Lysyl Oxidase 3 Is a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and Deacetylimination Modulation.

In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3-/- mice, Stat3 is constitutively acetylated and naive CD4+ T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

Irisin deficiency exacerbates diet-induced insulin resistance and cardiac dysfunction in type II diabetes in mice.

Irisin is involved in the regulation of a variety of physiological conditions, metabolism, and survival. We and others have demonstrated that irisin contributes critically to modulation of insulin resistance and the improvement of cardiac function. However, whether the deletion of irisin will regulate cardiac function and insulin sensitivity in type II diabetes remains unclear. We utilized the CRISPR/Cas-9 genome-editing system to delete irisin globally in mice and high-fat diet (HFD)-induced type II diabetes model. We found that irisin deficiency did not result in developmental abnormality during the adult stage, which illustrates normal cardiac function and insulin sensitivity assessed by glucose tolerance test in the absence of stress. The ultrastructural analysis of the transmission electronic microscope (TEM) indicated that deletion of irisin did not change the morphology of mitochondria in myocardium. Gene expression profiling showed that several key signaling pathways related to integrin signaling, extracellular matrix, and insulin-like growth factors signaling were coordinately downregulated by deletion of irisin. However, when mice were fed a high-fat diet and chow food for 16 wk, ablation of irisin in mice exposed to HFD resulted in much more severe insulin resistance, metabolic derangements, profound cardiac dysfunction, and hypertrophic response and remodeling as compared with wild-type control mice. Taken together, our results indicate that the loss of irisin exacerbates insulin resistance, metabolic disorders, and cardiac dysfunction in response to HFD and promotes myocardial remodeling and hypertrophic response. This evidence reveals the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.NEW & NOTEWORTHY By utilizing the CRISPR/Cas-9 genome-editing system and high-fat diet (HFD)-induced type II diabetes model, our results provide direct evidence showing that the loss of irisin exacerbates cardiac dysfunction and insulin resistance while promoting myocardial remodeling and a hypertrophic response in HFD-induced diabetes. This study provides new insight into understanding the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.

STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation.

T helper 17 (Th17)-cell differentiation triggered by interleukin-6 (IL-6) via STAT3 activation promotes inflammation in inflammatory bowel disease (IBD) patients. However, leukemia inhibitory factor (LIF), an IL-6 family cytokine, restricts inflammation by blocking Th17-cell differentiation via an unknown mechanism. Here, we report that microbiota dysregulation promotes LIF secretion by intestinal epithelial cells (IECs) in a mouse colitis model. LIF greatly activates STAT4 phosphorylation on multiple SPXX elements within the C-terminal transcription regulation domain. STAT4 and STAT3 act reciprocally on both canonical cis-inducible elements (SIEs) and noncanonical "AGG" elements at different loci. In lamina propria lymphocytes (LPLs), STAT4 activation by LIF blocks STAT3-dependent Il17a/Il17f promoter activation, whereas in IECs, LIF bypasses the extraordinarily low level of STAT4 to induce YAP gene expression via STAT3 activation. In addition, we found that the administration of LIF is sufficient to restore microbiome homeostasis. Thus, LIF effectively inhibits Th17 accumulation and promotes repair of damaged intestinal epithelium in inflamed colon, serves as a potential therapy for IBD.

Transcriptomics of acute myeloid leukaemia core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles.

While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.

Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.

INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvß5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvß5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvß5 contributes to the effects of irisin during the hemorrhagic response. METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  µg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvß5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin ß5 with nanoparticle delivery of integrin ß5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues. RESULTS: Hemorrhage induced reduction of integrin αvß5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin ß5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin ß5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvß5, or knockdown of integrin ß5. CONCLUSION: Integrin αvß5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis.

SET and MYND domain protein 2 (SMYD2) is a lysine methyltransferase that mediates histone H3 lysine 36 trimethylation (H3K36me3) and acts as a regulator of tumorgenesis and cystic growth. However, its role in renal fibrosis remains unknown. In this study, we found that SMYD2 was highly expressed in the murine kidney of renal fibrosis induced by unilateral ureteral obstruction, and primarily located in interstitial fibroblasts and renal tubular epithelial cells. Pharmacological inhibition of SMYD2 with AZ505, a highly selective inhibitor of SMYD2, protected against renal fibrosis and inhibited activation/proliferation of renal interstitial fibroblasts and conversion of epithelial cells to a profibrotic phenotype in this model. In cultured renal interstitial fibroblasts, treatment with AZ505 or silencing of SMYD2 by specific siRNA also inhibited serum- or TGF-ß1-induced activation and proliferation of renal interstitial fibroblasts. Mechanistic studies showed that SMYD2 inhibition reduced phosphorylation of several profibrotic signaling molecules, including Smad3, extracellular signal-regulated kinase 1/2, AKT, signal transducer and activator of transcription-3 and nuclear factor-κB in both injured kidney and cultured renal fibroblasts. AZ505 was also effective in suppressing renal expression of Snail and Twist, two transcriptional factors that mediate renal partial epithelial-mesenchymal transition and fibrosis. Conversely, AZ505 treatment prevented downregulation of Smad7, a renoprotective factor in vivo and in vitro. These results indicate that SMYD2 plays a critical role in mediating conversion of epithelial cells to a profibrotic phenotype, renal fibroblast activation and renal fibrogenesis, and suggest that SMYD2 may be a potential target for the treatment of chronic fibrosis in kidney disease.

