Critical Role of the cGAS-STING Pathway in Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin is an effective chemotherapy drug for treating various types of cancer. However, lethal cardiotoxicity severely limits its clinical use. Recent evidence has indicated that aberrant activation of the cytosolic DNA-sensing cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING (stimulator of interferon genes) pathway plays a critical role in cardiovascular destruction. Here, we investigate the involvement of this mechanism in doxorubicin-induced cardiotoxicity (DIC). METHODS: Mice were treated with low-dose doxorubicin to induce chronic DIC. The role of the cGAS-STING pathway in DIC was evaluated in cGAS-deficiency (cGAS-/-), Sting-deficiency (Sting-/-), and interferon regulatory factor 3 (Irf3)-deficiency (Irf3-/-) mice. Endothelial cell (EC)-specific conditional Sting deficiency (Stingflox/flox/Cdh5-CreERT) mice were used to assess the importance of this pathway in ECs during DIC. We also examined the direct effects of the cGAS-STING pathway on nicotinamide adenine dinucleotide (NAD) homeostasis in vitro and in vivo. RESULTS: In the chronic DIC model, we observed significant activation of the cGAS-STING pathway in cardiac ECs. Global cGAS, Sting, and Irf3 deficiency all markedly ameliorated DIC. EC-specific Sting deficiency significantly prevented DIC and endothelial dysfunction. Mechanistically, doxorubicin activated the cardiac EC cGAS-STING pathway and its target, IRF3, which directly induced CD38 expression. In cardiac ECs, the cGAS-STING pathway caused a reduction in NAD levels and subsequent mitochondrial dysfunction via the intracellular NAD glycohydrolase (NADase) activity of CD38. Furthermore, the cardiac EC cGAS-STING pathway also regulates NAD homeostasis and mitochondrial bioenergetics in cardiomyocytes through the ecto-NADase activity of CD38. We also demonstrated that pharmacological inhibition of TANK-binding kinase 1 or CD38 effectively ameliorated DIC without compromising the anticancer effects of doxorubicin. CONCLUSIONS: Our findings indicate a critical role of the cardiac EC cGAS-STING pathway in DIC. The cGAS-STING pathway may represent a novel therapeutic target for preventing DIC.

Myocardial infarction drives trained immunity of monocytes, accelerating atherosclerosis.

BACKGROUND AND AIMS: Survivors of acute coronary syndromes face an elevated risk of recurrent atherosclerosis-related vascular events despite advanced medical treatments. The underlying causes remain unclear. This study aims to investigate whether myocardial infarction (MI)-induced trained immunity in monocytes could sustain proatherogenic traits and expedite atherosclerosis. METHODS: Apolipoprotein-E deficient (ApoE-/-) mice and adoptive bone marrow transfer chimeric mice underwent MI or myocardial ischaemia-reperfusion (IR). A subsequent 12-week high-fat diet (HFD) regimen was implemented to elucidate the mechanism behind monocyte trained immunity. In addition, classical monocytes were analysed by flow cytometry in the blood of enrolled patients. RESULTS: In MI and IR mice, blood monocytes and bone marrow-derived macrophages exhibited elevated spleen tyrosine kinase (SYK), lysine methyltransferase 5A (KMT5A), and CCHC-type zinc finger nucleic acid-binding protein (CNBP) expression upon exposure to a HFD or oxidized LDL (oxLDL) stimulation. MI-induced trained immunity was transmissible by transplantation of bone marrow to accelerate atherosclerosis in naive recipients. KMT5A specifically recruited monomethylation of Lys20 of histone H4 (H4K20me) to the gene body of SYK and synergistically transactivated SYK with CNBP. In vivo small interfering RNA (siRNA) inhibition of KMT5A or CNBP potentially slowed post-MI atherosclerosis. Sympathetic denervation with 6-hydroxydopamine reduced atherosclerosis and inflammation after MI. Classical monocytes from ST-elevation MI (STEMI) patients with advanced coronary lesions expressed higher SYK and KMT5A gene levels. CONCLUSIONS: The findings underscore the crucial role of monocyte trained immunity in accelerated atherosclerosis after MI, implying that SYK in blood classical monocytes may serve as a predictive factor for the progression of atherosclerosis in STEMI patients.

