JOURNAL OF CELLULAR PHYSIOLOGY 153:196205 (1992)

Basic Fibroblast Growth Factor Increases JunctionalCommunication and Connexin 43 Expression in Microvascular Endothelial Cells Institute of Histology

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M.S. PEPPER* AND P. MEDA Embryology, Department of Morphology, University Medical Center, 12 I I Geneva 4, Switzerland

We have analyzed the effect of basic fibroblast growth factor (bFGF) on junctional communication (coupling) and connexin 43 (Cx43) expression in bovine microvascular endothelial (BME) cells. In control confluent cultures, the incidence of coupling, as assessed by the intercellular transfer of microinjected Lucifer Yellow, was limited to 1 3 % of injected cells, and decreased to 0% with time in culture. After exposure to bFGF i3ng/ml), the incidence of coupling was increascd in a time-dependent manner, reaching a maximum of 38% of microinjected cells after 10-1 2 hours. The extent of coupling, as assessed by scrape loading, was maximally increased 2.1-fold 8-9 hours atter addition of bFCF. bFCF also induced a 2-fold increase in Cx43 as assessed by Western blotting, and increased Cx43 immunolabelling at contacting interfaces of adjacent BME cells. Cx43 mRNA was likewise increased after exposure to bFGF in a time- and dose-dependent manner, with a maximal 6-7-fold increase after a 4 hour exposure to 3-l0ng/ml. Finally, the increase in coupling and Cx43 mRNA expression observed after mechanically wounding a confluent monolayer of BME cells was markedly rcduccd by antibodies to bFGF, which have previously been shown to inhibit migration. Taken together, these results indicate that exogenous and endogenous bFGF increase intercellular communication and Cx43 expression in niicrovascular endothelial cells. We propose that the bFGF-mediated increase in coupling i s necessary for the coordination of endothelial cells during angiogenesis and other vessel wall 0 IYY:! WiIey-Liss, Inc functions.

The vascular endothelium is comprised of a continuous monolayer of endothelial cells which provide a dynamic interface between the circulating blood and surrounding tissues. Adjacent endothelial cells are linked by intercellular tight junctions and gap junctions. Gap junctions are clusters of transmembranous channels composed of structurally related proteins known as connexins (Cx), which mediate the intercellular transfer of small molecules and ions between adjacent cells (reviewed in Bennett et al., 1991). A significant degree of heterogeneity exists in the size and distribution of gap junctions within the vascular tree (reviewed by Larson, 1988). Thus, large gap junctions are frequently found in endothelial cells of arteries, while smaller gap junctions are seen less frequently in veins. Gap junctions are usually undetectable in capillaries and post-capillary venules. These differences and the corresponding levels of coupling appear to be maintained in large vessel and microvascular endothelial cells in vitro (Pepper et al., 1992). A correlation has also been observed between the level of expression of Cx43, a major connexin of vessel-wall cells (Larson et al., 19901, and the levels of coupling in endothelial cells in vitro (Pepper et al., 1992). Although the role of endothelial cell coupling in vessel wall function remains largely unknown, i t has been suggested that homotypic endothelial cell communica0 1992 WILEY-LISS, INC.

tion may play a role in arteriolar vasodilation (Segal and Duling, 1986, 1987, 1989), and that heterotypic endothelial-smooth muscle cell coupling may play a role in the regulation of smooth muscle tone in larger vessels (reviewed by Davies et al., 1988). It has also been suggested that junctional coupling may provide a mechanism for coordinating endothelial cell migration during repair of the endothelial lining of large arteries (Larson and Haudenschild, 19881, and during angiogenesis (Pepper et al., 1989). Basic fibroblast growth factor (bFGF) is a broad spectrum heparin-binding polypeptide mitogen which also modulates several non-mitogenic functions in endothelial cells. Thus, bFGF is a potent angiogenic factor in vivo and in vitro, and induces endothelial cell migration and protease production (reviewed by Klagsbrun and DAmore, 1991). Systemically administered bFGF has also been shown to decrease arterial blood pressure in a n endothelium-dependent manner (Cuevas e t al., 1991). Since we have previously reported that coupling is increased in mechanically wounded monolayers of mi-

Received March ll, 1992; accepted May 20,1992. *To whom reprint requests/correspondence should be addressed.

bFGF INCREASES ENDOTHELIAL CELL COUPLING

crovascular endothelial cells (Pepper et al., 1989) in regions where bFGF-dependent migration (Sato and Rifkin, 1988) and protease production (Odekon et al., 1992; Pepper et al., 1987) are also increased, we wondered whether bFGF could be a n endogenous modulator of coupling. To test this hypothesis, the studies reported in this paper were performed to determine first whether exogenous bFGF is capable of modulating coupling and Cx43 expression in quiescent non-migrating endothelial cells, and second whether endogenous bFGF might mediate the increase in coupling and Cx43 expression which are seen when BME cells are induced to migrate in response to mechanical wounding in vitro (Pepper et al., 1989, 1992).

