|Home | About | Journals | Submit | Contact Us | Français|
Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGFxxxb) with significant potential effects on VEGF bioactivity. VEGFxxxb protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGFxxxb was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, “at risk,” and ARDS subjects. The effect of VEGF165 and VEGF165b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [3H]thymidine uptake. We found that VEGF165b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF121b and VEGF165b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF165 on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF165b (P < 0.01). These data demonstrate that the novel VEGFxxxb family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGFxxxb family may have a role in repair after lung injury.
the initial description of VEGF as a potent mitogen and permogen led to intense interest in this protein as a potential mediator in the development of ARDS. Initial animal studies demonstrated that overexpression of VEGF by adenoviral lung delivery induced an ARDS-like lung injury (14). However, observational data by several groups of workers demonstrated plasma VEGF levels rise and intrapulmonary levels fall in the early stages of lung injury with normalization of both during recovery (11, 18, 28, 29). This led us and others to consider that the predominant role of VEGF in this context may be lung repair (22). It has also been shown that VEGF protein is compartmentalized to high levels in normal human epithelial lining fluid (measured in fluid obtained at BAL) 500 times higher than plasma levels despite the fact that the VEGF-producing cells are not subject to hypoxia. Potential explanations for the apparent reductions in intrapulmonary VEGF levels in early ARDS are manifold and not mutually exclusive. Increased levels of soluble VEGFR-1(s-flt) have been demonstrated to contribute to these observations (23). Reduced levels of VEGF have also been described in normal smokers and patients with idiopathic pulmonary fibrosis (IPF), and other conditions in which damage to the alveolar epithelium may be present. Other potential explanations for these findings exist including changes in bound or soluble receptor density, physical changes in the alveolar-capillary membrane, and changes in VEGF isoform from soluble to more membrane-bound forms that we and others have previously explored (16, 20). The recognition of the VEGFxxxb isoform family led to another potential explanation for these observations. This family of VEGF isoforms is formed by splicing from exon 7 into the previously assumed 3′-untranslated region (exon 8b) of the VEGF mRNA. This new family of VEGF isoforms (VEGFxxxb) consists of peptides of the same length as other forms, but with a different COOH-terminal six amino acids, SLTRKD rather than CDKPRR (3).The receptor binding and dimerization domains are intact in these new isoforms. However, it stimulates a unique pattern of VEGF-R2 tyrosine residue phosphorylation, contrasting with those activated by conventional isoforms (12). The dominant exon 8b-containing isoform, VEGF165b, appears not only to be nonangiogenic but also actively anti-angiogenic (7 inhibiting the angiogenic VEGFxxx isoforms) (5, 12). This led us to speculate that the presence of this isoform family forms part of the explanation for both the observed bioactivity of VEGF in the normal human lung and the apparent variability of VEGF bioactivity in both experimental conditions and between studies.
As an initial step, we addressed the hypothesis that the VEGFxxxb family of isoforms would demonstrate functional effects that are of potential importance in lung injury and repair, and be expressed in normal human lung tissue and be reduced in ARDS.
Archival normal and ARDS lung tissue sections and paraffin blocks for which consent for research had been obtained, were utilized. Macroscopically normal lung tissue sections (~15 × 5 × 5 cm) were donated by 12 patients (6 females and 6 males) undergoing lobar resection for malignancy. The median age was 69. Ethical approval was obtained from the North Bristol and United Bristol Healthcare Trusts.
Primary human lung microvascular endothelial cells (HMVEC-L) cryopreserved from passage 4 were obtained from Clonetics (Cambrex, East Rutherford, NJ). They were cultured in EGM-2-MV medium without VEGF supplementation according to the manufacturer's instructions and used before passage 15 (Cambrex).
Primary lung epithelial cells were isolated as we have previously described (2, 32) from macroscopically normal lung tissue of patients undergoing lobectomy or pneumonectomy for lung cancer. Ethical approval was obtained from the North Bristol and United Bristol Healthcare Trusts.
