Airway basal cell vascular endothelial growth factor-mediated cross-talk regulates endothelial cell-dependent growth support of human airway basal cells

Abstract

Summary The human airway epithelium is a pseudostratified heterogenous layer comprised of ciliated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epithelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to express mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothelium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Endothelial MMP14 is required for endothelial dependent growth support of human airway basal cells

“…However, some ligands (FGF11-FGF14) do not bind FGFRs and have distinct modes of action (Olsen et al, 2003). Based on our prior work demonstrating crosstalk between endothelial cells and basal cells (Curradi et al, 2012), and the knowledge that FGF ligands play a role in regulating endothelial cell function, we assessed expression of FGF ligands in basal cells. RNA sequencing demonstrated that basal cells expressed only FGF2, FGF5, FGF11 and FGF13 (Fig.…”

“…Blocking of FGFR1 with a specific antibody suppresses endothelial-cell-dependent proliferation of basal cells Using our previously developed cytokine-and serum-free co-culture system, which utilizes modified primary human umbilical vein endothelial cells (HUVECs) constitutively expressing active Akt (HUVEC-Akt) (Curradi et al, 2012), we assessed the role of FGFR1-mediated signaling in regulating endothelial-cell-dependent proliferation of basal cells. Co-cultures of basal cells and endothelial cells were untreated, or incubated with control IgG or an antibody against FGFR1 (which blocks FGFligand-dependent signaling through this receptor) in the absence and presence of recombinant FGF2 or FGF5.…”

“…Western blot analysis was performed as described previously (Curradi et al, 2012) using NuPAGE 4 to 12% Bis-Tris gradient gels (Invitrogen). Primary antibodies against the following proteins were used: phosphorylated Akt (1:1000, catalog number 4060), Akt (1:1000, catalog number 9272), ERK1/2 (1:1000, catalog number 9102); phosphorylated ERK1/2 (1:1000, catalog number 9101); β-actin (1:1000; catalog number 4967) (all from Cell Signaling Technology), GAPDH (1:5000, catalog number SC-32233, Santa Cruz Biotechnology) and MMP14 (1:1000; catalog number ab51074, Abcam).…”

“…Co-culture assays were used to assess the ability of endothelial cells (HUVECAkt) to support basal cell proliferation in cytokine-and serum-free conditions as previously described (Curradi et al, 2012). To assess the role of FGFR1-mediated signaling on basal cell proliferation, human anti-FGFR1 neutralizing antibody (clone FR1-H7, ImClone, New York, NY) or IgG control was added at a final concentration of 1 µg/ml.…”

“…Previous work from our laboratory has demonstrated functional crosstalk between basal cells and endothelial cells, whereby endothelial cells, in response to basal-cell-secreted vascular endothelial factor A (VEGFA), could support growth of basal cells in co-culture (Curradi et al, 2012). The present study builds on these findings and demonstrates that, in addition to VEGFA, basal cells express multiple fibroblast growth factor (FGF) ligands, a family of growth factors known to regulate activation of endothelial cells (Presta et al, 2005;Lieu et al, 2011;Belov and Mohammadi, 2013;Carter et al, 2015).…”