Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts

Yidi Wu,1,* Qunzhou Zhang,1,* David K. Ann,2 Anita Akhondzadeh,1 Hai S. Duong,3 Diana V. Messadi,3 and Anh D. Le1,3

1Department of Oral and Maxillofacial Surgery, Charles R. Drew University of Medicine and Science, Los Angeles 90059; 2Department of Molecular Pharmacology and Toxicology and Medicine, University of Southern California, Los Angeles 90033; and 3School of Dentistry, University of California, Los Angeles, California, 90095

Submitted 15 May 2003 ; accepted in final form 25 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Keloids are characterized as an "overexuberant" healing response in which disequilibrium between production and catabolism of extracellular matrix (ECM) occurs. Previous studies from our laboratory and others demonstrate an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) expression in keloid tissues and cultured fibroblasts compared with normal bordering skin. These findings support the concept that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis. Vascular endothelial growth factor (VEGF) has been implicated as a critical factor in regulating angiogenesis and inflammation under both physiological and pathological conditions. This study was designed to assess whether VEGF plays a role in keloid fibrosis. We report that VEGF was expressed at higher levels in keloid tissues and their derived fibroblasts compared with their associated normal skin. We have further demonstrated that VEGF stimulated the expression of PAI-1, but not urokinase plasminogen activator (uPA), in keloid fibroblasts at both mRNA and protein levels, in a dose- and time-dependent manner. However, treatment of normal skin fibroblasts with VEGF exerted little effects on PAI-1 gene expression. Additionally, we have characterized for the first time that the extracellular signal-regulated kinase (ERK)1/2 signaling pathway is mainly involved in VEGF-induced PAI-1 expression and have demonstrated its potential as a target molecule for modulation of scar fibrosis. These findings suggest that VEGF may play an important role in keloid formation by altering ECM homeostasis toward a state of impaired degradation and excessive accumulation.

urokinase plasminogen activator; extracellular matrix; fibrosis


KELOID FORMATION IS A CONSEQUENCE of a derangement in the wound healing process characterized by an excessive accumulation of extracellular matrix (ECM). It usually develops in response to trauma of the skin, from either controlled injuries (e.g., earlobe piercing or surgery) or pathological events (e.g., acne, chickenpox, burn, or inflammation) (58). Clinically, keloid behaves like a benign tumor as it continues to grow beyond the boundaries of the original wound margins and rarely regresses as in a hypertrophic scar (40). In vitro, cultured fibroblasts derived from keloids exhibit an altered phenotype compared with normal fibroblasts and continue to produce high levels of collagen, fibronectin, and elastin in response to an enriched milieu of modulators such as growth factors and inflammatory cytokines (3, 4, 10, 36).

Previous studies using an in vitro three-dimensional fibrin gel system to simulate wound fibroplasia demonstrated that keloid fibroblasts exhibit an increase in collagen accumulation and an alteration in fibrin degradation, possibly because of an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) and a lower level of urokinase plasminogen activator (uPA) (5961). A marked increase in PAI-1 transcript and protein was also noted in fibroblasts derived from patients with either premature aging disorders (Werner syndrome) or keloid lesions (23). Our recent studies (66) detected a higher level of PAI-1 protein in keloid tissue homogenates than in normal bordering skins. These results support the hypothesis that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis (40). Moreover, numerous studies revealed an abundant accumulation of growth factors and cytokines in keloids (1, 31, 32, 63, 64), some of which can be upregulated by hypoxia (50). These factors, such as insulin and insulin-like growth factors (IGFs) (5, 12), fibroblast growth factor (FGF) (25), platelet-derived growth factor (PDGF) (47), transforming growth factor (TGF), and tumor necrosis factor-{alpha} (TNF-{alpha}) (49), in combination with local tissue hypoxia (16, 17, 27, 66), may play an essential role in the regulation of PAI-1 expression.

Vascular endothelial growth factor (VEGF), a well-known target gene of hypoxia-inducible factor-1 (HIF-1) (18, 38, 39), has been implicated as a master regulator in both physiological and pathological events such as angiogenesis and inflammation in tumor, rheumatoid arthritis, and wound healing (7, 13, 45, 51). It has been reported that VEGF plays an important role in the modulation of ECM proteolysis, an essential component of the angiogenic process, by increasing the expression of uPA and PAI-1 in endothelial cells (42, 44). Despite numerous studies characterizing its angiogenic property, data on the role of VEGF in ECM homeostasis and remodeling are still scanty. Furthermore, no biological effect of VEGF has been reported in the dermal fibroblast system because it is well characterized as an endothelium-specific growth factor (26, 65).