Briefly, human lung pieces were perfused with 0.9% NaCl and then inflated with 2.5 g/l trypsin in HBSS (Sigma-Aldrich). The digested tissue was minced and then filtered through a 40-μm mesh. The filtrate was centrifuged at 300 g for 10 min at room temperature. The cell pellet was then resuspended in DCCM-1 (React Scientific, Troon, UK) containing 100 μg/ml DNase I (Sigma-Aldrich) and plated into a large flask. Cells were incubated at 37°C to allow adhesion of contaminating leukocytes. After a 2-h incubation, the supernatant was centrifuged, and the cell pellet was resuspended in complete media [DCCM-1 10% NCS, 2 mM glutamine, 2 mg/l amphotericin B, and penicillin-streptomycin solution (10,000 U penicillin-G and 10 mg streptomycin/ml, Sigma-Aldrich)]. Cells were incubated at 37°C for another 2 h. The supernatant was then centrifuged, and the alveolar type II (ATII) cell-enriched pellet was resuspended in complete medium and seeded onto type I collagen-coated plastic (Vitrogen 100; Cohesion Technologies, Palo Alto, CA). ATII cell phenotype was confirmed as previously described (2, 32). ATII cells were used before day 5.
Lung tissues were snap-frozen in liquid nitrogen. They were chopped up on ice with a sterile scalpel blade in lysis buffer (10 mM Tris·Cl, pH 7.5, 1 mM EDTA, 1% Triton X-100, 10 μg/ml leupeptin, 20 μM E64, 200 μg/ml aprotinin, 10 μg/ml pepstatin A) and then homogenized on ice for 10 min and left to rotate at 4°C for 1 h. Cells were washed with cold PBS and protein extracted using RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA) containing protease and a phosphatase inhibitor cocktail (Sigma-Aldrich). The proteins extracted were then quantified using the BCA Protein Assay Kit (Perbio Science UK, Cramlington, Northumberland, UK).
Bronchoscopy was performed according to standard protocol in living subjects. The clinical characteristics are shown in Table 1. These were not the subjects from whom the lung tissues were obtained. After topical anesthesia with 2% lidocaine, BAL was performed with four 60-ml aliquots of buffered saline instilled into the middle lobe. The BAL fluid was aspirated into a siliconized glass bottle and stored on ice until processing. Samples were processed within 15 min of collection. The chilled BAL fluid was strained through a single layer of coarse gauze to remove clumps of mucus and then spun at 400 g for 5 min to recover cells. BAL fluid supernatant was collected and stored at −80°C until analysis. The BAL samples were subsequently concentrated using Amicon Centricon centrifugal filter devices (cutting weight 10 kDa, YM-10) at 4°C according to the manufacturer's instructions (Millipore, Bedford, MA).
Normal and ARDS lung tissue sections were examined (n = 8). Normal lung tissue implied that there was no lung involvement in the cause of death. ARDS was defined according to ATS/ERS criteria. Paraffinized 8-μm sections were dewaxed in serial xylene (BDH Laboratory Supplies, Poole, UK), rehydrated through ethanol solutions (BDH Laboratory Supplies), and pressure cooked in 0.01 M tri-sodium citrate (BDH Laboratory Supplies) buffer (pH 6) to facilitate antigen retrieval. Endogenous peroxidase was blocked with 3% hydrogen peroxide (BDH Laboratory Supplies) in methanol (BDH Laboratory Supplies). Sections were incubated in 2.5% horse blocking serum (Vectastain Universal Quick Kit; Vector Laboratories, Peterborough, UK) before avidin D and biotin blocking sera (Vector Laboratories). They were then incubated with 2 μg/ml mouse monoclonal anti-VEGFxxxb IgG1 neutralizing antibody (MAB3045, R&D Systems) or a normal mouse IgG1 (X0931, Dako) as a control. A horse biotinylated anti-mouse-IgG (BA2000, Vector Labs) was then added. Incubation with Vectastain ABC solution (PK4000, Vector Labs) followed by 3,3-diaminobenzidine peroxidase substrate solution (SK4100, Vector Labs) was followed by counterstaining in hematoxylin. Sections were examined on a Nikon Eclipse E-400 microscope, and images were captured using a Coolpix 995 digital camera and a DN-100 digital imaging system (Nikon Instruments, Surrey, UK).