Ablation of Sam68 in adult mice increases thermogenesis and energy expenditure.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.

Identification of histone deacetylase 8 as a novel therapeutic target for renal fibrosis.

Histone deacetylases (HDACs) have been shown to alleviate renal fibrosis, however, the role of individual HDAC isoforms in this process is poorly understood. In this study, we examined the role of HDAC8 in the development of renal fibrosis and partial epithelial-mesenchymal transitions (EMT). In a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), HDAC8 was primarily expressed in renal tubular epithelial cells and time-dependently upregulated. This occurred in parallel with the deacetylation of cortactin, a nonhistone substrate of HDAC8, and increased expression of three fibrotic markers: α-smooth muscle actin, collagen 1, and fibronectin. Administration of PCI34051, a highly selective inhibitor of HDAC8, restored acetylation of contactin and reduced expression of those proteins. PCI34051 treatment also reduced the number of renal tubular epithelial cells arrested at the G2/M phase of the cell cycle and suppressed phosphorylation of Smad3, STAT3, ß-catenin, and expression of Snail after ureteral obstruction. In contrast, HDAC8 inhibition reversed UUO-induced downregulation of BMP7 and Klotho, two renoprotective proteins. In cultured murine proximal tubular cells, treatment with PCI34051 or specific HDAC8 siRNA was also effective in inhibiting transforming growth factor ß1 (TGFß1)-induced deacetylation of contactin, EMT, phosphorylation of Smad3, STAT3, and ß-catenin, upregulation of Snail, and downregulation of BMP7 and Klotho. Collectively, these results suggest that HDAC8 activation is required for the EMT and renal fibrogenesis by activation of multiple profibrotic signaling and transcription factors, and suppression of antifibrotic proteins. Therefore, targeting HDAC8 may be novel therapeutic approach for treatment of renal fibrosis.

The Essential Role of PRAK in Preserving Cardiac Function and Insulin Resistance in High-Fat Diet-Induced Diabetes.

Regulated/activated protein kinase (PRAK) plays a crucial role in modulating biological function. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high-fat diet (HFD). Wild-type and PRAK-/- mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance tests and insulin tolerance tests were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Western blot was employed to determine cellular signaling pathway. HFD-induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared with wild-type littermates. As compared with wild-type mice, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high-fat diet intervention. High-fat diet intervention displayed a decline in fractional shortening and ejection fraction. However, deletion of PRAK exacerbated the decline in cardiac function as compared with wild-type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and ßMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared with wild-type controls. Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.

Irisin counteracts high glucose and fatty acid-induced cytotoxicity by preserving the AMPK-insulin receptor signaling axis in C2C12 myoblasts.

Irisin, a newly identified myokine, is critical to modulating body metabolism and biological homeostasis. However, whether irisin protects the skeletal muscles against metabolic stresses remains unknown. In this study, we determine the effect of irisin on high glucose and fatty acid-induced damages using irisin-overexpressed mouse C2C12 (irisin-C2C12) myoblasts and skeletal muscle from irisin-injected mice. Compared with empty vector-transfected control C2C12 cells, irisin overexpression resulted in a marked increase in cell viability and decrease in apoptosis under high-glucose stress. Progression of the cell cycle into the G2/M phase in the proliferative condition was also observed with irisin overexpression. Furthermore, glucose uptake, glycogen accumulation, and phosphorylation of AMPKα/insulin receptor (IR) ß-subunit/Erk1/2 in response to insulin stimulation were enhanced by irisin overexpression. In irisin-C2C12 myoblasts, these responses of phosphorylation were preserved under palmitate treatment, which induced insulin resistance in the control cells. These effects of irisin were reversed by inhibiting AMPK with compound C. In addition, high glucose-induced suppression of the mitochondrial membrane potential was also prevented by irisin. Moreover, suppression of IR in irisin-C2C12 myoblasts by cotransfection of shRNA against IR also mitigated the effects of irisin while not affecting AMPKα phosphorylation. As an in vivo study, soleus muscles from irisin-injected mice showed elevated phosphorylation of AMPKα and Erk1/2 and glycogen contents. Our results indicate that irisin counteracts the stresses generated by high glucose and fatty acid levels and irisin overexpression serves as a novel approach to elicit cellular protection. Furthermore, AMPK activation is a crucial factor that regulates insulin action as a downstream target.

