VEGF Blockade by Soluble VEGF Receptor-2 Improves the in vitro Chondrogenic Capacity of Human Mesenchymal Stem Cells and Nasal Chondrocytes

Introduction Vascular Endothelial Growth Factor (VEGF) is involved in regulating cartilage growth and endochondral ossification during growth plate development or in cartilage degenerative processes, e.g. in osteoarthritis or following mechanical overloading. Instead, VEGF expression is suppressed during hyaline cartilage formation and maintenance. It was previously proposed that in cartilage physiology and pathology VEGF could be involved not only in regulating angiogenesis, but also in autocrine signals. Based on these observations, in the present study we tested the hypothesis that blockade of the endogenous VEGF pathway will improve in vitro chondrogenesis. For this purpose, human bone marrow-derived mesenchymal stem cells (BMSC) and nasal chondrocytes (NC) were transduced to express soluble VEGF receptor-2 (sFlk-1) and cultured in hypoxic culture conditions, known to upregulate VEGF expression. Materials and Methods Cells were transduced with a retroviral vector carrying the cDNAs for sFlk-1 and a truncated form of CD8a as a cell-surface marker, or with a control vector carrying only CD8a. For each group a CD8a-positive population was FACSsorted. The functionality of the secreted sFlk-1 was tested by using human umbilical vein endothelial cells (HUVEC). The chondrogenic differentiation of BMSC or NC was tested in 3D pellet culture at 2% of O2 for 2 weeks . Results BMSC and NC were successfully transduced and sorted and the secreted sFlk-1 was functional since in its presence the mitogenic activity of VEGF concentrations up to 10 ng/mL on HUVEC was specifically inhibited (Fig. 1). Pellets generated from BMSC had higher GAG deposition when cells were transduced with sFlk-1. The pellets generated by NC always had higher GAG/DNA ratio than BMSC, but this was also improved by sFlk-1 production compared to naïve or CD8-transduced cells (1.6and 1.4-fold, respectively) (Fig. 2).