The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet-derived and basic fibroblast growth factors in human vascular smooth muscle cells

Teresa Padró, Rolf M. Mesters, Berno Dankbar, Heike Hintelmann, Ralf Bieker, Michael Kiehl, Wolfgang E. Berdel and Joachim Kienast*

Department of Medicine, Hematology and Oncology, University of Münster, Albert-Schweitzer Str. 33, D-48129 Münster, Germany

* Author for correspondence (e-mail: kienast{at}uni-muenster.de

Accepted 4 February 2002


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 Materials and Methods
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Emerging data suggest that urokinase-type plasminogen activator (UPA), beyond its role in pericellular proteolysis, may also act as a mitogen. We investigated the function of endogenous UPA in mediating the mitogenic effects of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) on human vascular smooth muscle cells (SMC). Growth-arrested SMC constitutively expressed UPA, but UPA expression and secretion increased several times upon stimulation with either PDGF or bFGF. Inhibition of endogenous UPA with a polyclonal antibody significantly reduced DNA synthesis and proliferation of PDGF or bFGF stimulated SMC, this effect already being evident when the cells entered S-phase. The proliferative activity of endogenous UPA was dependent on a functional catalytic domain as demonstrated by inhibition experiments with a specific monoclonal antibody (394OA) and p-aminobenzamidine, respectively. In contrast, neither plasmin generation nor binding of UPA to its receptor (CD87) were required for UPA-mediated mitogenic effects. The results demonstrate that endogenous UPA is not only overexpressed in SMC upon stimulation with PDGF/bFGF, but also mediates the mitogenic activity of the growth factors in a catalytic-domain-dependent manner. Specific inhibition of this UPA domain may represent an attractive target for pharmacological interventions in atherogenesis and restenosis after angioplasty.

Key words: Urokinase, Growth factors, Smooth muscle cells, Proliferation, Atherogenesis


    Introduction
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 Introduction
 Materials and Methods
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Proliferation of smooth muscle cells (SMC) is a crucial event in the formation of intimal thickening in vascular diseases, such as atherosclerosis and restenosis after angioplasty (Ross, 1993Go). Urokinase type plasminogen activator (UPA) is overexpressed in atherosclerotic as well as in restenotic lesions of human arteries, mostly in colocalization with SMC and macrophages (Padró et al., 1995Go; Lupu et al., 1995Go; Schneiderman et al., 1995Go; Raghunath et al., 1995Go; Kienast et al., 1998Go). In early atherosclerotic lesions, UPA expression is particularly related to proliferating SMC in the abluminal part of the neointima (Kienast et al., 1998Go). Studies in animal models have demonstrated a significant increase of UPA expression in vessel wall following arterial injury at the time of SMC proliferation (Clowes et al., 1990Go; More et al., 1995Go; Plekhanova et al., 2001Go). Furthermore, mice genetically deficient in UPA have a markedly attenuated response to vascular injury in terms of reduced neointima cell accumulation (Carmeliet et al., 1997Go). Similarly, UPA neutralizing antibodies greatly reduced neointima size and the number of neointimal SMC in rat arteries after balloon catheter injury (Plekhanova et al., 2001Go). UPA is a serine protease composed of three functionally autonomous domains, the N-terminal growth factor-like domain containing the binding site for the glycolipid-anchored UPA receptor (UPAR, CD87), the kringle domain, and the catalytic domain (serine protease region) (Blasi et al., 1987Go; Behrendt et al., 1995Go). UPA plays a pivotal role in plasmin-mediated pericellular proteolysis and therefore is considered a key factor in physiological and pathological processes in which matrix degradation and tissue remodeling are required (Vassalli et al., 1991Go; Andreasen et al., 1997Go). Beyond this activity, UPA is reported to generate biological effects characteristic of molecules with signal transduction properties including chemotaxis, migration, adhesion and mitogenesis (Schmitt et al., 2000Go). The generation of intracellular signals by UPA may occur via both UPAR-dependent (Blasi, 1999Go; Chapmann and Wei, 2001) or independent mechanisms (Bhat et al., 1999Go; Poliakov et al., 1999Go).

Growth factors and cytokines that regulate cell proliferation have been shown to promote UPA production in various tumoral and normal cell-types (Stopell et al., 1986Go; Dong-Le Bourhis et al., 1998Go; Sieuwerts et al., 1999Go; Fibbi et al., 1999Go; Kitange et al., 1999Go). In addition, there is growing evidence of an association between UPA expression and the mitogenic activity of the cells (Grimaldi et al., 1986Go; Zavizion et al., 1998Go; Hibino et al., 1999Go; Scott et al., 1987Go). Thus, expression of UPA could be a key step for the regulation of cell proliferation in response to growth factors. Indeed, in vitro studies have shown that UPA acts as an autocrine mitogen in human melanoma cells (Kirchheimer et al., 1989Go) and that growth-factor-induced proliferation of human hepatic cells is grossly dependent on UPA (Fibbi et al., 1999Go). In addition, exogenous UPA induces proliferation and growth of cells of various origins (Fibbi et al., 1999Go; De Petro et al., 1994Go; Koopman et al., 1998Go; Fischer et al., 1998Go: Fishmann et al., 1999) and recent studies have demonstrated that UPA can also elicit a mitogenic response in human vascular SMC (Kanse et al., 1997Go; Stepanova et al., 1999Go; Dumler et al., 1999Go). However, as yet it remains unclear whether this mitogenic response depends on the proteolytic activity of UPA or requires binding to its cellular receptor CD87.

Atherosclerotic and restenotic lesions are characterized by overexpression of growth factors and their receptors (Ross, 1993Go). Both, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are known to play an important role in vascular SMC proliferation (Ross et al., 1986Go; Lindner et al., 1991Go). bFGF has been found to upregulate UPA-expression in human and murine vascular SMC both at mRNA and protein levels (Wang et al., 1997Go; Herbert et al., 1997Go). Although a similar effect for PDGF on SMC could not be shown in the latter study (Herbert et al., 1997Go), PDGF has been reported to increase the expression of UPA in human hepatic cells (Fibbi et al., 1999Go).

Regardless of the close relationship between growth factors and UPA during cell proliferation, only one report in transgenic mice has dealt with the role of endogenous UPA on the PDGF-and bFGF-induced growth of vascular SMC (Herbert et al., 1997Go). Therefore, the present study was undertaken to investigate whether endogenous UPA contributes to the mitogenic response of human vascular SMC to PDGF and bFGF, and to characterize the structural and functional properties of UPA that are involved in its growth factor-like function.


    Materials and Methods
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 Materials and Methods
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Reagents
Dulbecco Modified Eagle's Medium (DMEM) containing stable glutamine and DMEM supplemented with 15 mM Hepes and Ham's nutrient mix F-12 (DMEM/Ham's F-12) were purchased from BioConcept (Germany). Bovine fetal calf serum (FCS) was obtained from Sigma (USA). Other cell culture supplements were purchased from Cascade Biologics (USA) and Roche Diagnostics (Germany). Human recombinant PDGF-BB and bFGF were obtained from Sigma and Roche Diagnostics, respectively. All anti-UPA and anti-UPA receptor antibodies were from American Diagnostics (USA) and the goat-, mouse- and rabbit-non immune IgG were obtained from Sigma. Tranexamic acid (trans-4-aminomethyl-cyclohexanecarboxylic acid), {epsilon}-aminocaproic acid (6-amino-n-hexanoic acid), aprotinin, and p-aminobenzamidine were purchased from Sigma. High molecular weight urokinase purified from human urine (HMW-UPA) was obtained from Calbiochem (USA). 4,5,6,7-tetrabromobenzotriazole (TBB) was a kind gift from David Shugar (Polish Academy of Sciences, Warsaw, Poland) and 5,6-dichloro-1-ß-D-ribofuranosyl benzimidazole (DRB) was purchased from Alexis Corporation (USA).