The role of Bach2 in regulating CD8 + T cell development and function.

Bach2 was initially discovered in B cells, where it was revealed to control the transcription involved in cell differentiation. Bach2 is intimately connected to CD8 + T lymphocytes in various differentiation states and subsets according to recent findings. Bach2 can regulate primitive T cells, stimulate the development and differentiation of memory CD8 + T cells, inhibit the differentiation of effector CD8 + T cells, and play a significant role in the exhaustion of CD8 + T cells. The appearance and development of diseases are tightly linked to irregular CD8 + T cell differentiation and function. Accordingly, Bach2 offers novel approaches and possible targets for the clinical treatment of associated disorders based on research on these pathways. Here, we summarize the role of Bach2 in the function and differentiation of CD8 + T cells and its potential clinical applications.

Legumain Is an Endogenous Modulator of Integrin αvß3 Triggering Vascular Degeneration, Dissection, and Rupture.

BACKGROUND: The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive. METHODS: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn-/-), macrophage-specific Lgmn knockout mice (LgmnF/F;LysMCre), and RR-11a-treated C57BL/6 mice were given BAPN (ß-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvß3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses. RESULTS: Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvß3 in VSMCs and blocked integrin αvß3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection. CONCLUSIONS: LGMN signaling may be a novel target for the prevention and treatment of TAD.

Impact and potential mechanism of effects of chronic moderate alcohol consumption on cardiac function in aldehyde dehydrogenase 2 gene heterozygous mice.

BACKGROUND: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme in alcohol metabolism. The ALDH2*2 mutations are found in approximately 45% of East Asians, with 40% being heterozygous (HE) ALDH2*1/*2 and 5% homozygous (HO) ALDH2*2/*2. Studies have shown that HO mice lack cardioprotective effects induced by moderate alcohol consumption. However, the impact of moderate alcohol consumption on cardiac function in HE mice is unknown. METHODS: In this study, HO, HE, and wild-type (WT) mice were subjected to a 6-week moderate alcohol drinking protocol, following which myocardial tissue and cardiomyocytes of the mice were extracted. RESULTS: We found that moderate alcohol exposure did not increase mortality, myocardial fibrosis, apoptosis, or inflammation in HE mice, which differs from the effects observed in HO mice. After exposure to the 6-week alcohol drinking protocol, there was impaired cardiac function, cardiomyocyte contractility, and intracellular Ca2+ homeostasis and mitochondrial function in both HE and HO mice as compared to WT mice. Moreover, these animals showed overt oxidative stress production and increased levels of the activated forms of calmodulin-dependent protein kinase II (CaMKII) and ryanodine receptor type 2 (RYR2) phosphorylation protein. CONCLUSION: We found that moderate alcohol exposure impaired cardiac function in HE mice, possibly by increasing reactive oxygen species (ROS)/CaMKII/RYR2-mediated Ca2+ handling abnormalities. Hence, we advocate that people with ALDH2*1/*2 genotypes rigorously avoid alcohol consumption to prevent potential cardiovascular harm induced by moderate alcohol consumption.

Interleukin-35 Promotes Macrophage Survival and Improves Wound Healing After Myocardial Infarction in Mice.

