MicroRNA-574-5p targeting HOXC6 expression inhibits the hepatocyte lipid uptake to alleviate non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of liver disease that has reached its last stage. Over the past few years, evidence for miRNAs' centrality in NAFLD pathogenesis has accumulated. According to some studies, miR-574-5p plays a role in lipid metabolism. However, research on the relationship between miR-574-5p and NAFLD is lacking. For in vivo experiments, we induced the NAFLD mice model with a high-fat diet (HFD). AgomiR-574-5p was injected intravenously into HFD-fed mice for eight weeks, and qPCR was used to identify the expression of miR-574-5p in the serum. In in vitro experiments, The treatment of L-O2 cells with a miR-574-5p mimic resulted in a significant reduction in lipid deposition, suggesting that miR-574-5p can inhibit lipid accumulation and lipid formation induced by OA. The dual-luciferase reporter gene assay revealed that miR-574-5p targets the 3' UTR region of HOXC6 directly. We discovered that OA-induced lipid accumulation in hepatocytes might be mediated through the miR-574-5p-HOXC6 signaling axis. Additional research is required in order to determine the specific mechanism by which HOXC6 downstream pathways are involved in the miR-574-5p induced lipid uptake.

CREG ameliorates embryonic stem cell differentiation into smooth muscle cells by modulation of TGF-ß expression.

Vascular smooth muscle cell (SMCs) differentiation is critical for cardiovascular development, but the mechanisms remain largely unknown. The overall aim of this study was to investigate the functional impact and mechanism of cellular repressor of E1A-stimulated genes (CREG) in SMC differentiation. Two embryonic stem cell (ESC) models were generated (1) the overexpression of CREG (CREG-OE), by transfection with Pcreg-IRECS2-EGFP vector, and (2) the knockout of CREG, by transfection with CREG shRNA (CREG-KO). Interesting, SMC-marker levels (SM α-actin, SM22, Calponin, and SM-MHC) dramatically increased in CREG-OE ESCs into the SMC while significantly decreased in CREG-KO ESCs during differentiation. After 14 days, and calcium ion concentrations in angiotensin II-stimulated embryoid bodies were increased in CREG-OE ESCs but reduced in CREG-KO ESCs. Consistently, the contractile capacity of SMC from CREG-OE ESC was increased, while the contractile capacity of SMC CREG1 from CREG-KO ESCs was significantly reduced. Furthermore, we demonstrated that CREG promotes differentiation of ESCs to SMCs and maturation of their function through the transforming growth factor-ß -smad2/3 pathway.

MiR-221-3p targets Hif-1α to inhibit angiogenesis in heart failure.

Angiogenesis is involved in ischemic heart disease as well as the prognosis of heart failure (HF), and endothelial cells are the main participants in angiogenesis. In this study, we found that miR-221-3p is highly expressed in vascular tissue, especially in endothelial cells, and increased miR-221-3p was observed in heart tissue of HF patients and transverse aortic constriction (TAC)-induced HF mice. To explore the role of miR-221-3p in endothelial cells, microRNA (miRNA) mimics and inhibitors were employed in vitro. Overexpression of miR-221-3p inhibited endothelial cell proliferation, migration, and cord formation in vitro, while inhibition of miR-221-3p showed the opposite effect. Anti-argonaute 2 (Ago2) coimmunoprecipitation, dual-luciferase reporter assay, and western blotting were performed to verify the target of miR-221-3p. Hypoxia-inducible factor-1α (HIF-1α) was identified as a miR-221-3p target, and the adverse effects of miR-221-3p on endothelial cells were alleviated by HIF-1α re-expression. In vivo, a mouse model of hindlimb ischemia (HLI) was developed to demonstrate the effect of miR-221-3p on angiogenesis. AntagomiR-221-3p increased HIF-1α expression and promoted angiogenesis in mouse ischemic hindlimbs. Using the TAC model, we clarified that antagomiR-221-3p improved cardiac function in HF mice by promoting cardiac angiogenesis. Furthermore, serum miR-221-3p was detected to be negatively correlated with heart function in chronic heart failure (CHF) patients. Our results conclude that miR-221-3p inhibits angiogenesis of endothelial cells by targeting HIF-1α and that inhibition of miR-221-3p improves cardiac function of TAC-induced HF mice. Furthermore, miR-221-3p might be a potential prognostic marker of HF.