Cell culture
Human vascular SMC were obtained as confluent monolayers in passage two from Technoclone (Austria). According to the manufacturer, cells were delivered as a pool of SMC isolated from the umbilical veins of 10 donors by the explant technique. SMC were identified by their characteristic `hill and valley' growth appearance and by immunostaining for SMC {alpha}-actin. Cells, routinely used from the third to the seventh passage, were grown in DMEM supplemented with 10% (v/v) heat inactivated FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B at 37°C in a humid atmosphere of 5% CO2 in air. Cells were passaged using 0.25% trypsin/0.02% EDTA (ethylenediaminetetraacetic acid). Culture medium was changed every 2-3 days and a confluent SMC monolayer was obtained after about 7 days.

DNA synthesis
Vascular SMC were sparsely plated into 96-well plates (1x103 cells/well) in the presence of 10% FCS in DMEM/Ham's F12. At 24 hours, cells were washed and rendered quiescent with 0.5% FCS for further 72 hours. After this period, proliferation was reinitiated with 10% FCS or with 1% FCS in the presence of either PDGF (20 ng/ml) or bFGF (10 ng/ml) without and with anti-UPA-antibodies, anti-UPAR-antibodies, p-aminobenzamidine (UPA-inhibitor), lysine analogues ({epsilon}-aminocaproic acid, tranexamic acid), or the plasmin inhibitor aprotinin. After 24 hours, 5-bromo-2-deoxyuridine (BrdU, 10 µM, Roche Diagnostics) was added, and the cultures were maintained for a further 24 hours. In other experiments, cells were labeled with BrdU for 24 hours starting at time of stimulation with or without PDGF and bFGF, with increasing concentrations (0-40 µM) of TBB or DRB (protein kinase CK2 inhibitors) in the absence or presence of p-aminobenzamidine or purified HMW-UPA. Incorporation of BrdU in the genomic DNA of proliferating cells was measured by a colorimetric immunoassay according to the manufacturer's instructions (Cell proliferation ELISA; Roche Diagnostics). In a separate set of experiments, the vascular SMC were exposed to BrdU for serial pulses of 6 or 12 hours each. The mitogenic activity of the cells was expressed by the BrdU-labeling index (BrdU-LI), which was calculated as follows:

Cell proliferation
Vascular SMC growth rates were measured by plating 1x104cells/well in 24-well plates. Growth-arrested cells (as described above) were stimulated with 10% FCS or with 1% FCS in the presence of either PDGF (20 ng/ml) or bFGF (10 ng/ml) with and without anti-UPA-antibodies or the UPA-inhibitor p-aminobenzamidine. Cells were maintained in culture for a period of 7 days with medium changes every 48 hours. At 24 hour intervals, cells were trypsinized and counted under the microscope in a Neubauer hemocytometer. Cell viability was monitored by Trypan Blue dye exclusion.

Preparation of SMC-conditioned medium (SMC-CM) and cell extracts
To evaluate PDGF- and bFGF-induced expression of UPA, SMC were cultured in 24-well cluster plates and treated with 1% FCS in the absence or presence of PDGF or bFGF, as described above, for time periods of up to 48 hours. After 24 and 48 hours, SMC-CM was collected, centrifuged at 3000 g for 5 minutes for removal of cell debris, and stored at -80°C until the assays were performed. For UPA antigen extraction, the cells were washed with phosphate-buffered saline (PBS) and harvested at 4°C using an acid acetate/Triton X-100 buffer (Padró et al., 1995Go). After two freeze-thawing cycles to break the cell membranes, samples were further incubated for 15 minutes at 4°C and finally centrifuged for 10 minutes at 5000 g. Supernatants were stored at -80°C until analysis. The protein content in cell extracts was determined by the bicinchoninic acid method using a commercial assay (Pierce Chemical, USA). To evaluate the levels of UPA mRNA in SMC, total cellular RNA was isolated using the guanidine isothiocyanate/phenol method (Chomczynski and Sacchi, 1987Go).

Reverse transcription-polymerase chain reaction (RT-PCR) analyses
For semi-quantitative UPA messenger RNA (mRNA) analysis, 300 ng total RNA was reverse transcribed. Complementary DNA (cDNA) was synthesized for 1 hour at 37°C using random hexamers and moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Germany). UPA transcripts were amplified using Taq polymerase (Promega) on a Hybaid thermocycler (MWG-Biotech, Germany) for 35 cycles (45 seconds at 94°C, 45 seconds at 58°C, 1 minute at 72°C and 5 minutes at 72°C). The following primer pairs were used for the amplification of human UPA cDNA: 5'-TCCCGGACTATACAGACCAT-3' (sense) and 5'-TCTCTTCCTTGGTGTGACTG-3' (antisense) (Iwasaka et al., 1996Go). In all PCR amplifications, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 347 base pairs) was co-amplified as an internal control for RNA integrity and quantification, using the GAPDH primers 5'-CCCTCCAAAATCAAGTGGGG-3' (sense) and 5'-CGCCACAGTTTCC-CGGAGGG-3' (antisense). The amplified PCR products were separated on 6% polyacrylamide gels and stained with ethidium bromide. For the estimation of UPA expression, the corresponding signals were normalized against GAPDH using a fluorometric analysis system (Gel Doc 1000; Bio-Rad Laboratories, Germany).

Quantification of UPA
UPA antigen was quantitatively determined in cell extracts and SMC-CM by a commercial ELISA (Tintelize, Biopool, Sweden). Functional UPA in SMC-CM (pro-UPA and two-chain active UPA) was measured by a biologic immunoassay (Chromolize, Biopool, Sweden). Calibration curves were prepared by dilution of UPA standard provided by the manufacturer either in culture medium or extraction buffer as required. UPA-activity was directly evaluated in a chromogenic assay using the substrate S-2444, as described by the providers (Chromogenix, Sweden).

Statistics
Data are presented as individual data plots or as medians and interquartile ranges (IQR). The statistical significance of overall differences between groups was analyzed by the Kruskal-Wallis one-way analysis of variance. If the test was significant, pairwise comparisons were done by the multiple-comparisons' criterion. Differences between two independent groups were analyzed by the Mann-Whitney rank sum test. The Wilcoxon matched-pair signed rank test was used for comparison of differences within pairs. A P value of 0.05 or less was considered significant.


    Results
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 Materials and Methods
 Results
 Discussion
 References
 
Mitogenic response of human vascular SMC to PDGF and bFGF
Exposure of vascular SMC to growth factors such as PDGF and bFGF is known to promote vascular SMC proliferation during neointima formation. In preliminary experiments we treated subconfluent growth-arrested human vascular SMC with increasing amounts of PDGF(-BB) or bFGF for 72 hours, which resulted in a dose-dependent increase of both DNA synthesis and cell proliferation (data not shown). Maximal effects were achieved with 20 ng/ml of PDGF and 10 ng/ml of bFGF. These concentrations were therefore used in all subsequent experiments. When quiescent SMC were treated with 1% FCS in the presence of PDGF or bFGF, DNA synthesis (as determined by 12-hour pulses for BrdU incorporation) was evident after 24 hours of stimulation (Fig. 1). In the PDGF- and bFGF-treated cells, BrdU incorporation into DNA progressively increased over a 48-hour period. Further experiments monitoring BrdU incorporation at 6-hour intervals confirmed that the majority of PDGF- and bFGF-treated SMC entered S-phase between 18 and 24 hours of stimulation (P=0.03 for PDGF- or bFGF-treated SMC versus controls, Mann-Whitney test; Table 1).