RATIONALE: Targeting inflammation has been shown to provide clinical benefit in the field of cardiovascular diseases. Although manipulating regulatory T-cell function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. IL (interleukin)-35, an immunosuppressive cytokine mainly produced by regulatory T cells, is a novel member of the IL-12 family and is composed of an EBI3 (Epstein-Barr virus-induced gene 3) subunit and a p35 subunit. However, the role of IL-35 in infarct healing remains elusive. OBJECTIVE: This study aimed to determine whether IL-35 signaling is involved in healing and cardiac remodeling after myocardial infarction (MI) and, if so, to elucidate the underlying molecular mechanisms. METHODS AND RESULTS: IL-35 subunits (EBI3 and p35), which are mainly expressed in regulatory T cells, were upregulated in mice after MI. After IL-35 inhibition, mice showed impaired infarct healing and aggravated cardiac remodeling, as demonstrated by a significant increase in mortality because of cardiac rupture, decreased wall thickness, and worse cardiac function compared with wild-type MI mice. IL-35 inhibition also led to decreased expression of α-SMA (α-smooth muscle actin) and collagen I/III in the hearts of mice after MI. Pharmacological inhibition of IL-35 suppressed the accumulation of Ly6Clow and major histocompatibility complex IIlow/C-C motif chemokine receptor type 2- (MHC IIlow CCR2-) macrophages in infarcted hearts. IL-35 activated transcription of CX3CR1 (C-X3-C motif chemokine receptor 1) and TGF (transforming growth factor) ß1 in macrophages by inducing GP130 signaling, via IL12Rß2 and phosphorylation of STAT1 (signal transducer and activator of transcription family) and STAT4 and subsequently promoted Ly6Clow macrophage survival and extracellular matrix deposition. Moreover, compared with control MI mice, IL-35-treated MI mice showed increased expression of α-SMA and collagen within scars, correlating with decreased left ventricular rupture rates. CONCLUSIONS: IL-35 reduces cardiac rupture, improves wound healing, and attenuates cardiac remodeling after MI by promoting reparative CX3CR1+Ly6Clow macrophage survival.

Systolic overload-induced pulmonary inflammation, fibrosis, oxidative stress and heart failure progression through interleukin-1ß.

Chronic heart failure is associated with increased interleukin-1ß (IL-1ß), leukocyte infiltration, and fibrosis in the heart and lungs. Here we further studied the role of IL-1ß in the transition from left heart failure to pulmonary hypertension and right ventricular hypertrophy in mice with existing left heart failure produced by transverse aortic constriction. We demonstrated that transverse aortic constriction-induced heart failure was associated with increased lung inflammation and cleaved IL-1ß, and inhibition of IL-1ß signaling using blocking antibodies of clone B122 effectively attenuated further decrease of left ventricular systolic function in mice with existing heart failure. We found that inhibition of IL-1ß attenuated lung inflammation, inflammasome activation, fibrosis, oxidative stress, and right ventricular hypertrophy. IL-1ß blocking antibodies of clone B122 also significantly attenuated lung T cell activation. Together, these data indicate that IL-1ß signaling exerts a causal role for heart failure progression, or the transition from left heart failure to lung remodeling and right heart hypertrophy.

Novel role of mitochondrial GTPases 1 in pathological cardiac hypertrophy.

While most mitochondrial proteins are encoded in the nucleus and translated on cytosolic/endoplasmic reticulum ribosomes, proteins encoded by mitochondrial DNA are translated on mitochondrial ribosomes. Mitochondrial GTPases 1 (MTG1) regulates mitochondrial ribosome assembly and translation, but its impact on cardiac adaptation to stress is unknown. Here, we found that MTG1 is dramatically elevated in hearts of dilated cardiomyopathy patients and in mice exposed to left ventricular pressure overload (AB). To examine the role of MTG1 in cardiac hypertrophy and heart failure, MTG1 loss/gain of function studies were performed in cultured cardiomyocytes and mice exposed to hypertrophic stress. MTG1 shRNA and adenoviral overexpression studies indicated that MTG1 expression attenuates angiotensin II-induced hypertrophy in cultured cardiomyocytes, while MTG1 KO mice exhibited no observable cardiac phenotype under basal conditions. MTG1 deficiency significantly exacerbated AB-induced cardiac hypertrophy, expression of hypertrophic stress markers, fibrosis, and LV dysfunction in comparison to WT mice. Conversely, transgenic cardiac MTG1 expression attenuated AB-induced hypertrophy and LV dysfunction. Mechanistically, MTG1 preserved mitochondrial respiratory chain complex activity during pressure overload, which further attenuated ROS generation. Moreover, we demonstrated that TAK1, P38 and JNK1/2 activity is downregulated in the MTG1 overexpression group. Importantly, dampening oxidative stress with N-acetylcysteine (NAC) lowered hypertrophy in MTG1 KO to WT levels. Collectively, our data indicate that MTG1 protects against pressure overload-induced cardiac hypertrophy and dysfunction by preserving mitochondrial function and reducing oxidative stress and downstream TAK1 stress signaling.

