Perlecan Domain V induces VEGf secretion in brain endothelial cells through integrin α5β1 and ERK-dependent signaling pathways.

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α(5)β(1) integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV\u27s angio-modulatory activity outside of the brain, binds poorly to α(5)β(1) and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV\u27s DGR sequence as an important element for the interaction of DV with α(5)β(1). Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV\u27s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV\u27s mechanism of action on BECs, and further support its potential as a novel stroke therapy

DV induces HIF-1α and increases eukaryotic initiation factor 4E.

<p>(A) HIF-1α immunoblot (upper) and OD quantification (lower) following DV exposure. Note the transient HIF-1α band at 5 minutes. N = 3, *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 in comparison between vehicle and DV treated cells. (B) Immunoblots (upper) of eukaryotic initiation factor 4E phosphorylation (peIF4E) and total eIF4E after treatment of BECs with combinations of DV, LY294002 and AktIV as labeled. Cells were treated with DV for 5 min. OD quantification (lower) of peIF4E as normalized to total eIF4E. N = 3, **<i>P</i><0.01 versus untreated cells, ^##<i>P</i><0.01 within LY294002 treated-group (C) Immunoblots (upper) of peIF4E and eIF4E from BECs treated with combinations of DV and U0126 as labeled demonstrating that U0126 significantly abolished DV-induced peIF4E hyperphosphorylation as OD quantified (lower). Cells were treated with DV for 5 min. N = 3, **<i>P</i><0.01 versus control, ^##<i>P</i><0.01 versus DV-treated group. (D) Immunoblots (upper) of phosphorylated c-jun (pc-jun) and total c-jun from BECs treated with DV +/− U0126 demonstrating that U0126 significantly inhibited DV-induced c-Jun phosphorylation as OD quantified (lower). Note the significant increase after 30 min following DV treatment. N = 3, **<i>P</i><0.01 in comparison to untreated cells, ^##<i>P</i><0.01 between U0126-treated and untreated cells.</p

DV increases both Akt and ERK phosphorylation.

<p>(A) Immunoblots (upper) and OD quantification (lower) for phosphorylated Akt (pAkt) and pan-Akt (Akt) from BECs in the presence of DV and/or 10 µM LY294002 (PI3K inhibitor). N = 3, *<i>P</i><0.05 and **<i>P</i><0.01 versus 0 min timepoint, ^##P<0.01 versus DV-only treated group (vehicle) (B) Immunoblots (upper) and OD quantification (lower) for pAkt and Akt in BECs treated with 10 µM AktIV (Akt inhibitor). N = 3, *<i>P</i><0.05 and **<i>P</i><0.01 versus 0 min timepoint. (C) Immunoblots (upper) and OD quantification (lower) for phosphorylated ERK (pERK) and pan-ERK (ERK) in BECs treated with 10 µM LY294002. N = 3, **<i>P</i><0.01 versus 0 min timepoint, ^#<i>P</i><0.01 versus DV-only treated group (vehicle). (D) Immunoblots (upper) and OD quantification (lower) for phosphorylated ERK (pERK) and pan-ERK (ERK) in BECs treated with 10 µM AktIV. N = 3, **<i>P</i><0.01 versus 0 min timepoint, ^#<i>P</i><0.01 versus DV-only treated group (vehicle).</p

DV increases VEGF-A at mRNA and protein level through an ERK-dependent pathway.

<p>(A) qPCR analysis of VEGF-A mRNA in BECs +/− DV and +/−10 µM LY294002 or 10 µM AktIV. GAPDH was used as internal control. N = 3, ***<i>P</i><0.001 in comparison between DV-treated and untreated cells, ^##<i>P</i><0.01 in comparison between inhibitors and untreated groups. (B) Similar analysis as in (A) demonstrating that 10 µM U0126 (MEK inhibitor) treatment significantly blunted DV-induced VEGF-A mRNA up-regulation. N = 3. ***<i>P</i><0.001 in comparison between DV-treated and untreated cells. (C) Secreted VEGF ELISA profile from BECs following treatment with DV, LY294002, or AktIV alone or in combination Note the significant up-regulation of secreted VEGF following DV, LY294002 or AktIV treatment. N = 3, ***<i>P</i><0.001 in comparison between DV-treated and untreated cells, ^##<i>P</i><0.01 in comparison between LY and AktIV treated groups versus untreated group. (D) U0126 treatment significantly inhibited VEGF secretion from BECs following DV treatment. N = 3. ***<i>P</i><0.001 in comparison between DV-treated and untreated cells.</p

DV binding to α₅β₁ is partially mediated through its DGR sequence.

<p>(A) Sequence schematic of the second EGF repeat within DV demonstrating the exact location, highlighted in yellow, where D3904A DV was mutated. (B) Coomassie stain of SDS PAGE of D3904A DV and LG3 protein preparations (left image for each as labeled) and anti-his immunoblot (right image for each as labeled) to recognize the 6xHis-tag present in the recombinant purified D3904A DV and LG3 (DV C-terminal fragment). In both analyses, D3904A DV yields a single 82 kDa band as does wild-type DV (not shown), and LG3 yields a single 25 kDa band. (C) Optical biosensor traces showing the association and dissociation of D3904A DV with immobilized α₅β₁ integrin at the concentrations listed (RU = relative units). (D) Quantification of proliferation of BECs after 24 h ± wild-type DV or D3904A DV at 100 nM or 300 nM concentrations in serum starved media as measured via MTS assay. N = 3, *<i>P</i><0.05 at 100 nM, <i>P</i> = 0.06 at 300 nM. (E) Optical biosensor traces showing the association and dissociation of LG3 with immobilized α₅β₁ integrin at the concentrations listed (RU = relative units). (F) LG3 (different concentrations of LG3 as labeled) BEC 24 h proliferation assay.</p

DV enhances brain endothelial cell capillary tube-like structure formation in neuronal co-culture in a VEGF-dependent fashion.

<p>(A) BECs or mouse dermal endothelial cells (DECs, red) were cultured on a bed of rat cerebellar granule neurons (green) in serum free medium for 6 hours +/− PBS vehicle control, VEGF, DV, or DV+VEGF neutralizing antibody. Scale bar is 50 µm. (B) Quantification of capillary tube-like structures as in A. *P<0.05 compared to corresponding control. (C) and (D) Quantification of percent BEC proliferation after 24 h (in reference to control, PBS vehicle treated condition arbitrarily set at 100%) treated as labeled. ***P<0.001.</p