HMVEC-L were seeded in 24-well plates (Greiner Bio-One, Stonehouse, UK) in EGM-2-MV medium at 7,000 cells/well. After 24 h, cells were washed with PBS and incubated in EBM-2 medium supplemented with 0.5% FBS (Cambrex) to starve overnight. Cells were then incubated with relevant recombinant protein. For the last 4 h of the 24-h incubation time, 37 kBq methyl-[3H]thymidine (Amersham Biosciences) was added to each well. The cells were washed with 5% TCA in PBS and then solubilized by adding 0.5 ml of 0.3 M NaOH (Sigma-Aldrich).
ATII were seeded in 24-well plates (Greiner Bio-One) in complete medium at 100,000 cells/well. After 48 h, cells were washed with HBSS (Sigma) and incubated in complete media. Recombinant proteins and 37 kBq methyl-[3H]thymidine (Amersham Biosciences) were added to each well. At 48 h of incubation at 37°C, the cells were washed with 5% TCA in PBS and then solubilized by adding 0.5 ml of 1 M NaOH (Sigma, Poole, UK).
Cell lysates were subsequently pipetted into scintillation vials (Fisher Scientific UK, Loughborough, Leicestershire, UK) containing 2 ml of scintillation liquid (Amersham Biosciences) and counted by a beta counter (Beckman Instruments, Beckman Coulter, High Wycombe, Buckinghamshire, UK).
HMVEC-L were seeded in six-well plates (Greiner Bio-One) (28,000/well). After 24 h, cells were washed with PBS and then incubated with EBM-2 medium supplemented with 0.5% FBS for HMVEC-L. After overnight starvation, cells were incubated with the various treatments. After a 48-h incubation, cells were washed with PBS and incubated with 200 μl of Trypsin-EDTA (Sigma-Aldrich).
ATII were seeded in 24-well plates (Greiner Bio-One) in complete medium at 100,000 cells/well. After 48 h, cells were washed with HBSS, and the protein of interest was added. After a 48-h incubation, cells were washed with PBS and incubated with 100 μl of Trypsin-EDTA (Sigma, Poole, UK). When cells were unattached, 200 μl of culture medium containing 10% FBS was added. Cells were then counted under the microscope using a hemacytometer.
After resuspension in 4× SDS sample buffer, the protein extracts were heated at 100°C for 5 min and subsequently resolved by SDS-PAGE in a 7.5% or 12% acrylamide gel. The proteins were subsequently transferred on polyvinylidene difluoride membrane (Bio-Rad). After blocking the membrane in 3% bovine serum albumin in PBS 0.05% Tween 20 (PBST) for 1 h at room temperature, membranes were incubated overnight at 4°C with the specified primary antibodies: mouse monoclonal anti-actin (clone AC-40, 1.5 mg/ml used at 1:10,000, Sigma-Aldrich), mouse monoclonal anti-tubulin α (clone DM1A, 200 μg/ml used at 1:1,000, Autogen Bioclear UK, Calne, Wilts, UK), mouse monoclonal anti-Pan VEGF (2.5 μg/ml, MAb293, R&D Systems), mouse monoclonal anti VEGFxxxb (R&D Systems, 0.8 μg/ml, MAB3045). After three washes in PBST, membranes were incubated in 5% milk in PBST with the corresponding HRP-conjugated secondary antibodies: goat anti-mouse (Pierce, 1.4 ng/ml), goat anti-rabbit (Pierce, 1.4 ng/ml), or donkey anti-goat (Santa Cruz, 40 ng/ml). After three washes in PBST, signal was detected by chemiluminescence using the SuperSignal West Femto Substrate (Perbio Science UK) using the ChemiDoc XRS system (Bio-Rad). When required, membranes were stripped with a 3% hydrogen peroxide solution in distilled water for 1 h at room temperature or with a stronger stripping buffer [100 mM 2-mercaptoethanol, 2% (wt/vol) SDS, 62.5 mM Tris·HCl, pH 6.7] for 30 min at room temperature.