Our recent studies demonstrated that PAI-1 and HIF-1{alpha} are expressed at higher levels in keloid tissues than in their associated normal skin and that exposure to hypoxia induces an increase in PAI-1 expression in keloid fibroblasts at both transcriptional and posttranscriptional levels (66), during which multiple signaling pathways including extracellular signal-regulated kinase (ERK)1/2, phosphatidylinositol 3-kinase (PI3-kinase)/Akt, and protein tyrosine kinases (PTKs) have been involved (67). These findings imply a role for VEGF, an immediate downstream target of HIF-1{alpha}, in the regulation of PAI-1 gene expression. To date, evidence of VEGF-mediated PAI-1 expression in dermal fibroblasts is still scarce. In this study, we demonstrate for the first time that keloid tissues intrinsically express a higher level of VEGF than their associated normal skins. We hypothesized that an increased VEGF level may contribute, at least in part, to the upregulation of PAI-1 expression in keloids, in particular, supporting a role of VEGF in tissue fibrosis. To this end, we investigated the effect of VEGF on the regulation of PAI-1 expression in keloid fibroblasts and its associated signaling pathways. Our results showed that VEGF stimulates PAI-1 expression in keloid fibroblasts at both mRNA and protein levels. We also provide evidence that the ERK1/2 or p42/44 mitogen-activated protein kinase (MAPK) signaling pathway is mainly involved in VEGF-induced PAI-1 expression, thus providing a potential target for modulation of scar fibrosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell origin and cell culture. The protocol for tissue collection was approved by the Institutional Review Board of the Charles R. Drew University of Medicine and Science. Keloid tissues were taken from patients who underwent excisional biopsies at the King Drew Medical Center via an elliptical incision, which includes a 2- to 4-mm peripheral rim of clinically normal skin. Clinically raised keloid lesions and associated normal skin borders were histologically confirmed before further studies. All keloid samples obtained in this study had not been previously treated and were not recurred cases. Fibroblast cells derived from normal skin and keloid tissues were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Rockville, MD) supplemented with 10% fetal bovine serum. All cultures were maintained at 37°C and 5% CO2-20% O2. Cells from passages 1–8 were used for experiments and routinely monitored for cell proliferation, morphology, and phenotype.

Treatment of keloid fibroblasts with VEGF. At 70% cell confluence, cells were starved for 24 h and fresh serum-free medium was added. Cells were treated with VEGF-165 (R&D Systems, Minneapolis, MN) at 50 ng/ml for different time intervals under normoxic conditions or with VEGF-165 at different concentrations for an indicated time period. To investigate the signaling mechanisms underlying VEGF-induced PAI-1 expression in keloid fibroblasts, cells were pretreated with a range of specific kinase inhibitors including PD-98059, U-0126, LY-294002, genistein, or calphostin C at the indicated concentrations without obvious cellular toxicities (67) and then exposed to VEGF-165 (50 ng/ml) for 15 min. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) for reverse transcription-polymerase chain reaction (RT-PCR) analysis on PAI-1 mRNA levels. Whole cell lysates were extracted to determine the levels of phosphorylated ERK1/2 or phosphorylated Akt, and conditioned media were collected to determine PAI-1 protein levels by Western blot analyses.

RT-PCR analysis of PAI-1 mRNA induced by VEGF. Expression of PAI-1 mRNA levels was addressed with the One-Step RT-PCR kit (Qiagen). For each sample, 1 µg of total RNA was used as template for reverse transcription and the housekeeping gene {beta}-actin was used as internal standard. Numbers of PCR cycles were chosen to ensure that amplification of PCR products was within an exponential range. Primers specific to PAI-1 were forward 5'-CGCCTCTTCCACAAATCAG-3' and reverse 5'-ATGCGGGCTGAGACTATGA-3'. {beta}-Actin-specific primers were forward 5'-TCATGAAGTGTGACGTTGACATCCGT-3' and reverse 5'-CCTAGAAGCATTTGCGGTGCACGATG-3'. The PCR conditions were 50°C for 45 min for reverse transcriptional synthesis of the first-strand cDNA followed by denaturing at 95°C for 2 min. For second-strand synthesis and PCR amplification, 25 cycles were performed: 94°C for 30 s, 60°C for 1 min, 72°C for 1 min. The PCR products were electrophoresed on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.