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Fig. 1. Time-dependent increase in DNA synthesis in cultured human vascular SMC stimulated with PDGF or bFGF. Quiescent cells in 96-well plates were treated with 1% FCS ([UNK]); 1% FCS+10 ng/ml bFGF ({blacktriangledown}); or 1% FCS+20 ng/ml PDGF ({blacktriangleup}). Cells were exposed to 4x12 hour BrdU pulses (10 µM) from 0-48 hours in the continuous presence of the growth factors. BrdU uptake was determined by ELISA. Data points represent absorbance levels (medians and IQR, n=6).

 

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Table 1. Effect of PDGF and bFGF on the kinetics of vascular SMC entry into S phase

 

Expression of UPA in vascular SMC after PDGF and bFGF stimulation
Quiescent human vascular SMC constitutively expressed UPA. Median concentrations of UPA antigen in cell lysates of growth-arrested cells did not vary significantly during culture for periods of up to 48 hours [0 hours: 12.0 (IQR, 10.9-21.3) ng/mg extracted protein; 24 hours: 18.9 (11.3-22.2) ng/mg extracted protein; 48 hours: 13.3 (11.2-17.3) ng/mg extracted protein].

To study the effects of PDGF and bFGF on UPA expression in human vascular SMC, growth-arrested cells were exposed to 1% FCS without (control) and with growth factors for periods of up to 48 hours as described in Materials and Methods. The steady-state levels of UPA antigen were not significantly altered when cells were treated with 1% FCS alone with median concentrations in cell lysates of 12.1 (7.1-19.1) ng/mg extracted protein after 24 hours and 10.8 (8.1-14.4) ng/mg extracted protein after 48 hours (Fig. 2A). In contrast, median UPA antigen levels in PDGF-stimulated SMC increased 1.9-times (IQR; 1.5-2.8) and 3.1-times (IQR; 2.7-4.4) over controls (1% FCS) after 24 and 48 hours in culture, respectively (P<0.01 for both time periods; Fig. 2A). More marked stimulation effects, although with a similar trend, were observed when human vascular SMC were treated with bFGF for up to 48 hours [median fold increase over controls: 24 hours, x3.7 (IQR; 2.5-7.3), P<0.001; 48 hours, x5.1 (IQR; 2.8-7.0), P<0.001, Fig. 2A].



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Fig. 2. Effect of PDGF and bFGF on UPA expression in human vascular SMC after 24 and 48 hours stimulation. Growth-arrested subconfluent SMC were cultured in DMEM/Ham's F-12 with 0.2% FCS or with 1% FCS in the absence or presence of PDGF (20 ng/ml) and bFGF (10 ng/ml). (A) UPA antigen levels in cell lysates. Data from a minimum of six independent experiments performed in triplicates are given as box-and-whisker plots; boxes denote interquartile ranges with median values shown as horizontal lines inside the boxes. Whiskers denote ranges; outliers beyond 1.5-times interquartile ranges are represented by individual symbols ([UNK]). UPA antigen levels in PDGF- or bFGF-stimulated cells were significantly higher than in cells treated with 1% FCS alone both after 24 hours (P=0.03 for PDGF and P=0.001 for bFGF; Mann-Whitney test) and 48 hours of stimulation (P<0.001 for PDGF and P<0.001 for bFGF; Mann-Whitney test). (B) UPA transcripts in unstimulated (1% FCS) and PDGF- or bFGF-stimulated SMC were analyzed by RT-PCR after 24 and 48 hours treatment. Levels of UPA messenger RNA were estimated by normalization against co-amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH); M, size marker.

 

Semiquantitative RT-PCR showed an increase in UPA transcripts in vascular SMC after exposure to PDGF and bFGF for 24 and 48 hours, respectively, which indicated PDGF- and bFGF-induced stimulation of UPA gene expression (Fig. 2B).

A similar trend as that described for UPA in cell lysates emerged for levels of UPA antigen in conditioned media. Control SMC (1% FCS) had consistently low levels of UPA antigen in culture supernatants over the 48 hour incubation period. After 48 hours in culture, UPA antigen in the conditioned media of growth factor stimulated vascular SMC increased by 7- to 11-times compared with the control SMC (Table 2). Functional UPA levels were measured by a biologic immunoassay that detects both active two-chain UPA and pro-UPA. We found that the pro-UPA form almost quantitatively accounted for total functional UPA in the conditioned media of both the control and the growth-factor-stimulated SMC (data not shown).


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Table 2. Levels of UPA antigen in the conditioned media of vascular SMC stimulated with PDGF (20 ng/ml) or bFGF (10 ng/ml)

 

Effect of polyclonal antibodies against UPA on DNA synthesis of PDGF and bFGF-stimulated vascular SMC
Since PDGF- and bFGF-induced UPA expression in SMC paralleled the induction of DNA-synthesis, we next investigated whether endogenous UPA was involved in the mitogenic response to these growth factors.

To this end, growth-arrested SMC were exposed to polyclonal goat anti-UPA IgG (398, American Diagnostica; 40 µg/ml) or goat non-immune IgG (40 µg/ml) for 15 minutes. Thereafter, cells were stimulated with PDGF or bFGF in the continuous presence of the neutralizing antibodies or the non-immune IgG for periods of up to 48 hours. In a first set of experiments, DNA-synthesis was analyzed by BrdU-uptake during the second 24 hour incubation period. Fig. 3 shows that in PDGF and bFGF stimulated SMC, the BrdU labeling-index (BrdU-LI, see Materials and Methods) reached median values of 0.86 (IQR, 0.75-1.10) and 0.70 (IQR, 0.57-0.75), respectively, compared with 0.26 (IQR, 0.20-0.33) in the control cells treated with 1% FCS alone (P<0.001 both for PDGF and bFGF plus vehicle, respectively, versus controls; Mann-Whitney test). In the presence of polyclonal anti-UPA IgG, there was a significant reduction in the BrdU-uptake induced by either PDGF (BrdU-LI: 0.65; P=0.004) or bFGF (BrdU-LI: 0.52; P=0.004). The BrdU-LI in growth factor stimulated SMC was not significantly affected by the presence of non-immune goat IgG (PDGF, 0.86; bFGF, 0.77).



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Fig. 3. UPA-mediated DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. Growth arrested cells were exposed to PDGF or bFGF (for 48 hours) in the absence or presence of polyclonal goat anti-UPA IgG (anti-UPA; 40 µg/ml) or goat nonimmune IgG (goat-IgG; 40 µg/ml). The BrdU incorporation during the second 24 hours incubation period was determined by ELISA. The BrdU-labeling index was calculated as the ratio of the corresponding value in 10% FCS-stimulated cells. Results, given as box-and-whisker plots, were obtained from triplicate determinations in five independent experiments. The Mann-Whitney test was employed to identify differences between groups (PDGF stimulation: anti-UPA, P=0.004; goat-IgG, P>0.05 versus vehicle; bFGF stimulation: anti-UPA, P=0.004; goat-IgG, P>0.05 versus vehicle). The control group represents cells treated with 1% FCS alone (PDGF plus vehicle versus control P<0.0001; bFGF plus vehicle versus control P<0.0001).

