Inhibition of elastin peptide-mediated angiogenic signaling mechanism(s) in choroidal endothelial cells by the α6(IV)NC1 collagen fragment

Abstract

Purpose: The inhibitory effects and mechanism(s) of type IV collagen α-6 chain-derived noncollagenous domain (α6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)-activated choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloproteinase (MT1-MMP) activation are explored.

Methods: Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for different time intervals with or without α6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and α6(IV)NC1 treatments. The MCECs also were subjected to EDPs, and specific inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) phosphorylation.

Results: Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting the proliferation of MCECs. The α6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by α6(IV)NC1. Further, EDP-activated MT1-MMP expression and FAK/phosphoinositide-3-kinase (PI-3K)/mammalian target of rapamycin (mToR)/Akt phosphorylation in MCECs, were reduced by α6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK- and Akt-specific inhibitors.

Conclusions: The EDPs and α6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The α6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates α6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye.

Keywords: angiogenesis; elastin-derived peptides; noncollagenous domains of type IV collagen.

Figure 1

Inhibition of MCEC survival by α6(IV)NC1. (A) Upper graph showing survival of MCECs without polymyxin-B treatments. (B) Lower graph showing MCECs treated with polymyxin-B. Status bars indicate average cell survival of MCECs (mean ± SD of 3 replicates) after 48 hours of treatment. Presence (+) and absence (−) of factor in respective treatments are indicated.

Figure 2

Inhibition of MCEC migration by α6(IV)NC1. Photographs showing MCECs from the underside of Boyden chamber membrane. Controls indicate cells migrating in EBM-2 (arrowheads). Relatively higher migration exhibited by MCECs towards wells containing (A) κE (upper right), (B) ME (upper right), and (E) VGVAPG (upper right) used as positive controls; and lowered migration exhibited by MCECs in treated wells containing 0.5 and 1.0 μM α6(IV)NC1 along with (A, C, E) EDPs (lower panels). Fields represent 5 of the replicates as observed at ×200 magnification with Olympus CK2 light microscope. Data graph representation of MCEC migration in presence or absence of EDPs and α6(IV)NC1 (B, D, F). Experiments were performed with three replicates and data in the graphs are represented as mean ± SD. Presence (+) and absence (−) of factor in respective treatments are indicated.

Figure 3

The α6(IV)NC1 inhibits EDP-enhanced in vitro wound closure. (A–H) Wells showing migration of MCECs into artificial scratch wound made on the surface of culture wells at 48 hours. Migration of cells to words scratch wound was higher in 2 μg/mL κE (A, E), 2 μg/mL ME- (B, F), and 200 ng/mL BP- (C, G), 2% serum medium + 1.0 μM α6(IV)NC1 (D, H)–treated wells, and relatively lower in 1.0 μM α6(IV)NC1-treated wells (2 μg/mL κE + 1.0 μM α6[IV]NC1 [I, L], 2 μg/mL ME + 1.0 μM α6[IV]NC1 [J, M], and 200 ng/mL BP + 1.0 μM α6[IV]NC1 [K, N]), respectively (black bars in the scratch wound indicate 0.5 mm). Average gap covered by MCECs in 48 hours in each treatment is represented in graphic format (O). Status bars (O) indicate difference between initial and final gap measured at three equidistant points in each image, represented as mean ± SD. Presence (+) and absence (−) of factor in respective treatments are indicated.

Figure 4

Inhibition of MCEC tube formation by α6(IV)NC1. Micrographs indicate incomplete and thin tubes in control wells containing only (A, C, E) EGM-2 (upper left panels), thick and higher number of tubes formed in presence of (A) κE (upper right), (C) ME (upper right), and (E) VGVAPG (upper right), respectively. Fragmented tubes and clumping of MCECs with incomplete tube formation evident in presence of 0.5 and 1.0 μM α6(IV)NC1 along with (A, C, E) EDPs (lower panels). Graphic representation of average number of tubes formed by MCECs in presence or absence of EDPs and α6(IV)NC1, represented in (B, D, F), respectively. Status bars indicate average of three measurements for each treatment represented as mean ± SD. Presence (+) and absence (−) of factor in respective treatments are indicated.

Figure 5

The α6(IV)NC1 inhibits EDP enhanced MT1-MMP levels in MCECs. Western blot analyses showing MT1-MMP levels in MCEC cell lysates treated in media containing combination of (A) κE and α6(IV)NC1 (upper lane), and (B) ME or BP and α6(IV)NC1 (upper lane), respectively. Presence (+) and absence (−) of factor in respective treatments are indicated. Lower Western blots in (A, B); β-actin is shown as loading control.

Figure 6

Inhibition of different bioactive peptides promoted kinase signaling in MCECs by α6(IV)NC1. (A) Inhibition of κ-E promoted kinase signaling in MCECs by α6(IV)NC1. Western blot analyses showing bands corresponding to p-FAK, p-Akt, p-mToR, and p-PI3K in MCEC lysates subjected to combinations of κE and α6(IV)NC1 treatments. Presence (+) and absence (−) of factor in respective treatments are indicated. (B) Inhibition of ME and BP promoted kinase signaling in MCECs by α6(IV)NC1. Western blot analyses showing bands corresponding to p-FAK, p-Akt, p-mToR, and p-PI3K in MCEC lysates subjected to combinations of ME, BP, and α6(IV)NC1 treatments. Presence (++/+) and absence (−) of factor in respective treatments are indicated. (A, B) GAPDH is shown as loading control.

Figure 7

Inhibition of κE- and BP-enhanced kinase signaling in MCECs by synthetic FAK and Akt inhibitors. (A) Upper Western blots showing bands corresponding to p-FAK from MCEC lysates subjected to κE, BP, and Y15 treatments. (B) Upper Western blots showing bands corresponding to p-Akt from MCEC lysates subjected to κE, BP, and GSK69093 treatments. (A, B) Presence (++/+) or absence of factor in respective treatments is indicated. Lower Western blots in (A, B) show GAPDH as loading control.