Mingyu Sun

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.

Nanoporous CREG-Eluting Stent Attenuates In-Stent Neointimal Formation in Porcine Coronary Arteries

Background The goal of this study was to evaluate the efficacy of a nanoporous CREG-eluting stent (CREGES) in inhibiting neointimal formation in a porcine coronary model. Methods In vitro proliferation assays were performed using isolated human endothelial and smooth muscle cells to investigate the cell-specific pharmacokinetic effects of CREG and sirolimus. We implanted CREGES, control sirolimus-eluting stents (SES) or bare metal stents (BMS) into pig coronary arteries. Histology and immunohistochemistry were performed to assess the efficacy of CREGES in inhibiting neointimal formation. Results CREG and sirolimus inhibited in vitro vascular smooth muscle cell proliferation to a similar degree. Interestingly, human endothelial cell proliferation was only significantly inhibited by sirolimus and was increased by CREG. CREGES attenuated neointimal formation after 4 weeks in porcine coronary model compared with BMS. No differences were found in the injury and inflammation scores among the groups. Scanning electron microscopy and CD31 staining by immunohistochemistry demonstrated an accelerated reendothelialization in the CREGES group compared with the SES or BMS control groups. Conclusions The current study suggests that CREGES reduces neointimal formation, promotes reendothelialization in porcine coronary stent model.

Glycosylation-independent binding to extracellular domains 11–13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic modulation of vascular smooth muscle cells

The bio-effects of cellular repressor of E1A-stimulated genes (CREG) have been proposed to depend on its N-glycosylation and binding to mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R). The present study aimed to investigate the detailed mode and specific sites for their binding and the functional relevance of this binding in the phenotypic modulation of vascular smooth muscle cells (SMCs). Wild-type and glycosylation mutant human CREG (wtCREG and mCREG) proteins were expressed and isolated from HEK293 cells. CREG knocked-down SMCs were used to evaluate their biological activity. Both wtCREG and mCREG arrest cell cycle progression of CREG knocked-down SMCs when added to the culture medium. In vitro binding assay revealed that CREG bound to M6P/IGF2R extracellular domains 7-10 and 11-13 in a glycosylation-dependent and -independent manner, respectively. Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody suggest that the binding to domains 11-13, as well as to 7-10, is adequate for CREG to modulate SMC proliferation. These data suggest that soluble CREG protein can exert its biological function via glycosylation-independent binding to the extracellular domains 11-13 of cell surface M6P/IGF2R, and thereby provide novel insights into CREG modulation of SMC phenotypic switching from contractile to proliferative.

CREG inhibits migration of human vascular smooth muscle cells by mediating IGF-II endocytosis.

We previously determined that the cellular repressor of E1A-stimulated genes, (CREG) plays a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). This study aimed to identify the role of CREG in modulating the migration of SMCs. Recombinant virus-mediated CREG expression inhibited the cellular migration of cultured SMCs associated with down-regulated activity of matrix metalloproteinase-9 (MMP-9). In contrast, CREG knockdown via the retroviral transfer of short hairpin RNAs promoted cellular migration. Enzyme-linked immunosorbent assay and endocytosis analysis revealed that CREG knockdown attenuated the internalization and increased secretion of insulin-like growth factor (IGF)-II. Western blot analysis demonstrated that both phosphoinositide 3-kinase (PI3K) and phosphatase Akt were enhanced in CREG knockdown SMCs. Furthermore, the effect of CREG knockdown on SMC migration was abrogated in a dose-dependent manner by the addition of either IGF-II neutralizing antibody or the PI3K inhibitor, LY294002. These results indicate that the CREG knockdown-mediated increase in IGF-II secretion promoted cellular migration in SMCs via the PI3K/Akt signal pathway. Additionally, blockage of IGF-II binding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF2R) by IGF2R antibody or recombinant IGF2R fragment attenuated the endocytosis of IGF-II in cells overexpressing CREG. This indicates that M6P/IGF2R is involved in the regulation of CREG-mediated IGF-II endocytosis. In summary, these data demonstrate for the first time that CREG plays a critical role in the inhibition of SMC migration, as well as maintaining SMCs in a mature phenotype. These results may provide a new therapeutic target for vascular disease associated with neointimal hyperplasia.