 

We further examined the effect of the polyclonal antibody 398 (anti-UPA IgG) on BrdU-incorporation by vascular SMC through the time period (12-24 hours) when quiescent cells stimulated with PDGF and bFGF entered S phase (Table 1). Polyclonal anti-UPA IgG consistently reduced the BrdU-labeling index during both the 12-18 hour and the 18-24 hour intervals of stimulation with either PDGF (P=0.03 for both time periods) or bFGF (P=0.03 for both time periods) (Fig. 4A,B).



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Fig. 4. Effect of polyclonal anti-UPA antibodies on S phase entry of vascular SMC stimulated with PDGF or bFGF. Growth arrested vascular SMC exposed to 20 ng/ml PDGF or 10 ng/ml bFGF in the absence or presence of anti-UPA antibodies (40 µg/ml) received 6 hour pulses of BrdU after (A) 12 hours and (B) 18 hours of stimulation. The BrdU-labeling index was calculated as described in Fig. 3. Results from five independent experiments are given as data points. Each individual point corresponds to the mean value of triplicate determinations. Points linked by lines represent data from the same experiment obtained in the absence or presence of anti-UPA antibodies. Analysis of significance for differences within pairs in each group was performed by the Wilcoxon matched-pair signed rank test (P=0.031 for each group).

 

DNA synthesis in PDGF- or bFGF-stimulated vascular SMC is dependent on the UPA-catalytic domain but does not require UPA-UPAR interaction
In order to characterize the structural and functional properties of UPA that are involved in PDGF- and bFGF-induced DNA synthesis, we examined the effects of a specific monoclonal antibody against the UPA-B chain blocking the catalytic site and enzymatic activity (mAb 394OA, American Diagnostica) and a monoclonal antibody against the UPA-A chain that blocks UPAR binding (mAb 3921, American Diagnostica). As described above, vascular SMC were stimulated with the growth factors in the absence or in the continuous presence of the monoclonal antibodies (40 µg/ml). Fig. 5 shows that median PDGF- and bFGF-induced DNA synthesis decreased by 70% (P<0.001) and 89% (P<0.001), respectively, in the presence of mAb 394OA. In contrast, BrdU-uptake was not significantly affected by mAb 3921 [BrdU-LI, PDGF: 0.91 (0.83-0.94) versus 0.96 (0.90-1.03) in controls; bFGF: 0.74 (0.71-0.79) versus 0.83 (0.80-0.85) in controls] (Fig. 5). In addition, a monoclonal antibody directed against the UPAR (mAb 3936, American Diagnostica; Fig. 5), which interferes with UPA binding, was also ineffective in preventing BrdU-incorporation into DNA of human vascular SMC stimulated with either PDGF [BrdU-LI: 0.89 (0.86-0.94)] or bFGF [BrdU-LI: 0.79 (0.68-0.80)].



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Fig. 5. Role of the catalytic and the growth factor domains of UPA in PDGF- or bFGF-stimulated DNA synthesis by vascular SMC. As described in Fig. 1, growth-arrested cells were exposed to PDGF or bFGF in the presence of 40 µg/ml mAb 394OA against the UPA B-chain (catalytic domain; crosshatched bars), 40 µg/ml mAb 3921 against the UPA A-chain (growth factor domain; right-hatched bars), 40 µg/ml mAb 3936 against the UPAR (inhibition UPA binding; left-hatched bars) or vehicle (open bars). Incorporation of BrdU was determined between 24 and 48 hours of stimulation with PDGF or bFGF and expressed as BrdU-labeling index. Results, given as box-and-whisker plots, are obtained from triplicate determinations in six independent experiments. P<0.001 for differences in BrdU-LI between cells treated with mAb 394OA and vehicle under stimulation with either PDGF (A) or bFGF (B).

 

In a subsequent series of experiments, BrdU-uptake in PDGF and bFGF stimulated vascular SMC was evaluated in the presence or absence of the UPA-inhibitor p-aminobenzamidine (Geratz and Cheng, 1975Go). Preliminary experiments demonstrated that p-aminobenzamidine inhibited UPA protease activity in a dose-dependent manner (15-1000 µM; evaluation by a chromogenic assay), with a Kd of 180 µM (data not shown). The presence of p-aminobenzamidine significantly reduced the PDGF- and bFGF-induced DNA synthesis in cultured SMC with a dose-dependent effect between 50 µM and 1000 µM (PDGF, 19-84% reduction; bFGF, 11-94% reduction; Fig. 6).



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Fig. 6. Effect of p-aminobenzamidine on DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. As described in Fig. 1, growth arrested cells were exposed to PDGF ({blacktriangleup}) or bFGF ({blacktriangledown}) in the presence of 0, 62.5, 250 and 1000 µM p-aminobenzamidine for 48 hours and incubated with BrdU for the second 24 hour period. BrdU-labeling indices are presented as medians (IQR) of four independent experiments performed in triplicate. Statistical analysis was performed by the Kruskal-Wallis test (P<0.0001). The multiple comparisons' criterion was employed to identify differences between groups (P<0.001 for 250 and 1000 µM versus 0 µM in PDGF- and bFGF-stimulated cells).

 

Role of the UPA catalytic domain on vascular SMC cell proliferation induced by PDGF and bFGF
The relevance of the catalytic domain of UPA for the mitogenic effects of PDGF and bFGF on human vascular SMC was also studied by cell growth-curves. SMC stimulated with PDGF or bFGF had median doubling times of 47 hours and 50 hours, respectively, and the median cell number increased 12-fold after 7 days of culture in the presence of PDGF (Fig. 7A) and 10-fold when cultured in the presence of bFGF (Fig. 7B). With regard to PDGF-induced SMC proliferation, doubling times were 1.2-times prolonged in the presence of polyclonal anti-UPA IgG (40 µg/ml; P=0.05), 2.3-times in the presence of mAb 394OA (40 µg/ml; P=0.03), and 1.4-times in the presence of p-aminobenzamidine (250 µM; P=0.03). In accordance with these findings, median cell numbers after 7 days in culture were reduced by 35%, 74% and 52%, respectively (P<0.01 for all treatments). Similarly, bFGF-stimulated SMC had longer doubling times in the presence of polyclonal anti-UPA IgG (x1.4, P=0.05), mAb 394OA (x2.8, P=0.03) and p-aminobenzamidine (x1.3, P=0.03). Median cell numbers were reduced by 50%, 79% and 41% (P<0.01 for all treatments) compared with the controls (bFGF alone) after 7 days in culture. Non-immune-goat IgG, non-immune-mouse-IgG and the vehicle of p-aminobenzamidine did not affect PDGF- or bFGF-induced cell growth (data not shown).



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Fig. 7. Inhibition of PDGF- or bFGF-stimulated SMC proliferation by antibodies against UPA and p-aminobenzamidine. Cell counts [medians (IQR) of 3-4 independent experiments] were performed over periods of 7 days on PDGF- (A) or bFGF- (B) stimulated SMC cultured in the absence (hexagon) or in the presence of 40 µg/ml polyclonal goat anti-UPA 398 ([UNK]), 40 µg/ml monoclonal mouse anti-UPA 394OA ({square}), or 250 µM of the UPA inhibitor p-aminobenzamidine ({triangleup}).