Briefly, Immulon 96-well plates (Thermo Fisher Scientific, Basingstoke, UK) were coated overnight with 200 ng of mouse monoclonal antibody anti-human VEGFxxxb (R&D Systems, at 2 μg/ml in 100 μl PBS) per well. To control for nonspecific signal, one-half of the plate was coated with a mouse monoclonal antibody anti-α tubulin (clone DM1A, 200 ng/well, Autogen Bioclear UK) and run under the same conditions. After three washes with PBST, the plates were blocked with 300 μl/well of Superblock according to the manufacturer's instructions (Perbio Science UK). Then, 100 μl/well of standard or sample, diluted in 50% of heat-inactivated FBS in PBS, was added and incubated for 2 h at room temperature. After three washes with PBST, 100 μl/well of a biotinylated goat polyclonal antibody anti-human Pan VEGF (AF293B, R&D Systems), at 50 ng/ml in PBS, was added. After a 2-h incubation and three washes with PBST, 100 μl/well of HRP-conjugated streptavidin diluted 1:200 in PBS was added and left at room temperature for 20 min, protected from light. Finally, after three washes with PBST, 100 μl/well of substrate solution was added (R&D Systems). The reaction was then stopped by addition of 1 M sulfuric acid, 50 μl/well. The optical density of each well was determined using a microplate reader set to 450 nm with a 570-nm wavelength correction.
Data were analyzed by ANOVA with the Tukey post hoc multiple comparison correction using GraphPad Prism version 4.0 software. A P value of < 0.05 was considered significant.
For immunohistochemistry, VEGFxxxb was widely detected in normal lung tissue within the alveolar space, with a predominant localization to the corner of the alveolus, where ATII are located (Fig. 1B). In contrast, in ARDS sections, the lung tissue expression was markedly altered. VEGFxxxb isoforms were found in ARDS tissue, mostly colocalized with inflammatory cells, but staining was weak in alveolar tissue per se (Fig. 1D). Negative controls for normal and ARDS are shown in Fig. 1, A and C, respectively.
This staining represents all VEGF and VEGFxxxb isoforms, both bound and unbound. Immunoblotting was used to resolve the different isoforms present. VEGF165 and VEGF165b are glycosylated to various extents resulting in molecular weights between 19 and 23 kDa for the monomer, and when they are strongly dimerized, 38–43 kDa. Figure 2A shows that several isoforms of the VEGFxxxb family were present in normal lung. Figure 2A shows that the fully glycosylated 165- and 121-amino acid forms of VEGF were identified, and, in some samples, unglycosylated protein was also seen. Figure 2B shows that VEGF165b and VEGF121b both formed part of this VEGF. In addition, VEGF165b isoforms were found in isolated normal ATII protein extracts and in BAL fluids (Fig. 2C).
A limited number of BAL samples from patients with or at-risk of ARDS (Table 1) were also sampled by ELISA that will specifically detect VEGFxxxb isoforms (n = 8 in each group). This suggests a differential expression with disease with significant changes between normal and at-risk subjects (P < 0.05, Fig. 3).
VEGF165 is well recognized as an endothelial cell mitogen. VEGF165b has been documented to inhibit human umbilical vein endothelial cell proliferation (3) and in vivo angiogenesis (33). To investigate whether VEGF165b had a similar effect on human pulmonary microvascular endothelial cells, tritiated thymidine incorporation and cell counts were measured. VEGF165 significantly stimulated thymidine incorporation in lung microvascular endothelial cells compared with control (10 ng/ml VEGF165, P < 0.01, 20 ng/ml VEGF165, P < 0.001; Fig. 4A). Furthermore, the concomitant addition of VEGF165b significantly decreased the VEGF165-induced thymidine incorporation (10 ng/ml VEGF165 and VEGF165b, P < 0.001, 20 ng/ml VEGF165 and VEGF165b, P < 0.001 compared with VEGF165 alone; Fig. 4A). Cell number results indicate that VEGF165 significantly increased the number of endothelial cells compared with control cells (comparable to serum in which VEGF will be present constitutively) after 48-h incubation control (10 ng/ml VEGF165, P < 0.01, 20 ng/ml VEGF165, P < 0.01; Fig. 4B). VEGF165b on its own did not significantly modify the number of endothelial cells compared with untreated cells. Moreover, the presence of VEGF165b significantly reduced cell number when combined with VEGF165 compared with those treated with VEGF165 alone (10 ng/ml VEGF165 and VEGF165b, 20 ng/ml VEGF165 and VEGF165b, P < 0.01; Fig. 4B). Together, these results indicate that VEGF165b decreased the ratio of thymidine incorporation per cell in VEGF165-stimulated pulmonary microvascular endothelial cells and hence inhibited lung endothelial cell proliferation.
