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 < 0.05). The heart weight to tibia length ratio of EGFP ESCs or wtCREG ESCs was decreased at day 28 (1.5 ± 0.1 and 1.4 ± 0.1) in comparison with age-matched control hearts (1.6 ± 0.2, p < 0.05). TTC analysis performed at 28 days after MI shows a highly significant reduction of the infarct size in the EGFP ESCs and wtCREG ESCs groups, as compared with the DMEM and sham mice (p < 0.05). Haemodynamic assessment by Millar catheterization demonstrated a significant increase in +dP/dt and –dP/dt, as well as a significant drop of LVEDP at 21 days after MI in mice transplanted with wtCREG ESCs, as compared with mice transplanted with EGFP ESCs or DMEM (p < 0.05). Intriguingly, animals transplanted with EGFP ESCs also showed a significant improvement of these cardiac functional parameters, as compared with those transplanted with DMEM (p < 0.05). WtCREG animals had a significantly greater decrease in end-diastolic and end-systolic dimensions in comparison with DMEM and EGFP animals, reflecting decelerated dilatative remodelling in the wtCREG. This was associated with marked LV function improvement as reflected by increaced LVEF and LVFS. Results from our experiments show a remarkable decrease of both myocardial fibrosis and cardiomyocyte and noncardiomyocyte apoptosis in wtCREG ESCs-injected mice. A significant release of VEGF was observed not only in the wtCREG ESCs animals (41.3 ± 12.6 pg/ml), but also in the EGFP ESCs animals (50.0 ± 4.1). Over-expression of CREG (wtCREG ESCs) decreased ASK1 protein level by 48% (p < 0.05). Upon elevation of CREG levels, also decreased JNK1/2 and p38 expression levels in the cardiomyocytes. Here, we found increased Bax and decreased bcl-2 expression at 2 weeks post-MI.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.