Zinc chloride affects chondrogenesis via VEGF signaling

Insulin mimetics, including zinc containing compounds, have previously been shown to influence chondrogenesis as it relates to healing of fractures in various preclinical models. However, the mechanism by which these compounds drive chondrogenic differentiation is yet undefined. Here, via next-generation sequencing (NGS) and in vitro functional validation, we show that Zinc Chloride (ZnCl₂) induces expression of both chondrogenic genes (Sox9, Runx1, collagen) as well as genes associated with VEGF-mediated signal transduction, including VEGF receptors 1 and 2 and their ligands; VEGF-A and VEGF-B. Noticeably, although insulin was able to also induce expression of these pro-angiogenic and pro-chondrogenic genes, the impact of insulin on expression of VEGF receptor and ligand genes was marginal when compared to that of ZnCl_2. Furthermore, while the VEGFR antagonist, Axitinib, was able to attenuate the pro-chondrogenic effects of both insulin and ZnCl₂; a reduction in gene and protein expression was most profoundly observed when the antagonist was applied to cells treated with ZnCl_2. Taken together, these data suggest an important role for the VEGF-mediated signal transduction pathways in the positive effects observed when applying zinc-based compounds as adjuvants for chondrogenesis-mediated fracture healing. In this regard, further mechanistic evaluation of ZnCl₂ and other zinc-containing insulin mimetics may support rational design of therapies targeted for disease indications associated with impaired fracture healing.