The identification of VEGF165b within the human lung and our substantiation of its effect on HMVEC-L led us to explore its effect on the previously identified VEGF165-induced proliferation of ATII (20). Figure 5A shows that 5 ng/ml VEGF165 significantly increased thymidine incorporation into ATII cells. In contrast, VEGF165b significantly reduced the rate of thymidine incorporation compared with untreated cells (P < 0.01 at 5 and 10 ng, P < 0.05 at 20 ng/ml). Furthermore, the concomitant addition of VEGF165b significantly decreased the VEGF165-induced thymidine incorporation (5 ng, 10 and 20 ng/ml VEGF165 and VEGF165b, P < 0.001, compared with VEGF165 alone; only 5 ng/ml shown; Fig. 5A). Cell number results indicate that VEGF165 significantly increased the number of ATII cells as previously described (20) compared with control cells after 48-h incubation control (5 ng/ml VEGF165, P < 0.01, 20 ng/ml VEGF165, P < 0.01; Fig. 5B). VEGF165b on its own did not significantly modify the number of ATII cells compared with untreated cells. However, the presence of VEGF165b did significantly reduce the VEGF165-stimulated increase in cell number (10 ng/ml VEGF165 and VEGF165b, P < 0.01, 20 ng/ml VEGF165 and VEGF165b, P < 0.01; Fig. 5B). These results indicate that VEGF165b decreased the ratio of thymidine incorporation per cell, in VEGF165-stimulated ATII, and hence inhibited ATII proliferation.
The presence of high levels of VEGF in the normal alveolar space has led several authors to consider the role of VEGF in loss or change in the alveolar capillary membrane in many forms of lung disease including ARDS (1, 11, 18, 28, 29). We have previously reported changes in total VEGF levels detected in the alveolar space and plasma of patients with ARDS (28, 29). Other groups have reported similar data, and reduced levels of VEGF have also been reported in other forms of lung disease (1, 11, 18). The VEGF levels in the normal alveolus are significant, twice the concentration previously shown to induce angiogenesis (4). However, in healthy lung, these processes are extremely restricted (13). This apparent paradox has led to controversy about the role of VEGF in the normal and diseased lung (22).
VEGF exerts its biological effect on vascular endothelium through specific receptors, VEGFR-1 and VEGFR-2, and coreceptors, neuropilin-1 and neuropilin-2 (10, 11, 19, 26, 27). In addition, alternative splicing of the VEGF transcripts from exons 6 to 8 leads to the generation of several different isoforms with variable heparin binding, receptor affinity, and bioavailability depending on their length and heparin-binding moieties. VEGF121, VEGF165, and VEGF189 are the main members of the originally described VEGF isoform family, VEGFxxx (8, 25, 30). Exon 8a (present in all conventional proangiogenic, propermeability isoforms) is necessary for the stimulation of endothelial cell mitosis and angiogenesis in vivo (7). VEGF165 (lacking exon 6 but not 7) has some heparin-binding affinity and so upon secretion exists in equilibrium between soluble and cell-associated forms. We have previously explored changes in VEGFxxx isoform and receptor expression as mechanisms for regulating VEGF bioactivity (21).
However, a further family of VEGF isoforms has been identified that is formed by splicing from exon 7 into the previously assumed 3′-untranslated region (exon 8b) of the VEGF mRNA. This new family of VEGF isoforms (VEGFxxxb) consists of peptides of the same length as other forms, but with a different COOH-terminal six amino acids, SLTRKD rather than CDKPRR (3).The receptor binding and dimerization domains are intact in these new isoforms. However, it stimulates a unique pattern of VEGF-R2 tyrosine residue phosphorylation, contrasting with those activated by conventional isoforms (12). The dominant exon 8b-containing isoform, VEGF165b, appears not only to be nonangiogenic but also actively antiangiogenic (5, 7, 12). This led us to speculate that the presence of this isoform family forms part of the explanation for both the observed bioactivity of VEGF in the normal human lung and the apparent variability of VEGF bioactivity in both experimental conditions and between studies.