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either normal rabbit gamma-globulins or rabbit antirhbFGF gamma-globulins (200 pgiml), was added to the monolayers. Coupling between migrating cells was assessed 24 hours later as described below. Cell migration was assessed in the same dishes used for coupling studies by counting the number of cells bordering the scrape-loading line which had crossed the original wound edge. To determine the effect of anti-rhbFGF antibodies on wound-induced Cx43 mRNA expression, confluent monolayers in 100 mm petri dishes were multiple-wounded in the presence of antibodies as follows: medium was removed, the monolayers were washed once with serum-free alpha-MEM, and fresh serum-free alpha-MEM containing 0.1% gelatin and normal rabbit gamma-globulins or rabbit anti-rhbFGF gamma-globulins (200 pg/ml) was added to the monolayers. Approximately 50 parallel wounds were then created with a pointed 1.5 mm-wide rubber policeman, the dish rotated through go", and a n additional 50 parallel wounds created perpendicular to the first set. Total cellular RNA was extracted 4 hours later as described below. The preparation and characterization of the rabbit anti-rhbFGF antibodies have been fully described (Dennis and Rifkin, 1990). Gamma-globulin fractions prepared from normal rabbit serum and rabbit antirhbFGF antiserum by two precipitations with 50% ammonium sulfate were dialysed against distilled water or PBS and stored at -20°C.

MATERIALS AND METHODS Cell culture Bovine microvascular endothelial (BME) cells isolated from the adrenal cortex according to the procedure of Folkman et al. (1979) and cloned as described by Furie et al. (1984) were generously provided by Drs. M.B. Furie and S.C. Silverstein (Columbia University, New York). The cells were grown in minimal essential medium (MEM) alpha modification (Gibco AG, Basel, Switzerland), supplemented with 15% heat-inactivated donor calf serum (Flow Laboratories, Baar, Switzerland), penicillin (500 Uiml), and streptomycin (100 pgi ml). This medium is hereafter referred to as complete medium. The endothelial nature of these cells was conDetermination of junctional firmed by uptake of l,l'-dioctadecy1-3-3-3'-3'-tetracommunication (coupling) methyl-indocarbocyanine perchlorate-labelled acetylated low density lipoprotein (DiI-Ac-LDL, Paesel and Coupling was determined by two approaches. In the Lorei, Frankfurt, Germany) and immunostaining with first, individual endothelial cells were injected with the a rabbit antiserum against bovine (Furie et al., 1984) gap junction-permeant tracer Lucifer Yellow (Sigma or human (Nordic Immunology, Tilburg, The Nether- Chemical Co., St Louis, MO) a s described (Pepper et al., lands) factor VIII-related antigen. Cells were routinely 1989). The incidence of coupling was expressed as the subcultured at a split ratio of 1:4 or 1:5 in 1.5% gelatin- percentage of injections showing intercellular dye coated tissue culture flasks (Falcon Labware, Becton transfer. The extent of dye transfer was expressed as Dickinson Company, Lincoln Park, NJ) and used be- the number of Lucifer Yellow-labelled cells (including tween passages 14 and 20. For dye coupling and immu- the injected cell) per microinjection. In the second apnofluorescence studies, cells were seeded into gelatin- proach, monolayers were scrape-loaded in the presence coated 35 mm tissue culture dishes (Falcon) or onto of Lucifer Yellow and dextran rhodamine (Molecular glass coverslips in 35 mm dishes, at 1-2 x lo5 cells per Probes, Eugene, OR), using a mini glass-cutter (Pepper dish. For Cx43 protein and mRNA studies, cells were et al., 1989). For rhbFGF-treated monolayers, the exseeded into gelatin-coated 100 mm tissue culture dishes tent of coupling was calculated a s the total number of (Falcon) at 5-10 x lo5 cells per dish. Complete medium Lucifer Yellow-labelled cells per 350 pm fielathe numwas changed every 2-3 days, and all experimental ma- ber of labelled cells lying immediately adjacent to the nipulations were performed upon reaching confluence scrape-loading line in the same field (Pepper et al., (after 5-7 days). The last medium change was always 1992). Measurements were from 3 consecutive 350 pm 24 hours before starting the experiment. fields on either side of the scrape loading line from a t least 2 petri dishes per condition (i.e., n 3 12 fields) and Experimental conditions were pooled from two separate experiments. For In experiments in which the effect of recombinant wounding experiments, monolayers were scraped perhuman bFGF (rhbFGF, kindly provided by Dr. P. pendicular to the original endothelial wound and the Sarmientos, Farmitalia Carlo Erba, Milan, Italy) was extent of coupling between migrating cells was evaluto be tested, the growth factor was added directly to ated for cells bordering the scrape line which had complete medium of confluent cultures. To determine crossed the original wound edge (Pepper et al., 1989). the effect of anti-rhbFGF antibodies (kindly provided Labelling of only the first layer of cells lining the by Drs. Y. Sat0 and D.B. Rifkin, New York University scrape indicated the absence of coupling (these cells Medical Center, New York) on coupling and migration, were also labelled with dextran rhodamine, indicating confluent monolayers in 35 mm petri dishes were me- that they had been permeabilized during the scrapechanically wounded with a blade to mark the original loading procedure). The presence of coupling was indiwound edge (Burk, 19731,washed 2-3 times, and fresh cated by the labelling of at least the first two rows of serum-free alpha-MEM, containing 0.1% gelatin and cells along the scrape (the second and subsequent rows

