CREG Modulates Apoptosis Of Human Vascular Smooth Muscle Cells Via P38/JNK MAPK Signaling Pathway

BackgroundApoptosis is a physiological cell death process involved in many pathological conditions. VSMC apoptosis is observed in the advanced atherosclerotic lesion as well as the injured blood vessel. VSMC apoptosis is a key pathologic feature of atherosclerosis and angioplasty-induced restenosis and plays an important role in determining the course of atherogenesis. Accordingly, modulating VSMC apoptosis might have applicational perspective in attenuating the progression of atherosclerotic plaques and arterial restenosis after balloon angioplasty.CREG is a recently established secreted glycoprotein that has been shown to antagonize transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. We and other studies have shown that the secreted CREG can inhibit several kinds of cellular proliferation such as VSMCs, NIH3T3 and human embryonal carcinoma cells in vitro. Previous studies also have shown that CREG is significantly upregulated at both the mRNA and protein level during the phenotypic conversion of proliferative VSMCs to quiescent differentiated ones in vitro. Furthermore, CREG is downregulated in the vascular media after balloon injury to the rat carotid artery. Adenovirus-mediated CREG overexpression in balloon-injured rabbit carotid arteries inhibits VSMCs proliferation and attenuates neointimal hyperplasia. Since CREG is highly expressed in normal adult tissues and keeps cells in a homeostatic state, the findings support the hypothesis that CREG may play a key role in protecting VSMCs from apoptosis. In this study, we used cultured human VSMCs and balloon-injuried human internal thoracic artery model to examine the effect of CREG on the apoptosis of VSMCs.Materials and methodsVSMCs were isolated from human internal thoracic artery medial smooth muscle cells. The retrovirus was added to cells with MOI (multiplicity of infection) of ~20 pfu/cell in complete medium plus polybrene (8μg/mL) for 72h (viral titer based on pLNCX-GFP). Stable cell lines were established by selection with G418 (500μg/mL) for 2 weeks. To generate CREG knockdown VSMCs, cells were infected with retrovirus expressing CREG shRNAs and selected with 6μg/mL puromycin. Cell lysates and medium were harvested and CREG expression was evaluated by Western analysis. We assessed the role of CREG on cellular apoptosis in stable VSMC lines.Human internal thoracic arteries were obtained at surgery from subjects with coronary artery disease (CAD) who underwent coronary artery bypass grafting. Vascular tissue pieces were grown in DMEM (Invitrogen) with 4 mM glutamine supplemented with 10 % fetal calf serum (FCS) and 100 U/mL penicillin and 100 U/mL streptomycin at 37℃in a humidified atmosphere of 5 % CO2 and 95 % air. The arteries were injured by a 6F Fogarty balloon embolectomy catheter (Baxter). CREG protein was continually infused into the injured artery segments. The tissues were extracted at 7, 14, 28 and 42 days after balloon injury and CREG protein treatment. The uninjured thoracic artery served as control.ResultsCREG overexpression inhibits cultured VSMCs apoptosis and enhances cellular differentiationRetroviral vector pLNCX-hCREG or pLNCX-hCREG/shRNA was used to infect cultured human VSMCs and to generate stable clones that overexpressing or knocking-down the expression of CREG (named CREG-SN cells or CREG-AS cells respectively). CREG in VSMC lysates displays different bands, possibly due to the levels of glycosylation. Meanwhile, the cells overexpressing CREG converted to a spindle shape, a characteristic of the mature VSMC phenotype when cultured in 10 %FCS. In contrast, the GFP-transduced cells (named Ctl cells) exhibited a classic"hill-and-valley"growth pattern at and those CREG knockdown cells assumed spread morphology. Western blotting revealed that cell lysates from CREG-SN was detected no obviously increase in CREG expression compared to Ctl cells, whereas the CREG protein secreted to cell medium was detected ~6-fold increase in