Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity - PubMed (original) (raw)

Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity

Elma Kurtagic et al. Am J Physiol Lung Cell Mol Physiol. 2009 Mar.

Abstract

Excessive neutrophil elastase (NE) activity and altered vascular endothelial growth factor (VEGF) signaling have independently been implicated in the development and progression of pulmonary emphysema. In the present study, we investigated the potential link between NE and VEGF. We noted that VEGF(165) is a substrate for NE. Digestion of purified VEGF(165) with NE generated a partially degraded disulfide-linked fragment of VEGF. Mass spectrometric analysis revealed that NE likely cleaves VEGF(165) at both the NH(2) and COOH termini to produce VEGF fragment chains approximately 5 kDa reduced in size. NE treatment of VEGF-laden endothelial cell cultures and smooth muscle cells endogenously expressing VEGF generated VEGF fragments similar to those observed with purified VEGF(165). NE-generated VEGF fragment showed significantly reduced binding to VEGF receptor 2 and heparin yet retained the ability to bind to VEGF receptor 1. Interestingly, VEGF fragment showed altered signaling in pulmonary artery endothelial cells compared with intact VEGF(165). Specifically, treatment with VEGF fragment did not activate extracellular-regulated kinases 1 and 2 (ERK1/2), yet resulted in enhanced activation of protein kinase B (Akt). Treatment of monocyte/macrophage RAW 264.7 cells with VEGF fragment, on the other hand, led to both Akt and ERK1/2 activation, increased VEGFR1 expression, and stimulated chemotaxis. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment and activation via VEGF receptor 1.

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Figures

Fig. 1.

125I-labeled vascular endothelial growth factor (VEGF) degradation by neutrophil elastase (NE). A: 0.82 nM 125I-VEGF was incubated with and without 20 μg/ml NE in 44 mM sodium bicarbonate buffer, pH 7.4 for the indicated time at 37°C. The reaction was stopped by adding 1 μM di-isopropyl fluorophosphate (DFP), and the samples were subjected to 15% SDS-PAGE under reducing conditions, followed by PhosphorImager analysis. B: 125I-VEGF (0.82 nM) was incubated with the indicated concentration of NE for 30 min at 37°C and applied to SDS-PAGE as in A. C: 15% SDS-PAGE of NE-digested (20 μg/ml; 30 min at 37°C) 125I-VEGF was conducted under reducing and nonreducing conditions. 125I-protein bands were visualized by PhosphorImager analysis. bME, β-mercaptoethanol.

Fig. 2.

VEGF fragment characterization and mass spectrometry. A and B: carrier-free VEGF165 (100 μg/ml) was incubated with and without 20 μg/ml NE in 44 mM sodium bicarbonate buffer, pH 7.4 for 30 min at 37°C. The reaction was stopped by adding 1 μM DFP, and the samples were reduced and cysteine blocked by treatment with dithiotheritol and iodoacetamide. Samples were resolved on 12% SDS-PAGE under reducing conditions, and protein bands were visualized either by silver staining (A) or Coomassie blue staining for mass spectrometry analysis (B). VEGF bands in Coomassie blue-stained gels were excised and subjected to in-gel trypsin digestion, and then analyzed by mass spectrometry. Sequences recovered from mock-treated VEGF and NE-treated VEGF are underlined in bold.

Fig. 3.

125I-VEGF165 binding and release from endothelial cells. A: confluent cultures of bovine aortic endothelial cells were incubated with 0.23 nM 125I-VEGF165 at 4°C for 2 h, and unbound 125I-VEGF165 was removed by washing the cells three times in binding buffer. The 125I-VEGF165-bound cells were incubated in 44 mM sodium bicarbonate buffer, pH 7.4, ±0.5 μg/ml porcine pancreatic elastase (PPE) for the indicated time. Released 125I-VEGF165 was counted using a γ-counter. •, 125I-VEGF released with PPE; ○, 125I-VEGF released in bicarbonate buffer. Each data point represents the mean of triplicate determinations ± SD. Statistical analysis revealed a significant difference between PPE and buffer-treated cells (p < 0.01). The experiment was repeated more than three times with similar results. B: 44 mM sodium bicarbonate buffer, pH 7.4, containing released 125I-VEGF165 was collected from the cells from three separate wells after the indicated incubation period and was subjected to 15% SDS-PAGE and PhosphorImager analysis.