This physical characteristic makes their discrimination difficult experimentally. VEGF165b and VEGF121b protein have been clearly identified. Interestingly, VEGF165b binds the generic VEGF receptors (VEGF-R1 and VEGF-R2) but not neuropilin-1 (NRP-1), the binding to which now appears to be exon 8a dependant (6). Therefore, VEGF165b is not simply a competitive inhibitor. VEGF165b binds to VEGF-R2 with equal affinity to VEGF165, but it also phosphorylates VEGF-R2 but in a qualitatively unique way (15). VEGF165b fails to phosphorylate the VEGF-R2 intracellular tyrosine residues that mediate the angiogenic response (15). In addition, it has been recently shown that NRP-1-deficient mice result in changes to air space size, which was further increased in the presence of cigarette smoke proposed to be related to changes in epithelial cell death (17). Thus the lack of VEGFxxxb binding may be particularly significant in this context. A qualitative switch in VEGF isoform expression from VEGFxxxb to VEGFxxx expression as seen in malignancy (31) and eye disease (24) could also contribute to the well-described changes in VEGF bioactivity observed in the lung.
We therefore assessed expression of VEGFxxxb in archival normal and ARDS lung tissue where we found significant expression in normal lung tissue, mainly in the alveolar epithelium and macrophages. In contrast, there was minimal expression in the ARDS tissue, other than in infiltrating inflammatory cells. Identical immunochemical findings were found in both archival necropsy tissue in the ARDS subjects and from both archival normal tissue and snap-frozen fresh pneumonectomy sections.
Obtaining ARDS lung tissue is limited by the lack of surgical biopsies of this disease in our clinical practice, and, theoretically, necropsy lung tissue might introduce selection bias for a more severe spectrum of ARDS, as intrapulmonary VEGF levels are known to be lower in nonsurvivors with ARDS (29). Surgical lung biopsy from living ARDS patients would have been preferred, but this is now seldom performed in the United Kingdom. There was no evidence of any significant lung disease in the normal necropsy lung tissue, but it is conceivable that the extrapulmonary disease process contributing to death might have affected VEGF levels.
We also undertook immunoblotting of snap-frozen whole lung lysate and freshly isolated ATII cells, which demonstrated significant expression of several members of the VEGFxxxb family. Finally, we made a preliminary assessment of VEGFxxxb levels in a limited number of BAL fluids, which suggested a disease-related change.
The mitogenic properties of VEGF165 are well described in the systemic vasculature. The presence of such a molecule in the lung where angiogenesis is a restricted process requires explanation. However, other properties of this molecule have become increasingly identified, in particular, its identification as an epithelial mitogen and survival factor (9). However, as previously described (3), conventional methods for detection are measuring pan-VEGF and do not differentiate the VEGFxxxb family. Indeed, our data suggest that they are not equipotent with inhibition of mitogenesis occurring at a less than one-to-one ratio. The presence of such very significant amounts of VEGFxxxb in the normal lung suggested that it may have an inhibitory role in pulmonary angiogenesis.
Our studies using primary cultured pulmonary endothelial cells have shown not only that VEGF165b has no proliferative effect on human pulmonary endothelial cells but also inhibits the proliferative effect of VEGF165. This has been previously demonstrated in human umbilical vein endothelial cells, but in view of the role of hypoxia-inducible factor as a transcription factor of VEGF, we considered it critical to establish the response in primary human lung cells. We have also shown that VEGF165b inhibits VEGF165-induced proliferation of primary ATII cells.
These data lead us to suggest that members of the VEGFxxxb family are a major set of isoforms in normal lung in contrast to disease states such as ARDS. The methodological difficulties in differentiating these families will require a reevaluation of our knowledge base of these proteins. The regulation of isoform switching may be of critical importance to the outcome of disease, a further intrinsic method of determining VEGF bioactivity.
This work was funded by the Wellcome Trust (074702) and the British Heart Foundation (BS/006/005).
The authors have no competing interests. Profs. Bates and Harper are named inventors on a patent describing therapeutic potential of the VEGFxxxb isoforms.
We thank Haydn Kendall and Sharon Standen for technical advice.