Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0–20 μg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR. Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium. Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.