 

Role of plasmin generation in the UPA mediated PDGF- or bFGF-induced DNA synthesis in vascular SMC
To examine whether plasmin generation is required for the catalytic-domain-dependent mediator function of UPA in PDGF- and bFGF-induced SMC proliferation, the effects of the plasmin inhibitor aprotinin and of the lysine analogues tranexamic acid and {epsilon}-aminocaproic acid were tested. Lysine analogues have been shown to interfere with the binding of plasminogen to the cell surface and, therefore, prevent the generation of plasmin (Dunn and Goa, 1999Go; Vldavsky, 1987). Table 3 shows that the addition of 10 mM tranexamic acid, 10 mM {epsilon}-aminocaproic or 100 µM aprotinin to the culture medium had no effect on DNA synthesis, indicating that the UPA-mediated mitogenic activity of PDGF and bFGF did not occur through the generation of plasmin.


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Table 3. Effect of lysine analogues and aprotinin on DNA synthesis (expressed as BrdU-labeling index) in PDGF- or bFGF-stimulated vascular SMC

 

Simultaneous inhibition of UPA and CK2 activity reduced PDGF- or bFGF-induced DNA synthesis in vascular SMC in an additive manner
Protein kinase CK2 (formally casein kinase II) is an abundant serine/threonine kinase located in different cellular compartments including the plasma membrane (Faust and Montenarh, 2000Go). CK2 is overexpressed in proliferating tissues (for a review, see Issinger, 1993Go) and CK2 activity has been correlated with cell proliferation (Lebrin et al., 2001Go). Furthermore, a recent report has implicated CK2 in the mitogenic response of human vascular SMC to recombinant UPA (Dumler et al., 1999Go). Based on this evidence, we next investigated whether CK2 also plays a role in mediating UPA-dependent cell proliferation following growth factor stimulation. To this end, DNA synthesis assays in vascular SMC were performed as described above using PDGF or bFGF stimulation in the absence or continuous presence of the selective CK2 inhibitors, TBB or DRB (Shugar et al., 1994; Sarno et al., 2001Go). The data obtained from these experiments demonstrated that blocking CK2 led to inhibition of the PDGF- and bFGF-induced mitogenic response in vascular SMC. TBB (4-10 µM) reduced BrdU incorporation in a dose-dependent manner up to inhibition levels higher than 99% at a concentration of 10 µM, both in PDGF- and bFGF-stimulated cells (Fig. 8). Significant, albeit much less pronounced inhibition was also observed by the presence of DRB (40 µM DRB, PDGF: 64% reduction BrdU-LI; bFGF: 63% reduction BrdU-LI, data not shown). Therefore, further experiments were performed with TBB, which showed a reduction in DNA synthesis in vascular SMC at concentrations reported as highly selective for inhibition of CK2 activity by others (Sarno et al., 2001Go). Concomitant addition of the UPA inhibitor p-aminobenzamidine (100 and 200 µM) markedly enhanced the effect of TBB in preventing the mitogenic activity of PDGF (12%, 45% and 57% inhibition at 4 µM TBB in the presence of 0, 100 and 200 µM p-aminobenzamidine, respectively; P<0.005 for overall group differences; Fig. 8A) and bFGF (37%, 89% and 84% inhibition at 4 µM TBB in the presence of 0, 100 and 200 µM p-aminobenzamidine, respectively; P<0.005 for overall group differences; Fig. 8B) in human vascular SMC. In contrast, when HMW-UPA was added to the culture medium of the TBB-treated cells, median levels of BrdU-uptake tended to recover to approximately 90% of the median levels obtained in cells treated with PDGF or bFGF alone (Table 4).



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Fig. 8. Effect of simultaneous inhibition of UPA and CK2 activities on DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. Growth arrested cells were exposed to PDGF (A) or bFGF (B) in the presence of 0, 4, 6, 8 or 10 µM 4,5,6,7-tetrabromobenzotriazole (TBB) with or without the simultaneous presence of p-aminobenzamidine (p-AMB) at doses of 100 and 200 µM. Incorporation of BrdU was determined between 0 and 24 hours of stimulation with PDGF or bFGF and expressed as BrdU-labeling index. Results, given as box-and-whisker plots, are obtained from triplicate determinations in a minimum of four independent experiments. BrdU-labeling index in PDGF- or bFGF-stimulated cells was significantly reduced in all groups receiving TBB with or without p-AMB (Kruskal-Wallis test and multiple comparison's criterion for pairwise comparisons, P<0.01 for all groups receiving TBB and/or p-AMB compared with groups receiving vehicle alone). Groups treated simultaneously with TBB and p-AMB had significantly lower BrdU-LI than groups treated with TBB alone (Kruskal-Wallis test and multiple comparison's criterion for pairwise comparisons, P<0.01 for all comparisons between TBB/p-AMB-treated groups versus the corresponding TBB-treated groups unless inhibition was complete with TBB alone).

 

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Table 4. Effect of human HMW-UPA on DNA synthesis (expressed as BrdU-labeling index) in vascular SMC stimulated with PDGF or bFGF in the presence of TBB

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proliferation of SMC, a crucial event in neointima formation, is thought to be regulated by endogenous growth factors such as PDGF and bFGF that are liberated at the time of vascular injury or produced by cell types present in the vessel wall lesions (Ross, 1993Go; Ross et al., 1986Go; Lindner et al., 1991Go; Vldavsky et al., 1987Go; Rosenberg, 1993Go). Along with growth factors, components of the plasminogen activator/plasmin system are also considered to play a regulatory role in SMC proliferation during neointima formation (Lupu et al., 1995Go; Schneiderman et al., 1995Go; Raghunath et al., 1995Go; Kienast et al., 1998Go; Clowes et al., 1990Go; More et al., 1995Go; Carmeliet et al., 1997Go; Herbert et al., 1994Go; Brunner and Preissner, 1994Go). The first evidence for the involvement of plasminogen activators came from data by Clowes et al. showing that UPA was overexpressed in the vessel wall following arterial injury at the time of SMC proliferation (Clowes et al., 1990Go). These authors suggested that UPA might act on SMC growth through its ability to activate plasminogen, but did not take into account a direct effect of UPA on SMC proliferation. More recent studies have shown that exogenous UPA may act as a mitogen for several tumor cells as well as normal cell-types in vitro (Bhat et al., 1999Go; Fibbi et al., 1999Go; De Petro et al., 1994Go; Koopman et al., 1998Go; Fischer et al., 1998Go; Kanse et al., 1997Go; Stepanova et al., 1999Go; Dumler et al., 1999Go). The present study expands these observations and demonstrates that endogenous UPA is an important mediator of DNA synthesis and SMC growth induced by mitogens such as PDGF or bFGF.

Upregulation of UPA expression by a diverse set of growth factors, including bFGF and PDGF, has been described for a wide variety of carcinoma cells (reviewed by Aguirre Ghiso et al., 1999Go) as well as nontumor cells (Fibbi et al., 1999Go; Miralles et al., 1998Go; Rusnati et al., 1997Go). As yet, however, only limited information is available on the effects of PDGF and bFGF on UPA expression and secretion by vascular SMC. Using SMC of human umbilical vein origin, we found that quiescent SMC constitutively expressed UPA. However, mRNA and protein expression of UPA was several times increased in proliferating cells upon stimulation with either PDGF or bFGF. Furthermore, SMC stimulated with PDGF or bFGF secreted 5- to 11-times more UPA than control cells. These observations are in line with the report by Clowes et al. showing UPA overexpression in the neointima formed after vascular injury (Clowes et al., 1990Go). A stimulatory effect of PDGF and bFGF on UPA antigen levels also has been described by Wang and co-workers in SMC of human aortic origin (Wang et al., 1997Go). In contrast, PDGF did not have an effect on UPA levels in murine vascular SMC (Herbert et al., 1997Go) and neither PDGF nor bFGF affected UPA levels in cultured arterial SMC from baboons (Kenagy and Clowes, 1995Go). These divergent results are likely to reflect important differences between species in the regulation of UPA expression in vascular SMC.

