ECs express a number of integrin receptors.
34 These include 4x β1 (α1, α2, α3, and α5), αvβ3, and αvβ5 integrins. FN is recognized by α5β1 and α
Vβ3. The remaining integrins expressed by ECs interact with other matrix proteins such as collagen (α1β1, α2β1), laminin (α1β1, α2β1, α3β1), and vitronectin (αvβ5). The sequence of FN recognized by α5β1 and α
Vβ3 integrins is a tripeptide (Arg-Gly-Asp; RGD).In addition to the RGD site, α5β1 can also bind FN through a proximal site (32 Å; RGD).
35 We used a combination of antibodies and integrin-binding mimetics to determine whether ED-B FN increases VEGF expression through the engagement of integrins. Pretreatment of ECs with either α2β1 or α5β1 antibody prevented HG-induced and ED-B peptide-induced increase in VEGF mRNA and protein (
Fig. 3). Pretreatment of HG- and ED-B peptide-exposed cells with IgG isotype control produced no changes at 0.5 μg/mL concentration (data not shown). Because of the limitation of reagents available for specific α-β integrin subunit combinations, we proceeded to test the involvement of integrins by using specific neutralizing antibodies against the β subunits (β1, β3, and β5) that inhibit the function of the integrin regardless of the α subunit partner. Our results showed that only β1 integrin neutralization prevents the increase in VEGF (Figs.
4a,
4b). Next, we determined whether β1 integrin inhibition would prevent VEGF expression by immobilized ED-B FN. To do this, we coated the culture plates with 1 μg/mL ED-B peptide and seeded the cells. Addition of β1 neutralizing antibody prevented VEGF expression (
Fig. 4c). No changes were observed in cells treated with β3 or β5 neutralizing antibodies.
We wanted to know whether changes we saw in VEGF were due to altered subcellular distribution of β1 integrin. We then performed immunofluorescence staining for β1 integrin in ECs exposed to HG. Our results showed no significant changes in the localization of β1 integrin upon HG exposure (
Fig. 4d). Finally, we knocked down the expression of β1 integrin in ECs and observed similar results as with the neutralizing antibodies (
Figs. 5a,
5b). Both HG- and ED-B peptide-mediated VEGF expression were completely normalized when β1 integrin was silenced.
So far, our results showed that HG increases ED-B FN, which then leads to VEGF expression through the β1 integrin. To show that these changes were mediated by direct binding of ED-B FN to integrin, we used integrin-binding peptides to competitively block the interaction between β1 integrin and ED-B FN. DGEA peptide is recognized by the α2β1 integrin, and the RGDS peptide is recognized by α5β1. Pretreatment with these peptides prevented HG-induced increase in VEGF mRNA and protein (
Figs. 6a,
6b).In contrast, KDGE (scrambled peptide; negative control for DGEA) showed no effect on VEGF expression at the mRNA or protein level (
Figs. 6a,
6b). As a functional readout of these effects, we assayed for tube formation by ECs. Exposure of ECs to HG increases tube morphogenesis (branching), which was prevented by DGEA and RGDS peptide, but not KDGE peptide (
Fig. 6c). These results confirmed that the interaction between ED-B FN and α2β1/ α5β1 was necessary for the increased VEGF expression.