Perlecan Domain V Induces VEGF Secretion in Brain Endothelial Cells Through ?5?1 Integrin Dependent Mechanism a Novel Insight in Brain Tissue Recovery Following Ischemia
Clarke, Douglas Nelson
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Stroke is the leading cause of long term disability and the third leading cause of death in the United States. Perlecan plays a significant role in brain development by sequestering and delivering growth factors to developing neuronal precursor cells in a neurovascular niche. Previous results demonstrated that perlecan proteolysis results in the cleavage of perlecan?s most C-terminal domain five (DV) in the post-ischemic brain. As post-stroke angiogenesis is an important step in post-stroke brain repair, I focused on the mechanism of DV?s role in brain angiogenesis in vitro. I first demonstrated that DV significantly increased brain endothelial (BE) cell migration, proliferation and tube-like formation suggesting DV is a pro-angiogenic factor for BE cells. I next investigated VEGF secretion from BE cells in the presence of DV. DV significantly increased VEGF secretion into the cell media, which was both dose and time dependent. Using quantitative real-time PCR, DV induced a maximal nine-fold increase in VEGF expression, compared to control, indicating DV is an upstream regulator of VEGF transcription. DV treated cells show an increase in phosphorylation of ERK-(1/2) that could be blocked by the pharmacological inhibitor U0126. This inhibitor could also block DV?s effect on VEGF mRNA expression and secretion indicating ERK is involved with DV?s effect on VEGF regulation. Optical sensor binding assays confirmed that DV binds to the ?5?1 integrin with a Kd of 160nM, and cells treated with DV showed a visual representation of integrin ?5?1-DV colocalization. Furthermore, shRNA-mediated knockdown of integrin ?5 blocked DV?s effect on VEGF mRNA expression, indicating integrin ?5 is involved with DV?s regulation of VEGF expression. In conclusion, these results demonstrate that DV has an unexpected proangiogenic effect in brain angiogenesis. This occurs via a previously unreported interaction between DV and the ?5?1 integrin, resulting in the activation of the ERK, eIF4A and HIF1? signaling pathway and an ultimate increase in VEGF mRNA expression and VEGF secretion. As DV is generated post-stroke, these results suggest a novel mechanism by which brain tissue recovery following ischemia is influenced by processed fragments from the extracellular matrix.