Abstract 5443: Osteopontin Stimulates Secretion of Angiogenic Proteins in Endothelial Progenitor Cells
Patients with Type 1 Diabetes Mellitus (T1DM) often demonstrate an ineffective angiogenic response to ischemia. This may be due to a decrease in the number and function of their endothelial progenitor cells (EPC). EPC are promising cells for autologous cell transplantation in the treatment of T1DM hence it is imperative to understand the underlying cause of their dysfunction. Therefore, we identified proteins that are dysregulated in EPC from patients with T1DM. Osteopontin (OPN), a secreted protein involved in cell migration, cell survival, and tumour growth was found to be down regulated. Thus, we hypothesized that OPN down regulation in T1DM EPC contributes to a decreased angiogenic response. To elucidate the role of OPN in EPC, EPC were isolated from OPN knockout (KO) mice and wild type mice (WT) for a matrigel tubule assay to assess in vitro angiogenic potential. KO EPC induced significantly less tubule formation than WT EPC (p<.05, n=3). Knockout EPC that were pre-incubated with recombinant OPN induced tubule formation at levels similar to WT and significantly higher than KO cells that were not incubated with OPN (p<.05, n=3). Further, conditioned media (CM) from WT cells induced tubule formation to the same levels as the EPCs themselves suggesting that secreted proteins are responsible for the angiogenic effect. Interestingly, when KO EPC were pre-incubated with OPN, washed, and fed with new media the CM media induced tubule formation to WT levels(n=3), even though there was no OPN directly in the media. Hence, we further hypothesized that OPN is acting on EPC to induce the secretion of angiogenic cytokines. To test this, a chemi-array was performed on EPC from WT, KO and KO exposed to OPN. WT EPCs expressed FGFα at a much higher level than KO cells. Expression of FGF was restored to WT levels when KO cells were pre-incubated with OPN. Further, WT cells expressed Il-6 and TGF-α whereas KO cells did not express these proteins at detectable levels. Interestingly, when KO cells are exposed to OPN both IL-6 and TGF-α are expressed at WT levels. Taken together, this data suggests that OPN expression increases the angiogenic potential of EPCs via an autocrine mechanism whereby OPN is secreted by the EPC and subsequently induces the expression of a variety of angiogenic proteins.