Cellular Repressor Of E1A-stimulated Genes Expression Research Articles

CREG Protects Retinal Ganglion Cells loss and Retinal Function Impairment Against ischemia-reperfusion Injury in mice via Akt Signaling Pathway.

The irreversible death of retinal ganglion cells (RGCs) plays an important role in the pathogenesis of glaucoma. Cellular repressor of E1A-stimulated genes (CREG), a secreted glycoprotein involved in cellular proliferation and differentiation, has been shown to protect against myocardial and renal ischemia-reperfusion damage. However, the role of CREG in retinal ischemia-reperfusion injury (RIRI) remains unknown. In this study, we aimed to explore the effect of CREG on RGCs apoptosis after RIRI. We used male C57BL/6J mice to establish the RIRI model. Recombinant CREG was injected at 1day before RIRI. The expression and distribution of CREG were examined by immunofluorescence staining and western blotting. RGCs survival was assessed by immunofluorescence staining of flat-mounted retinas. Retinal apoptosis was measured by the staining of TdT-mediated dUTP nick-end labeling and cleaved caspase-3. Electroretinogram (ERG)analysis and optomotor response were conducted to evaluate retinal function and visual acuity. The expressions of Akt, phospho-Akt (p-Akt), Bax, and Bcl-2 were analyzed by western blotting to determine the signaling pathways of CREG. We found that CREG expression was decreased after RIRI, and intravitreal injection of CREG attenuated RGCs loss and retinal apoptosis. Besides, the amplitudes of a-wave, b-wave, and photopic negative response (PhNR) in ERG, as well as visual function, were significantly restored after treatment with CERG. Furthermore, intravitreal injection of CREG upregulated p-Akt and Bcl-2 expression and downregulated Bax expression. Our results demonstrated that CREG protected RGCs from RIRI and alleviated retinal apoptosis by activating Akt signaling. In addition, CREG also improved retinal function and visual acuity.

CREG mitigates neonatal HIE injury through survival promotion and apoptosis inhibition in hippocampal neurons via activating AKT signaling.

Neonatal hypoxic ischemic encephalopathy (Neonatal HIE) is a common but serious disease caused by perinatal asphyxia injury in newborns. Elevated neuronal apoptosis plays an important role in the injury process post hypoxia ischemia of the brain, which accurate mechanism is still worthy to be studied. Cellular repressor of E1A-stimulated genes (CREG) possesses the protective effect in ischemia-reperfusion in multiple organs, including livers and hearts. The main purpose of this work was to investigate whether CREG was involved in alleviating neonatal HIE and explore the possible mechanisms. We found that CREG expression was downregulated in the hippocampus of neonatal HIE rats as well as oxygen-glucose deprivation/reperfusion (OGD/R)-treated hippocampal neurons. Besides, CREG overexpression promoted survival while inhibited apoptosis in OGD/R-induced hippocampal neurons accompanied by AKT signaling activation, which could be reversed by CREG silence. In addition, the protective effects of CREG overexpression could be antagonized by AKT deactivation, indicating the function of CREG was attributed by regulating AKT pathway. Collectedly, we demonstrated that CREG protected hippocampal neurons from hypoxic ischemia-induced injury through regulating survival and apoptosis via activating AKT signaling pathway.

CREG improves cardiac function by regulating cardiomyocytes' autophagy in diabetic myocardial infarction rats.

We aimed to observe the changes of cardiac function, cell morphology, cellular repressor of E1A-stimulated genes (CREG) and LC3-II after myocardial infarction (MI) in non-diabetic and diabetic rats, and to explore the relationship between myocardial damage and CREG and autophagy in diabetic rats. Diabetic rat models were prepared by intraperitoneal injection of low concentration (50 mg/kg) streptozotocin (STZ). MI models were established in normal rats and diabetic rats. The cardiac function of each group was detected by echocardiography. The pathological results of myocardial tissue in the infarcted area were observed under light microscope. The expression of CREG was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Myocardial protein CREG LC3-II expression was measured by Western blot. Autophagy levels were also detected at the cellular level. Construction of CREG overexpressing adenovirus transfected H9c2 cells, and injection of rats with AAV-CREG to achieve the purpose of overexpressing CREG. In vitro and in vivo experiments were conducted to explore the effects of CREG on autophagy and cardiac function in a diabetic MI model. Compared with the non-diabetic sham (NS) group, there were no marked differences in cardiac function and CREG levels in the diabetic sham (DS) group. Compared with the NS group, the cardiac function of the non-diabetic myocardial infarction (NI) group and the diabetic infarction myocardial (DI) group were reduced, and the levels of autophagy were increased. However, the cardiac function of the DI group was worse than that of the NI group, and the autophagy level of the DI group was lower than the NI group. The results at the cellular level were similar to the experiments in vivo. The overexpression of CREG in vivo or in vitro can increase autophagy levels and improve cardiac function. The exacerbation of myocardial injury after MI in diabetic rats may be related to the inhibition of CREG in myocardial cells of infarcted area by diabetes.