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Time (hours) Fig. 1. bFGF-induced increase in coupling in confluent monolayers of BME cells as determined by microinjection. A: microinjection reveals lack of coupling in a control culture (a and b),as indicated by the retention of Lucifer Yellow within the microinjected cell (arrow). Addition of bFGF (3 ngiml) for 7 hours increased coupling, as demonstrated by the diffusion of Lucifer Yellow from the microinjected cell (arrow) to 4 adjacent cells (c and d). a and c are fluorescence micrographs of the same fields shown in b and d, which are phase contrast views of control and bFGF-treated BME cultures, respectively. Bar = 50 km. B: kinetics of bFGF-induced increase in the incidence of coupling in confluent BME monolayers, as determined by microinjection. Incidence of coupling decreased with time in controls (triangles), and increased in bFGF (3 ngim1)-treated cultures (circles), with a maximal induction after 10-12 hours.

F 4-5 hrs

C F 8-9 hrs

C

F

24 hrs

Fig. 2. bFGF-induced increase in coupling in confluent monolayers of BME cells as determined by scrape loading. A: Scrape loading reveals the virtual absence of coupling in control BME cultures in which Lucifer Yellow is confined to a single row of cells on either side of the scrape line (a).After exposure to bFGF (10 ngiml) for 4 hours, coupling is markedly increased, as revealed by the transfer of Lucifer Yellow from cells lying immediately adjacent to the scrape line to one or more laterally-lying cells (b). a and b are combined phase contrast and fluorescence micrographs. Bar = 200 pm. B: Quantitation of the bFGF 13 ngim1)-induced increase in the extent of coupling in confluent BME monolayers, as determined by scrape loading. When compared to controls (C), the extent of coupling is increased in bFGF-treated cultures (F)with a maximal 2.1-fold increase after 8-9 hours. Results are the mean ? SEM of the total number of Lucifer Yellow-labelled cells per 350 pm field'the number of labelled cells lying immediately adjacent to the scrape loading line in the same field. Measurements are from 3 consecutive 350 K r n fields on either side of the scrape loading line from at least 2 petri dishes per condition (i.e., n >> 121, and are pooled from two separate experiments.

bFGF INCREASES ENDOTHELIAL CELL COUPLING TABLE 1. Incidence and extent of coupling in confluent monolayers of control and bE'CF-treated BME cells' Control 0 hours 6-8 hours

10-12 hours 24-26 hours

Incidence Extent Incidence Extent Incidence Extent Incidence Extent

12.59 (118) 1.2 0.2 9.18 cvllj

*

1.3

* 0.3

0% 1.0 ? 0 05% 1.0 I 0

(017) (015)

bFGF

31.6% (6119) 2.0 -t 0.4 38.5% (5113) 2.1 0.6

*

11.1%

(1/9)

1.1 2 0.1

'Incidence of coupling: values are the F;: ofmicrumjected cells demonstrating intercellul a r transl'er of Lucifer Yellow. Values In parenthesis arc nnmher of cells showing couplinghiumber of cells Injected. Extent of' coupling: values are the mean + SEM ofthe niimber of Lucifrr Yellow-labelled cells (including the injected cell) per microinjcction.

being labelled exclusively with Lucifer Yellow). Microinjected and scrape-loaded cultures were photographed under combined phase-contrast and fluorescence illumination. Mean values of control and rhbFGF-treated or wounded cultures were compared using Student's unpaired t-test.