Overexpression of Kininogen-1 aggravates oxidative stress and mitochondrial dysfunction in DOX-induced cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a widely used cancer chemotherapeutic drug with cardiotoxicity effect limiting its clinical use. DOX induced cardiotoxicity is mediated by oxidative stress and mitochondrial damage. Kininogen-1(KNG1) is an important pro-inflammatory and pro-oxidant factor, and studies have found that it can aggravate lung and brain damage. However, it has not been known in terms of cardiotoxicity. Therefore, the purpose of this study is to understand the mechanism of KNG1 in DOX-induced heart injury. METHODS: C57 mice were selected for intraperitoneal injection of DOX. The model was successfully established, and fresh ventricular tissues were isolated from the ctrl group and the DOX group for mass spectrometry analysis to screen for differentially expressed proteins. Nuclear Factor-Like 2 (Nrf2), Heme Oxygenase 1 (HO-1), 4-Hydroxynonenal (4-HNE) were used to evaluate oxidative stress level, Cytochrome C Oxidase Subunit 4 (COX4) was used to evaluate mitochondria function. Mitochondrial inner membrane potential (ΔΨm) was monitored with JC-1 fluorescence. RESULTS: KNG1 was identified as a core gene which was highly expressed in the DOX myocardial injury model. Following this, an overexpression adenovirus was constructed, and KNG1 was overexpressed in vivo (mice) and in vitro (neonatal mouse cardiomyocytes (NMCMs)). It was found that overexpression of KNG1 can aggravate heart oxidative stress and mitochondrial damage. Besides, a knockdown KNG1 model was constructed, and the low expression of KNG1 was performed in cytology. It was found that knockdown of KNG1 can improve cardiomyocyte oxidative stress and mitochondrial damage caused by DOX. Nrf2 is an important antioxidant factor. Further, following KNG1 knock down, Nrf2 was also knocked down, and found that its cardiomyocyte protective effect was weakened. CONCLUSION: The overexpression of KNG1 aggravates the oxidative stress and mitochondrial damage of the heart in vivo and in vitro, which might play a role by regulating Nrf2, providing a therapeutic target for DOX-induced cardiotoxicity.

TRPV5 attenuates abdominal aortic aneurysm in mice by regulating KLF4-dependent phenotype switch of aortic vascular smooth muscle cells.

Abdominal aortic aneurysm (AAA) is a fatal vascular disease with insidious symptoms. However, the mechanism behind its development remains unclear. The transient receptor potential vanilloid (TRPV) family has crucial protective effects against cardiovascular diseases, but the role of TRPV5 in AAA has yet to be reported. In this study, ApoE-/- mice were intraperitoneally injected with AAV-GFP or AAV-TRPV5. After 30 days, mice were further administered with angiotensin II (Ang II, 1.44 mg/kg/day) by using osmotic pumps to induce the AAA model or Saline for 28 days, (i.e., Saline + AAV-GFP, Saline + AAV-TRPV5, Ang II + AAV-GFP and Ang II + AAV-TRPV5 groups were established). Compared with the control group, the incidence of AAA and the maximal diameter of the abdominal aorta markedly decreased in Ang II + AAV-TRPV5, which was detected by vascular ultrasound at 28 day. Meanwhile, less collagen and elastin degradation were observed in the Ang II + AAV-TRPV5 group by using Masson and Elastin stains. Moreover, more α-SMA and less MMP2 was observed in the abdominal aortas collected at 28 day by immunohistochemistry. In vitro, primary mouse vascular smooth muscle cells (VSMCs) were treated with Ang II (1 µM) to induce phenotype switch. Sh-TRPV5 and AdTRPV5 were used to transfect VSMCs. PCR and Western blotting were used to access the expression of contractile marker, including α-SMA and SM-22α. The results showed that the mRNA and protein level of α-SMA and SM-22α were decreased under the stimulation of Ang II, but could be attenuated by TRPV5 overexpression. The cell scratch assay demonstrated that the migration ability of VSMCs was increased in Ang II treated group and could be ameliorated by TRPV5 overexpression. Above all, VSMCs transformed from the contractile into secretory phenotype under Ang II stimuli, but could be rescued by TRPV5 overexpression. Furthermore, TRPV5 overexpression suppressed the increased expression of KLF4 induced by Ang II treatment in VSMCs. The data demonstrated that TRPV5 could inhibit AAA formation and play a critical role in the VSMC phenotype switch by downregulating KLF4, suggesting TRPV5 as a new strategy for treating AAA.