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition.

Disruptor of telomeric silencing-1 like (DOT1L) protein specifically catalyzes the methylation of histone H3 on Lys79 (H3K79) and is implicated in tumors. But its role in tissue fibrosis remains unclear. Here we demonstrated that injury to the kidney increased DOT1L expression and H3K79 dimethylation in renal tubular epithelial cells and myofibroblasts in a murine model of unilateral ureteral obstruction. Administration of EPZ5676, a highly selective inhibitor of DOT1L, attenuated renal fibrosis. Treatment with EPZ5676 or DOT1L small interfering RNA also inhibited TGF-ß1 and serum-induced activation of renal interstitial fibroblasts and epithelial-mesenchymal transition (EMT) in vitro. Moreover, blocking DOT1L abrogated injury-induced epithelial G2/M arrest; reduced expression of Snail, Twist, and Notch1; and inactivated several profibrotic signaling molecules in the injured kidney, including Smad3, epidermal growth factor receptor, platelet-derived growth factor receptor, signal transducer and activator of transcription 3, protein kinase B, and NF-κB. Conversely, DOT1L inhibition increased expression of phosphatase and tensin homolog, a protein associated with dephosphorylation of tyrosine kinase receptors, and prevented decline in levels of Klotho and Smad7, 2 renoprotective factors. Thus, our data indicate that targeting DOT1L attenuates renal fibrosis through inhibition of renal fibroblasts and EMT by suppressing activation of multiple profibrotic signaling pathways while retaining expression of renoprotective factors.-Liu, L., Zou, J., Guan, Y., Zhang, Y., Zhang, W., Zhou, X., Xiong, C., Tolbert, E., Zhao, T. C., Bayliss, G., Zhuang, S. Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition.

Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

In this study, we examined the effect of MC1568, a selective class IIa histone deacetylase (HDAC) inhibitor, on the development and progression of renal fibrosis in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO). All 4 class IIa HDAC isoforms, in particular HDAC4, were up-regulated in renal epithelial cells of the injured kidney. Administration of MC1568 immediately after UUO injury reduced expression of α-smooth muscle actin (α-SMA), fibronectin, and collagen 1. MC1568 treatment or small interfering RNA-mediated silencing of HDAC4 also suppressed expression of those proteins in cultured renal epithelial cells. Mechanistically, MC1568 abrogated UUO-induced phosphorylation of Smad3, NF-κB, and up-regulation of integrin ɑVß6 in the kidney and inhibited TGF-ß1-induced responses in cultured renal epithelial cells. MC1568 also increased renal expression of klotho, bone morphogenetic protein 7, and Smad7. Moreover, delayed administration of MC1568 at 3 d after ureteral obstruction reversed the expression of α-SMA, fibronectin, and collagen 1 and increased expression of matrix metalloproteinase (MMP)-2 and -9. Collectively, these results suggest that selectively targeting class IIa HDAC isoforms (in particular HDAC4) may inhibit development and progression of renal fibrosis by suppressing activation and expression of multiple profibrotic molecules and increasing expression of antifibrotic proteins and MMPs.-Xiong, C., Guan, Y., Zhou, X., Liu, L., Zhuang, M. A., Zhang, W., Zhang, Y., Masucci, M. V., Bayliss, G., Zhao, T. C., Zhuang, S. Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

E2F1 Suppresses Oxidative Metabolism and Endothelial Differentiation of Bone Marrow Progenitor Cells.

RATIONALE: The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury. OBJECTIVE: We sought to define the role of E2F1 in the regulation of BM PC function. METHODS AND RESULTS: Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction. CONCLUSION: Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy.

Sam68 impedes the recovery of arterial injury by augmenting inflammatory response.

OBJECTIVE: The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α-induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury. APPROACH AND RESULTS: The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68-/-) and WT mice. Sam68-/- mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68-/- mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1ß, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68-/- vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction. CONCLUSIONS: Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

p38-Regulated/activated protein kinase plays a pivotal role in protecting heart against ischemia-reperfusion injury and preserving cardiac performance.

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK-/- and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK-/- mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK-/- mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK-/- mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

Irisin promotes cardiac progenitor cell-induced myocardial repair and functional improvement in infarcted heart.