Identification of Cx43 Immunofluorescence. Confluent monolayers of BME cells on glass coverslips were incubated in the presence or absence of rhbFGF (3 ngiml) for 7 hours, fixed for 3 minutes in acetone (-8O0C), rinsed in cold (4°Cj phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin, and processed for indirect immunofluorescence (Meda et al., 1991). The cultures were incubated for 2 hours at room temperature with an affinity-purified rabbit polyclonal anti-Cx43 antibody either directed against a peptide comprising residues 252-271 of heart Cx43 (Beyer et al., 1989), diluted 1:100, or directed against a peptide comprising residues 314-322 of heart Cx43 (El Aoumari et al., 1990), diluted 1 5 0 . The second incubation was carried out for one hour a t room temperature using fluorescein-conjugated goat anti-rabbit IgG antibodies (BioSys, Compiegne, France), diluted 1:200 in PBS. After rinsing, the cells were mounted in 0.02% paraphenylenediamine in PBS-glycerol (12,v:v) and photographed with a n Axiophot micrnscope (Zeiss, Oberkochen, Germany) fitted with filters for fluorescein detection. Controls included omission ofthe first antibody or exposure of monolayers during the first incubation to one of the following reagents: a) purified non-immune rabbit IgG; b) affinitypurified rabbit polyclonal antibodies against Cx32, diluted 1:100; or c) affinity-purified rabbit polyclonal antibodies against liver Cx26, diluted 1:50 (the latter two antibodies were kindly provided by Drs. 0. Traub and K. Willecke). None of these incubations resulted in specific staining of endothelial cell membranes (not shown). Western blot. rhbFGF (3 ngiml) was added t o the medium of confluent monolayers of BME cells for 5-6 hours. Crude membranes were prepared from control and bFGF-treated cultures a s follows: monolayers were rinsed thrce times in PBS and the cells scraped with a rubber policeman into sonication buffer consisting of 20 mM Tris-HC1, pH 8.0, supplemented with 20 mM EDTA, 1 bg/ml pepstatin A, 1 pgiml antipain, 1 mM benzamidine, 200 KIUiml aprotinin, 2 mM PMSF, and

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1mM DFP (all reagents from Sigma Chemical Co., St. Louis, MO). 1 mM sodium orthovanadate (Ventron, Karlsruhe, Germany) was also added to the sonication buffer in 2 experiments. Cells were collected and homogenized by sonication, and the sonicate was centrifuged a t 3,OOOg for 10 minutes at 4°C. The resulting supernatant was collected and centrifuged for 60 minutes at 100,OOOg and 4°C. Pelleted material, hereafter referred to as crude membrane preparation, was resuspended in PBS, distributed in small aliquots, and stored a t -80°C. Protein content was determined by the Bradford method (Bradford, 1976). Samples of crude membrane preparations (50 pg proteinilane) were fractionated by electrophoresis in a 12% polyacrylamide gel and immunoblotted as previously described (Meda et al., 1991). To this end, electrophoresed samples were transferred onto 0.22 pm nitrocellulose membranes (Schleicher & Schuell, Feldbach, Switzerland) for 18 hours, at a constant voltage of 25V in the presence of 0.02% SDS. After checking for efficient transfer by Ponceau Red S staining, the nitrocellulose membranes were saturated for 8 hours at room temperature in BLOTTO solution (40 mM Tris-HC1, 0.1% Tween 20, and 4% dried milk) and then incubated overnight at 4°C with affinity purified antibodies against residues 314322 of heart Cx43 (El Aoumari et al., 1990) diluted 1200 in BLOTTO. After repeated rinsing in BLOTTO, the nitrocellulose immunoblots were incubated for 60 minutes a t room temperature with a biotinylated goat anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories, Inc.; diluted 1:1,000), rinsed again, and incubated for one hour at room temperature with peroxidase-labelled streptavidin (Jackson Immunoresearch Laboratories, Inc.; diluted 1:2,500). Peroxidase activity was detected with 4-chloronaphthol as previously described (Meda et al., 1991). The same procedure was followed to analyze crude membrane preparations from adult male Sprague-Dawley rat hearts, which were used as internal standards in each experiment. Controls included omission of the affinity-purified antibody during the first incubation, and use of either a preimmune serum or of affinity-purified antibodies that had been preincubated with the peptide used for immunization during the first incubation step. None of these incubations resulted in the labelling of a n endothelial cell protein (not shown). Absorbance of the bands revealed on nitrocellulose strips was measured with a n LKB Ultroscan laser densitometer (Pharmacia LKB Biotechnology, Broma, Sweden) and is expressed a s percentage of the signal found in rat heart on the same filter.