Cellular repressor E1A-stimulated genes controls phenotypic switching of adventitial fibroblasts by blocking p38MAPK activation

AIMS Phenotypic modulation of adventitial fibroblasts (AFs) plays an important role in the pathogenesis of proliferative vascular diseases. The current study aimed to identify the role of cellular repressor E1A-stimulated genes (CREG), a critical mediator in the maintenance of vascular homeostasis, in AF phenotypic modulation and adventitial remodeling. METHOD AND RESULTS Using in situ double-immunofluorescence staining, we ascertained that CREG expression was significantly down-regulated in the adventitia after vascular injury, and its expression pattern was conversely correlated with the expression of smooth muscle α-actin (α-SMA), a marker for differentiation of AFs into myofibroblasts. In vitro data confirmed the association of CREG in angiotensin II (Ang II)-induced AF differentiation. Additionally, overexpression of CREG attenuated Ang II-induced α-SMA expression in AFs. CREGoverexpressing AFs showed decreased levels of proliferation on days 2-5 following stimulation by Ang II compared with controls, with changes in the cell cycle profile as shown by BrdU incorporation assay and fluorescence activated cell sorting analysis. Moreover, wound healing assay and transwell migration model demonstrated that upregulation of CREG expression inhibited Ang II-induced AF migration. We found that CREG-mediated its counterbalancing effects in Ang II-induced phenotypic modulation, proliferation and migration by inhibition of the p38MAPK signaling pathway, validated by pharmacological blockade of p38MAPK with SB 203580 and by overexpression of p38MAPK with transfectants expressing constitutively active p38αMAPK. CONCLUSION Our findings suggest that CREG is a novel AF phenotypic modulator in a p38MAPK-dependent manner. Modulating CREG on the local vascular wall may become a new therapeutic target against proliferative vascular diseases.

Cellular repressor of E1A-stimulated gene overexpression in bone mesenchymal stem cells protects against rat myocardial infarction

BACKGROUND Bone mesenchymal stem cell (BMSC) therapy has modest success in ischemic heart disease but has been limited by poor survival in diseased microenvironments. Cellular repressor of E1A-stimulated genes (CREG) can prevent BMSCs from apoptosis in vitro; however, the effects of CREG-modified BMSCs on ischemic heart disease and the related mechanism remain undefined. Therefore, we designed to study the cardioprotective effects of CREG overexpression in BMSCs ((CREG)BMSCs) after transplantation into infarcted heart of rats. METHODS In vivo studies, 50 μl PBS or 1.5×10(6)(Norm)BMSCs, (GFP)BMSCs or (CREG)BMSCs were implanted intramyocardially in myocardial infarction rat models. 3 or 14 days later, cardiac function, fibrosis, apoptosis and angiogenesis were analyzed by echocardiography, masson, western blot and immunofluorescence staining, respectively. ELISA, western blot and matrigel assay were used in vitro to detect vascular endothelial growth factor (VEGF) secretion, signaling molecule expression, and angiogenic tube formation. RESULTS In vivo, prolonged cardiac function (14d LVEF: 50.87 ± 0.94%; LVFS: 23.41 ± 1.12%), decreased fibrosis (14d Fibrotic area: 27.37 ± 1.03%) and apoptosis and increased angiogenesis were observed in (CREG)BMSCs, compared with other groups. In vivo and in vitro, VEGF secretion from (CREG)BMSCs was markedly enhanced. In vitro, angiogenic tube formation in (CREG)BMSC supernatants significantly increased. Moreover, CREG activated hypoxia-inducible factor-1α (HIF-1α), but not HIF-1β. Knockdown of HIF-1α with siRNA decreased VEGF secretion and angiogenic tube formation. Notably, CREG did not influence HIF-1α mRNA synthesis but inhibited the expression of Von Hippel-Lindau (VHL), a key protein that regulates HIF-1α degradation. CONCLUSIONS The (CREG)BMSC transplantation, directly or indirectly, may promote VEGFs anti-apoptosis and angiogenesis via the inhibition of VHL-mediated HIF-1α degradation, consequently protecting against myocardial infarction.

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.