Thrombopoietic effects of CCAAT/enhancer-binding protein ß on the early-stage differentiation of megakaryocytes.

CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that is involved in adipocytic and monocytic differentiation. However, the physiological role of C/EBPß in megakaryocytes (MKs) is not clear. In this study, we investigated the effects of C/EBPß on the early-stage differentiation of MKs, and explored the potential mechanisms of action. We established a cytosine arabinoside-induced thrombocytopenia mouse model using C57BL/6 mice. In the thrombocytopenia mice, the platelet count was found to be decreased, and the mRNA and protein expression levels of C/EBPß in MKs were also reduced. Furthermore, the maturation of Dami (MKs cell line) cells was induced by phorbol 12-myristate 13-acetate. When C/EBPß was silenced in Dami cells by transfection using C/EBPß-small interfering RNA, the expression of MKs-specific markers CD41 and CD62P, was dramatically decreased, resulting in morphological changes and differentiation retardation in low ploidy, which were evaluated using flow cytometry, real-time polymerase chain reaction, western blot, and confocal microscopy. The mitogen activated protein kinase-extracellular signal-regulated kinase signaling pathway was found to be required for the differentiation of MKs; knockdown of C/EBPß in MEK/ERK1/2 pathway attenuated MKs differentiation. Overexpression of C/EBPß in MEK/ERK1/2 pathway inhibited by U0126 did not promote MKs differentiation. To the best of our knowledge, C/EBPß plays an important role in MKs differentiation and polyploidy cell cycle control. Taken together, C/EBPß may have thrombopoietic effects in the differentiation of MKs, and may assist in the development of treatments for various disorders.

Low-dose nicotine promotes autophagy of cardiomyocytes by upregulating HO-1 expression.

Nicotine as a major component of addiction in cigarettes has been reported to play protective roles in some pathological processes. It is reported that activation of the nicotinic acetylcholine receptor also has a cardioprotective effect. Thus, in our study, we investigated the effect and mechanism of nicotine on the autophagy of cardiomyocytes, and whether nicotine protects cardiomyocytes against palmitic acid (PA) injury. The results indicated that low-dose nicotine promoted neonatal mouse cardiac myocytes (NMCMs) autophagy and accelerated autophagic flux while inhibiting NMCMs apoptosis, but high-dose nicotine inhibited autophagy and promoted apoptosis. Moreover, low-dose nicotine upregulated heme oxygenase-1 (HO-1) expression and knocking down HO-1 abolished the effects of nicotine on the autophagy and apoptosis of NMCMs. Methyllycaconitine citrate (α7-nAChR blocker, MLA) inhibited HO-1 expression and the effects of nicotine on autophagy and apoptosis of NMCMs. Furthermore, low-dose nicotine improved the inhibited autophagy and increased apoptosis induced by palmitic acid (PA) in NMCMs and these effects were reversed by knocking down HO-1. In conclusion, our data suggested that low-dose nicotine promoted autophagy and inhibited apoptosis of cardiomyocytes by upregulating HO-1.

Recombinant Cellular Repressor of E1A-Stimulated Genes Protects against Renal Fibrosis in Dahl Salt-Sensitive Rats.