Acetaldehyde dehydrogenase 2 deficiency exacerbates cardiac fibrosis by promoting mobilization and homing of bone marrow fibroblast progenitor cells.

Cardiac fibrosis is a common feature of various cardiovascular diseases. Previous studies showed that acetaldehyde dehydrogenase 2 (ALDH2) deficiency exacerbated pressure overload-induced heart failure. However, the role and mechanisms of cardiac fibrosis in this process remain largely unknown. This study aimed to investigate the effect of ALDH2 deficiency on cardiac fibrosis in transverse aortic constriction (TAC) induced pressure overload model in mice. Echocardiography and histological analysis revealed cardiac dysfunction and enhanced cardiac fibrosis in TAC-operated animals; ALDH2 deficiency further aggravated these changes. ALDH2 chimeric mice were generated by bone marrow (BM) transplantation of WT mice into the lethally irradiated ALDH2KO mice. The proportion of circulating fibroblast progenitor cells (FPCs) and ROS level in BM after TAC were significantly higher in ALDH2KO mice than in ALDH2 chimeric mice. Furthermore, FPCs were isolated and cultured for in vitro mechanistic studies. The results showed that the stem cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4) axis played a major role in the recruitment of FPCs. In conclusion, our research reveals that increased bone marrow FPCs mobilization and myocardial homing contribute to the enhanced cardiac fibrosis and dysfunction induced by TAC in ALDH2 KO mice via exacerbating accumulation of ROS in BM and myocardial SDF-1 expression.

Megakaryocytic Leukemia 1 Bridges Epigenetic Activation of NADPH Oxidase in Macrophages to Cardiac Ischemia-Reperfusion Injury.

BACKGROUND: Excessive accumulation of reactive oxygen species (ROS), catalyzed by the NADPH oxidases (NOX), is involved in the pathogenesis of ischemia-reperfusion (IR) injury. The underlying epigenetic mechanism remains elusive. METHODS: We evaluated the potential role of megakaryocytic leukemia 1 (MKL1), as a bridge linking epigenetic activation of NOX to ROS production and cardiac ischemia-reperfusion injury. RESULTS: Following IR injury, MKL1-deficient (knockout) mice exhibited smaller myocardial infarction along with improved heart function compared with wild-type littermates. Similarly, pharmaceutical inhibition of MKL1 with CCG-1423 also attenuated myocardial infarction and improved heart function in mice. Amelioration of IR injury as a result of MKL1 deletion or inhibition was accompanied by reduced ROS in vivo and in vitro. In response to IR, MKL1 levels were specifically elevated in macrophages, but not in cardiomyocytes, in the heart. Of note, macrophage-specific deletion (MϕcKO), instead of cardiomyocyte-restricted ablation (CMcKO), of MKL1 in mice led to similar improvements of infarct size, heart function, and myocardial ROS generation. Reporter assay and chromatin immunoprecipitation assay revealed that MKL1 directly bound to the promoters of NOX genes to activate NOX transcription. Mechanistically, MKL1 recruited the histone acetyltransferase MOF (male absent on the first) to modify the chromatin structure surrounding the NOX promoters. Knockdown of MOF in macrophages blocked hypoxia/reoxygenation-induced NOX transactivation and ROS accumulation. Of importance, pharmaceutical inhibition of MOF with MG149 significantly downregulated NOX1/NOX4 expression, dampened ROS production, and normalized myocardial function in mice exposed to IR injury. Finally, administration of a specific NOX1/4 inhibitor GKT137831 dampened ROS generation and rescued heart function after IR in mice. CONCLUSIONS: Our data delineate an MKL1-MOF-NOX axis in macrophages that contributes to IR injury, and as such we have provided novel therapeutic targets in the treatment of ischemic heart disease.