ASSA14-03-25 Cellular repressor of E1A-stimulated genes antagonises inflammation and promotes autophagy via lysosome biogenesis in mouse macrophages

Objective Macrophage inflammation plays an important role in the pathogenesis of atherosclerosis. In this study, we investigated the involvement of cellular repressor of E1A-stimulated genes (CREG) in tumour necrosis factor-α (TNF-α)-induced macrophage inflammation, and explored its inhibitory capacity and mechanisms to assess its potential as a therapeutic reagent for atherosclerosis. Methods and results We confirmed that CREG played an important role in TNF-α-induced macrophage inflammation and had anti-inflammatory effects in RAW 264.7 mouse macrophages induced by TNF-α, using enzyme-linked immunosorbent assays and western blotting. Gain-of-function and loss-of-function experiments revealed that CREG promoted autophagy in TNF-α-induced RAW 264.7 cells. Using the autophagy inhibitors 3-methyladenine and bafilomycin A, we demonstrated that autophagy played an important role in attenuating TNF-α-induced inflammation. Immunofluorescence analysis and western blotting showed that CREG protein stimulated the expression and maturation of cathepsin B and cathepsin L and induced the biogenesis of lysosomes, while CREG deficiency reduced lysosome biogenesis. Exogenous CREG protein was located in lysosomes, as shown by confocal microscopy and immunoprecipitation analysis. CREG protein played a critical role in the distribution but not in the expression of mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR), as demonstrated by western blotting and immunofluorescence analysis. In vivo experiments indicated that CREG protein alleviated the development of aortic atherosclerosis and affected inflammation and autophagy in aortas of ApoE−/− mice. Conclusion CREG inhibits macrophage inflammation and promotes autophagy mediated by lysosome biogenesis, which is related to the distribution of M6P/IGFIIR. CREG may represent a new therapeutic target against atherosclerosis.

ASSA14-03-26 Cellular repressor of E1A stimulated genes antagonise inflammation in RAW 264.7 cells via autophagy-lysosome pathway

Background Macrophage inflammation plays an important role in the pathogenesis of atherosclerosis. In this study, we investigated the involvement of cellular repressor of E1A-stimulated genes (CREG) in tumour necrosis factor-α (TNF-α)-induced macrophage inflammation, and explored its inhibitory capacity and mechanisms to assess its potential as a therapeutic reagent for atherosclerosis. Methods RAW 264.7 mouse macrophage-like cells were transfected with CREG siRNA to down-regulate the expression of CREG. The expression of CREG, cathepsin B, cathepsin L, LAMP1, IGFIIR, LC3, Beclin 1, p62 and Tubulin was identified by Western blot. The amounts of IL-6 and MCP-1 secreted into the cell culture supernate and expressed in the tissue lysates were determined using ELISA kits. Colocalization of anti-His and anti-cathepsin B/ cathepsin L/ M6P/IGFIIR/ LAMP1 was determined by immunofluorescence analysis and confocal microscopy. Lysosomal visualisation was carried out using the Lysotracker Red staining. Accumulation of autophagosomes and autolysosomes were detected in CREG-treated and CREG down-regulated cells using electron microscopy. The interaction between the exogenous recombinant CREG protein and cathepsin B, cathepsin L and IGFIIR were studied with immunoprecipitation analysis. Male ApoE−/− mice (n = 40, age 10 weeks, 20–25 g) were fed a high-fat, high-cholesterol diet containing 21% fat and 1.3% cholesterol for 16 weeks. The mice underwent sham operation or were infused with CREG protein (Abcam, UK) at a rate of 30 μg/kg/d during the 4–12 weeks by an osmotic mini-pump. Aortas were collected for oil red O and hematoxylin-eosin staining to assess atherosclerotic plaques. The expression of CD68 and MCP-1 in the atherosclerotic plaques was detected by the immunohistochemical staining. Results We confirmed that CREG played an important role in TNF-α-induced macrophage inflammation and had anti-inflammatory effects in RAW 264.7 mouse macrophages induced by TNF-α. Results of ELISA assays showed that the amount of IL-6 and MCP-1 secreted into the cell culture supernate decreased in a dose-dependent manner in the RAW 264.7 cells supplemented with exogenous recombinant CREG protein and increased in those knocking down of CREG expression by siRNA. Gain-of-function and loss-of-function experiments revealed that CREG promoted autophagy in TNF-α-induced RAW 264.7 cells. The numbers of autolysosomes increased in RAW 264.7 cells following treatment with CREG protein while less autolysosome were seen in those transfected with CREG siRNA. Meanwhile, exogenous CREG protein induced autophagy with significant increased autophagosome-bound LC3-II and Beclin 1 abundance and decreased the level of p62. Increment of LC3-II and Beclin 1 and accumulation of p62 in CREG down-regulated cells suggested accumulation of autophagosomes and impairment of autophagy. Using the autophagy inhibitors 3-methyladenine and bafilomycin A, we demonstrated that autophagy played an important role in attenuating TNF-α-induced inflammation. Immunofluorescence analysis and western blotting showed that CREG protein stimulated the expression and maturation of cathepsin B and cathepsin L and induced the biogenesis of lysosomes, while CREG deficiency reduced lysosome biogenesis. Since lysosomal activity is essential to autophagy, we deduce that CREG may mediate the regulation of autophagy via its effects on lysosomes. Exogenous CREG protein was located in lysosomes, as shown by confocal microscopy and immunoprecipitation analysis. CREG protein played a critical role in the distribution but not in the expression of mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR), as demonstrated by western blotting and immunofluorescence analysis. In vivo experiments indicated that CREG protein alleviated the development of aortic atherosclerosis and affected inflammation and autophagy in aortas of ApoE−/− mice. Haematoxylin-eosin staining showed that the aortic lesion area was significantly reduced in the CREG-treated mice compared with those in the control group. The expression of MCP-1 in arterial tissue detected by ELISA assays and CD68, a marker of active macrophages and MCP-1 in atherosclerotic lesions of aortic root sections determined by immunohistochemical staining were all decreased in CREG-treated group. Furthermore, Western analysis of aorta lysates showed increased autophagosome-bound LC3-II abundance and p62 consumption in CREG-treated group. Conclusion CREG inhibits macrophage inflammation and promotes autophagy mediated by lysosome biogenesis, which is related to the distribution of M6P/IGFIIR. CREG may represent a new therapeutic target against atherosclerosis.

