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Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Submitted 30 April 2003 ; accepted in final form 10 November 2003

	ABSTRACT

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In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-, IL-1, and PGE₂. We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.

extracellular matrix; signal transduction; asthma; extracellular signal-related kinase; airway smooth muscle

VEGF may contribute to the pathophysiology of asthma in several ways. First, elevated levels of VEGF, a known endothelial cell mitogen, could promote bronchial vascular remodeling. Second, the ability to induce vascular permeability could induce extravasation of plasma proteins and subsequent tissue edema. VEGF might also indirectly recruit inflammatory leukocytes by inducing expression of IL-8 and ICAM-1 and by enhancing transendothelial migration (20, 27, 35). Finally, other biological effects of VEGF are likely to be important in the pathological changes associated with airway remodeling, such as the ability to induce expression of matrix metalloproteinases in both endothelial cells and vascular smooth muscle cells (38, 41) and the ability to induce expression of connective tissue growth factor and collagen (1, 37). Alterations in the ECM may have significant effects on smooth muscle cell phenotype and function. Certain substrates, such as fibronectin, appear to promote ASM cell proliferation, whereas others, such as laminin, are inhibitory (7). In addition, the presence of fibronectin may affect smooth muscle cell contractility by altering the expression of contractile proteins (7).

The regulation of VEGF expression in ASM is not completely characterized. Recent reports describe the secretion of VEGF in response to bradykinin and PGE₂ (15). Th2 cytokines, such as IL-4 and IL-13, have also been shown to increase ASM cell expression of VEGF (40); interestingly, these cytokines have only a modest effect on bronchial fibroblast secretion of VEGF (36). Other cytokines, such as IL-1 and IL-6, have been shown to upregulate VEGF protein expression, but these effects appear to be species and tissue specific (4). In contrast, there are no studies examining the effects of VEGF on ASM cell signal transduction or synthetic function.

We hypothesized that cytokines induce expression of VEGF and that autocrine secretion of VEGF modulates the synthetic responses of ASM cells, as defined by secretion of matrix proteins. We found that IL-1 and TGF-, but not TNF-, increased secretion of VEGF by ASM cells. Treatment of ASM cells with VEGF promoted secretion of soluble fibronectin but did not induce cell migration or proliferation. These data suggest that VEGF is an important modulator of smooth muscle cell function and likely contributes to airway remodeling in diseases such as asthma.

	METHODS

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Reagents. Human VEGF₁₆₅ and placenta growth factor (PlGF) were purchased from R&D Systems (Minneapolis, MN). PDGF and EGF were purchased from Calbiochem (San Diego, CA). PD-98059 and U-0126 were purchased from Cell Signaling Technology (Beverly, MA).

Human ASM cell culture. Human ASM cells isolated from the trachealis muscle of transplant donors were maintained in Ham's F-12 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. These cells retain smooth muscle-specific actin staining and responsiveness to contractile agonists, as previously described (31). Confluent ASM cells were growth arrested in serum-free Ham's F-12 containing 0.1% BSA for 48 h before experiments. Third- and fourth-passage cells were used in all experiments.

PCR. Growth-arrested ASM cells were stimulated for 12 h as indicated, and steady-state mRNA was isolated using RNAeasy columns (Qiagen, Valencia, CA). cDNA was prepared from 2 µg of mRNA by reverse transcription, using Superscript II (Stratagene, La Jolla, CA), according to the manufacturer's instructions. Oligonucleotide primers specific for human VEGF (forward 5'-CGA AGT GGT GAA GTT CAT GGA TG-3'; reverse 5'-TTC TGT ATC AGT CTT TCC TGGTGA-3') or GAPDH (forward 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'; reverse 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; see Refs. 14 and 22) were used to amplify the cDNA.

ELISA. Conditioned media were assayed using a commercially available VEGF ELISA, according to the manufacturer's instructions (R&D Systems). Soluble fibronectin was also assayed by ELISA, as previously described (16).

Transfection of human ASM cells. Transfection studies were performed using GeneJammer Transfection Reagent (Stratagene). Briefly, human ASM cells were transfected with 10 µg of the reporter plasmid pFN510-Luc (kind gift of Dr. N. S. Nahman, Ohio State University, Columbus, OH) or control plasmid pGL3 (Promega, Madison, WI), and 5 µg of a pSV--galactosidase control vector was used to normalize transfection efficiencies (Promega). After transfection (48 h), the cells were rendered quiescent in medium containing 0.1% FBS for 24 h and exposed to 10 ng/ml VEGF or 10 ng/ml TGF- for the indicated times. Cells were then harvested, and luciferase and -galactosidase activities were measured according to the manufacturer's instructions (Promega). The luciferase activity in cell extracts was normalized for -galactosidase activity. Data are expressed as a percentage of increase according to the formula {[(normalized luciferase values of treated cells)/(normalized luciferase values of control cells)] - 1} x 100.

