Vascular endothelial growth factor drives autocrine epithelial cell proliferation and survival in chronic rhinosinusitis with nasal polyposis

Abstract

Rationale: The pathogenesis of nasal polyps in chronic rhinosinusitis is poorly understood.

Objectives: These studies seek to implicate a functional role for vascular endothelial growth factor (VEGF) in perpetuating primary nasal epithelial cell overgrowth, a key feature of hyperplastic polyps.

Methods: Comparison of VEGF and receptor expression was assessed by ELISA of nasal lavage, immunohistochemistry of sinus tissue, flow cytometry of nasal epithelial cells, and ELISA of supernatants. VEGF-dependent cell growth and apoptosis were assessed with blocking antibodies to VEGF, their receptors, or small interfering RNA knockdown of neuropilin-1 by cell proliferation assays and flow cytometric binding of annexin V.

Measurements and main results: VEGF protein was sevenfold higher in nasal lavage from patients with polyposis compared with control subjects (P < 0.001). We also report elevated expression of VEGF (P < 0.012), receptors VEGFR2 and phospho-VEGFR2 (both P < 0.04), and identification of VEGF coreceptor neuropilin-1 in these tissues. Nasal epithelial cells from patients with polyps demonstrated faster growth rates (P < 0.005). Exposure of cells to blocking antibodies against VEGF resulted in inhibition of cell growth (P < 0.05). VEGF receptor blockade required blockade of neuropilin-1 (P < 0.05) and resulted in increased apoptosis (P < 0.001) and inhibition of autocrine epithelial VEGF production (P < 0.05).

Conclusions: These data demonstrate that VEGF is a novel biomarker for chronic rhinosinusitis with hyperplastic sinonasal polyposis that functions in an autocrine feed-forward manner to promote nasal epithelial cell growth and to inhibit apoptosis. These findings implicate a previously unrecognized and novel role of VEGF functioning through neuropilin-1 on nonneoplastic primary human airway epithelial cells, to amplify cell growth, contributing to exuberant hyperplastic polyposis.

Figure 1.

Comparison of soluble growth factors in nasal lavages of patients with untreated chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP), patients with CRSwNP 1 month postoperatively, patients with CRS alone, and normal control subjects. Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in nasal lavages were assayed according to the manufacturer's instructions (antibodies and kits from R&D Systems). The lower limit of detection was 5 pg/ml. Values represent the means and SEM. *P < 0.001 versus control, CRS alone, and CRSwNP postoperative (PostOP) subjects by Kruskal-Wallis test. Human subject data are shown in Table 1.

Figure 2.

Vascular endothelial growth factor (VEGF) is overexpressed by epithelial cells in sinonasal tissue. (A) Surgical sinonasal tissue from chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) mucosa (right, n = 5) and normal control sinus mucosa (left, n = 5) were immediately placed in 4% paraformaldehyde and processed for immunohistochemical staining of VEGF as described in Methods. Score shown in parentheses is the brown-colored staining intensity expressed as mean intensity × 10³ per cell ± SEM, determined with Image-Pro software. Each inset represents the matching IgG control. Data are representative of n = 5 subjects in each group and are shown at an original magnification of ×10. ^tP < 0.012 by Kruskal-Wallis test. (B) Flow cytometric analysis of cell surface VEGF on cultured primary nasal airway epithelial cells (PNECs) from normal control subjects and subjects with CRSwNP. *P < 0.05 versus control subjects by Kruskal-Wallis test. (C) Soluble VEGF (isoforms 165 and 121) in cell supernatants of PNECs were measured by ELISA according to the manufacturer's instructions (R&D Systems). Lower limit of detection was 5 pg/ml. **P < 0.02 versus control subjects by Kruskal-Wallis test.

Figure 3.

Receptors for vascular endothelial growth factor (VEGF) are abundantly expressed by epithelial cells in sinonasal tissue. (A) Expression of VEGF receptor-2 (VEGFR2), phospho-VEGFR2, and neuropilin-1 (NP1) by epithelial cells in sinonasal tissue. Surgical sinonasal tissue from chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) mucosa (n = 5) and normal control sinus mucosa (n = 5) were immediately placed in 4% paraformaldehyde and processed for immunohistochemical staining of VEGFR2, phospho-VEGFR2, and NP1, as described in Methods. Scores shown in parentheses represent the brown-colored staining intensity expressed as mean intensity × 10³/cell ± SEM determined with Image-Pro software. Data are shown at an original magnification of ×10. *P < 0.04 versus control by Kruskal-Wallis test, n = 6 for each group. (B) Flow cytometric analysis of cell surface NP1 expression on primary nasal airway epithelial cells (PNECs) from normal control subjects and subjects with CRSwNP.

Figure 4.