BACKGROUND: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that attenuates angiotensin II-induced hypertension, alleviates myocardial fibrosis, and improves heart function. However, the role of CREG in high-salt (HS) diet-induced hypertensive nephropathy is unclear. METHODS: To determine the effects and molecular mechanisms of CREG in HS diet-induced hypertensive nephropathy, we established a hypertensive nephropathy animal model in Dahl salt-sensitive (SS) rats fed a HS diet (8% NaCl, n = 20) for 8 weeks. At week 4 of HS loading, these rats were administered recombinant CREG (reCREG; 35 µg/kg·day, n = 5) and saline (n = 5) via subcutaneously implanted pumps and were also administered the vasodilator hydralazine (20 mg/kg·day, n = 5) in drinking water. We used hematoxylin and eosin staining, Masson's trichrome staining, immunohistochemical labeling, western blotting, RT-PCR, and Tunel staining to determine the signaling pathways of CREG in HS diet-induced hypertensive nephropathy. RESULTS: After 8 weeks of HS intake, the Dahl SS rats developed renal dysfunction and severe renal fibrosis associated with reductions of 78 and 67% in CREG expression, respectively, at both mRNA and protein levels in the kidney. Administration of reCREG improved renal function and relieved renal fibrosis. Administration of CREG also inhibited monocyte infiltration and reduced apoptosis in the kidney cells. CREG overexpression upregulated forkhead box P1 expression and inhibited the transforming growth factor-ß1 signaling pathway. CONCLUSION: Our study shows that CREG protected the kidney against HS-diet-induced renal damage and provides new insights into the mechanisms underlying kidney injury.

MiR-207 inhibits autophagy and promotes apoptosis of cardiomyocytes by directly targeting LAMP2 in type 2 diabetic cardiomyopathy.

Autophagy dysfunction plays a critical role in diabetic cardiomyopathy (DCM). MiR-207 regulates the expression of lysosomal-associated membrane protein 2 (LAMP2), an autophagy-related protein, following ischemic stroke in neurocytes. However, the roles and mechanisms of miR-207 in DCM remain unknown. Therefore, in this study, we aim to investigate the roles and mechanisms of miR-207 in type 2 DCM. The results showed that autophagic dysfunction with increased LC3II and P62 expression and decreased LAMP2 expression, and increased cell apoptosis with up-regulated cleaved-caspase3 expression were noted in the myocardium of type 2 DCM mice and neonatal mouse cardiac myocytes (NMCMs) stimulated with PA. In addition, miR-207 was significantly upregulated in the myocardium of DCM mice and NMCMs stimulated with PA. In NMCMs, miR-207 inhibited LAMP2 mRNA and protein expression. MiR-207 mimics significantly inhibited autophagy by increasing P62 and LC3II expression and promoted cell apoptosis by increasing cleaved-caspase3 expression, and these effects were reversed by LAMP2 overexpression. In conclusion, miR-207 inhibited autophagy and promoted apoptosis of cardiomyocytes by directly targeting LAMP2, which participated in the development of type 2 diabetic cardiomyopathy.

Nicotine promotes the differentiation of C2C12 myoblasts and improves skeletal muscle regeneration in obese mice.

Nicotine is the main addictive substance in tobacco. It has been reported that nicotine can improve obesity and promote body weight loss in humans and rodents. In addition, obesity is associated with many chronic diseases. Many studies have demonstrated that the skeletal muscle regenerative capacity is impaired in obese mice. However, the effect of nicotine on skeletal muscle regeneration under obese conditions remains unclear. Thus, in the present study, we examined the effects of nicotine on the differentiation of C2C12 myoblasts in vitro and on skeletal muscle regeneration in obese mice in vivo. The results showed that nicotine promoted C2C12 myoblast differentiation by upregulating myogenic regulatory factors, including MyoD and Myogenin. Nicotine also activated the PI3K/Akt signaling pathway, while blocking PI3K with the inhibitor LY294002 abrogated the effects of nicotine on the differentiation of C2C12 cells. Furthermore, nicotine was injected into the cardiotoxin (CTX)-injured skeletal muscles of obese mice. The results showed that the skeletal muscles injected with nicotine regenerated more quickly than the skeletal muscles injected with saline. Taken together, our data suggested that nicotine promoted the differentiation of C2C12 cells through activation of the PI3K/Akt pathway and rescued the impaired skeletal muscle regeneration in obese mice.

SNRK (Sucrose Nonfermenting 1-Related Kinase) Promotes Angiogenesis In Vivo.