CREG-SN cells than in Ctl cells. Meanwhile, the expression of CREG was detected a siginaficant reduction of CREG protein expression by ~80% compared to the Ctl cells both in cell medium and in cellular lysates from CREG-AS cells (P<0.01).The CREG-AS cells were treated with different concentrations of STS (50, 100 and 200 nmol/L) or VP-16 (20, 50 and 100μmol/L) for various periods (0, 4, 8, 12 and 24h). The results indicated that treatment with STS (200 nmol/L) and VP-16 (100μmol/L) for 24h greatly increased the number of apoptotic cells by flow cytometry. To observe the effect of CREG on VSMC apoptosis, three groups of cells were stimulated with STS (200 nmol/L) or VP-16 (100μmol/L) for 0,8, 12 and 24h, respectively. The amount of CREG and cleaved caspase-3 was quantified at designated time points by Western blotting. The results showed that CREG knockdown upregulated cleaved caspase-3 content, whereas CREG overexpression lowered the levels of cleaved caspase-3. We also observed that the number of cells undergoing apoptosis remarkably increased in response to STS or VP-16 provocation at different time points (0, 8 and 12h) in CREG-AS cells compared to in the Ctl cells, whereas apoptotic cells significantly reduced in CREG-SN cells compared to in the Ctl cells by annexin V/PI dual-color flow cytometry. Furthermore, the percentage of TUNEL-positive cells in CREG-AS cells with STS or VP-16 at 12h markedly increased compared to in CREG-SN cells. Western analysis also revealed that CREG overexpression increased VSMC differentiation markers SMα-actin and SM myosin heavy chains (SM MHC) content and reduced cell-associated Fibronectin(FN)and Laminin-1(LN). After STS or VP-16 provocation, the expression of CREG was relative to VSMC differentiation markers, and inversely in parallel with proliferating cell associated protein. The results suggest that CREG participates in regulating VSMC apoptosis and the maintaining differentiated VSMC phenotype.CREG attenuates the induction of apoptosis through p38/JNK MAPK pathways in cultured VSMCsAfter STS or VP-16 stimulation, although there was no obvious change in the total p38, JNK and Akt amount, the phosphorylation of p38, JNK and Akt in CREG-AS cells significantly increased by Western blotting. The data in CREG-SN cells revealed a little increase in the amount of phosphorylated p38, JNK and Akt, which were maintained at a very low level.In order to further elucidate the effect of CREG on the involvement of signal transduction pathways in VSMC apoptosis, the specific inhibitor SB203580, SP600125 and LY294002 were used. CREG-AS cells were pretreated for 30 ~ 60 min with SB203580 (10, 20 and 50μmol/L) or SP600125 (10, 20 and 50μmol/L) or LY294002 (10, 20 and 50μmol/L), then the cells were stimulated with STS or VP-16 for 12h respectively. Using flow cytometry to estimate the number of apoptotic cells, treatment with SB203580 (10μmol/L) and SP600125 (10μmol/L) significantly lowered the number of STS-induced apoptotic cells from 41.33±3.8 % to 20.39±1.5 % and 29.05±2.3 % (P<0.01), and similarly reduced in VP-16-induced apoptotic cells from 53.45±5.36 % to 26.78±3.1 % and 38.05±3.3 % (P<0.01). But pretreatment with LY294002 was not associated with an increased apoptotic index in CREG-AS cells.We transfected CREG-AS cells with the plasmids p38αCA and p38αAGF (named p38αCA-CREG-AS cells or p38AGF-CREG-AS cells,respectively). After transfection was carried out for 48h, cell lysates were harvested and phosphorylated p38 expression was evaluated by Western analysis. Furthermore, CREG-AS cells were divided into three groups according to the transfection and p38 inhibitor pretreatment or not (CREG-AS cells, p38 AGF-CREG-AS cells and SB203580-CREG-AS cells respectively). After SB203580-CREG-AS cells were treated with the specific p38 inhibitor SB203580 for 30 min, these groups were provocated by STS at designed times. As shown in these blots, p38 activation diminished in parallel with decrease in the amount of cleaved caspases-3 in p38AGF-CREG-AS cells and SB203580-CREG-AS cells. Flow