Primary nasal airway epithelial cells (PNECs) from subjects with chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) display faster growth rates in vitro. (A) Comparison of time to confluence of cultures of PNECs from normal control subjects and subjects with CRSwNP. PNECs from normal control subjects and subjects with CRSwNP were cultured as described in Methods. Results are expressed as number of days to confluency of culture from day of seeding into 6-well plates and were assessed in triplicate. (B) Comparison of cell proliferation rates of PNECs from normal control subjects and subjects with CRSwNP. Cell number was determined by normalization of DNA content to a standard curve, using the CyQUANT cell proliferation assay as described in Methods. Each circle indicates an experiment from a single donor. Bars represent the mean. *P < 0.008 or **P < 0.005 versus control group by Kruskal-Wallis test.

Figure 5.

Sinonasal epithelial cell growth is vascular endothelial growth factor (VEGF) dependent. (A) Effects of anti-VEGF antibody and recombinant epidermal growth factor (EGF) exposure on growth rates of primary nasal airway epithelial cells (PNECs) from subjects with chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP). (B) Effects of anti–neuropilin-1 (NP1), anti–VEGF receptor-1 (VEGFR1), and anti-VEGFR2 antibody exposures on growth rates of PNECs from subjects with CRSwNP. (C) Effects of combination of anti-NP1, anti-VEGFR1, and anti-VEGFR2 antibody exposures on growth rates of PNECs from subjects with CRSwNP. PNECs from subjects with CRSwNP were seeded at 5,000 cells per well of a 96-well plate and exposed to the designated blocking antibodies, recombinant EGF (R&D Systems), or IgG control antibody as described in Methods. Cell number was determined after normalization of DNA fluorescence to a standard curve, using the CyQUANT cell proliferation assay as described in Methods. Each curve represents the mean of five experiments, each from an individual donor with CRSwNP. Each condition and time point was measured in quadruplicate. The SEM ranged between 12 and 27% of the mean. *P < 0.05, **P < 0.02, versus control condition at 96 hours by analysis of variance and post-hoc Bonferroni test.

Figure 6.

Functional blocking of neuropilin-1 (NP1) results in apoptosis. Primary nasal airway epithelial cells (PNECs) from subjects with chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) (n = 5) were grown to 90% confluence and incubated for 48 hours with blocking antibodies to NP1 (1 μg/ml), vascular endothelial growth factor receptor-1 (VEGFR1) (R1, 10 μg/ml), VEGFR2 (R2, 1 μg/ml), IgG control (1 μg/ml), or medium control and processed for flow cytometric analysis of annexin V–fluorescein isothiocyanate staining (R&D Systems). Results in (A) are expressed as mean (and SEM) percentage of cells staining positive for annexin. (B) Light microscopic views of PNECs (original magnification, ×20). Open arrowheads point to cell membrane blebs. *P < 0.001, **P < 0.02, ***P < 0.05 versus control condition by analysis of variance and post-hoc Bonferroni test.

Figure 7.

Functional blocking of neuropilin-1 (NP1) results in inhibition of autocrine vascular endothelial growth factor (VEGF) expression by primary nasal airway epithelial cells (PNECs). PNECs from subjects with chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) (n = 3) were incubated for 48 hours with blocking antibodies to NP1 (1 μg/ml), VEGF receptor-1 (VEGFR1) (R1, 10 μg/ml), VEGFR2 (R2, 1 μg/ml), IgG control (1 μg/ml), or medium control. Cell supernatants were then harvested and assayed for VEGF by ELISA as described in Methods. *P < 0.05 versus control condition by analysis of variance and post-hoc Bonferroni test.

Figure 8.

Small interfering RNA (siRNA) knockdown of neuropilin-1 (NP1) results in inhibition of autocrine vascular endothelial growth factor (VEGF) expression by primary nasal airway epithelial cells (PNECs). PNECs from subjects with chronic rhinosinusitis with hyperplastic sinonasal polyposis (CRSwNP) were transfected with three nonoverlapping siRNA sequences against NP1 or scrambled negative control for 24 hours as described in Methods. Untransfected medium control was also used (Control). (A) Flow cytometric analysis of cell surface VEGF. Data represent the mean fluorescence intensity (MFI) and SEM of n = 4 experiments. *P < 0.05 versus medium control by ANOVA with post-hoc Bonferroni test. (B) Fluorescence and matching light microscopy micrographs of PNECs transfected with rhodamine-tagged control siRNA. (C) Real-time polymerase chain reaction analysis of NP1 mRNA. (D) Flow cytometric analysis of cell surface NP1. Data represent means and SEM of n = 3 experiments. Analysis of variance with post-hoc Bonferroni test resulted in **P < 0.02 for all three NP1 siRNAs versus medium control or negative control siRNA (siControl). ***P < 0.05 for siRNA1 (siNP1 #1)or siRNA2 (siNP #2) for NP1 versus medium control. ⁺P < 0.05 for siRNA2 for NP1 versus negative control siRNA.