Fig. 4.

Endogenous VEGF and VEGF fragments are released from smooth muscle cells (SMCs). SMCs were maintained in culture for 4 wk and then subjected to treatment with 44 mM NaHCO3 with or without 5 μg/ml PPE (A and B) or 5 μg/ml NE (C and D) for 30 min at 37°C. Elastase digests were collected, and 1 μM was DFP added. Digests were centrifuged (800 g, 10 min at 4°C) and concentrated (10,000 MWCO centrifugal devices). PPE (A and B) and NE (C and D) digest samples were subjected to 15% SDS-PAGE and analyzed by immunoblot with anti-VEGF polyclonal antibody raised to full-length VEGF165 (no. 06–565; A and C) or anti-VEGF polyclonal antibody raised to the internal region of VEGF (no. 07-1376; B and D).

Fig. 5.

Evaluating the specificity of VEGF degradation. A: 50 ng/ml of 125I-VEGF165, 125I-fibroblast growth factor 2 (FGF2), 125I-platelet-derived growth factor (PDGF), and 125I-tumor necrosis factor (TNF)-α were incubated with 20 μg/ml NE in 44 mM sodium bicarbonate buffer, pH 7.4, at 37°C for 30 min. The reaction was stopped by boiling in reducing SDS-PAGE sample buffer, and samples were subjected to 15% SDS-PAGE, followed by gel fixation and PhosphorImager analysis. B: 125I-VEGF165 was treated with 10 μg/ml activated matrix metalloproteinase (MMP) 9 [activation by 4-aminophenylmercuric acetate (APMA) (72)] for the indicated times at 37°C, and the reaction was terminated by boiling in reducing SDS-PAGE sample buffer. Samples were subjected to 15% SDS-PAGE, followed by gel fixation and PhosphorImager analysis.

Fig. 6.

VEGF fragment (VEGFf) binding to heparin. A: a batch of VEGFf was prepared by incubating 125I-VEGF165 (33.3 μg/ml) with NE (20 μg/ml) in 44 mM sodium bicarbonate buffer, pH 7.4 for 30 min at 37°C. The reaction was stopped by adding 1 μM DFP, and the samples were dialyzed exhaustively (10-kDa MWCO) against PBS at 4°C. A fraction was analyzed by 15% SDS-PAGE followed by gel fixation and PhosphorImager visualization. B: 96-well plates were coated with 1 μg/ml heparin by overnight incubation in PBS. Plates were incubated with 125I-VEGF and 125I-VEGFf (0.11, 0.23, 0.68, and 1.14 nM) in binding buffer (0.15 M NaCl, 25 mM HEPES, pH 7.5) for 2 h at 4°C, and bound VEGF/VEGFf was extracted with 1 M NaCl, 25 mM HEPES (pH 7.5), and 0.5% Triton X-100. Samples were counted in a gamma counter. •, 125I-VEGF bound; ○, 125I-VEGFf bound. Each data point is the mean of quadruplicate determinations ± SD. The binding of intact VEGF and VEGFf to heparin was significantly different (P < 0.01). Similar results were observed in three separate experiments. C: 125I-VEGFf with various NaCl concentrations in PBS (0.15, 0.24, 0.3, and 0.5 M) was incubated with heparin-Sepharose beads (1:1 slurry) for 1 h at 4°C while rotating. Heparin-bound VEGF was separated from the unbound VEGFf by centrifugation at 1,000 g for 3 min. Supernatants were collected and analyzed by 15% SDS-PAGE and phosphor screen visualization.

Fig. 7.

Binding of VEGF and VEGFf to VEGF receptor (VEGFR) 1 and VEGFR2. 125I-VEGF165 (A) and 125I-VEGFf (B) were incubated with Fc-VEGFR1 or -R2 (0.1 nM) for 2 h; complexes were pulled down with magnetic protein-A beads, and the quantity of 125I-VEGF/VEGFf was counted in the gamma counter. filled bars. VEGF/VEGFf associated with the protein A beads with no receptors present; open bars, VEGF/VEGFf bound to R2; gray bars, VEGF/VEGFf bound to R1. Each data point represents the mean of triplicate determinations ± SD. Similar results were observed in four separate experiments. A: binding of VEGF to R1 and R2 was significantly different than when no receptors were present (P < 0.05). B: binding of VEGFf to R1 was significantly different than when no receptors were present (P < 0.05). There was no statistically significant binding of VEGFf to R2 compared with that observed with no receptor [P = not significant (NS)].