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-β1 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.

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

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.

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. 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. 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.

Up-Regulation of CREG Expression by the Transcription Factor GATA1 Inhibits High Glucose- and High Palmitate-Induced Apoptosis in Human Umbilical Vein Endothelial Cells.

BackgroundEndothelial cell (EC) apoptosis plays a vital role in the pathogenesis of atherosclerosis in patients with diabetes mellitus (DM), but the underlying mechanism remains unclear. Cellular repressor of E1A-stimulated genes (CREG) is a novel gene reported to be involved in maintaining the homeostasis of ECs. Therefore, in the present study, we investigated the role of CREG in high glucose/high palmitate-induced EC apoptosis and to decipher the upstream regulatory mechanism underlying the transcriptional regulation of CREG.MethodsThe expression of CREG and the rate of apoptosis were assessed in lower-limb atherosclerotic lesions from patients with type 2 DM (T2DM). Primary human umbilical vein endothelial cells (HUVECs) were isolated and cultured in a high glucose/high palmitate medium (25 mmol/L D-glucose, 0.4 mmol/L palmitate), and the over-expression and knock-down of CREG were performed in HUVECs to determine the role of CREG in EC apoptosis. The upstream regulatory mechanism of CREG was identified using a promoter-binding transcription-factor profiling array, chromatin immunoprecipitation (ChIP) assay and a mutation analysis.ResultsCompared with normal arteries from non-diabetic patients, reduced CREG expression and increased apoptosis were found in the endothelium of atherosclerotic lesions from patients with T2DM. In vitro treatment of HUVECs with a high glucose/high palmitate medium also resulted in decreased CREG expression and increased apoptosis. Moreover, high glucose/high palmitate induced-HUVEC apoptosis was increased by the knock-down of CREG and rescued by the over-expression of CREG. We also demonstrated that GATA1 was able to bind to the promoter of the human CREG gene. A deletion mutation at -297/-292 in the CREG promoter disrupted GATA1 binding and reduced the activation of CREG transcription by approximately 83.3%. Finally, the overexpression of GATA1 abrogated the high glucose/high palmitate-induced apoptosis in HUVECs.ConclusionsThe over-expression of CREG inhibits high glucose/high palmitate-induced apoptosis in HUVECs. CREG is transcriptionally upregulated by GATA1. Thus, CREG might be a potential therapeutic target for intervention of vascular complications related to diabetes.

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

AimsMacrophage 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 resultsImmunostaining 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. ConclusionCREG inhibits inflammation and promotes autophagy mediated by lysosome formation; it might be a potential therapeutic target in atherosclerosis.

ASSA14-03-23 CREG over-expression in BMSC protect against myocardial infarction via VHL/HIF-1α/VEGF pathway mediated vascularisation

