Interaction between mesenchymal stem cells and endothelial cells restores endothelial permeability via paracrine hepatocyte growth factor in vitro

Qi-Hong Chen, Ai-Ran Liu, Hai-Bo Qiu, Yi Yang

Mesenchymal stem cells (MSCs) have potent stabilising effects on vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secrete more factors by MSC-endothelial cell (MSC-EC) interactions. We hypothesised that MSC-EC interactions restore endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF. We investigated the endothelial permeability induced by LPS under two co-culture conditions. Human pulmonary microvascular endothelial cells (HPMECs) were added into the upper chambers of cell-culture inserts, while two different co-culture conditions were used in the lower side of the transwells, as follows: 1) MSC-EC interaction group: MSCs and HPMECs contact co-culture; 2) MSC group: MSCs only. The endothelial paracellular and transcellular permeability in the upper side of transwells were detected. Then, the concentration of HGF was measured in the culture medium using an Enzyme Linked Immunosorbent Assay (ELISA) kit, followed by neutralisation of HGF with anti-HGF antibody in the co-culture medium. In addition, Adherens junction and cytoskeleton proteins expression were measured by Western blot and immunofluorescence. HPMECs proliferation was analyzed by Bromodeoxyuridine incorporation assay. The paracellular permeability significantly increased after LPS stimulation in a dose-dependent and time-dependent manner. Meanwhile, MSC-EC interaction more significantly decreased endothelial paracellular and transcellular permeability induced by LPS. Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group. However, neutralising HGF with anti-HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability. Compared to the MSC group, MSC-EC interaction increased VE-cadherin and occludin protein expression, reduced caveolin-1 protein expression in HPMECs and restored remodelling of F-actin and junctional localisation of vascular endothelial cadherin (VE)-cadherin. Furthermore proliferation ratio in MSC-EC interaction group was higher than that of MSC group. However, the effects of MSCs was significantly blocked by anti-HGF antibody. These data suggested that MSC-EC interaction decreased endothelial permeability induced by LPS, which was mainly attributed to HGF secreted by MSCs. The main mechanisms by which HGF restored the integrity of endothelial monolayers was remodelling of endothelial intercellular junctions, decreasing caveolin-1 protein expression and inducing proliferation in HPMECs.