Brg1 trans-activates endothelium-derived colony stimulating factor to promote calcium chloride induced abdominal aortic aneurysm in mice.

Endothelial cell derived secretive factors play pivotal roles maintaining the homeostasis by influencing the behaviors of other cells. When dysregulated, these factors may contribute to the disruption of physiological integrity and promote disease genesis in a number of different tissues and organs. In the present study we investigated how targeted deletion of brahma related gene 1 (Brg1), a chromatin remodeling protein, in endothelium might affect the pathogenesis of abdominal aortic aneurysm (AAA) induced by calcium chloride (CaCl2). We report here that compared to the wild type (WT) littermates, endothelial conditional Brg1 knockout (ecKO) mice exhibited an attenuated phenotype of AAA. Immunostaining and quantitative PCR analyses showed that vascular inflammation was suppressed in ecKO mice as opposed to WT mice likely due to diminished recruitment of macrophages. Further examination revealed that Brg1 deficiency led to a reduction in colony stimulating factor 1 (CSF1) levels. In cultured endothelial cells, Brg1 cooperated with histone H3K9 demethylase KDM3A to activate CSF1 transcription and macrophage recruitment thereby perpetuating vascular inflammation. Depletion of BRG1 or KDM3A in endothelial cells dampened CSF1 production and attenuated macrophage chemotaxis. Therefore, our data suggest that epigenetic activation of CSF1 transcription by Brg1 may contribute to AAA pathogenesis.

Cardiomyocyte dimethylarginine dimethylaminohydrolase1 attenuates left-ventricular remodeling after acute myocardial infarction: involvement in oxidative stress and apoptosis.

Asymmetric dimethylarginine (ADMA) is a risk factor for heart diseases. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes are key proteins for ADMA degradation. Endothelial DDAH1 is a vital regulator of angiogenesis. DDAH1 is also expressed in cardiomyocytes. However, the role of DDAH1 in cardiomyocytes needs further clarification. Herein, we used an inducible cardiac-specific DDAH1 knockdown mouse (cardiac DDAH1-/-) to investigate the role of cardiomyocyte DDAH1 in left-ventricular (LV) remodeling after acute myocardial infarction (AMI). DDAH1flox/flox and α-MHCMerCreMer mice were used to generate cardiac DDAH1-/- mice. Deletion of DDAH1 in cardiomyocytes was confirmed by Western blotting. No significant differences were observed in plasma ADMA levels and LV function between cardiac DDAH1-/- mice and control mice. Cardiac DDAH1-/- mice showed aggravated LV remodeling 4 weeks after AMI, as demonstrated by a large infarct area and impaired LV function. The rate of cardiomyocyte apoptosis and level of oxidative stress were higher in the LV tissue of cardiac DDAH1-/- mice than in that of control mice. However, treatment of cardiomyocytes with exogenous ADMA had no effect on reactive oxygen species (ROS) levels or apoptosis sensitivity. Cardiac DDAH1-/- LV tissue showed downregulated superoxide dismutase2 (SOD2) expression, and treatment of DDAH1-/- cardiomyocytes with the SOD mimic tempol significantly attenuated apoptosis and ROS levels under hypoxic conditions. Tempol administration also attenuated oxidative stress and apoptosis in cardiac DDAH1-/- LV tissue and partially alleviated LV remodeling after AMI. DDAH1 in cardiomyocytes plays a vital role in attenuating LV remodeling after AMI by regulating intracellular ROS levels and apoptosis sensitivity via a SOD2-dependent pathway.