RNA preparation and Northern blot analysis. Total RNA was isolated from cultured cells with guanidinium thiocyanate, and 15 µg was submitted on a 1.4% agarose gel containing formaldehyde and transferred onto nylon membranes (Amersham Pharmacia Biosciences, Piscataway, NJ). A cDNA fragment complementary to human VEGF and HIF-1{alpha} was amplified with RT-PCR, subcloned into the pGEM-T Easy Vector System (Promega, Madison, WI), labeled with [{alpha}-32P]dCTP by using the Rediprime II Random Prime DNA labeling system (Amersham Pharmacia Biosciences), and purified through Sephadex G-50 Quick Spin columns (Tris-EDTA buffer) (Roche Diagnostics, Indianapolis, IN). After overnight hybridization at 42°C, membranes were washed, exposed to an intensifying screen in cassette (Kodak, Rochester, NY) for 24 h, and analyzed with a phosphorimaging scanner (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Quantitation of autoradiographs was carried out with the ImageQuant system, using {beta}-actin levels as loading controls.

Western blot analyses. For assay of VEGF protein expression in tissues, keloid or normal skin tissue homogenates were prepared as described previously (52). For determination of phosphorylated ERK1/2 and Akt, cells were solubilized in lysis buffer (in mM: 50 Tris·HCl, pH 7.5, 150 NaCl, 5 EDTA, and 50 NaF with 200 µM Na3VO4 and 0.5% Triton X-100) supplemented with 10 mM dithiothreitol (DTT), 200 µM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktails (Sigma). Total protein concentrations of tissue homogenates or whole cell lysates were determined with the Bio-Rad bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The conditioned media were collected for determination of PAI-1 protein and activity. Equal amounts of protein sampled from each tissue homogenate, whole cell lysate, or conditioned medium were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biosciences). After blocking with Tris-buffered saline (TBS)-5% skim milk, the membranes were incubated overnight at 4°C with antibodies against human VEGF (Santa Cruz), PAI-1 (American Diagnostica, Greenwich, CT), phosphorylated ERK1/2 (Thr202/Tyr204), or total and phospho-Akt (Ser473) (New England Biolabs, Beverly, MA). Membranes were subsequently washed, incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; Pierce) for 1 h at room temperature, and visualized with enhanced chemiluminescence (ECL) detection.

Reverse zymogram analysis of PAI-1 activity in conditioned media. Conditioned media were collected for zymographic assay of PAI-1 activity. Samples were separated by SDS-PAGE, and zymographic analysis was done with a plasminogen-containing substrate gel. Human urokinase (0.5 U/ml, Sigma) was added to the substrate gel for reverse zymographic analysis. Zymograms and reverse zymograms were scanned after incubation at 37°C (60).

Data analysis. For statistical analysis, a paired Student's test was used, with significant differences determined as P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VEGF is elevated in keloid tissues and their derived cultured fibroblasts. To study whether VEGF contributes to the ongoing growth of keloid scars, we used tissue homogenate lysates obtained from fresh biopsied specimens as well as their derived cultured fibroblasts. Analyses of three representative sets of keloid lesions compared with normal skin controls showed a consistent increase in VEGF protein level in keloid tissues (Fig. 1A). Elevated VEGF mRNA expression was also demonstrated by Northern blot analyses in primary cultured keloid fibroblasts derived directly from the same tissue samples (Fig. 1B). The consistent findings of elevated VEGF in keloid tissues and tissue-derived cells confirm that fundamental in vivo biological differences are recapitulated by an in vitro system.