Identification of Cx43 mRNA RNA extraction. Total cellular RNA was extracted according to a modification of the method of Glisin et al. (1974) a s previously described (Pepper et al., 1992). Plasmid construction and in vitro transcription. pSP65Cx43-3'AS was constructed by subcloning a 509 base-pair Sad-EcoRI fragment from the 3 ' coding region of G2, a 1.4 kb rat heart gap junction cDNA (Beyer e t al., 19871, into pSP65 (Melton e t al., 1984). pSP64cGAPDH was constructed by subcloning a 1.1kb chicken muscle glyceraldehyde-3-phosphatedehydrogenase (GAPDH) cDNA (Dugaiczyk e t al., 1983) into

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periments which assessed Cx43 mRNA levels at this time point. Filters were exposed to Kodak XAR-5 films a t - 80°C between intensifying screens. Autoradiographs were scanned with a GenoScan laser scanner (Genofit, Geneva, Switzerland).

RESULTS bFGF increases coupling between BME cells Coupling between BME cells in a confluent monolayer was quantitated by the intercellular transfer of microinjected (Fig. 1Aj or scrape-loaded (Fig. 2A) Lucifer Yellow. The incidence of coupling expressed as the percentage of cells demonstrating intercellular transfer of Lucifer Yellow was determined by microinjection, while the extent of coupling, expressed as the number of cells labelled by Lucifer Yellow (including the loaded cell), was determined by microinjection and scrape loading. In control cultures, the incidence of coupling was limited to 12.5% of cells a t the start of the experiment (approximately day 7 after plating and 24 hours after the last medium change), decreased to 0% by 10-12 hours, and remained at 0% until the end of the experiment (24 hours) (Fig. 1B and Table 1).After addition of rhbFGF (3 ngiml), the incidence of coupling increased in a time-dependent manner. Thus, coupling increased to 31.6% of microinjected cells after 6-8 hours, reached a maximum of 38.5% after 10-12 hours, and gradually approached baseline levels thereafter (Fig. 1A,B, and Table 1).When evaluated by scrapeFig. 3. bFGF induces Cx43 immunoreactivity in confluent BME loading, the extent of coupling increased significantly monolayers. Representative fluorescence micrographs of a control cul- after exposure to rhbFGF (Fig. 2A). Thus, the number ture (a)sho-ing the virtual absence of Cx43-imrnunoreactivity, and a of coupled cells increased (P < 0.001) 1.8- and 2.1-fold culture exposed to bFGF ( 3 ng/ml) for 7 hours (b) in which punctate after 6 5 and 8-9 hours exposure to 3 ngiml rhbFGF, Cx43-immunoreactivity was induced along the lateral borders of adjarespectively, and approached control values after 24 cent BME cells. Bar = 35 wm. hours (Fig. 2B), thereby mimicking the increase in the incidence of coupling observed by microinjection. A significant (P < 0.001) increase in the extent of coupling the PstI site of pSP64 (Melton et al., 1984). pSP65Cx43- was also observed after 4-5 and 8-9 hours exposure to 3'AS and pSP64cGAPDH were linearized with Sac1 10 or 30 ng/ml rhbFGF (results not shown). Although a and EcoRI, respectively, and used as templates for bac- similar rhbFGF-induced increase in the extent of couteriophage SP6 RNA polymerase. Transcription was pling was observed by microinjection, these values did not reach statistical significance (Table 1). performed a s described by Busso et al. (1986). Northern blot hybridization. Total cellular RNA bFGF increases Cx43 protein and mRNA in was denatured with glyoxal, electrophoresed in a 1.2% monolayers of BME cells agarose gel (5 kg RNA per lane), and transferred overAntibodies to two different portions of the cytoplasnight onto nylon membranes (Hybond, Amersham) as described by Thomas (1980). Filters were baked under mic tail of rat heart Cx43 were used to detect Cx43 in vacuum at 80°C for 2 hours, exposed to UV light (302 BME cells, and one of them (to residues 314-322) was nm) for 30 seconds, and stained with methylene blue to also used to quantitate the amount of Cx43 protein in determine RNA integrity. Prehybridization, hybridiza- BME cell membranes. In control cultures, the contacttion, and post-hybridization washes were as previously ing borders of BME cells were rarely decorated by a described (Pepper et al., 1992). All filters were rehy- sparse punctate Cx43 immunofluorescence labelling, bridized with a chicken muscle GAPDH cRNA probe to while the extent of immunolabelling markedly innormalize for the amount of RNA loaded. Although we creased after exposure to rhbFGF (3 ngiml) for 7 hours have observed a marginal and late (12-24 hours) bFGF- (Fig. 3 ) . By Western blot analysis, antibodies to Cx43 induced increase in GAPDH mRNA in BME cells recognize a protein with a greater electrophoretic mo(Fig. 5), GAPDH was selected from other potential con- bility than heart Cx43 in crude membrane preparatrol mRNAs for which a greater bFGF-induced increase tions of BME cells (Fig. 4A). When compared to conwas observed. Since GAPDH mRNA was not increased trols, this 41 kDa protein was increased 2.0-fold after 4 hours exposure to bFGF, a t which point the (median of 7 experiments) after 5-6 hours exposure to bFGF-induced increase in Cx43 mRNA was maximal, rhbFGF (3 ngiml) (Fig. 4A,B). In the controls of 3 of the GAPDH was considered to be a n appropriate control for 7 experiments performed, the antibodies to Cx43 also the dose-curve (Fig. 6) and multiple-wound (Fig. 8) ex- immunolabelled a second band which had a n apparent