Flow cytometry. Cell surface expression of the VEGF receptors was detected using goat anti-human kinase domain region (KDR) or flt-1 (R&D Systems), followed by FITC-conjugated rabbit anti-goat IgG (Jackson ImmunoResearch, West Point, PA). Fluorescence intensity was analyzed using a FACScan and CellQuest Software (Becton-Dickinson, San Jose, CA).

Migration assay. ASM cells (5 x 10⁵) were placed in the upper well of a modified Boyden chamber. VEGF₁₆₅ (10-50 ng/ml) or PDGF (10 ng/ml) was placed in the lower well, and a migration assay was performed, as previously described (13).

Proliferation. Growth-arrested ASM cells were stimulated with increasing concentrations of VEGF in the absence or presence of EGF (10 ng/ml) for 36 h. Incorporation of [³H]thymidine was measured as previously described (31).

Immunoblotting. After stimulation, cells were lysed in buffer containing 1% Triton X-100, protease, and phosphatase inhibitors, as previously described (17). Equal amounts of protein were resolved on a 10% gel by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tween-3% milk and incubated with antibodies specific for phospho-ERK or phospho-p38 (Cell Signaling Technology). The blots were stripped and reprobed with anti-ERK to verify equal loading. To analyze VEGF receptor phosphorylation, proteins were resolved on an 8% gel. Membranes were incubated with antiphosphotyrosine antibodies (4G10; Upstate Biotechnology, Waltham, MA). Equal loading of protein was confirmed by stripping and reprobing with anti-KDR. Bands were visualized by chemiluminescence (Amersham, Piscataway, NJ).

Statistical analysis. Data were analyzed using the Student's paired t-test. P values <0.05 were sufficient to reject the null hypothesis for all analyses.

	RESULTS

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ASM cells express VEGF receptors. VEGF mediates its effects by activating two high-affinity receptors, VEGF receptor-1 (flt-1) and VEGF receptor-2 (KDR/Flk-1; see Ref. 24). The data supporting the expression of both VEGF receptors on vascular smooth muscle are conflicting (6, 38). Because there have been no studies of VEGF receptor expression in ASM cells, we performed flow cytometric analysis after staining with antibodies specific for flt-1 and KDR. Interestingly, we found that ASM cells expressed high levels of both VEGF receptors (Fig. 1). Expression of these receptors was not changed after incubation with TNF-, IL-1, TGF-, or PGE₂ (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Airway smooth muscle (ASM) cells express vascular endothelial growth factor (VEGF) receptors. ASM cells were stained with isotype-matched control antibodies (dashed lines) or those specific for kinase domain region (KDR; left) and flt-1 (right; filled areas). Receptor expression was analyzed by flow cytometry, as described in METHODS. The data are representative of 3 experiments.

VEGF induces fibronectin secretion. Fibronectin is abundant in the lung ECM and is increased both in patients with asthma and in animal models of asthma (26, 30). Because studies in other mesenchymal cells suggested that VEGF induced secretion of ECM proteins (1, 37), we investigated VEGF effects on ASM cell matrix secretion. We hypothesized that VEGF stimulates expression of matrix proteoglycans, such as fibronectin, given its important role in angiogenesis and wound healing (4). Over 48 h, VEGF induced a time-dependent increase in the amount of fibronectin released in the media compared with basal, although this response was less robust than that induced by TGF- (Fig. 2). In contrast, VEGF did not induce an increase in collagen production (data not shown), suggesting specificity of the response. Interestingly, higher concentrations of VEGF did not increase fibronectin secretion, suggesting the activation of a negative regulatory mechanism.

View larger version (20K): [in this window] [in a new window]   Fig. 2. VEGF induces fibronectin secretion by ASM cells. Top: growth-arrested ASM cells were maintained in serum-free media (open bars) or stimulated with VEGF (25 ng/ml, gray bars) or transforming growth factor (TGF)- (10 ng/ml, hatched bars) for 24 and 48 h. Bottom: growth-arrested ASM cells were stimulated with increasing concentrations of VEGF for 48 h. Data are expressed as the mean concentration ± SD and are representative of 3 separate experiments. *P < 0.02 and **P < 0.01 compared with unstimulated.