SUV39H1 mediated SIRT1 trans-repression contributes to cardiac ischemia-reperfusion injury.

Ischemic reperfusion (I/R) contributes to deleterious cardiac remodeling and heart failure. The deacetylase SIRT1 has been shown to protect the heart from I/R injury. We examined the mechanism whereby I/R injury represses SIRT1 transcription in the myocardium. There was accumulation of trimethylated histone H3K9 on the proximal SIRT1 promoter in the myocardium in mice following I/R injury and in cultured cardiomyocytes exposed to hypoxia-reoxygenation (H/R). In accordance, the H3K9 trimethyltransferase SUV39H1 bound to the SIRT1 promoter and repressed SIRT1 transcription. SUV39H1 expression was up-regulated in the myocardium in mice following I/R insults and in H/R-treated cardiomyocytes paralleling SIRT1 down-regulation. Silencing SUV39H1 expression or suppression of SUV39H1 activity erased H3K9Me3 from the SIRT1 promoter and normalized SIRT1 levels in cardiomyocytes. Meanwhile, SUV39H1 deficiency or inhibition attenuated I/R-induced infarction and improved heart function in mice likely through influencing ROS levels in a SIRT1-dependent manner. Therefore, our data uncover a novel mechanism for SIRT1 trans-repression during cardiac I/R injury and present SUV39H1 as a druggable target for the development of therapeutic strategies against ischemic heart disease.

Role of bone marrow-derived CD11c+ dendritic cells in systolic overload-induced left ventricular inflammation, fibrosis and hypertrophy.

Inflammatory responses play an important role in the development of left ventricular (LV) hypertrophy and dysfunction. Recent studies demonstrated that increased T-cell infiltration and T-cell activation contribute to LV hypertrophy and dysfunction. Dendritic cells (DCs) are professional antigen-presenting cells that orchestrate immune responses, especially by modulating T-cell function. In this study, we investigated the role of bone marrow-derived CD11c+ DCs in transverse aortic constriction (TAC)-induced LV fibrosis and hypertrophy in mice. We observed that TAC increased the number of CD11c+ cells and the percentage of CD11c+ MHCII+ (major histocompatibility complex class II molecule positive) DCs in the LV, spleen and peripheral blood in mice. Using bone marrow chimeras and an inducible CD11c+ DC ablation model, we found that depletion of bone marrow-derived CD11c+ DCs significantly attenuated LV fibrosis and hypertrophy in mice exposed to 24 weeks of moderate TAC. CD11c+ DC ablation significantly reduced TAC-induced myocardial inflammation as indicated by reduced myocardial CD45+ cells, CD11b+ cells, CD8+ T cells and activated effector CD8+CD44+ T cells in LV tissues. Moreover, pulsing of autologous DCs with LV homogenates from TAC mice promoted T-cell proliferation. These data indicate that bone marrow-derived CD11c+ DCs play a maladaptive role in hemodynamic overload-induced cardiac inflammation, hypertrophy and fibrosis through the presentation of cardiac self-antigens to T cells.

MRTF-A mediates LPS-induced pro-inflammatory transcription by interacting with the COMPASS complex.

