Secreted Wnt molecules are involved in regulating morphogenesis and cardiogenesis (Wnt16>Wnt5>Wnt3) in CD117+/LIN- cardiac stem cells (RCSC) via canonical or non-canonical signalling. We found enhanced expression of cardiac transcription factors and structural molecules e.g. GATA4, cTnT and a-s-actinin in Wnt16 co-cultured RCSC. This study provides evidence of the cardiac reparative properties of Wnt16 expressing-RCSC involving matrix metalloproteinases (MMP) and their inhibitors (TIMP).
Methods: Wnt16-GFP-RCSC transfection efficiency was confirmed with flow-cytometry (FC), immuno–blotting (IB) and -fluorescence. Rats were divided into 3 groups (media control, untreated RCSC and Wnt16-GFP-RCSC, n=5/group). Hearts were exposed to 30min ischemia before injection of ~1x106 cells 2 mm apart, into infarct-border zone (BZ), followed by harvesting at 48h and 4w for histology and assay of cardio-protective, pro-apoptotic and tissue remodelling factors.
Results: FC showed 96.23±0.5% of Wnt16-GFP-RCSC as Wnt16+. At 48h, hearts injected with these cells had smaller infarcts compared to media control (ischemic area 30.8+/-1.4%, infarct 17.9+/-1.9% of LV vs 50.8+/-2.7%, 37.9+/-3.9%, p<0.05). At 4w, Sirius red LV fibrotic area was 20+/-1.35% with Wnt16-GFP-RCSC, 35+/-1.8% with untreated RCSC and 52+/-2.1% in media control (p<0.05). Wnt16-GFP-RCSC treatment showed elevated expression of TIMP-1 and -4 (2.5- and 3.6 fold increase), and down-regulation of MMP-2, -3, and -9 (1.3, 6.2, and 4.8 fold). Significant increase of pro-apoptotic molecules (Bcl-2 and Bax), angiogenic (Flk-1 and VEGF) and cardio-protective factors (phos-GSK3b and NF-kappab) was shown on IB.Conclusion: Wnt16-expressing RCSC showed enhanced reparative effects in ischemic hearts by increasing angiogenic, pro-apoptotic and cardio-protective molecules, and by regulating tissue remodelling factors.