cytometric analysis showed that cellular apoptosis was substantially inhibited in p38AGF-CREG-AS cells and SB203580-CREG-AS cells from 36.23±5.8 % to 17.7±3.6 % and 24.39±2.7 % at 12h (P<0.01). We also found that the AGF mutant of p38 and the specific p38 inhibitor decreased the level of phosphorylated JNK, especially in p38AGF-CREG-AS cells. These results indicate that these p38 fusion proteins are functionally active in regulating JNK activity and induced JNK phosphorylation contributes positively to VSMC apoptosis.CREG enhances VSMC differentiation and inhibits cellular apoptosis via p38/JNK MAPK pathways in balloon-injured arteryIn the present study, we observed that CREG expression closely correlated with VSMC apoptosis and cellular differentiation in vitro. To detect whether CREG might participate in cellular apoptosis and whether its expression might involved in phenotypic modulation of VSMCs in vivo, we introduced balloon injury to the human internal thoracic artery and harvested the injured arteries on days 7, 14, 28 and 42. We tested the effects of CREG on cellular apoptosis and VSMC differentiation at designed time points. Western analysis revealed that CREG was markedly downregulated in the human atherosclerotic artery. Although CREG revealed a little increase, its expression maintained at a very low level inversely correlated with cellular apoptosis as revealed by cleaved caspase-3 after vascular injury. This is also reflected by the reduction of VSMC differentiation markers SMα-actin and SM MHC in parallel with the expression of CREG after vascular injure. These data strongly suggest that CREG is associated with quiescent mature VSMCs and that CREG downregulation may facilitate cellular apoptosis and dedifferentiation. To further clarify the effects of CREG on cellular apoptosis and phenotypic modulation, treatment with CREG protein (600 ng/mL and 1200 ng/mL) significantly lowered the amount of cleaved caspase-3 and increased in the expression of SMα-actin and SM MHC in the injured artery. Immunohistochemistry also revealed that cleaved caspase-3 was markedly upregulated at the fourth week after balloon injury. Addition of CREG protein in medium abrogated the expression of cleaved caspase-3 in injured artery wall. Meanwhile, HE staining also indentified that the neointimal area was increased at the fourth week after injury. Inversely, treatment with CREG protein markedly reduced neointimal area after balloon injury. These data strongly suggest that CREG expression in the injury artery may atteuate VSMC apotosis and inhibit the neointimal formation associated with maintainence of the quiescent mature VSMCs.To test whether CREG might regulate cellular apoptosis by these signaling pathways in the injured artery, we performed Western analysis using antibodies that specifically reacts with active, phosphorylated ERK1/2, p38, JNK, Akt and so on. After 2 weeks, balloon injury to the internal thoracic artery induced significant activation of p38, JNK and Akt, not ERK1/2. Treatment with CREG protein markedly reduced the amount of phosphorylated p38, JNK and Akt as compared to the injured artery, especially in the group of treatment with CREG protein (600 ng/mL). Furthermore, the specific inhibitor SB203580 (10μmol/L), SP600125 (10μmol/L) and LY294002 (50μmol/L) were used in the balloon-injured artery for 7, 14 and 28d,respectively. Western blot showed that treatment with SB203580 and SP600125 in the injured artery significantly lowered the levels of cleaved caspase-3, and that stimulation with LY294002 did not increase the activation of caspase-3. The blots also showed that the inactivation of p38 decreased JNK phosphorylation. These results suggest that CREG-mediated inhibition of p38 and JNK activation and cooperative activities among p38 and JNK pathways are involved in mediating cellular apoptosis in the injured arterial wall.ConclusionThese results demonstrate for the first time that CREG plays a key role in modulating VSMC apoptosis by p38/JNK MAPK signaling transduction pathways in vitro and in vivo.