In several types of tumor cells, the expression of UPA is associated with a proliferative state (Aguirre Ghiso et al., 1999Go). Synthesis of UPA has also been found to be highly regulated during transition from the quiescent to the proliferative state in nontumor murine (Grimaldi et al., 1986Go) and bovine (Zavizion et al., 1998Go) cells. Furthermore, UPA activity and DNA synthesis increase in parallel in venular endothelial cells upon stimulation with bFGF (Ziche et al., 1997Go). Presta et al. suggested that two independent pathways are involved in the mitogenic and the UPA-inducing activity of bFGF (Presta et al., 1992Go). From our studies we cannot exclude this possibility. However, our data suggest a causal relationship between the expression of UPA in human vascular SMC stimulated with bFGF and the ability of these cells to proliferate. Indeed, we demonstrated that blocking endogenous UPA with a polyclonal antibody significantly reduced DNA synthesis and cell growth in PDGF- and bFGF-stimulated SMC. This decrease in DNA synthesis was already observed at the time when quiescent SMC started to proliferate upon stimulation with the growth factors. That UPA may act as a mediator of PDGF- and bFGF-induced proliferation has already been suggested by observations in human hepatic cells (Fibbi et al., 1999Go). Using SMC isolated from transgenic mice deficient in UPA, Herbert et al. also provided evidence of a role for UPA in bFGF-induced cell growth (Herbert et al., 1997Go). However, in contrast to our results, UPA did not appear to be involved in PDGF-induced proliferation of the murine SMC.

An important finding in our study is that DNA synthesis and cell growth in PDGF-and bFGF-stimulated vascular SMC were significantly abrogated in the presence of a monoclonal antibody against the catalytic domain of UPA as well as in the presence of the low-molecular-weight UPA inhibitor p-aminobenzamidine. In contrast, lysine analogues that interfere with plasminogen activation and the plasmin inhibitor aprotinin did not affect mitogen-induced DNA synthesis. In line with these observations, only functional UPA was found to act as a mitogen on human fibroblasts (De Petro et al., 1994Go) and melanoma cells (Kirchheimer et al., 1989Go). Despite the importance of a functional catalytic domain of UPA, plasmin generation was shown not to be required for the mitogenic effects of exogenous UPA (De Petro et al., 1994Go; Koopman et al., 1998Go; Herbert et al., 1997Go). The present study demonstrates a direct association between the presence of functionally active UPA and the mitogenic effects of PDGF and bFGF on human vascular SMC, which is independent of plasmin formation.

PDGF and bFGF, at concentrations that stimulate UPA expression, also potentiate the expression and occupancy of its high affinity binding receptor UPAR (Fibbi et al., 1999Go; Reuning et al., 1994Go). In addition, the low molecular weight forms of UPA, containing the catalytic domain but being devoid of the receptor binding region, do not exhibit mitogenic activity (Koopman et al., 1998Go; Fischer et al., 1998Go; Kanse et al., 1997Go). These findings would support the view that the binding of UPA to UPAR is essential for the induction of mitogenesis. However, currently available information suggests that the importance of the UPA-UPAR interaction is highly cell-type-specific with respect to cell proliferation. In our study, blocking of either the UPA high affinity binding site on UPAR or the N-terminal region of UPA with specific monoclonal antibodies did not alter the mitogenic effect of PDGF and bFGF on vascular SMC. These findings suggest a role for UPA in the proliferative response of human vascular SMC to these growth factors that is independent of its high affinity binding to UPAR. In agreement with our results, Carmeliet et al., using a model with targeted disruption of the UPAR gene, demonstrated that binding of UPA to UPAR is not required for medial and neointimal SMC proliferation in the injured vessel wall (Carmeliet et al., 1998Go).

Our results do not exclude that binding of UPA to cell surface sites other than UPAR is required for its role in the proliferative response of SMC to PDGF and bFGF. For instance, melanoma cells have been found to show a mitogenic response to exogenous UPA through a mechanism mediated by low affinity binding of UPA to the cell surface rather than binding to UPAR (Koopman et al., 1998Go). Evidence for novel cell-surface UPA binding proteins has also recently been reported for platelets (Jiang et al., 1996Go) and leukemic cell lines (Longstaff et al., 1999Go).

The precise molecular mechanisms involved in the UPA-induced mitogenesis are currently unclear. UPA has been demonstrated to mediate cleavage and activation of high molecular weight isoforms of vascular endothelial growth factor (VEGF), which otherwise remain devoid of mitogenic activity (Plouet et al., 1997Go). In a series of experiments in which we pretreated PDGF and bFGF with human HMW-UPA, the results did not provide any evidence that a similar proteolytic activation was required for the induction of SMC proliferation by the recombinant forms of PDGF and bFGF (data not shown).

Results by Dumler et al. endorse the notion that in human vascular cells the mitogenic response to exogenous UPA occurs via activation of the protein kinase CK2 (Dumler et al., 1999Go). In the present study, the UPA-inhibitor p-aminobenzamidine and 4,5,6,7-tetrabromobenzotriazole (TBB), a compound known as a selective inhibitor of CK2 (Sarno et al., 2001Go), showed an additive effect in reducing DNA synthesis induced by PDGF or bFGF. In turn, the addition of active human HMW-UPA tended to counteract the decrease in DNA synthesis induced by the CK2 inhibitor. Although preliminary, these findings raise the possibility that CK2 might also play a role mediating UPA-dependent cell proliferation following growth factor stimulation. Dumler et al. demonstrated that UPAR, CK2 and nucleolin are colocalized on the cell surface of different cell types including vascular SMC. The authors further suggested a role for this complex in the proliferative response of vascular SMC to exogenous UPA (Dumler et al., 1999Go). Based on our results, we can hypothesize that UPA-mediated cell proliferation following PDGF and bFGF stimulation is likely to act through a UPAR-independent CK2-activation mechanism, which still needs to be precisely characterized. Interestingly, Bath et al. have provided evidence that UPA induces tyrosine phosphorylation of a 78 kDa protein via a proteolysis-dependent, but UPAR-independent mechanism. The authors have suggested a link between activation of the 78 kDa protein and cell proliferation in UPA-treated cells (Bhat et al., 1999Go).

In summary, we have demonstrated that UPA is an important mediator of the mitogenic effects of PDGF and bFGF on human vascular SMC. This proliferative activity of UPA in vascular SMC does not involve UPAR binding or plasmin generation, but does require a functional catalytic domain. According to these findings, the UPA catalytic domain may represent an attractive target for pharmacological interventions in atherogenesis or restenosis following angioplasty. Specific inhibition of the UPA catalytic domain may be beneficial not only in preventing plaque destabilization and rupture, but also in preventing SMC proliferation during neointima formation. Furthermore, inhibition of the catalytic site of UPA might by a way of antagonizing mitogenic effects of angiogenic cytokines such as bFGF (e.g. during tumor neovascularization).


