Abstract 487: Heart fibroblasts mediate the effect of High Mobility Group Box 1 Protein on cardiac stem cells
Introduction. We have previously shown that High Mobility Group Box-1 Protein (HMGB1) injection into the infarcted mouse heart induces resident cardiac c-kit+ stem cell (CSC) proliferation and differentiation toward the myocardial lineage. However, in contrast to the in vivo studies, in vitro experiments have shown no effect of HMGB1 on CSC proliferation. Here we examined whether HMGB1 modulates CSC proliferation and differentiation in a paracrine manner through cardiac fibroblasts (cFb) activation.
Methods and Results. cFbs were obtained from human auricle and identified for the expression of vimentin and smooth muscle actin. CSC, magnetically sorted for c-kit antigen, were obtained from adult CD1 mice. Western blot analysis revealed that cFbs expressed the HMGB1 receptors RAGE and Toll-like receptors 2 and 4. HMGB1 (5, 10, 100, 200 ng/ml) induced a dose-dependent chemotactic effect on cFbs but had no effect on proliferation. Moreover, HMGB1 was not a profibrotic stimulus since it failed to promote the appearance of myofibroblast phenotype. The conditioned medium (CM) of unstimulated (CM-Con) and HMGB1-(10–100 ng/ml) stimulated (CM-HMGB1) cFbs was analyzed for the presence of growth factors, cytokines and chemokines using Luminex technology and ELISA assays. HMGB1 (10 ng/ml) enhanced cFb secretion of VEGF, PlGF, Mip-1α, IFN-γ, GM-CSF, Il-10, Il-1β, Il-4, Il-1ra, Il-9 and TNF-α. Further, CM-HMGB1, compared to CM-Con, enhanced about 2-fold CSC migration in a Boyden chamber assay (n=5; p<0.05) and induced CSC differentiation towards the endothelial lineage as detected by AcLDL-DiI incorporation (23.7±2.5% vs 14.5±1.8%; n=6, p<0.01). Finally, 48 hours CM-HMGB1 treatment stimulated CSC proliferation (16.8±2.2 x104 vs 11.9±1.0x104 cells/ml, n=5, P<0.05) and BrdU incorporation (6.9±1.5% vs 2.1±0.2% of BrdU positive nuclei, n=3, P<0.05). Importantly, HMGB1 alone had no significant effect on CSC BrDU incorporation (0.5±0.2% of BrdU positive nuclei, n=5) or differentiation into endothelial cells (10.6±1.5% of AcLDL-DiI positive cells).
Conclusions. HMGB1 stimulates growth factor, cytokine and chemokine release by cFbs which, in turn, modulate CSC proliferation and differentiation.