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Fig. 1. Keloid tissues and derived fibroblasts express higher levels of vascular endothelial growth factor (VEGF) than normal skin. A: Western blot analysis of VEGF proteins in tissue homogenates obtained from 3 representative tissue samples. Normal skin control (NSK): lanes 1, 2, and 3 are different samples. Keloid lesion (KEL): lanes 4, 5, and 6 are different samples. B: Northern blot analysis of VEGF mRNAs using dermal fibroblasts isolated from the keloid and normal skin tissues described in A.

 

VEGF causes dose- and time-dependent induction of PAI-1 expression in keloid fibroblasts. To test whether VEGF is involved in the regulation of the plasminogen system in normal skin and keloid fibroblasts, we treated dermal fibroblasts with different concentrations of VEGF (ng/ml) and assayed PAI-1 protein levels in the conditioned media by Western blot. Our results have convincingly shown that after 24-h exposure to VEGF under normoxic conditions, a two- to threefold increase in PAI-1 protein level in keloid fibroblasts is detected in a dose-dependent manner, with a maximal effect at 10–100 ng/ml (Fig. 2A, top, and B). On the contrary, no obvious changes in PAI-1 protein levels were observed in media obtained from normal skin fibroblasts after treatment with different concentrations of VEGF for up to 24 h (Fig. 2A, bottom, and B). Next, we studied the effect of VEGF on PAI-1 mRNA expression in keloid fibroblasts by RT-PCR. The results showed that treatment of keloid fibroblasts with VEGF induced a two- to fivefold increase in PAI-1 mRNA expression in a dose-dependent manner (Fig. 2, C and E). Consistent with findings from Western blot analyses, no obvious changes in PAI-1 mRNA levels were detected in normal fibroblasts exposed to different concentrations of VEGF for up to 6 h (Fig. 2, D and E). We questioned whether VEGF has any effect on the proteolytic activity of the plasminogen system, specifically uPA, in dermal fibroblasts. As shown in Fig. 2, F and G, no apparent changes in uPA protein levels were detected in the conditioned media of either normal or keloid fibroblasts after exposure to different concentrations of VEGF for 24 h.



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Fig. 2. Effect of VEGF on plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA) expression in keloid and normal skin fibroblasts. A: dose response of VEGF treatment on PAI-1 protein expression in conditioned media after exposure to different concentrations of VEGF for 24 h as determined by Western blot; results were normalized to equal protein concentrations of whole cell lysates. KEL, keloid fibroblasts; NSK, normal skin fibroblasts. B: densitometric analysis of PAI-1 levels in A. C and D: semiquantified RT-PCR analysis of PAI-1 mRNA levels in keloid fibroblasts (C) and normal cells (D) after exposure to different concentrations of VEGF for 6 h. E: densitometric analysis of RT-PCR products in C and D. F: dose response of VEGF treatment on uPA protein expression as determined by Western blot. G: densitometric analysis of uPA levels in F. Data are representative of 3 independent experiments.

 

We then characterized the time course effect of VEGF exposure on PAI-1 expression in keloid fibroblasts. Our results indicated that VEGF (50 ng/ml) stimulated PAI-1 mRNA expression in a time-dependent manner (Fig. 3A), whereas no obvious changes in PAI-1 mRNA level were observed in non-VEGF-treated cells (Fig. 3B). The time course of VEGF-stimulated PAI-1 protein expressions was slower compared with that of RNA levels, with a saturating effect at 24–40 h after treatment with VEGF (Fig. 3, C vs. A). The PAI-1 protein levels and activities in the conditioned media of VEGF-treated cells remained consistently higher throughout the time course study than those in non-VEGF-treated controls (Fig. 3, C and D). These results suggest that VEGF affects PAI-1 expression in keloid fibroblasts at both mRNA and protein levels.



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Fig. 3. Time course of VEGF treatment on PAI-1 expression in keloid fibroblasts. A and B: after semiconfluent cells were cultured in the presence (A) or absence (B) of VEGF (50 ng/ml) for different time intervals, PAI-1 mRNA levels were determined by RT-PCR. C: Western blot analysis of PAI-1 protein levels in conditioned media in the presence (+) or absence (–) of VEGF (50 ng/ml) treatment at different time intervals; results were normalized to equal total protein concentrations of whole cell lysates. D: zymographic analysis of PAI-1 activity in the conditioned media. Data are representative of 3 independent experiments.