<h3>Background</h3> We hypothesised that mesenchymal stem cells (MSCs) overexpressing Cellular Repressor of E1A-stimulated Genes (CREG) showed improved survival and engraftment in the infarcted heart and promoted vascularisation through paracrine release of vascular endothelial growth factor (VEGF) and activation of the HIF-1α signal pathway. <h3>Methods</h3> Mononuclear cells derived from rat bone marrow were isolated by density-gradient centrifugation and were cultured on fibronectin-coated plates, supplied with bovine pituitary extract. After the marrow-derived mesenchymal stem cells over-expressed CREG, we compare with the production of VEGF by ELISA and the protein expression of HIF-1α in different groups by western blot under hypoxia. Rat bone marrow–derived MSCs were used as nontransduced (^NormMSCs) or transduced with adenoviral-GFP vector (^GFPMSCs) or vector encoding for GFP-CREG (^CREGMSCs). <h3>Results</h3> For <i>in vivo</i> studies, 50 μl of DMEM without cells (group 1) or containing 1.5×10^6NormMSCs (group 2) or ^GFPMSCs (group 3) or ^CREGMSCs (group 4) were implanted intramyocardially in rat model of permanent coronary artery occlusion. one week later, immunoblot on rat heart tissue (n = 4 per group) showed highest survival of ^CREGMSCs (p &lt; 0.06 vs ^NormMSCs and ^GFPMSCs)(n = 6 per group). Confocal imaging after immunostaining for CD31 showed extensive angiomyogenesis in the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 4 as compared with group 1 (p &lt; 0.05). <i>In vitro</i>, under hypoxia the secretion of VEGF are most in the supernatant from the MSCs over-expression CREG. And the supernatants from BMSC over-expressed CREG exhibited a significant increase in angiogenic tube formation. In addition, the expression of CREG begins to appear after hypoxia. Molecular studies revealed that under hypoxia CREG active HIF-1α, but not HIF-2α, HIF-1β and we use the inhibition of HIF-1α confirm it. Further, we found that CREG can not influence the HIF-1α mRNA synthesis but it can inhibit the expression of pVHL which is the key protein regulation degradation of HIF-1α. <h3>Conclusions</h3> The strategies of CREG transgene expression inhibit pVHL and induced HIF-1α signalling and culminated in extensive angiomyogenesis in the infarcted heart.

ASSA14-03-28 Cellular repressor of E1A-stimulated genes improves heart function in a mouse model of MI

Backgroud Cellular repressor of E1A-stimulated genes (CREG) is a mannose-6-phosphate-containing secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. In humans, the potential therapeutic role of embryonic stem cells (ESCs) in ischaemic heart disease is subject to intense investigation. Particularly, the contribution of ESCs to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in mouse model, and monitored the effects of ESCs transplantation of overexpression of CREG on cardiac function. Methods pCXN2-Flag-wtCREG, pCXN2-Flag-mutCREG and pCXN2-Flag-EGFP plasmids were transfected into ESCs by lipofectamine 2000. Coronary artery ligation to induce MI model in seven- to nine-week-old mice was developed by a novel and rapid surgical method. wtCREG, mutCREG and EGFP ESCs or DMEM were then injected into the peri-ischaemic area. Four groups of mice were analysed for haemodynamic and pathologic parameters 1 and 2 months after MI and injection. Results The heart weight to body weight ratio was also significantly decreased at day 28 (5.9 ± 0.5 and 5.5 ± 0.4) in comparison with control hearts (7.0 ± 0.5, p Conclusions Therefore, the expression of CREG improves cardiac functions and inhibits birosis and apoptosis. These data suggest that a key mechanism of the protective effects of ASK1 in reducing ischaemic injury is via maintaining the classic proapoptotic factor Bax in an inactive state.

CREG Promotes the Proliferation of Human Umbilical Vein Endothelial Cells through the ERK/Cyclin E Signaling Pathway

Cellular repressor of E1A-stimulated genes (CREG) is a recently discovered secreted glycoprotein involved in homeostatic modulation. We previously reported that CREG is abundantly expressed in the adult vascular endothelium and dramatically downregulated in atherosclerotic lesions. In addition, CREG participates in the regulation of apoptosis, inflammation and wound healing of vascular endothelial cells. In the present study, we attempted to investigate the effect of CREG on the proliferation of vascular endothelial cells and to decipher the underlying molecular mechanisms. Overexpression of CREG in human umbilical vein endothelial cells (HUVEC) was obtained by infection with adenovirus carrying CREG. HUVEC proliferation was investigated by flow cytometry and 5-bromo-2′-deoxy-uridine (BrdU) incorporation assays. The expressions of cyclins, cyclin-dependent kinases and signaling molecules were also examined. In CREG-overexpressing cells, we observed a marked increase in the proportion of the S and G2 population and a decrease in the G0/G1 phase population. The number of BrdU positively-stained cells also increased, obviously. Furthermore, silencing of CREG expression by specific short hairpin RNA effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVEC). CREG overexpression induced the expression of cyclin E in both protein and mRNA levels to regulate cell cycle progression. Further investigation using inhibitor blocking analysis identified that ERK activation mediated the CREG modulation of the proliferation and cyclin E expression in HUVEC. In addition, blocking vascular endothelial growth factor (VEGF) in CREG-overexpressed HUVEC and supplementation of VEGF in CREG knocked-down HUVEC identified that the pro-proliferative effect of CREG was partially mediated by VEGF-induced ERK/cyclin E activation. These results suggest a novel role of CREG to promote HUVEC proliferation through the ERK/cyclin E signaling pathway.

