in vitro. Our findings suggest that OSMR-ß deficiency in macrophages improved high-fat diet-induced atherogenesis and plaque vulnerability. Mech-anistically, the protective effect of OSMR-ß deficiency on atherosclerosis may be partially attributed to the inhibition of the JAK2/STAT3 activation in macrophages, whereas OSM stimulation can activate the signaling pathway.

Dickkopf-3 Ablation Attenuates the Development of Atherosclerosis in ApoE-Deficient Mice.

BACKGROUND: Dickkopf-3 (DKK3) is a negative regulator of the Wnt/ß-catenin signaling pathway, which is involved in inflammation. However, little is known about the relationship between DKK3 expression and the progression of atherosclerosis. The aim of the present study was to define the role of DKK3 and its potential mechanism in the development of atherosclerosis. METHODS AND RESULTS: Immunofluorescence analysis showed that DKK3 was strongly expressed in macrophages of atherosclerotic plaques from patients with coronary heart disease and in hyperlipidemic mice. The expression level was significantly increased in atherogenesis. DKK3-/-ApoE-/- mice exhibited a significant decrease in atherosclerotic lesions in the entire aorta, aortic sinus, and brachiocephalic arteries. Transplantation of bone marrow from DKK3-/-ApoE-/- mice into lethally irradiated ApoE-/- recipients resulted in a reduction of atherosclerotic lesions, compared with the lesions in recipients transplanted with ApoE-/- donor cells, suggesting that the effect of DKK3 deficiency was largely mediated by bone marrow-derived cells. A reduction in the necrotic core size, accompanied by increased collagen content and smooth muscle cells and decreased accumulation of macrophages and lipids, contributed to the stability of plaques in DKK3-/-ApoE-/- mice. Furthermore, multiple proinflammatory cytokines exhibited marked decreases in DKK3-/-ApoE-/- mice. Finally, we observed that DKK3 ablation increased ß-catenin expression in the nuclei of macrophages both in vivo and in vitro. CONCLUSIONS: DKK3 expression in macrophages is involved in the pathogenesis of atherosclerosis through modulation of inflammation and inactivation of the Wnt/ß-catenin pathway.

Vinexin ß Ablation Inhibits Atherosclerosis in Apolipoprotein E-Deficient Mice by Inactivating the Akt-Nuclear Factor κB Inflammatory Axis.

BACKGROUND: Vinexin ß is a novel adaptor protein that regulates cellular adhesion, cytoskeletal reorganization, signal transduction, and transcription; however, the exact role that vinexin ß plays in atherosclerosis remains unknown. METHODS AND RESULTS: Immunoblot analysis showed that vinexin ß expression is upregulated in the atherosclerotic lesions of both patients with coronary heart disease and hyperlipemic apolipoprotein E-deficient mice and is primarily localized in macrophages indicated by immunofluorescence staining. The high-fat diet-induced double-knockout mice exhibited lower aortic plaque burdens than apolipoprotein E-/- littermates and decreased macrophage content. Vinexin ß deficiency improved plaque stability by attenuating lipid accumulation and increasing smooth muscle cell content and collagen. Moreover, the bone marrow transplant experiment demonstrated that vinexin ß deficiency exerts atheroprotective effects in hematopoietic cells. Consistent with these changes, the mRNA expression of proinflammatory cytokines were downregulated in vinexin ß-/- apolipoprotein E-/- mice, whereas the anti-inflammatory M2 macrophage markers were upregulated. The immunohistochemical staining and in vitro experiments showed that deficiency of vinexin ß inhibited the accumulation of monocytes and the migration of macrophages induced by tumor necrosis factor α-stimulated human umbilical vein endothelial cells as well as macrophage proliferation. Finally, the inhibitory effects exerted by vinexin ß deficiency on foam cell formation, nuclear factor κB activation, and inflammatory cytokine expression were largely reversed by constitutive Akt activation, whereas the increased expression of the nuclear factor κB subset promoted by adenoviral vinexin ß was dramatically suppressed by inhibition of AKT. CONCLUSIONS: Vinexin ß deficiency attenuates atherogenesis primarily by suppressing vascular inflammation and inactivating Akt-nuclear factor κB signaling. Our data suggest that vinexin ß could be a therapeutic target for the treatment of atherosclerosis.