 

VEGF stimulates activation of ERK1/2 signaling pathway in keloid fibroblasts. To examine the signaling mechanisms underlying VEGF-induced PAI-1 expression in keloid fibroblasts, we challenged cells with different concentrations of VEGF for 15 min and determined phosphorylated ERK1/2 levels with Western blots. As shown in Fig. 4, A and B, VEGF stimulated phosphorylation of ERK1/2 in a dose-dependent manner, with a saturating effect at 25–50 ng/ml. We next investigated the activation patterns of ERK1/2 in cells exposed to VEGF at 50 ng/ml for various time intervals. The data shown in Fig. 4, C and D, revealed that VEGF induced a rapid and transient-peaked activation of ERK1/2 within <15 min (5–15 min) after VEGF stimulation. To test whether VEGF exerts similar effects on activation of PI3-kinase/Akt and ERK5, also known as big MAPK 1 (BMK1), keloid fibroblasts were exposed to VEGF (50 ng/ml) for different time periods and whole cell lysates were analyzed for phosphorylated Akt levels (Ser473) and ERK5 with Western blots. Although a similar transient activation pattern of Akt was detected after treatment with VEGF, only a mild increase in phosphorylated activity peak was observed at 15–30 min (Fig. 4, C and D). However, no obvious increase in phosphorylated ERK5 was detected in keloid fibroblasts after treatment with VEGF for different time intervals (Fig. 4, C and D).



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Fig. 4. VEGF stimulates phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and Akt in keloid fibroblasts. A: dose-dependent effect of VEGF on ERK1/2 activation. After cells were serum starved for 24 h, fresh medium and different concentrations of VEGF (ng/ml) were added for 15 min and whole cell lysates were analyzed by Western blot. B: densitometric analysis of A. C: time course of VEGF treatment on the activation of ERK1/2, ERK5[big MAPK 1(BMK1)], and Akt. Serum-starved cells were treated with VEGF (50 ng/ml) for the indicated time intervals, and whole cell lysates were analyzed by Western blot. D: densitometric analysis of C. Data are representative of 3 independent experiments.

 

VEGF induces PAI-1 gene expression mainly through activating ERK1/2 signaling pathway in keloid fibroblasts. To further explore the intracellular pathways by which VEGF stimulates PAI-1 gene expression in keloid fibroblasts, cells were pretreated for 1 h with a variety of selective protein kinase inhibitors followed by exposure to VEGF (50 ng/ml) for 24 h. Western blot analyses of the treated conditioned media showed that pretreatment with genistein, a broad-spectrum potent inhibitor of tyrosine kinases, or PD-98059, specific inhibitors of ERK1/2, significantly suppressed the basal as well as the VEGF-induced PAI-1 expression in a dose-dependent manner (Fig. 5, A–C). Recent studies by Suzaki et al. (57) have demonstrated that PD-98059 inhibits not only ERK1/2 but also ERK5. Therefore, the involvement of ERK1/2 in VEGF-mediated upregulation of PAI-1 expression in keloid fibroblasts was addressed with U-0126, another specific inhibitor of ERK1/2. As expected, a similar dose-dependent suppression of VEGF-induced PAI-1 expression was achieved after pretreatment with U-0126 (Fig. 5, A and C). In addition, we explored whether PKC and PI3-kinase pathways are involved in VEGF-mediated PAI-1 expression in keloid fibroblasts. Our results showed that pretreatment with different concentrations of calphostin C, a selective inhibitor of PKC, led to a moderate decrease in VEGF-induced PAI-1 expression (Fig. 5, A and C). However, blocking the PI3-kinase/Akt pathway with LY-294002 at different concentrations only slightly reduced VEGF-stimulated PAI-1 protein level (Fig. 5, A and B). Next, we studied the effects of these protein kinase inhibitors on the expression of PAI-1 transcripts induced by VEGF. Our results showed that pretreatment of keloid fibroblasts with PD-98059 (50 µM) and genistein (50 µM) attenuated VEGF-induced PAI-1 mRNA expression by 83.53% and 76.16%, respectively (P < 0.01; Fig. 5, D and E) and pretreatment with calphostin C (1 µM) decreased VEGF-induced PAI-1 mRNA expression by 54.26% (P < 0.05). However, pretreatment of the cells with LY-294002 (50 µM) only slightly attenuated VEGF-induced PAI-1 mRNA expression (P > 0.05).



