Working Toward Better Tissue Repair Therapies: Exposure of MSC to Hypoxic Conditions In Vitro Increases Levels of the Hepatocyte Growth Factor Receptor c-met, Improves Motility, and Induces Phosphorylation of the Pro-Survival Protein AKT.
Authors: Rosova Ivana , Meyerrose Todd E. , Nolta Jan A. 
Published: April 2020

Abstract
Necrosis, apoptosis, and fibrosis are characteristics of tissue damage/injuries such as cardiac ischemia and liver damage. In most instances, a loss of blood supply due to death of endothelial cells results, creating a hypoxic environment at the damage sites. In addition, a flux of growth factors and chemokines are induced as a “rescue” signal to recruit exogenous and/or proximal stem cells into proliferation and differentiation. One such soluble factor reported to have both mitogenic and motogenic effect on stem cells for liver and cardiac regeneration is the hepatocyte growth factor (HGF), also known as “scatter factor”. Our lab has previously demonstrated that administration of HGF in vivo following human hematopoietic stem cell transplantation into an immune deficient mouse model of liver injury greatly enhances recruitment of human stem cells to sites of liver damage (Wang et al, Blood 2003). In the current studies, we addressed the role of HGF in promoting human bone marrow-derived mesenchymal stem cells (MSC) to sites of tissue damage such as liver or cardiac ischemia. In addition to exploiting the beneficial effect of HGF, we also evaluated the possible additive effect of hypoxia in stem cell regeneration based on the following hypothesis - that exposure of MSC to hypoxic conditions prior to transplantation will enhance the levels of c-met and amplify the signaling cascades downstream of HGF/c-met. To answer the question of whether MSCs have increased motility in hypoxic conditions, human bone marrow derived MSC were cultured in hypoxic (2 to 3% oxygen) vs. normoxic conditions (20–21% O2) in the presence or absence of 25ng/ml HGF, and scratch tests were performed to assess the scattering potential of MSC. There was an increase in total c-met protein, by immunohistochemical analysis, and increased migration of MSC under hypoxic conditions with HGF, as compared to normoxic conditions with HGF. Protein studies were designed to measure c-met induction/stabilization and downstream signals following ligand binding. By immunoprecipitation followed by immunoblotting with specific phosphorylation antibodies, we showed that hypoxic conditions + HGF stimulation induced a higher level of total cellular phosphotyrosine activity in MSC. Downstream of HGF/c-met, we observed an amplification of AKT phosphorylation when comparing HGF stimulation under normoxic vs. hypoxic conditions. In contrast, MAPK phosphorylation was moderately, but not significantly, different between hypoxic vs. normoxic conditions. Our data from these functional and molecular studies suggest that pre-treatment of MSC under hypoxic conditions might not only increase c-met to enhance HGF-mediated chemotactic recruitment to sites of tissue damage but may also enhance the survival of these stem cells upon arrival at the damaged site, through increasing the levels of phosphorylation of the pro-survival protein AKT.