Because ASM cells express two VEGF receptors, we attempted to determine which receptor mediated the effects of VEGF on fibronectin secretion. Stimulation of cells with PlGF, a ligand specific for flt-1 (24), induced a significant increase in fibronectin secretion (Fig. 3), whereas neutralizing anti-KDR antibodies had no effect on fibronectin expression (data not shown). Together, these data suggest that flt-1 is a primary mediator of VEGF-induced fibronectin secretion.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Flt-1 mediates VEGF-induced fibronectin secretion. Growth-arrested ASM cells were stimulated with VEGF (10 ng/ml), placenta growth factor (PlGF, 10 ng/ml), or TGF- (10 ng/ml) for 48 h. Conditioned media were collected and analyzed for fibronectin by ELISA. Data are expressed as the mean concentration ± SD and are representative of 3 separate experiments. *P < 0.02 compared with control.

To determine the mechanism of VEGF-induced fibronectin production, ASM cells were transfected with a fibronectin promoter reporter construct. As expected, we observed basal luciferase expression, consistent with constitutive fibronectin expression by ASM cells. Stimulation of ASM cells with VEGF induced an increase in luciferase activity as early as 6 h that was maximal at 12 h and decreased by 24 h (Fig. 4A). PlGF induced a similar increase in luciferase activity (Fig. 4B). These data support our hypothesis that VEGF alters the production of matrix components by ASM cells, in part, through transcriptional regulation.

View larger version (15K): [in this window] [in a new window]   Fig. 4. VEGF enhances expression of a fibronectin promoter construct. A: ASM cells were transfected with pFN510-luc or empty vector (ctl) and then stimulated with VEGF (10 ng/ml) for the indicated times or with TGF- (10 ng/ml) for 12 h. Luciferase activity was determined as described in METHODS. B: ASM cells were transfected as in A and then stimulated with VEGF, PlGF, or TGF- (all at 10 ng/ml) for 12 h. Data represent the mean of triplicate determinations and are expressed as the percentage increase over basal activity (n = 3).

VEGF induces ERK activation. VEGF has been shown to activate multiple signaling pathways, including the MAPK family, in endothelial and vascular smooth muscle cells (reviewed in Ref. 24). After stimulation of ASM cells with VEGF, we observed a dose- and time-dependent increase in the phosphorylated forms of ERK (Fig. 5, A and B), with maximal activation at 15 min that decreased by 30 min. Activation of ERK was inhibited by the mitogen/extracellular signal-regulated kinase-1 inhibitors PD-98059 and U-0126 (Fig. 5B). In contrast, we found that VEGF had no effect on p38 MAPK activation (data not shown). Similarly, PlGF also induced a dose- and time-dependent increase in ERK phosphorylation (Fig. 5, A and B). Similar to the effects on fibronectin secretion, higher concentrations of VEGF or PlGF were not as effective in activating ERK. We found that neutralizing antibodies specific for KDR had no effect on VEGF-induced ERK activation (Fig. 5B). The efficacy of the antibody was confirmed by demonstrating that it blocked VEGF-induced receptor phosphorylation (Fig. 5C). We could not perform parallel experiments testing the effects of flt-1, since blocking antibodies to human flt-1 are not yet commercially available.

View larger version (44K): [in this window] [in a new window]   Fig. 5. VEGF induces ERK activation. A: growth-arrested ASM cells were stimulated with VEGF or PlGF at the indicated concentrations (in ng/ml) for 15 min. B: growth-arrested ASM cells were stimulated with VEGF (V) or PlGF (both at 10 ng/ml) for the indicated times (in min). Where indicated, cells were pretreated with PD-98059 (PD; 30 µM), U-0126 (U; 10 µM), or anti-KDR (2 µg/ml), followed by VEGF stimulation for 15 min. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted for phospho (p)-ERK and then stripped and reprobed for ERK. Data are representative of 3 separate experiments. C: cells were stimulated with VEGF (10 ng/ml) for 5 min in the presence or absence of anti-KDR. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted for anti-phosphotyrosine (PY). C, control.

Given the observation that VEGF induced ERK activation, we investigated whether fibronectin secretion was ERK dependent. We found that PD-98059 and U-0126 completely inhibited VEGF-induced fibronectin secretion (Fig. 6). The doses of PD-98059 and U-0126 were chosen based on our previous studies. In contrast, these compounds had only a minimal effect on basal fibronectin secretion, suggesting differential regulatory mechanisms for basal and stimulated fibronectin secretion.

