Objectives 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 ischaemia is not well established. In our studies, we induced myocardial infarct (MI) in mouse model, and monitored the effects of CREG modified ESCs transplantation on cardiac function.
Methods pCXN2-Flag-wtCREG, pCXN2-Flag-mutCREG and pCXN2-Flag-EGFP plasmids were transfected into ESCs by lipofectamine 2000. Myocardial infarction was induced by coronary artery ligation in seven- to nine-week-old mice. A total of 2×105 ESC over-expressing wild type CREG (wtCREG), glycosylation mutant CREG (mutCREG) and EGFP or 20 μl PBS were injected into the peri-ischaemic area. Four groups of mice were analysed for hemodynamic and pathologic parameters 1 and 2 months after MI and injection. Heart functions were assessed by small animal ultrasound system. Left ventricular pressure was measured by catheterisation through right carotid artery. Fibrosis and collagen synthesis were assessed by Massson staining. Apoptosis was determined by TUNEL assay. Protein possibly involved in signalling pathway was detected by Western Blot.
Results wtCREG and mutCREG-ESCs significantly improved murine cardiac function after MI, as compared with EGFP-ESCs or PBS. The beneficial effect of wtCREG and mutCREG-ESCs may mostly be ascribed to their notable resistance to apoptosis and fibrosis, and to their anti-inflammatory action, since cardiomyogenesis was limited. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MAPK-ERK1)/2-dependent signalling cascade. In addition, CREG expression attenuated fibrosis and collagen synthesis through blocking MAPKK-ERK1/2-dependent Smad2/3 activation in vivo.
Conclusions Therefore, the expression of CREG improves cardiac functions and inhibits inflammation and fibrosis through blocking MAPK-ERK1/2-dependent signalling.