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Fig. 4. bFGF induces Cx43 in confluent BME monolayers. A: Western blot analysis of cell membranes using polyclonal antibodies against the cytoplasmic portion of Cx43 revealed the presence o f an immunoreactive band in control BME cells (Lane 3) with a higher electrophoretic mobility (approximately 41 kDa) than the 43 kDa band seen in rat heart membranes (Lane 2). After addition of bFGF (3 ng/ml) for 6 hours (Lane 4), the intensity o f the Cx43 immunoreactive band was increased. (The 86 kDa band seen in Lane 2 is likely to be a rat heart Cx43 doublet.) As a control for the loading of heart and BME cell membranes, the same nitrocellulose strips shown in Lanes 2 4 are

shown in Lanes 5-7 after staining with Ponceau Red S. Molecular weight standards are shown in Lane 1. B: Quantitation of Cx43 protein in control and bFGF-treated confluent BME monolayers. Cx43immunoreactive bands of Western blots from control cultures and cultures treated with bPGF (3 ng/ml) for 5-6 hours were quantitated by densitometric scanning. The results of seven separate experiments are shown. Cx43 was increased in six of the seven experiments. Median values (arrows) revealed a 2.0-fold increase in Cx43 after bFGF treatment. Values are B absorbance relative to that of rat heart Cx43 used a s an internal control in each experiment.

molecular weight of 42-43 kDa and a n intensity of 12.5% (median value) relative to the 41 kDa species; the 42-43 kDa band was also detected in bFGF-treated cultures in 5 out of 7 experiments (Fig. 4A), with a n intensity of 22.5% (median value) relative the 41 kDa species (results not shown). When compared to controls, the 42-43 kDa species was increased 4.8-fold (median of 3 experiments) after exposure to bFGF (results not shown). Similar results were obtained in two experiments in which a n ‘“I-labelled second antibody was used instead of the streptavidin-biotin-peroxidaseamplification system (not shown). Northern blots of total cellular RNA were hybridized with a 500 base-pair cRNA probe spanning the 3’ coding region of rat Cx43 (Beyer et al., 1987). Levels of Cx43 mRNA were quantitated by densitometric scanning and were normalized to GAPDH mRNA in the same samples. This revealed a time- and dose-dependent increase in BME Cx43 mRNA in response to rhbFGF. Thus, rhbFGF (30 ngiml) increased Cx43 mRNA for at least 8 hours, with a maximal 5.4-fold increase after 4 hours (Fig. 5A,B). A dose-response analysis a t 4 hours revealed a maximal f3-7-fold increase in Cx43 mRNA in response to 3-10 ngiml of rhbFGF (Fig. 6A,B).

Increased coupling and Cx43 mRNA expression in migrating BME cells is p r e v e n t e d b y anti-bFGF antibodies Addition of anti-bFGF antibodies to mechanically wounded BME cell monolayers immediately after wounding reduced the increase in coupling normally seen between migrating cells after 24 hours by 59% (P< 0.03) (Fig. 7 and Table 2). In the same experiments, the number of cells migrating across the original wound edge was reduced by 44% (P < 0.001) (Fig. 7 and Table 2). To determine whether antibodies to bFGF could also affect Cx43 mRNA, monolayers were multiple-wounded in the presence of anti-rhbFGF antibodies, and total cellular RNA extracted 4 hours later. Northern blot hybridization revealed that the increase in Cx43 mRNA which is observed after multiple wounding was reduced by 50.3 2 6.7% (3 experiments) in the presence of the antibodies (Fig. 8). Levels of Cx43 mRNA quantitated by densitometric scanning were normalized to GAPDH mRNA in the same samples.