OBJECTIVE: SNRK (sucrose nonfermenting 1-related kinase) is a novel member of the AMPK (adenosine monophosphate-activated protein kinase)-related superfamily that is activated in the process of angiogenesis. Currently, little is known about the function of SNRK in angiogenesis in the physiological and pathological conditions. APPROACH AND RESULTS: In this study, in Snrk global heterozygous knockout mice, retina angiogenesis and neovessel formation after hindlimb ischemia were suppressed. Consistently, mice with endothelial cell (EC)-specific Snrk deletion exhibited impaired retina angiogenesis, and delayed perfusion recovery and exacerbated muscle apoptosis in ischemic hindlimbs, compared with those of littermate wide-type mice. Endothelial SNRK expression was increased in the extremity vessel samples from nonischemic human. In ECs cultured in hypoxic conditions, HIF1α (hypoxia inducible factor 1α) bound to the SNRK promoter to upregulate SNRK expression. In the nuclei of hypoxic ECs, SNRK complexed with SP1 (specificity protein 1), and together, they bound to an SP1-binding motif in the ITGB1 (ß1 integrin) promoter, resulting in enhanced ITGB1 expression and promoted EC migration. Furthermore, SNRK or SP1 deficiency in ECs ameliorated hypoxia-induced ITGB1 expression and, consequently, inhibited EC migration and angiogenesis. CONCLUSIONS: Taken together, our data have revealed that SNRK/SP1-ITGB1 signaling axis promotes angiogenesis in vivo.

Transplantation of CREG modified embryonic stem cells improves cardiac function after myocardial infarction in mice.

Engraftment of embryonic stem cells (ESC) has been proposed as a potential therapeutic approach for post-infarction cardiac dysfunction. However, only mild function improvement has been achieved due to low survival rate and paracrine dysfunction of transplanted stem cells. Cellular repressor of E1A stimulated genes (CREG) has been reported to be a secreted glycoprotein implicated in promoting survival and differentiation of many cell types. Therefore we hypothesized that transplantation of genetically modified ESC with CREG (CREG-ESC) can improve cardiac function after myocardial infarction in mice. A total of 2 × 105 CREG-ESC or EGFP-ESC were engrafted into the border zone in a myocardial infarction model in mice. Cardiac function, infarct size and fibrosis at 4 weeks, survival of transplanted ESC, apoptosis and cytokine level of heart tissue, and teratoma formation were assessed in vivo. Apoptosis of ESC under inflammatory stimuli and cardiac differentiation of ESC were investigated in vitro. After 4 weeks, we found transplantation of CREG-ESC could significantly improve cardiac function, ameliorate cardiac remodeling, and reduce infarct size and fibrosis area. Transplantation of CREG-ESC remarkably increased ESC survival in the border zone and inhibited apoptosis of cardiomyocytes. Furthermore, the decrease of inflammatory factors (IL-1ß, IL-6 and TNF-α) and increase of anti-inflammatory factors (TGF-ß, bFGF and VEGF165) in the border zone were higher in CREG-ESC transplanted hearts. Safety evaluation showed that all transplantation at 2 × 105 per heart dose produced no teratoma. Surprisingly, the mice with 3.0 × 106 CREG-ESC transplantation was demonstrated teratoma free without cardiac rhythm disturbances in contrast to 100% teratoma formation and rhythm abnormality for the same dose of EGFP-ESC transplantation. In addition, overexpression of CREG inhibits ESC apoptosis and enhanced their differentiation into cardiomyocytes in vitro. Transplantation of CREG-modified ESC exhibits a favorable survival pattern in infarcted hearts, which translates into a substantial preservation of cardiac function after acute myocardial infarction.

Chemokine CC-motif ligand 2 participates in platelet function and arterial thrombosis by regulating PKCα-P38MAPK-HSP27 pathway.