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by The ILK/AKT/mTOR/VEGF 165 signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor 165, neutralizing antibody of vascular endothelial growth factor 165 and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF 165 signaling pathway.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation. CREG is widely expressed in adult tissues such as the brain, heart, lungs, liver, intestines and kidneys in mice. We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus (dpc) using immunohistochemical staining with diaminobenzidine, western blotting and reverse transcription-polymerase chain reaction. CREG expression was first detected in mouse embryos at 4.5 dpc. It was expressed at almost all stages up to 18.5 dpc. The level of CREG was found to increase gradually and was highest at 18.5 dpc. Western blotting showed that the CREG protein was expressed at higher levels in the brain, heart, intestines and kidneys than in the lungs and liver at 18.5 dpc. In 9.5 dpc embryos, CREG was expressed only in the endothelial cells of blood vessels, after the vascular lumen had formed. With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures; the expression of smooth muscle α-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels. CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc. These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Cellular repressor of E1A-stimulated genes inhibits human vascular smooth muscle cell apoptosis via blocking P38/JNK MAP kinase activation

Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes restenosis following vascular injury. The current study examined the effects of cellular repressor of E1A-stimulated genes (CREG), a novel glycoprotein inhibiting transcription activation, on the regulation of VSMC apoptosis. Serum starvation or treatment of human VSMCs with apoptosis inducers (STS or VP-16) significantly reduced CREG expression and caused caspase-3 activation. CREG downregulation and caspase-3 activation were inversely related, suggesting that reduced CREG expression may contribute to VSMC apoptosis. Both loss-of-function (CREG-DW produced by retroviruses expressing CREG shRNAs) and gain-of-function (CREG-UP produced by retroviral infection with vector pLNCX-CREG) studies were performed to confirm this hypothesis. CREG-DW significantly increased VSMC apoptosis, whereas CREG-UP significantly reduced apoptosis. Moreover, p38 and JNK mitogen-activated protein kinases were significantly upregulated in CREG-DW and significantly reduced in CREG-UP VSMCs. More importantly, CREG-DW-induced VSMC apoptosis was blocked by the p38-specific inhibitor SB203580 or by overexpression of a dominant-negative P38α (p38α AGF). Balloon injury-induced vascular caspase-3 activation was significantly inhibited by treatment with recombinant CREG protein. These results demonstrated for the first time that CREG plays a key role in modulating VSMC apoptosis through the p38 and JNK signal transduction pathways, both in vitro and in situ.

Cellular repressor of E1A‐stimulated genes attenuates cardiac hypertrophy and fibrosis

Cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro. However, such a role has not been determined in vivo. In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo. The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK-ERK1/2-dependent Smad 2/3 activation in vitro and in vivo. Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK-ERK1/2-dependent signalling.

Secreted CREG inhibits cell proliferation mediated by mannose 6‐phosphate/insulin‐like growth factor II receptor in NIH3T3 fibroblasts

Cellular repressor of E1A-stimulated genes (CREG) is a recently described glycoprotein that plays a critical role in keeping cells or tissues in mature, homeostatic states. To understand the relationship between CREG and its membrane receptor, mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), we first generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors, which produced an approximately 80% decrease in CREG levels both in the lysate and in the media. We used fluorescence activated cell sorting and a bromide deoxyuridine incorporation assay to identify whether CREG knockdown promoted the cell proliferation associated with the increase of IGF-II in NIH3T3 fibroblasts. Proliferation was markedly inhibited in a concentration-dependent manner by re-addition of recombinant CREG protein into the media, and this was mediated by the membrane receptor M6P/IGF2R. We subsequently confirmed the direct interaction of CREG and M6P/IGF2R by both immunoprecipitation-Western blotting and immunofluorescence staining. We found that expression of CREG correlated with localization of the receptor in NIH3T3 fibroblasts but did not affect its expression. Our findings indicated that CREG might act as a functional regulator of M6P/IGF2R to facilitate binding and trafficking of IGF-II endocytosis, leading to growth inhibition.