Fig. 8.

VEGFf activities on endothelial cells. A: serum-starved bovine pulmonary artery endothelial cells (bPAEC) were treated with VEGF or VEGFf (10× = 0.23 nM or 1× = 0.023 nM) or both for 10 min. Cells were extracted from duplicate wells and analyzed by immunoblot for activated extracellular signal-regulated kinase (ERK) 1/2 [phosphorylated (p) ERK1/2] and total ERK1/2. pERK1/2/ERK1/2 indicates the average density ratio of the activated pERK and total ERK bands for each condition. B: serum-starved bPAEC were treated with VEGF and VEGFf (0.23 nM each) for 10 min at 37°C. The cells from duplicate wells were extracted and analyzed by immunoblot for phosphorylated protein kinase B (Akt) and total Akt levels. C: bPAEC were treated with VEGF and VEGFf (0.45 nM each), and the CASE Akt enzyme-linked immunosorbent assay (ELISA) was used to determine Akt activation. Each data point is the mean of triplicate determinations ± SE. ANOVA followed by multiple-comparison _t_-tests reveal significant differences between all treatment groups (P < 0.01).

Fig. 9.

VEGFf activities on RAW 264.7 cells. A: RAW 264.7 cells were treated for 1.5 h with VEGFR kinase inhibitor III (10 μM) or left untreated and then incubated with VEGF and VEGFf (0.23 nM each) for 10 min. Cells from duplicate wells were extracted and analyzed by immunoblot for pERK1/2 and total ERK1/2. B: RAW 264.7 cells were treated with VEGFf (0.23 and 0.45 nM) and placental growth factor (PlGF, 0.45 and 0.23 nM) for 10 min; cells from duplicate wells were extracted and analyzed for pERK1/2 and total ERK1/2. C: RAW 264.7 cells were treated with VEGF and VEGFf (0.45 nM each), and Akt activation was measured using the CASE Akt ELISA (n = 6; each point is average ± SE): ANOVA (P < 0.01); VEGF and VEGFf vs. control (P < 0.01); VEGF vs. VEGFf (P < 0.01). D: RAW 264.7 cells were treated with 0.45 nM VEGF or VEGFf for 30 h; mRNA was isolated, and real-time PCR was performed using ABI TaqMan gene expression assays: VEGFR1 and the eukaryotic 18S rRNA endogenous control (n = 9; each point is average ± SE): ANOVA (P < 0.01); VEGF and VEGFf vs. control (P < 0.001); VEGF vs. VEGFf (P < 0.05). E: RAW 264.7 cells (5,000 cells/well) were seeded on the upper membrane of Transwell cell migration chambers. VEGF, VEGFf, PlGF, and TNF-α (0.45, 0.45, 0.45, and 1 nM, respectively) were added to the lower chamber. The cells were incubated for 4 h at 37°C, and migrated cells were detected using phosphorescence (n = 4; each point is average ± SE): ANOVA (P < 0.05) all growth factors vs. control (P < 0.05). Similar results were obtained in four separate experiments.

Fig. 10.

Schematic representation of the proposed elastase-mediated VEGF cleavage and release of VEGFf. In this representation, VEGF is bound within the extracellular matrix (ECM). Upon stress or injury, neutrophils are recruited to the site of tissue damage and secrete elastase. Elastase cleaves VEGF, altering its ECM binding (reduced heparin binding), and releases it to act on surrounding cells. The released VEGFf would potentially bind to VEGFR1 on endothelial cells, leading to Akt activation. VEGFf having no affinity for VEGFR2 will not activate its downstream events, yet might alter the distribution of intact VEGF on VEGFR1 vs. VEGFR2 (data not shown). VEGFf might also act on macrophage/monocytic cells through VEGFR1, leading to ERK1/2 and Akt activation. Activation of VEGFR1 by VEGFf could mediate macrophage recruitment to the site of injury, where macrophages would be able to participate in tissue repair/remodeling. This schematic representation is simplified and is not intended to exclude the possibility that other proteases (e.g., MMPs) may also influence VEGF release during tissue injury and inflammation.

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Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity - PubMed (original) (raw)