BACKGROUND: Studies indicate that chemokine CC-motif ligand 2 (CCL2) is involved in inflammation and atherosclerosis. However, the roles and mechanisms of CCL2 on platelet function and arterial thrombosis are unknown. METHODS: The expressions of CCL2 or CCR2 in the plasma, platelets and coronary thrombus of ST-elevated myocardial infarction (STEMI) patients were examined by ELISA, Western blot, immunohistochemistry and immunofluorescence. The roles of CCL2 on platelet aggregation, activation and secretion were examined by light transmission aggregometry, flow cytometry and ELISA. RESULTS: The expressions of CCL2 or CCR2 in the plasma or platelets of STEMI patients with platelet high response were higher than those with platelet normal response; In vitro, exogenous recombinant human CCL2 markedly increased platelet aggregation, activation and granule secretion, which were abolished by CCL2 neutralizing antibody or CCR2 inhibiter. CCL2 increased the phosphorylation levels of PKCα (Thr638), P38MAPK (Thr180/Tyr182) and HSP27 (S78/S82) in human platelets, which were abrogated by PKCα inhibitor (RO 318220) or P38MAPK inhibitor (SB 203580). RO 318220 or SB 203580 diminished CCL2-induced platelet function. In CCL2-/- mice, platelet aggregation and secretion were attenuated; the phosphorylation of PKCα, P38MAPK and HSP27 were decreased. In a carotid arterial thrombus mouse model, CCL2-/- mice displayed a significantly extended carotid artery occlusion time compared with wild type. CONCLUSIONS: CCL2 played important roles in regulating platelet function and arterial thrombosis through the PKCα-P38MAPK-HSP27 pathway, which might provide theoretical basis for searching new antiplatelet drugs and the treatment for cardiovascular diseases.

The novel intracellular protein CREG inhibits hepatic steatosis, obesity, and insulin resistance.

Cellular repressor of E1A-stimulated genes (CREG), a novel cellular glycoprotein, has been identified as a suppressor of various cardiovascular diseases because of its capacity to reduce hyperplasia, maintain vascular homeostasis, and promote endothelial restoration. However, the effects and mechanism of CREG in metabolic disorder and hepatic steatosis remain unknown. Here, we report that hepatocyte-specific CREG deletion dramatically exacerbates high-fat diet and leptin deficiency-induced (ob/ob) adverse effects such as obesity, hepatic steatosis, and metabolic disorders, whereas a beneficial effect is conferred by CREG overexpression. Additional experiments demonstrated that c-Jun N-terminal kinase 1 (JNK1) but not JNK2 is largely responsible for the protective effect of CREG on the aforementioned pathologies. Notably, JNK1 inhibition strongly prevents the adverse effects of CREG deletion on steatosis and related metabolic disorders. Mechanistically, CREG interacts directly with apoptosis signal-regulating kinase 1 (ASK1) and inhibits its phosphorylation, thereby blocking the downstream MKK4/7-JNK1 signaling pathway and leading to significantly alleviated obesity, insulin resistance, and hepatic steatosis. Importantly, dramatically reduced CREG expression and hyperactivated JNK1 signaling was observed in the livers of nonalcoholic fatty liver disease (NAFLD) patients, suggesting that CREG might be a promising therapeutic target for NAFLD and related metabolic diseases. CONCLUSION: The results of our study provides evidence that CREG is a robust suppressor of hepatic steatosis and metabolic disorders through its direct interaction with ASK1 and the resultant inactivation of ASK1-JNK1 signaling. This study offers insights into NAFLD pathogenesis and its complicated pathologies, such as obesity and insulin resistance, and paves the way for disease treatment through targeting CREG. (Hepatology 2017;66:834-854).

Cellular Repressor of E1A-Stimulated Genes Is a Critical Determinant of Vascular Remodeling in Response to Angiotensin II.

OBJECTIVE: Cellular repressor of E1A-stimulated genes (CREG) is a lysosomal glycoprotein implicated in maintaining vascular homeostasis. Here, we have hypothesized that CREG is a critical target of intervention for the prevention of hypertensive vascular remodeling. APPROACH AND RESULTS: CREG gene expression was significantly decreased accompanied by an upregulated expression of angiotensin II (Ang II) in remodeled vascular tissues of high salt-induced Dahl salt-sensitive rats and Ang II-induced mice. In particular, the downregulation of CREG gene was Ang II specific and independent from blood pressure. Prominent medial hypertrophy and vascular fibrosis in both thoracic aortas and mesenteric arteries were observed in CREG+/- mice infused with Ang II than in CREG+/+ mice, but blunted response in CREG+/+ mice received recombinant human CREG protein, suggesting that changes in CREG expression account for the different phenotype between genotypes. Within a tiled promoter array, E26 transformation-specific-1 binds to CREG promoter at high stringency with the stimulation of Ang II. Moreover, the Ang II-induced E26 transformation-specific-1 directly interacted with the CREG promoter (-1179 and -271 bp) and inhibited its transcription in vascular smooth muscle cells. Selective, pharmacological inhibition of E26 transformation-specific-1 led to restoration of CREG expression in aortas and rescue of experimental vascular remodeling by systemic administration of dominant negative E26 transformation-specific-1 membrane-permeable peptides. CONCLUSIONS: CREG is a novel mediator of vascular remodeling in response to Ang II and may be an attractive therapeutic target for prevention of vascular diseases.