Irisin, a newly identified hormone and cardiokine, is critical for modulating body metabolism. New evidence indicates that irisin protects the heart against myocardial ischemic injury. However, whether irisin enhances cardiac progenitor cell (CPC)-induced cardiac repair remains unknown. This study examines the effect of irisin on CPC-induced cardiac repair when these cells are introduced into the infarcted myocardium. Nkx2.5+ CPC stable cells were isolated from mouse embryonic stem cells. Nkx2.5 + CPCs (0.5 × 10 6 ) were reintroduced into the infarcted myocardium using PEGlylated fibrin delivery. The mouse myocardial infarction model was created by permanent ligation of the left anterior descending (LAD) artery. Nkx2.5 + CPCs were pretreated with irisin at a concentration of 5 ng/ml in vitro for 24 hr before transplantation. Myocardial functions were evaluated by echocardiographic measurement. Eight weeks after engraftment, Nkx2.5 + CPCs improved ventricular function as evident by an increase in ejection fraction and fractional shortening. These findings are concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Transplantation of Nkx2.5 + CPCs promoted cardiac regeneration and neovascularization, which were increased with the pretreatment of Nkx2.5 + CPCs with irisin. Furthermore, irisin treatment promoted myocyte proliferation as indicated by proliferative markers Ki67 and phosphorylated histone 3 and decreased apoptosis. Additionally, irisin resulted in a marked reduction of histone deacetylase 4 and increased p38 acetylation in cultured CPCs. These results indicate that irisin promoted Nkx2.5 + CPC-induced cardiac regeneration and functional improvement and that irisin serves as a novel therapeutic approach for stem cells in cardiac repair.

Targeting histone methyltransferase enhancer of zeste homolog-2 inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

Enhancer of zeste homolog-2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. Its role in renal epithelial-mesenchymal transition (EMT) remains unknown. In this study, we found that EZH2 and H3K27me3 were highly expressed in mouse kidney with unilateral ureteral obstruction and cultured mouse kidney proximal tubular (TKPT) cells undergoing EMT. Inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) attenuated renal fibrosis, which was associated with preserving E-cadherin expression and inhibiting Vimentin up-regulation in the obstructed kidney. Treatment with 3-DZNeP or transfection of EZH2 siRNA also inhibited TGF-ß1-induced EMT of TKPT cells. Injury to the kidney or cultured TKPT cells resulted in up-regulation of Snail-l family transcriptional repressor (Snail)-1 and Twist family basic helix-loop-helix (BHLH) transcription factor (Twist)-1, which are 2 transcription factors, and down-regulation of phosphatase and tensin homolog, a protein tyrosine phosphatase associated with inhibition of PI3K-protein kinase B (AKT) signaling; EZH2 inhibition or silencing reversed all those responses. 3-DZNeP was also effective in suppressing epithelial arrest at the G2/M phase and dephosphorylating AKT and ß-catenin in vivo and in vitro. These data indicate that EZH2 activation contributes to renal EMT and fibrosis through activation of multiple signaling pathways and suggest that EZH2 has potential as a therapeutic target for treatment of renal fibrosis.-Zhou, X., Xiong, C., Tolbert, E., Zhao, T. C., Bayliss, G., Zhuang, S. Targeting histone methyltransferase enhancer of zeste homolog-2 inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

Myocyte-specific overexpressing HDAC4 promotes myocardial ischemia/reperfusion injury.

BACKGROUND: Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. METHODS: In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. RESULTS: Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. CONCLUSIONS: Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury.

Irisin plays a pivotal role to protect the heart against ischemia and reperfusion injury.

Irisin, a newly identified hormone, is critical to modulating body metabolism, thermogenesis and reducing oxidative stresses. However, whether irisin protects the heart against myocardial ischemia and reperfusion (I/R) injury remains unknown. In this study, we determine the effect of irisin on myocardial I/R injury in the Langendorff perfused heart and cultured myocytes. Adult C57/BL6 mice were treated with irisin (100 mg/kg) or vehicle for 30 min to elicit preconditioning. The isolated hearts were subjected to 30 min ischemia followed by 30 min reperfusion. Left ventricular function was measured and infarction size were determined using by tetrazolium staining. Western blot was employed to determine myocardial SOD-1, active-caspase 3, annexin V, p38, and phospho-p38. H9c2 cardiomyoblasts were exposed to hypoxia and reoxygenation for assessment of the effects of irisin on mitochondrial respiration and mitochondrial permeability transition pore (mPTP). Irisin treatment produced remarkable improvements in ventricular functional recovery, as evident by the increase in RPP and attenuation in LVEDP. As compared to the vehicle treatment, irisin resulted in a marked reduction of myocardial infarct size. Notably, irisin treatment increased SOD-1 and p38 phosphorylation, but suppressed levels of active-caspase 3, cleaved PARP, and annexin V. In cardiomyoblasts exposed to hypoxia/reoxygenation, irisin treatment significantly attenuated hypoxia/reoxygenation (H/R), as indicated by the reduction of lactate dehydrogenase (LDH) leakage and apoptotic cardiomyocytes. Furthermore, irisin treatments suppressed the opening of mPTP, mitochondrial swelling, and protected mitochondria function. Our results indicate that irisin serves as a novel approach to eliciting cardioprotection, which is associated with the improvement of mitochondrial function.