ASSA14-03-24 CREG1 upregulates Rab7 expression to activate autophagy and ameliorate cardiac damage

Background 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 tissue homeostasis that has been shown to antagonise injury of tissues or cells. Recent studies showed that CREG1 is a lysosomal protein that undergoes proteolytic maturation by lysosomal cysteine proteases in the course of its biosynthesis. CREG1 contains a mannose 6-phosphate (M6P) recognition marker, and depends on interactions with M6P receptors for efficient delivery to lysosomes. CREG1 is implicated in the regulation of lysosome functions. In the present study, we aimed to investigate the regulatory role of CREG1 in cardiac autophagy, and to clarify autophagy activation mechanisms. Methods Male heterozygous Creg1+/− mice were generated and bred at the Southern model animal centre. Age-matched male transgene-negative wild-type (WT) littermates were used as controls. Two- to six-week-old Creg1+/− and WT mice were subjected to an infusion of Ang II (2.5 g/kg per day) for 28 days. In another series of experiments (reversal experiment), 4-week-old male Creg1+/− mice were subjected to an infusion of AngII for 28 days, meanwhile was treated with recombinant CREG1 protein (5 mg/kg per day, IP injections) or with chloroquine (5 or 10 mg/kg per day) for 14 days (starting day 1 and continuing until day 14). To measure fibrosis, Masson’s trichrome staining was performed on paraffin sections for all experimental animals. To measure autophagic flux, western blot for analyses of LC3-II and p62 levels was employed on the myocardium samples. RT-PCR was adopted for the total RNA. Primary culture of cardiac myocytes and Adenoviral Infection were all used in this study. Results We generated a Creg1 haploinsufficiency (Creg1+/−) mouse model, and identified that Creg1 deficiency aggravates myocardiac fibrosis in response to ageing or angiotensin II (Ang II). Conversely, exogenous infusion of recombinant Creg1 protein completely reversed cardiac damage. Creg1 deficiency in Creg1+/− mouse hearts showed a marked accumulation of autophagesomes that acquired LC3II and beclin-1, and a decrease in autophagic flux as indicated by the level of p62. Inversely, restoration of Creg1 activity activated 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 cardiacmyocytes, overexpression of Creg1 with concurrent resveratrol treatment significantly increased autophagy, while silencing Creg1 blocked the resveratrol-induced autophagy. Conclusions These results suggest that rapid, Creg1-induced activation of autophagy is required to maintain heart function in the face of stress-induced myocardial damage. Both in vitro and in vivo studies identified that Creg1 deficiency reduced the expression of Rab7, thereby influencing the maturation of lysosomes and the formation of autophagolysosomes and imparing autophagy. These findings also suggest that activating autophagy via Creg1 may be a viable therapeutic strategy for improving cardiac performance under pathologic conditions.