    Acknowledgments
 
We thank D. Shugar for kindly providing the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole. This work was supported by an institutional grant from the Interdisciplinary Clinical Research Center at the University of Münster.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Aguirre Ghiso, J. A., Alonso, D. F., Farias, E. F., Gomez, D. E. and Bal de Kier Joffè, E. (1999). Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur. J. Biochem. 263,295 -304.[Abstract/Free Full Text]

Andreasen, P. A., Kjoller, L., Christensen, L. and Duffy, M. J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1-22[Medline]

Behrendt, N., Ronne, E. and Dano, K. (1995). The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface. Biol. Chem. Hoppe Seyler. 376,269 -279.[Medline]

Bhat, G. J., Gunaje, J. J. and Idell, S. (1999). Urokinase-type plasminogen activator induces tyrosine phosphorylation of a 78-kDa protein in H-157 cells. Am. J. Physiol. 277,L301 -L309.[Abstract/Free Full Text]

Blasi, F. (1999). Proteolysis, cell adhesion, chemotaxis, and invasiveness are regulated by UPA-uPAR- PAI-1 system. Thromb. Haemost. 82, 298-304.

Blasi, F., Vassalli, J. D. and Dano, K. (1987). Urokinase-type plasminogen activator: proenzyme, receptor and inhibitors. J. Cell Biol. 104,801 -804.[Medline]

Brunner, G. and Preissner, K. T. (1994). Pericellular enzymatic hydrolysis: implications for the regulation of cell proliferation in the vessel wall and the bone marrow. Blood Coagul. Fibrinolysis 5,625 -639.[Medline]

Carmeliet, P., Moons, L., Herbert, J. M., Crawley, J., Lupu, F., Lijnen, R. and Collen, D. (1997). Urokinase but not tissue type plasminogen activator mediates arterial neointima formation in mice. Circ. Res. 81,829 -839.[Abstract/Free Full Text]

Carmeliet, P., Moons, L., Dewerchin, M., Rosenberg, S., Herbert, J. M., Lupu, F. and Collen, D. (1998). Receptor-independent role of urokinase-type plasminogen activator in pericellular plasmin and matrix metalloproteinase proteolysis during vascular wound healing in mice. J. Cell Biol. 140,233 -245.[Abstract/Free Full Text]

Chapman, H. A. and Wei, Y. (2001). Protease crosstalk with integrins: the urokinase receptor paradigm. Thromb. Haemost. 86,124 -129.[Medline]

Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156 -159.[Medline]

Clowes, A. W., Clowes, M. M., Au, Y. P. T., Reidy, M. A. and Belin, D. (1990). Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery. Circ. Res. 67, 61-67.[Abstract]

De Petro, G., Copeta, A. and Barlat, S. (1994). Urokinase-type and tissue-type plasminogen activators as growth factors of human fibroblasts. Exp. Cell. Res. 213,286 -294.[Medline]

Dong-Le Bourhis, X., Lambrecht, V. and Boilly, B. (1998). Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells. Br. J. Cancer 73,396 -403.

Dumler, I., Stepanova, V., Jerke, U., Mayboroda, O. A., Vogel, F., Bouvet, P., Tkachuk, V., Haller, H. and Gulba, D. C. (1999). Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin. Curr. Biol. 9,1468 -1476.[Medline]

Dunn, C. J. and Goa, K. L. (1999). Tranexamic acid: a review of its use in surgery and other indications. Drugs 57,1005 -1032.[Medline]

Faust, M. and Montenarh, M. (2000). Subcellular localization of protein kinase CK2. A key to its function? Cell Tissue Res. 301,329 -340.[Medline]

Fibbi, G., Pucci, M., Grappone, C., Pellegrini, G., Salzano, R., Casini, A., Milani, S. and Del Rosso, M. (1999). Functions of the fibrinolytic system in human ito cells and its control by bysic fibroblast and platelet-derived growth factor. Hepatology 29,868 -878.[Medline]

Fischer, K., Lutz, V., Wilhelm, O., Schmitt, M., Graeff, H., Heiss, P., Nishiguchi, T., Herbeck, N., Kessler, H., Luther, T., Magdolen, V. and Reuning, U. (1998). Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function. FEBS Lett. 438,101 -105.[Medline]

Fishman, D. A., Kearns, A., Larsh, S., Enghild, J. J. and Stack, M. S. (1999). Autocrine regulation of growth stimulation in human epithelial ovarian carcinoma by serine-proteinase-catalysed release of the urinary-type-plasminogen-activator N-terminal fragment. Biochem. J. 341,765 -769.[Medline]

Geratz, J. D. and Cheng, M. C. F. (1975). The inhibition of urokinase by aromatic diamidines. Thromb. Diath. Haemorrh. 33,230 -243.[Medline]

Grimaldi, G., Di Fiori, P., Locatelli, E. K., Falko, Y. and Blasi, F. (1986). Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells. EMBO J. 5, 855-861.[Abstract]

Herbert, J. M., Lamarche, I., Prabonnaud, V., Dol, F. and Gauthier, T. (1994). Tissue-type plasminogen activator is a potent mitogen for human aortic smooth muscle cells. J. Biol. Chem. 269,3076 -3080.[Abstract/Free Full Text]

Herbert, J. M., Lamarche, I. and Carmeliet, P. (1997). Urokinase and tissue-type plasminogen activator are required for the mitogenic and chemotatic effects of bovine fibroblast growth factor and platelet-derived growth factor-BB for vascular smooth muscle cells. J. Biol. Chem. 272,23585 -23591.[Abstract/Free Full Text]

Hibino, T., Matsuda, Y., Takahashi, T. and Goetinck, P. F. (1999). Suppression of keratinocyte proliferation by plasminogen activator inhibitor-2. J. Invest. Dermatol. 112, 85-90.[Abstract/Free Full Text]

Issinger, O. G. (1993). Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol. Ther. 59,1 -30[Medline]

Iwasaka, C., Tanaka, K., Abe, M. and Sato, Y. (1996). Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix matalloproteinase-1 and the migration of vascular endothelial cells. J. Cell. Physiol. 169,522 -531.[Medline]

Jiang, Y., Pannell, R., Liu, J. N. and Gurewich, V. (1996). Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes. Blood 87,2775 -2781.[Abstract/Free Full Text]

Kanse, S. M., Benzakour, O., Kanthou, C., Kost, C., Lijnen, R. and Preissner, K. T. (1997). Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders. Arterioscler. Thromb. Vasc. Biol. 17,2848 -2854.[Abstract/Free Full Text]

Kenagy, R. D. and Clowes, A. W. (1995). Regulation of baboon arterial smooth muscle cell plasminogen activators by heparin and growth factors. Thromb. Res. 77, 55-61.[Medline]

Kienast, J., Padró, T., Steins, M., Li, C. X., Schmid, K. W., Hammel, D., Scheld, H. H. and van de Loo, J. C. W. (1998). Expression of urokinase-type plasminogen activator in atherosclerotic coronary arteries from human heart explants. Thromb. Haemost. 79,579 -586.[Medline]

Kirchheimer, J. C., Wojta, J., Christ, G. and Binder, B. R. (1989). Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line. Proc. Natl. Acad. Sci. USA 86,5424 -5428.[Abstract]

Kitange, G., Shibata, S., Tokunaga, Y., Yagi, N., Yasunaga, A., Kishikawa, M. and Naito, S. (1999). Ets-1 transcription factor-mediated urokinase-type plasminogen activator expression and invasion in glioma cells stimulated by serum and basic fibroblast growth factors. Lab. Invest. 79,407 -416.[Medline]

Koopman, J. L., Slomp, J., de Bart, A. C. W., Quax, P. H. A. and Verheijen, J. H. (1998). Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor. J. Biol. Chem. 273,33267 -33272.[Abstract/Free Full Text]

Lebrin, F., Chambaz, E. M. and Laurence, B. (2001). A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit {alpha}. Oncogene 20,2010 -2022.[Medline]