View larger version (11K): [in this window] [in a new window]   Fig. 6. VEGF-induced fibronectin secretion is ERK dependent. Cells were stimulated with VEGF (25 ng/ml) in the absence or presence or PD-98059 (30 µM) or U-0126 (10 µM) for 48 h. Conditioned media were collected and analyzed for fibronectin by ELISA. Data are expressed as the mean concentration ± SD and are representative of 3 separate experiments. P < 0.02 compared with unstimulated (*) and compared with VEGF (***).

VEGF is not sufficient to induce ASM cell migration or proliferation. VEGF is known to stimulate both endothelial and vascular smooth muscle cell migration (4). To study this process in ASM, cells were harvested and placed in the upper well of a modified Boyden chamber and allowed to migrate in the presence of increasing doses of VEGF. After 4 h, we found no increase over basal levels in the number of cells migrating in response to VEGF (Fig. 7). In contrast, there was a significant increase in the number of cells migrating in response to PDGF (Fig. 7). These data are in contrast to similar studies performed using vascular smooth muscle cells, suggesting fundamental differences between these two cell types (6, 13, 39). Similarly, we found that VEGF had no effect on ASM cell proliferation (Fig. 7), which is consistent with data that VEGF is only mitogenic for endothelial cells (4). The transient, rather than sustained, ERK activation is also consistent with VEGF not promoting ASM cell proliferation (29).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effects of VEGF on ASM migration and proliferation. Top: growth-arrested cells were placed in a Boyden chamber, in the presence or absence of VEGF or PDGF (10 ng/ml) in the lower chamber. Data are expressed as the degree of increase in cell migration and are representative of 3 experiments. Bottom: growth-arrested cells were stimulated with VEGF at the indicated concentrations or with EGF (10 ng/ml). Incorporation of [3H]thymidine was determined as described in METHODS. Data are expressed as the mean counts/min ± SD and are representative of 3 experiments.

ASM cells express VEGF. There are several isoforms of VEGF generated by alternative exon splicing (4). We performed RT-PCR using mRNA derived from unstimulated ASM cells and found that ASM expressed steady-state levels of mRNA for VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆ (Fig. 8). VEGF mRNA expression was increased after stimulation with TGF-, IL-1, and PDGF (Fig. 8). In contrast, TNF- had no effect on VEGF mRNA expression (data not shown). To investigate the kinetics of VEGF protein secretion in response to cytokines and growth factors, growth-arrested ASM cells were cultured in the presence of the inflammatory mediators TNF- or IL-1, PDGF (a smooth muscle cell mitogen), or the growth modulators TGF- or PGE₂. We found that IL-1 and TGF- induced VEGF secretion in a dose- and time-dependent manner (Fig. 9). In contrast to IL-1, TNF- stimulated only a slight increase in VEGF secretion above basal (data not shown). PDGF and, to a lesser extent, PGE₂ also induced VEGF secretion (Fig. 9), although the effects of PDGF and PGE₂ were additive.

View larger version (55K): [in this window] [in a new window]   Fig. 8. ASM cells express mRNA for multiple VEGF isoforms. Steady-state mRNA was isolated from unstimulated (basal) or TGF- (10 ng/ml)-, IL-1-(2 ng/ml)-, or PDGF (10 ng/ml)-treated cells, as described in METHODS. RT-PCR was performed with specific VEGF or GAPDH primers (14, 22). Arrows denote isoforms (n = 3).

View larger version (11K): [in this window] [in a new window]   Fig. 9. Regulation of VEGF secretion by ASM. ASM cells were growth arrested in serum-free media for 48 h. A: cells were stimulated with TGF- (0.01-10 ng/ml) for 24 h or with TGF- (1 ng/ml) for the indicated times. B: cells were stimulated with IL-1 (0.02-20 ng/ml) for 24 h or IL-1 (2 ng/ml) for the indicated times. C: cells were stimulated with PDGF (0.1-10 ng/ml) or PGE2 (0.1-10 µM) for 24 h. Conditioned media were collected, and VEGF was measured using an ELISA. Data are expressed as mean ± SD and are representative of 3 experiments.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Virtually nothing is known regarding the effects of VEGF or its receptors on human ASM cells. We report the novel finding that ASM cells express KDR and flt-1, the primary VEGF receptors. In addition, we found that VEGF induced a significant increase in fibronectin secretion by ASM cells. The increase in fibronectin production was accompanied by parallel changes in the activity of a fibronectin-luciferase reporter construct and in the expression of steady-state fibronectin mRNA (data not shown); these findings are consistent with the known regulation of fibronectin expression in fibroblasts and other cell types. VEGF had no effect on smooth muscle cell secretion of collagen, suggesting specificity of the response. Further studies are necessary, however, to determine the effect of VEGF on other matrix components such as tenascin or laminin. The findings of enhanced ERK activation and fibronectin secretion were mimicked by the flt-1-specific ligand PlGF. This observation, coupled with the inability of anti-KDR antibodies to block ERK activation or fibronectin secretion, suggests that flt-1 may be the primary mediator of VEGF effects on ASM cells.