Chronic inflammation underscores the pathogenesis of a range of human diseases. Lipopolysaccharide (LPS) elicits strong pro-inflammatory responses in macrophages through the transcription factor NF-κB. The epigenetic mechanism underlying LPS-induced pro-inflammatory transcription is not fully understood. Herein, we describe a role for myocardin-related transcription factor A (MRTF-A, also known as MKL1) in this process. MRTF-A overexpression enhanced NF-κB-dependent pro-inflammatory transcription, whereas MRTF-A silencing inhibited this process. MRTF-A deficiency also reduced the synthesis of pro-inflammatory mediators in a mouse model of colitis. LPS promoted the recruitment of MRTF-A to the promoters of pro-inflammatory genes in an NF-κB-dependent manner. Reciprocally, MRTF-A influenced the nuclear enrichment and target binding of NF-κB. Mechanistically, MRTF-A was necessary for the accumulation of active histone modifications on NF-κB target promoters by communicating with the histone H3K4 methyltransferase complex (COMPASS). Silencing of individual members of COMPASS, including ASH2, WDR5 and SET1 (also known as SETD1A), downregulated the production of pro-inflammatory mediators and impaired the NF-κB kinetics. In summary, our work has uncovered a previously unknown function for MRTF-A and provided insights into the rationalized development of anti-inflammatory therapeutic strategies.

Histone Methyltransferase SET1 Mediates Angiotensin II-Induced Endothelin-1 Transcription and Cardiac Hypertrophy in Mice.

OBJECTIVE: Endothelin-1 is a potent vasoconstrictor derived from vascular endothelium. Elevated endothelin-1 levels are observed in a host of cardiovascular pathologies including cardiomyopathy. The epigenetic mechanism responsible for endothelin-1 induction in these pathological processes remains elusive. APPROACH AND RESULTS: We report here that induction of endothelin-1 expression in endothelial cells by angiotensin II (Ang II) was accompanied by the accumulation of histone H3K4 trimethylation, a preeminent histone modification for transcriptional activation, on the endothelin-1 promoter. In the meantime, Ang II stimulated the expression and the occupancy of Suv, Ez, and Trithorax domain 1 (SET1), a mammalian histone H3K4 trimethyltransferase, on the endothelin-1 promoter, both in vitro and in vivo. SET1 was recruited to the endothelin-1 promoter by activating protein 1 (c-Jun/c-Fos) and synergized with activating protein 1 to activate endothelin-1 transcription in response to Ang II treatment. Knockdown of SET1 in endothelial cells blocked Ang II-induced endothelin-1 synthesis and abrogated hypertrophy of cultured cardiomyocyte. Finally, endothelial-specific depletion of SET1 in mice attenuated Ang II-induced pathological hypertrophy and cardiac fibrosis. CONCLUSIONS: Our data suggest that SET1 epigenetically activates endothelin-1 transcription in endothelial cells, thereby contributing to Ang II-induced cardiac hypertrophy. As such, screening of small-molecule compound that inhibits SET1 activity will likely offer a new therapeutic solution to the treatment of cardiomyopathy.

A crosstalk between chromatin remodeling and histone H3K4 methyltransferase complexes in endothelial cells regulates angiotensin II-induced cardiac hypertrophy.

Angiotensin II (Ang II) induces cardiac hypertrophy and fibrosis in part by stimulating endothelin (ET-1) transcription. The involvement of the epigenetic machinery in this process is largely undefined. In the present study, we examined the epigenetic maneuvering underlying cardiac hypertrophy and fibrosis following ET-1 transactivation by Ang II. In response to Ang II stimulation, core components of the mammalian chromatin remodeling complex (Brahma-related gene 1, or Brg1, and Brahma or Brm) and histone H3K4 methylation complex (Ash2, absent, small, or homeotic discs 2, or Ash2 and WD domain repeat 5, or Wdr5) were recruited to the ET-1 promoter region in endothelial cells. Over-expression of Brg1/Brm or Ash2/Wdr5 enhanced while depletion of Brg1/Brm or Ash2/Wdr5 attenuated Ang II-induced ET-1 transactivation. Endothelial-specific knockdown of Brg1/Brm or Ash2/Wdr5 ameliorated cardiac hypertrophy both in vitro and in vivo. More important, Brg1/Brm interacted with Ash2/Wdr5 on the ET-1 promoter to catalyze H3K4 methylation. The crosstalk between Brg11/Brm and Ash2/Wdr5 was mediated by myocardin-related transcription factor A (MRTF-A). In conclusion, our data have unveiled an epigenetic complex that links ET-1 transactivation in endothelial cells to Ang II-induced cardiac hypertrophy and fibrosis.

