Poly-N-acetyl glucosamine nanofibers regulate endothelial cell movement and angiogenesis: dependency on integrin activation of Ets1

Abstract

Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-derived materials effectively achieve hemostasis during surgical procedures. Treatment of cutaneous wounds with pGlcNAc in a diabetic mouse animal model causes marked increases in cell proliferation and angiogenesis. We sought to understand the effect of the pGlcNAc fibers on primary endothelial cells (EC) in culture and found that pGlcNAc induces EC motility. Cell motility induced by pGlcNAc fibers is blocked by antibodies directed against alphaVbeta3 and alpha5beta1 integrins, both known to play important roles in the regulation of EC motility, in vitroand in vivo. pGlcNAc treatment activates mitogen-activated protein kinase and increases Ets1, vascular endothelial growth factor (VEGF) and interleukin 1 (IL-1) expression. pGlcNAc activity is not secondary to its induction of VEGF; inhibition of the VEGF receptor does not inhibit the pGlcNAc-induced expression of Ets1 nor does pGlcNAc cause the activation of VEGF receptor. Both dominant negative and RNA interference inhibition of Ets1 blocks pGlcNAc-induced EC motility. Antibody blockade of integrin results in the inhibition of pGlcNAc-induced Ets1 expression. These findings support the hypothesis that pGlcNAc fibers induce integrin activation which results in the regulation of EC motility and thus in angiogenesis via a pathway dependent on the Ets1 transcription factor and demonstrate that Ets1 is a downstream mediator of integrin activation.

Fig. 1

pGlcNAc treatment prevents cell death and stimulates EC motility. a EC were serum starved for 48 h (SS-48) in the presence or absence of pGlcNAc (NAG), at the concentrations indicated, or VEGF (20 ng/ml). ‘Control’ indicates the number of cells plated. Cell counts were performed using a hemacytometer. b ‘Scratch’ wounding assays were performed under conditions of serum starvation (SS) in the presence or absence of VEGF (20 ng/ml) or pGlcNAc (NAG) at 5 and 10 μg/ml. Photographs (×10) were taken, and the distance migrated into the wound was calculated using 6 different fields, 3 independent times. c Quantitation of transwell assays of EC migrating toward fibronectin under conditions of serum starvation (SS) in the presence or absence of VEGF (20 ng/ml) or pGlcNAc (NAG) at the concentrations indicated or with the addition of both (VEGF at 20 ng/ml and pGlcNAc at 5 and 10 μg/ml). Each assay was performed in triplicate, 3 independent times. d EC were plated onto growth factor-reduced Matrigel-coated plates under serum-starved conditions and treated with or without VEGF (20 ng/ml) or pGlcNAc (NAG, 10 μg/ml) and assessed for induction of cord formation within 6 h after plating. Matrigel assays were performed in duplicate, at least 3 independent times.

Fig. 2

pGlcNAc treatment stimulates MAPK and Ets1 expression. a Western blot analysis of total protein isolated from serum-starved EC (SS) treated with VEGF (V, 20 ng/ml) or pGlcNAc (NAG,10 μg/ml) for 15 min. Blots were probed with antibodies against phosphorylated Erk1 and Erk2 (P-Erk1/2). Blots were reprobed with antibodies against total Erk1 and Erk2. b Semiquantitative RT-PCR of total RNA isolated from serum-starved EC (SS) treated with or without VEGF (V) or pGlcNAc (NAG, 10 μg/ml) for 3 h. Primers were directed against Ets1, metallothionein 2A (MT) and S26 as an internal control. c QPCR of total RNA as described in b and assessed for Ets1 expression. d Western blot analysis of total protein isolated from serum-starved EC (SS) treated with pGlcNAc (NAG, 5 and 10 μg/ml) for 3 h and probed using antibodies directed against Ets1 and the p85 subunit of PI3 kinase as an internal control.

Fig. 3

pGlcNAc activity is independent of its induction of VEGF expression. a Semiquantitative RT-PCR of total RNA isolated from serum-starved EC (SS) treated with or without pGlcNAc (NAG, 5 or 10 μg/ml) for 12 h. Primers were directed against VEGF, IL-1, IL-8 and S26. The graph represents QPCR confirming the pGlcNAc-induction of VEGF and IL-1. b QPCR of total RNA isolated from serum-starved cells (SS) either stimulated with VEGF (V, 20 ng/ml) or pGlcNAc (NAG, 10 μg/ml) following a 15-min pretreatment with SU5416 (10 μm) using primers directed against Ets1 and S26 as an internal control. QPCR was performed at least in duplicate, 2 independent times. c Western blot analysis of total protein isolated from serum-starved EC (SS) treated with VEGF (20 ng/ml) or pGlcNAc (NAG,10 μg/ml) for the times indicated. Blots were probed with antibodies against phosphorylated VEGFR2 (P-VEGFR2) followed by antibodies against total VEGFR2 and p85 as an internal control.

Fig. 4

pGlcNAc-induced cell motility is integrin dependent. a Quantitation of the number of cells migrated through transwells coated with fibronectin after pretreatment with antibodies (ab) directed against αVβ₃ or α₅β₁ (CD49e) for 15 min prior to stimulation with pGlcNAc (NAG, 10 μg/ml). SS = Serum-starved EC. b As in a, except the transwells were coated with vitronectin. Each assay was performed in triplicate, 3 independent times. c Western blot analysis of total protein isolated from serum-starved EC (SS) treated with pGlcNAc (NAG, 10 μg/ml) for the times indicated. Blots were probed with antibodies (ab) against phosphorylated FAK (P-FAK) and the p85 subunit of PI3 kinase as a loading control.

Fig. 5

pGlcNAc-induced cell motility is Ets dependent. a Quantitation of the number of cells migrated through transwells coated with fibronectin after transfection with either dn-Ets or with Ets1 RNAi (siEts1), plated under serum-starved conditions (SS) and stimulated with pGlcNAc (NAG, 10 μg/ml). Each assay was performed in triplicate, 3 independent times. b Western blot analysis of Ets1 expression in cells transfected with increasing amounts of a dn-Ets expression plasmid. c Semiquantitative RT-PCR of total RNA isolated from cells transfected with 2 different amounts of Ets1 small interfering RNA (siEts1). S26 is used as a loading control. C = ■■■■.

Fig. 6

Ets1 expression is regulated by integrin activation. a Semiquantitative RT-PCR of total RNA isolated from serum-starved EC (SS) pretreated with or without antibodies directed against α₅β₁ or αVβ₃ or with normal rabbit serum (NR) prior to stimulation with pGlcNAc (NAG, 10 μg/ml) and performed using primers directed against Ets1 or S26 as an internal control. b Western blot analysis of total protein from cells treated similarly to those in a and probed with an antibody directed against Ets1 and p85 as an internal control.