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Fig. 5. Effects of specific inhibitors for various protein kinases on VEGF-induced PAI-1 expression in keloid fibroblasts. A: Western blot analyses of PAI-1 protein levels in the conditioned medium after pretreatment with different concentrations of specific inhibitors for various protein kinases, followed by stimulation with VEGF (50 ng/ml) for 24 h. Con, control. B and C: densitometric analysis of Western blot results in A, which are described as relative values to the reference point of VEGF (–) with no drug treatment (arbitrarily set as 1.0). D: after pretreatment with PD-98059 (PD; 50 µM), U-0126 (50 µM), LY-294002 (LY, 50 µM), genistein (Gen, 50 µM) or calphostin C (Cal, 1 µM) for 1 h, cells were exposed to VEGF (50 ng/ml) for 6 h and PAI-1 mRNA levels were determined by RT-PCR. E: densitometric analysis of D. Data are representative of 3 independent experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies described an abundance of growth factors and cytokines (31, 32, 63, 64) in the keloid milieu. During the inflammatory stage of wound healing, macrophages and neutrophils are activated and proinflammatory cytokines are released; among these is VEGF. VEGF, which makes blood vessels hyperpermeable, favors the continued induction of tissue granulation of the healing wounds (8, 31, 41). To study whether VEGF contributes to the ongoing growth of keloid scars we used tissue homogenate lysates obtained from fresh biopsied specimens, and we demonstrate for the first time that these tissues consistently express higher level of VEGF protein than normal skin (Fig. 1A). Likewise, an elevated basal level of VEGF mRNA was observed in their derived in vitro-cultured keloid fibroblasts compared with associated normal skin controls (Fig. 1B).

Numerous studies have characterized VEGF expression as regulated by several growth factors, cytokines, and, most importantly, hypoxic stress, in a variety of normal and cancer cell types (21, 38, 48, 55, 56, 62). Hypoxia-induced upregulation of VEGF expression is due to both transcriptional activation and an increase in mRNA stability (18, 24, 33, 35). Transcriptional activation of VEGF expression is mediated by the active HIF-1{alpha} via its binding to the consensus hypoxia-responsive elements (HRE) in the promoter of VEGF (18, 24, 35), whereas hypoxia-induced increase in message stability is likely conferred by RNA-binding proteins (34, 43, 53). Previous histological studies described significant microvascular occlusions (28, 29), a highly collagenous and avascular center of keloid lesions (2), which may contribute to a hypoxic microenvironment in hypertrophic scars and keloids. Our recent findings (66) demonstrated that keloid tissues intrinsically express a higher level of HIF-1{alpha} protein than their associated peripheral normal skin. This finding not only provided direct evidence for the existence of an hypoxic microenvironment as described previously in keloids (2830) but may provide explanations, at least in part, for the mechanisms underlying the elevated basal level of VEGF in keloid tissues reported in the present study (Fig. 1A).

Initially recognized as a tumor-released factor that increases vascular permeability to circulating macromolecules, VEGF was subsequently found to be a selective mitogen and chemotactic factor for endothelial cells in vitro (11, 26) and a potent inducer of angiogenesis in vivo (8, 9, 14, 41). Besides its pivotal role in angiogenesis (54), VEGF also contributes to ECM metabolism by regulating the expression and activity of plasminogen activators [uPA and tissue-type plasminogen activator (tPA)], urokinase receptor (37), and PAI-1 in endothelial cells (42, 44). An altered balance of proteolytic and antiproteolytic activities has been implicated in several fibrotic diseases, including keloids (23, 46, 60). Previous studies reported an intrinsically high level of PAI-1 in keloid fibroblasts and concluded a possible role of PAI-1 in impaired collagen degradation (60, 66). In this study, we demonstrated for the first time that VEGF stimulates the expression of PAI-1, not uPA, at both mRNA and protein levels, in a dose- and time-dependent manner in keloid fibroblasts; a similar stimulatory effect of VEGF on PAI-1 expression was not observed in normal skin fibroblasts (Figs. 2 and 3). VEGF appears to exert its stimulatory effect on PAI-1 protein expression at a lower concentration than it does on mRNA upregulation (Fig. 2, A and B vs. C and E). This observation may simply reflect the differential sensitivities of the techniques and antibody and/or probe used in the experiments. In summary, our findings provide evidence that VEGF may possibly play a role in keloid formation and tissue fibrosis via altering the balance of ECM homeostasis toward a state of excessive accumulation in dermal fibroblasts. Despite numerous studies on the stimulatory effect of VEGF on PAI-1 activity in endothelial cell system, as yet no similar biological function has been described in dermal fibroblasts.