DISCUSSION The studies reported in this paper have revealed that bFGF increases coupling and Cx43 expression in mono-

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Fig. 5. Kinetics of induction of Cx43 mRNA in bFGF-treated confluent BME monolayers. BME cells were treated with bFGF (30 ng/ml) for the times indicated. A: Graphic representation of B; circles, cultures exposed to 30 ng/ml bFGF; triangles, control cultures. B: Northern blot of total cellular RNA (5pg per lane) probed with a :'2P-labelled rat Cx43 cRNA. The same filter was hybridized with a "2P-labelled chicken GAPDH cRNA probe. Values from densitometric scanning in A are absorbance expressed relative to controls at time = 0, normalized to levels of GAPDH mRNA present on the same filter. 285 and 18s ribosomal RNAs are indicated in B

layers of microvascular endothelial cells, in a dose- and time-dependent manner. Although the magnitude of the bFGF-induced changes of the individual parameters measured was limited to a 2-5-fold increase, the parallel changes in all of these parameters clearly indicate a bFGF-mediated effect. Our results also demonstrate that anti-bFGF antibodies inhibit the increase in coupling and Cx43 expression which are seen when microvascular endothelial cells are induced to migrate after mechanically wounding a confluent monolayer (Pepper et al., 1989, 1992), indicating that endogenous bFGF may account for most of the functional and biochemical coupling-related changes displayed by these migrating cells.

GAPDH Fig. 6. Dose-response curve of Cx43 mRNA induction in bFGFtreated confluent BME monolayers. BME cells were treated with hFGF a t the indicated concentrations for 4 hours. A Graphic representation of B, a Northern blot of total cellular RNA 15 pg per lane) probed with a 32P-labelledrat Cx43 cRNA. The same filter was hybridized with a "2P-labelled chicken GAPDH cRNA probe. Values from densitometric scanning in A are absorbance expressed relative to untreated cultures, normalized to levels of GAPDH mRNA present on the same filter. 28s and 18s ribosomal RNAs are indicated in B.

Generally speaking, coupling can be regulated by two major mechanisms: first, through the synthesis, degradation, and incorporation of connexins into the plasma membrane, and second, via local regulation of the opening and closing of junctional channels. Our findings demonstrate that upon exposure to exogenous bFGF, coupling and Cx43 expression are modulated in parallel in microvascular endothelial cells. This suggests that the bFGF-induced increase in coupling is mainly regulated by the insertion of newly synthesized Cx43 into the plasma membrane of these cells, although our data do not exclude the possibility that the conductance and permeability of existing gap junctions may also be increased upon exposure to bFGF. In this context, i t is interesting to note that bFGF-like pep-

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bFGF INCREASES ENDOTHELIAL CELL COUPLlNG

Control 28sCx43-

18S* C N A

C N A

GAPDH Fig. 8. Antibodies to bFGF inhibit the increase in Cx43 mRNA in multiple-wounded BME monolayers. BME monolayers were multiplewounded (MiW) in the presence or absence (C) of normal rabbit (N) or anti-rhbFGF (A) gamma-globulins, and total cellular RNA was extracted 4 hours later as described in Materials and Methods. Antibodies were also added for 4 hours to non-wounded (control) monolayers. The Northern blot (5 pg total cellular RNA per lane) was hybridized with a 3”P-labelled rat Cx43 cRNA. The same filter was subsequently hybridized with a 3’P-labelled chicken GAPDH cRNA probe. 285 and 185 ribosomal RNAs are indicated. Fig. 7. Antibodies to bFGF inhibit the wound-induced increase in coupling between migrating BME cells. Wounded BME monolayers were incubated in the presence of a, normal rabbit gamma-globulins (NRG) or b, anti-bFGF gamma-globulins for 24 hours after wounding. a: In the presence of NRG, coupling, as assessed by scrape loading, was increased between cells that had migrated across the original wound edge (we).b: In contrast, both the increase in coupling and cell migration across the wound edge were markedly inhibited in the presence of anti-bFGF antibodies. Bar = 200 )*m in a and b.

TABLE 2. Eflect of antihFGF antibodies on wound-induced coupling and migration in BME cells’

Control anti-bFGF

Cell migration

Incidence of coupling

0.7 (n = 17) 3.6 L 0.4* (n = 26)

76.2 i 1.2 (n - 30) 31.5 i 13.3*”

6.4

?