Endothelial MRTF-A mediates angiotensin II induced cardiac hypertrophy.

Angiotensin II (Ang II) stimulates endothelin (ET-1) transcription, which contributes to cardiac hypertrophy and fibrosis. We have previously reported that myocardin related transcription factor A (MRTF-A) is indispensable for ET-1 transcription in vascular endothelial cells under hypoxic conditions, indicating that MRTF-A might mediate Ang II-induced pathological hypertrophy. Here we report that Ang II augmented the expression of MRTF-A in cultured endothelial cells and in the lungs of mice with cardiac hypertrophy. Over-expression of MRTF-A enhanced, whereas depletion of MRTF-A attenuated, transcriptional activation of ET-1 gene by Ang II. MRTF-A deficiency ameliorated Ang II induced cardiac hypertrophy and fibrosis in mice paralleling diminished synthesis and release of ET-1. Mechanistically, MRTF-A was recruited to the ET-1 promoter by c-Jun/c-Fos (AP-1) in response to Ang II treatment. Once bound, MRTF-A altered the chromatin structure by modulating histone acetylation and H3K4 methylation on the ET-1 promoter. More importantly, mice with endothelial-specific MRTF-A silencing by lentiviral particles phenocopied mice with systemic MRTF-A deletion in terms of Ang II-induced pathological hypertrophy. In conclusion, we data have unveiled a MRTF-A-containing complex that links ET-1 transactivation in endothelial cells to cardiac hypertrophy and fibrosis by Ang II.

Myocardin related transcription factor A programs epigenetic activation of hepatic stellate cells.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSCs) represents a key process in liver injury and, in the absence of intervention, leads to irreversible cirrhosis contributing significantly to the mortality of patients with liver disease. A missing link in the current understanding of HSC activation is the involvement of the epigenetic machinery. We investigated the role of the myocardin related transcription factor A (MRTF-A) in HSC activation. METHODS: Liver fibrosis was induced in wild type (WT) and MRTF-A deficient (KO) mice by CCl4 injection. Expression of mRNA and protein was measured by real-time PCR, Western blotting, and immunohistochemistry. Protein binding to DNA was assayed by chromatin immunoprecipitation (ChIP). Knockdown of endogenous proteins was mediated by either small interfering RNA (siRNA) or short hairpin RNA (shRNA), carried by lentiviral particles. RESULTS: KO mice exhibited resistance to CCl4-induced liver fibrosis compared to WT littermates. The expression of activated HSC signature genes was suppressed in the absence of MRTF-A. ChIP assays revealed that MRTF-A deficiency led to the erasure of key histone modifications, associated with transcriptional activation, such as H3K4 di- and tri-methylation, on the promoter regions of fibrogenic genes. Mechanistically, MRTF-A recruited a histone methyltransferase complex (COMPASS) to the promoters of fibrogenic genes to activate transcription. Silencing of individual COMPASS components dampened transactivation of fibrogenic genes in vitro and blocked liver fibrosis in mice. Oestradiol suppressed HSC activation by dampening the expression and binding activity of COMPASS. CONCLUSIONS: Our data illustrate a novel mechanism that connects MRTF-A dependent histone H3K4 methylation to HSC activation.

Proinflammatory stimuli engage Brahma related gene 1 and Brahma in endothelial injury.

RATIONALE: Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. OBJECTIVE: Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. METHODS AND RESULTS: In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor κB/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17ß-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe(-/-) mice on a Western diet. CONCLUSIONS: Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.