Generally, VEGF exerts its multiple endothelial cell-specific functions by binding to two receptor tyrosine kinases, VEGF receptor (VEGFR)-1/Flt-1 and VEGFR-2/KDR, expressed mainly in endothelial cells (15). After binding to its specific receptors, VEGF activates multiple early signaling cascades, including ERK1/2 (p42/p44 MAPK), PI3-kinase-dependent Akt/PKB pathway, and phospholipase C-{gamma} (PLC-{gamma}), which lead to a net increase in PKC activity and mobilization of intracellular Ca2+ (6, 19, 20, 22, 65). The relative contribution of these pathways to VEGF-induced PAI-1 expression remains unclear, even in the best-characterized endothelial cell system. Here, we report for the first time that VEGF initiates a time- and dose-dependent transient activation of ERK1/2 but not ERK5 in keloid fibroblasts (Fig. 4) and that pretreatment of keloid fibroblasts with PD-98059 or U-0126, a selective inhibitor for ERK1/2, abolishes the VEGF-induced expression of PAI-1 mRNA and protein (Fig. 5, A, C, D, and E). In addition, our studies have shown that pretreatment with genistein, a broad-spectrum inhibitor for receptor and nonreceptor tyrosine kinases, also potently inhibits the expression of PAI-1 mRNA and protein in VEGF-treated cells (Fig. 5, A, B, D, and E). However, pretreatment of keloid fibroblasts with calphostin C (1 µM), a selective inhibitor of PKC, only moderately decreased PAI-1 mRNA and protein level by 54.26%. In endothelial cells, activation of the PI3-kinase/Akt signaling pathway is nearly as strong as ERK1/2 and is essential for a number of VEGF-mediated biological effects (6, 19, 65). In contrast, exposure of keloid fibroblasts to VEGF only slightly stimulated Akt activation (Fig. 4, C and D) and pretreatment with LY-294002, a selective inhibitor of PI3-kinase/Akt, had no obvious suppressive effects on VEGF-induced PAI-1 protein and mRNA expression in these cells (P > 0.05; Fig. 5, A, B, D, and E). Together, these results suggest that VEGF treatment mainly activates ERK1/2, which, together with the activated PTKs, may play a pivotal role in VEGF-induced upregulation of PAI-1 expression in keloid fibroblasts. On the other hand, the Akt signaling pathway is not activated in keloid fibroblasts in response to VEGF and therefore not involved in VEGF-induced PAI-1 expression.

Furthermore, it is worth noting that an interesting finding in our present study includes the differential response of these derived dermal fibroblasts to VEGF. In keloid fibroblasts, VEGF stimulated PAI-1 upregulation in a dose-dependent manner; however, little to minimal stimulatory effects were observed in normal skin fibroblasts. Likewise, VEGF triggers a different activation pattern of signaling pathways in keloid fibroblasts compared with endothelial cells (6, 19, 65). Such differential responses to VEGF may represent differences due to distinct cell types, biological functions (receptor heterogeneity) of VEGFR, or their characteristic intracellular signaling components. Additional studies are needed to delineate the detailed upstream cascades of signal transduction pathways triggered by VEGF, especially the expression and characteristics of VEGF receptors, in fibroblasts derived from keloids and their associated normal skin, which may provide further explanations of the differential responses of keloid fibroblasts to VEGF.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant 1S11-AR-47359 (to A. D. Le).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Le, Dept. of Oral and Maxillofacial Surgery, Charles R. Drew Univ. of Medicine and Science, 1731 E. 120th St., Hawkins Bldg Rm. 3092A, Los Angeles, CA 90059 (E-mail: anle{at}cdrewu.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.

* Y. Wu and Q Zhang contributed equally to this work. Back


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 ABSTRACT
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 DISCUSSION
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