Alterations in the composition of the ECM may have important implications for airway inflammation in asthma. The deposition of fibronectin and collagen can affect the biomechanical properties of the airway wall (32) and can alter cell contractility (8). Data suggest that matrix proteins such as fibronectin have significant effects on the phenotype and survival of ASM cells (5, 7) and enhance cell cycle progression in response to growth factors (2). In contrast, fibronectin inhibits eosinophil migration and signaling (9). Finally, although Th2 cytokines such as IL-13 can alter smooth muscle cell expression of eotaxin and contractile proteins (19, 28), these cytokines have little effect on lung fibroblast secretion of fibronectin (23).

We found that VEGF stimulated ERK activation, which was necessary for fibronectin release. In contrast, basal secretion of fibronectin was unaffected by ERK inhibition, suggesting that autocrine secretion of VEGF is not a primary regulator of fibronectin synthesis in unstimulated smooth muscle cells. Given that VEGF induced only a transient increase in ERK phosphorylation, it was not surprising that VEGF did not act as a smooth muscle cell mitogen. Studies have shown that agents that promote sustained ERK activation induce ASM cell proliferation (29). It remains to be determined whether VEGF can synergize with other mitogens to augment cell proliferation, as has been described after VCAM-1 engagement on ASM cells (17).

Based on the wide range of effects of VEGF on endothelial and vascular smooth muscle cells, we anticipated that VEGF would promote ASM cell migration. Instead, we found that VEGF did not induce ASM cell migration, in contrast to its effects on pulmonary vascular smooth muscle cells (13). There are many possible explanations for this discrepancy, including relative levels of receptor expression or cell specificity regarding downstream signaling proteins, such as phosphatidylinositol 3-kinase, CrkII, pp60src, or focal adhesion kinase (3, 24). Finally, there may be organ specificity to the migration response to VEGF, as has been described for angiogenesis (34).

We confirmed that ASM cells express mRNA for the four common VEGF isoforms (15) and found that VEGF expression was significantly increased by important modulators of airway function. One critical difference between the VEGF isoforms lies in their ability to bind to heparin-like moieties expressed by ECM proteoglycans. VEGF₁₂₁ is a freely diffusible protein; VEGF₁₆₅ is secreted, but some remains bound to the ECM, whereas VEGF₁₈₉ and VEGF₂₀₆ are completely sequestered in the ECM but can be released by heparinases or proteases (33). These matrix-bound isoforms could potentially be released in diseases such as asthma or chronic obstructive pulmonary disease that display dysregulated protease activation (25). We have previously demonstrated that mast cell-derived chymase degrades the smooth muscle cell-associated matrix (18). Future studies will determine whether chymase alters the synthesis and/or release of the various VEGF isoforms by ASM, particularly those that are bound to ECM. Alternatively, matrix-bound VEGF isoforms may be important for smooth muscle cell survival, as has been demonstrated for endothelial cells (12).

In summary, VEGF is secreted by ASM cells in response to a variety of inflammatory mediators and growth factors. We now report that VEGF directly stimulates secretion of fibronectin from ASM cells in an ERK-dependent manner, while having no effect smooth muscle cell proliferation or migration. Our data suggest that flt-1, rather than KDR, may be the primary mediator of these responses in ASM cells. We conclude that VEGF is an important component of the process of airway remodeling in patients with chronic severe asthma, by contributing not only to angiogenesis, but also to changes in the composition of the ECM.

	ACKNOWLEDGMENTS

 
GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-64042 (A. L. Lazaar) and HL-071106 (V. P. Krymskaya), the University Research Foundation of the University of Pennsylvania (A. L. Lazaar), and an American Lung Association Career Investigator Award (A. L. Lazaar).

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. L. Lazaar, Pulmonary, Allergy, and Critical Care Division, Univ. of Pennsylvania School of Medicine, 852 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104 (E-mail: alazaar{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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