Adenovirus-mediated intra-arterial delivery of cellular repressor of E1A-stimulated genes inhibits neointima formation in rabbits after balloon injury

This study examined the effect on neointimal hyperplasia of adenovirus-mediated delivery of cellular repressor of E1A-stimulated genes (CREG) to the artery after balloon injury. Sixty rabbits were randomized into three groups and underwent balloon injury in the left common carotid arteries. The injured arterial segment was isolated by two inflated balloon catheters. Saline or recombinant adenovirus expressing CREG or green fluorescent protein was injected into the lumen of the isolated arterial segments and incubated for 30 minutes. The rabbits were euthanized for immunohistochemistry, Western blotting, and morphometric analysis at 3, 7, 14, and 28 days after balloon injury and in vivo gene transfer (5 rabbits for each time point). Common carotid artery angiography was performed before euthanasia. Immunohistochemistry and Western blot analysis demonstrated that CREG expression was significantly down-regulated in the acute phase of vascular injury and was gradually restored in the resolution phase. The changes of CREG expression were in parallel with those of the smooth muscle cell (SMC) differentiation markers SM alpha-actin and SM myosin heavy chain in the injured arteries. Adenovirus-mediated CREG transfer markedly increased CREG expression in the injured artery. Consequently, morphometric analysis revealed an approximate 50% reduction in the neointima and the intima/media ratio in CREG-transferred arteries compared with the saline and green fluorescent protein controls. Assay with 5-bromo-2-deoxyuridine showed that CREG transfer significantly inhibited SMC proliferation. In contrast, endothelialization of the injured artery was not affected by CREG transduction as assessed by CD31 immunostaining. These data suggest that forced expression of CREG in the artery wall after acute vascular injury inhibits SMC proliferation, induces cellular differentiation, and attenuates neointimal hyperplasia. CREG delivery may have therapeutic potential for the prevention of restenosis after vascular angioplasty.

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.

CREG, a new regulator of ERK1/2 in cardiac hypertrophy.

The cellular repressor of E1A-stimulated genes (CREG), a mannose-6-phosphate-containing secreted glycoprotein, enhances differentiation and inhibits proliferation. In this study, our aim was to understand the role of CREG in cardiac hypertrophy. Two models of cardiac hypertrophy were used: the in vivo pressure-overloaded rat cardiac hypertrophy and the in vitro stretched neonatal rat cardiomyocyte hypertrophy. CREG's function in cardiac cells was investigated after over-expression or antisense inhibition of CREG. We found reduced CREG expression in rat hearts after the in vivo overload, as shown by Northern blot analysis. CREG over-expression inhibited cardiac cell growth, as demonstrated by reduced protein content, cell area and ERK1/2 level in cultured neonatal rat cardiomyocytes, and by the reduced proliferation of cultured neonatal rat cardiac fibroblasts. Additionally, over-expression of CREG dampened the stretched cardiomyocyte hypertrophy through ERK1/2. On the other hand, the opposite effects were observed when CREG expression was decreased using antisense. This modulation of CREG expression resulted in no changes in the cardiomyocyte expressions of the hypertrophic or apoptotic signaling molecules such as protein kinase C (PKC) epsilon, PKC beta1, PKC alpha, PKC beta2, PKC delta, JNK1/2, p38, p53, Bax, Bcl2 and Fas. CREG was found to inhibit cardiac cell growth as a novel regulator of ERK1/2 and might participate in the development of cardiac hypertrophy under pressure overload. The insight that CREG inhibited the growth in rat hearts in vivo and in cardiac cells in vitro provides new clues for further investigation of the mechanism of heart remodeling.