Lindner, V., Lappi, D. A., Baird, A., Majack, R. A. and Reidy, M. A. (1991). Role of basic fibroblast growth factor in vascular lesion formation. Circ. Res. 68,106 -113.[Abstract]

Longstaff, C., Merton, E., Fabregas, P. and Félez, J. (1999). Characterization of cell-associated plasminogen activation catalyzed by urokinase-type plasminogen activator, but independent of urokinase receptor (UPAR, CD87). Blood 93,3839 -3846.[Abstract/Free Full Text]

Lupu, F., Heim, D. A., Bachmann, F., Hurni, M., Kakkar, V. V. and Kruithof, E. K. O. (1995). Plasminogen activator expression in human atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 15,1444 -1455.[Abstract/Free Full Text]

Miralles, F., Ron, D., Baiget, M., Félez, J. and Muñoz-Cánoves, P. (1998). Differential regulation of urokinase-type plasminogen activator expression by basic fibroblast growth factor and serum in myogenesis. J. Biol. Chem. 273,2052 -2058.[Abstract/Free Full Text]

More, R. S., Underwood, M. J., Brack, M. J., de, Bono, D. P. and Gershlick, A. H. (1995). Changes in vessel wall plasminogen activator activity and smooth muscle cell proliferation and activation after arterial injury. Cardiovasc. Res. 29, 22-26.[Medline]

Padró, T., Emeis, J. J., Steins, M., Schmid, K. W. and Kienast, J. (1995). Quantification of plasminogen activators and their inhibitors in the aortic vessel wall in relation to the presence and severity of atherosclerotic disease. Arterioscler. Thromb. Vasc. Biol. 15,893 -902.[Abstract/Free Full Text]

Plekhanova, O., Parfyonova, Y., Bibilashvily, R., Domogatskii, S., Stepanova, V., Gulba, D. C., Agrotis, A., Bobik, A. and Tkachuk, V. (2001). Urokinase plasminogen activator augments cell proliferation and neointima formation in injured arteries via protolytic mechanisms. Atherosclerosis 159,297 -306.[Medline]

Plouet, J., Moro, F., Bertagnolli, S., Coldeboeuf, N., Mazarguil, H., Clamens, S. and Bayard, F. (1997). Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect. J. Biol. Chem. 272,13390 -13396.[Abstract/Free Full Text]

Poliakov, A. A., Mukhina, S. A., Traktouev, D. O., Bibilashvily, R. S., Gursky, Y. G., Minashkin, M. M., Stepanova, V. V. and Tkachuk, V. A. (1999). Chemotactic effect of urokinase plasminogen activator: a major role for mechanisms independent of its proteolytic activity or growth factor domains. J. Recept. Signal Transduct. Res. 19,939 -951.[Medline]

Presta, M., Statuto, M., Isacchi, A., Caccia, P., Pozzi, A., Gualandris, A., Rusnati, M., Bergonzoni, L. and Sarmientos, P. (1992). Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region. Biophys. Res. Commun. 185,1098 -1107.[Medline]

Raghunath, P. N., Tomaszewski, J. E., Brady, S. T., Caron, R. J., Okada, S. S. and Barnathan, E. S. (1995). Plasminogen activator system in human coronary atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 15,1432 -1443.[Abstract/Free Full Text]

Reuning, U., Little, S. P., Dixon, E. P. and Bang, N. U. (1994). Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels. Blood 84,3700 -3708.[Abstract/Free Full Text]

Rosenberg, R. D. (1993). Vascular smooth muscle cell proliferation: basic investigations and new therapeutic approaches. Thromb. Haemost. 70,10 -16.[Medline]

Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362,801 -809.[Medline]

Ross, R., Reines, E. W. and Bowen-Pope, D. F. (1986). The biology of platelet-derived growth factor. Cell 46,155 -169.[Medline]

Rusnati, M., Tanghetti, E., Dell'Era, P., Gualandris, A. and Presta, M. (1997). {alpha}vß3 integrin mediatesthe cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Mol. Biol. Cell 8,2449 -2461.[Abstract/Free Full Text]

Sarno, S., Reddy, H., Meggio, F., Ruzzene, M., Davies, S. P., Donella-Deana, A., Shugar, D. and Pinna, L. A. (2001). Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (`casein kinase-2'). FEBS Lett. 496, 44-48.[Medline]

Schmitt, M., Wilhelm, O. G., Reuning, U., Krüger, A., Harbeck, N., Lengyel, E., Graeff, H., Gänsbacher, B., Kessler, H., Bürgle, M. et al. (2000). The urokinase plasminogen activator system as a novel target for tumor therapy. Fibrinolysis Proteolysis 14,114 -132.

Schneiderman, J., Bordin, G. M., Engelberg, I., Adar, R., Seiffert, D., Thinnes, T., Bernstein, E. F., Dilley, R. B. and Loskutoff, D. (1995). Expression of fibrinolytic genes in atherosclerotic abdominal aortic aneurysm wall. J. Clin. Invest. 96,639 -645.[Medline]

Scott, F. M., de Serrano, V. S. and Castellino, F. J. (1987). Appearance of plasminogen activator activity during a syncronous cycle of a rat adenocarcioma cell line, PA-III. Exp. Cell. Res. 169,39 -46.[Medline]

Shugar, D. (1994). Development of inhibitors of protein kinases CK1 and CK2 and some related aspects, including donor and acceptor specificities and viral protein kinases. Cell. Mol. Biol. Res. 40,411 -419.[Medline]

Sieuwerts, A. M., Klijn, J. G., Henzen-Logmans, C. and Foeckens, J. A. (1999). Cytokine-regulated urokinase-type plasminogen activator (UPA) production by human breast fibroblasts in vitro. Breast. Cancer Res. Treat. 55, 9-20.[Medline]

Stepanova, V., Mukhina, S., Köhler, E., Resink, T. J., Erne, P. and Tkachuk, V. A. (1999). Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms. Mol. Cell. Biochem. 195,199 -206.[Medline]

Stopell, M. P., Verde, P., Grimaldi, G., Locatelli, E. K. and Blasi, F. (1986). Increase in urokinase plasminogen activator mRNA synthesis in human carcinoma cells is a primary effect of the potent tumor-promoter phorbol myristate acetate. J. Cell Biol. 102,1235 -1244.[Abstract]

Vassalli, J. D., Sappino, A. P. and Belin, D. (1991). The plasminogen activator/plasmin system. J. Clin. Invest. 88,1067 -1072.[Medline]

Vldavsky, I., Friedman, R., Sullivan, R., Sasse, J. H. and Klagsbrun, M. (1987). Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell-associated and platelet derived growth factor-like protein which is secreted. J. Cell. Physiol. 131,402 -428.[Medline]

Wang, W., Chen, H. J., Schwartz, A., Cannon, P. J. and Rabbani, L. E. (1997). T cell lymphokines modulate bFGF-induced smooth muscle cell fibrinolysis and migration. Am. J. Physiol. 72,C392 -C398.

Zavizion, B., White, J. H. and Bramley, A. J. (1998). Cell cycle-dependent fluctuation of urokinase-type plasminogen activator, its receptor, and inhibitors in cultured bovine mammary epithelial and myoepithelial cells. Biochem. Biophys. Acta. 1403,141 -150.[Medline]

Ziche, M., Parenti, A., Ledda, F., Dell'Era, P., Granger, H. J., Maggi, C. A. and Presta, M. (1997). Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. Circ. Res. 80,845 -852.[Abstract/Free Full Text]


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