CREG protects from myocardial ischemia/reperfusion injury by regulating myocardial autophagy and apoptosis.

AIMS: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that regulates tissue and cell homeostasis and has been shown to antagonize heart fibrosis, which indicates a potential protective effect of CREG against cardiomyocyte chronic damage. However, little is known about the role of CREG in myocardial tissue acute injury, in this study, we aimed to investigate the role of CREG in myocardial ischemia/reperfusion (MI/R) injury and clarify the mechanism of action. METHODS AND RESULTS: Wild-type Creg (Creg+/+), heterozygous Creg (Creg+/-) mice and mice pretreated with infusion of recombinant 0.3mg/kg·d CREG protein (reCreg+/+) were subjected to 30min of left ascending coronary ischemia and 24h of reperfusion. Evan's Blue-triphenyl- tetrazolium chloride (TTC) solution and echocardiography analysis were used to evaluate the effects of CREG on MI/R mice. The underlying mechanisms were further determined by cultured myocardial cells in vitro. Our findings revealed that the level of CREG protein in mouse hearts was significantly decreased after mice were subjected to MI/R. Moreover, Creg+/- mice had larger infarction size 2h after reperfusion and worse cardiac function 28days after MI/R injury compared to that in Creg+/+ mice. However, reCreg+/+ mice could maintain CREG at a high level even after MI/R injury, and mitigated infarction size and improved cardiac function significantly. In Creg+/- mice, myocardial autophagy was dysfunctional characterized by accumulation of LC3A and p62, while apoptotic cell number increase was detected by cleaved caspase-3 blotting and TUNEL staining. Conversely, decreased apoptosis and activated autophagy were detected in reCreg+/+ mice. Furthermore, chloroquine, a kind of autophagy blocker, was used to demonstrate recombinant CREG protected cardiomyocytes against apoptosis mediated by activating autophagy both in vivo and in vitro. Finally, we found CREG was involved into lysosomal protein transfer and improve cellular autophagy. CONCLUSION: CREG protects heart against MI/R injury-induced cardiomyocytes apoptosis by activating lysosomal autophagy. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren and Megan Yingmei Zhang.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis. Stem Cells 2016;34:2648-2660.

CREG1 ameliorates myocardial fibrosis associated with autophagy activation and Rab7 expression.

In cardiomyocytes subjected to stress, autophagy activation is a critical survival mechanism that preserves cellular energy status while degrading damaged proteins and organelles. However, little is known about the mechanisms that govern this autophagic response. Cellular repressor of E1A genes (CREG1) is an evolutionarily conserved lysosomal protein, and an important new factor in regulating tissues homeostasis that has been shown to antagonize injury of tissues or cells. In the present study, we aimed to investigate the regulatory role of CREG1 in cardiac autophagy, and to clarify autophagy activation mechanisms. First, we generated a CREG1 haploinsufficiency (Creg1(+/-)) mouse model, and identified that CREG1 deficiency aggravates myocardial fibrosis in response to aging or angiotensin II (Ang II). Conversely, exogenous infusion of recombinant CREG1 protein complete reversed cardiac damage. CERG1 deficiency in Creg1(+/-) mouse heart showed a market accumulation of autophagosome that acquired LC3II and beclin-1, and a decrease in autophagic flux clearance as indicated by upregulating the level of p62. Inversely, restoration of CREG1 activates cardiac autophagy, Furthermore, chloroquine, an inhibitor of lysosomal acidification, was used to confirm that CREG1 protected the heart tissue against Ang II-induced fibrosis by activating autophagy. Using adenoviral infection of primary cardiomyocytes, overexpression of CREG1 with concurrent resveratrol treatment significantly increased autophagy, while silencing CREG1 blocked the resveratrol-induced autophagy. These results suggest that CREG1-induced autophagy is required to maintain heart function in the face of stress-induced myocardiac damage. Both in vitro and in vivo studies identified that CREG1 deficiency influenced the maturation of lysosomes and reduced the espression of Rab7, which might be involved in CREG1-induced cardiomyocyte autophagy. These findings suggest that autophagy activation via CREG1 may be a viable therapeutic strategy autophagy for improving cardiac performance under pathologic conditions. This article is part of a Special Issue entitled: autophagy and protein quality control in cardiometabolic diseases.

Association between insulin receptor substrate-1 polymorphisms and high platelet reactivity with clopidogrel therapy in coronary artery disease patients with type 2 diabetes mellitus.

BACKGROUND: The mechanisms leading to the high on-treatment platelet reactivity in diabetes patients are not fully elucidated. The genetic factors may be associated with the diminished antiplatelet efficacy of dual antiplatelet therapy. We investigated the possible association between insulin receptor substrate-1 (IRS-1) polymorphisms and high platelet reactivity in coronary artery disease (CAD) patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 674 CAD patients with T2DM were enrolled in this study. Platelet aggregation and platelet activation were assessed with light transmission aggregometry and flow cytometry analysis, respectively. Participants were divided into high platelet reactivity (HPR) group and non-HPR group according to their maximal platelet aggregation. Genotypes were identified by polymerase chain reaction and direct sequencing of genomic DNA. The association between IRS-1 genetic variants and platelet function was assessed. RESULTS: There were 233 participants in the HPR group and 441 participants in the non-HPR group. G allele frequencies of rs13431554 were 27.7 % for the HPR group and 18.6 % for the non-HPR group (p < 0.001). Adenosine diphosphate and arachidonic acid induced platelet aggregation were significantly higher in G allele carriers compared with non-carriers (56.8 ± 16.2 vs 52.0 ± 17.9 %, p < 0.01, 28.9 ± 18.6 vs 25.2 ± 17.8 %, p < 0.01, respectively). We observed that P-selectin expression and PAC-1 binding were higher in G allele carriers compared with non-carriers (40.8 ± 12.4 vs 36.2 ± 13.8, p = 0.01; 43.7 ± 15.9 vs 38.7 ± 19.9, p = 0.03, respectively). CONCLUSION: The G allele of rs13431554 in the IRS-1 gene was associated with a hyperreactive platelet phenotype in the CAD patients with T2DM.

Cellular repressor of E1A-stimulated genes inhibits inflammation to decrease atherosclerosis in ApoE(-/-) mice.

AIMS: Macrophage inflammation response is important in the pathogenesis of atherosclerosis. We investigated the role and mechanism of cellular repressor of E1A-stimulated genes (CREG) in regulating TNF-α induced inflammation response in macrophages and explore whether CREG might be a therapeutic target for atherosclerosis. METHOD AND RESULTS: Immunostaining and western blotting showed that expression of CREG was reduced in human atherosclerotic coronary artery. In vivo experiments demonstrated that supplementation of recombinant CREG protein to ApoE(-/-) mice fed with high fat diet alleviated aortic atherosclerosis development and inflammation. In vitro, macrophage from ApoE(-/-) mice fed with high fat diet had lower level of CREG compared to control mice fed with normal diet. Immunohistochemical staining and western blotting further confirmed that CREG inhibited inflammatory response of macrophages induced by TNF-α. Supplementation of exogenous recombinant CREG protein or CREG gene silencing showed that CREG promoted autophagy in TNF-α treated macrophages. The use of autophagy inhibitors, 3-methyladenine and bafilomycin A, identified that CREG attenuated TNF-α induced inflammation by activate autophagy. In addition, supplementation of exogenous CREG protein stimulated expression and maturity of cathepsin B and cathepsin L and induced lysosome formation, whereas CREG deficiency reduced lysosomal formation. CONCLUSION: CREG inhibits inflammation and promotes autophagy mediated by lysosome formation; it might be a potential therapeutic target in atherosclerosis.