(n = 48)

‘Cell migration: values are the meaii SEM of the number of cells bordering the scrape loading hne, which had crossed the original wound cdge; n - the total number of scrape line sidcs analyzed in 3 separate experiments. Incidence of coupling: values are the mean z SEM of the % of scrape-loaded cells n~hichhad crossed the original wound edge and which demonstrated intercellular transfer of 1.ucifer Yellow; n = t.he total numlwr of cells assessed in 3 separate experiments. *P c’ 0.001, **Pc’ 0.03, a s cnmparcd to control values using Students unpaired t-lest. +

tides have been co-localized with Cx43-containing gap junctions in astrocytes and cardiomyocytes (Kardami et al., 1991; Yamamoto et al., 19911, a n observation which was taken to suggest that bFGF may directly influence junctional channels and thereby regulate junctional-dependent tissue functions. We have observed that exposure to bFGF slightly modified the electrophoretic mobility of BME cell Cx43, which is comparable to what has been reported after phosphorylation of this connexin in other endothelial (Larson et al., 1991) and non-endothelial cells (Musil et al.,

1990). Since phosphorylation is the only post-translational modification which has so far been shown to affect connexins (Willeke et al., 1991) and correlate with alterations in gap junction assembly a n d o r activity (Musil et al., 1990,1991),it is possible that our observations are at least in part due to bFGF-induced posttranslational changes of Cx43, a major connexin expressed by microvascular endothelial cells in vitro (Larson et al., 1990; Pepper et al., 1992). In this context, it is important to note that FGF stimulates protein phosphorylation in endothelial cells (Burgess et al., 19901. Whether bFGF also modulates the expression of newly identified connexins such a s Cx40 (Haefliger et al., 19921, which is expressed by endothelial cells in vivo (R. Bruzzone, J.-A. Haefliger, R.L. Gimlich, and D.L. Paul, personal communication), remains to be determined. It is currently thought that bFGF, which lacks a signal peptide required for secretion via the classic secretory pathway (Abraham et al., 1986; Jaye et al., 19861, is released as a consequence of sublethal cell injury or cell death (Gajdusek and Carbon, 1989; McNeil et al., 1989; Muthukrishnan et al., 1991), or by as yet undefined mechanisms independent of these parameters (Kandel et al., 1991; Mignatti e t al., 1991). Antibody inhibition studies have revealed that mechanical wounding of endothelial cells induces bFGF-dependent division and migration of the cells lining the wound edge (Sato and Rifkin, 1988). We have also demonstrated that BME cells migrating from the wound edge are moving from a state of high to low density (Pepper et al., 1992). It is therefore possible that both bFGFinduced cell division and migration, as well a s de-

204

PEPPER AND MEDA

creased cell density, contribute to the increased cou- response to bFGF may mediate the significant degree of pling and Cx43 expression seen after wounding heterogeneity which exists in the size and distribution monolayers of microvascular endothelial cells in vitro of gap junctions within the vascular tree. (Pepper et al., 1989, 1992). However, we have previously demonstrated that the wound-induced increase in ACKNOWLEDGMENTS coupling and Cx43 expression are replication-indepenWe are grateful to Drs. R. Montesano and L. Orci for dent, and can be accounted for only in part by a detheir useful comments and continuing support, Drs. crease in cell density (Pepper et al., 1989, 1992). This M.B. Furie and S.C. Silverstein for providing the BME suggests that migration andlor additional factors are cells, Dr. P. Sarmientos for the rhbFGF, Drs. E. Beyer, responsible for the increased coupling and Cx43 expresD. Gros for the antibodies against A. El Aoumari, and sion after wounding. The studies reported in this paper indicate that bFGF, released as a consequence of Cx43, and Drs. Y. Sat0 and D.B. Rifkin for the antibodwounding, is one such factor. However, since the ies against rhbFGF. We thank A. Charollais, C. Diwound-induced increase in coupling is dependent on Sanza, M. Guisolan, L. Iuliano, and P. Ruga for techcell migration (Pepper et al., 1989), and since anti- nical assistance, and J.-P. Gerber and P.-A. Ruttiman bFGF antibodies inhibit endothelial cell migration for photographic work. This work was supported by (Sato and Rifkin, 1988), our results do not allow us to grants from the Swiss National Science Foundation determine whether the reduction in coupling in the (31-26625.89 and 32-34090.92) and a grant in aid from presence of these antibodies is a cause or a consequence The Sir Jules Thorn Charitable Overseas Trust. of the inhibition of cell migration. Our in vitro studies LlTERATURE CITED do nonetheless provide evidence for the involvement of endogenous bFGF in the control of coupling and Cx43 Abraham, J.A., Mergia, A,, Whang, G.L., Tumolo, A,, Friedman, J., Hjerrild, K.A., Gospodarowicz, D., and Fiddes, J . (1986) Nucleotide expression in migrating microvascular endothelial sequence of a bovine clone encoding the angiogenic protein, basic cells. Taken together with previous reports documentfibroblast growth factor. Science, 233:545-548. ing a cytokine-mediated modulation of coupling in non- Bennett, M.L.V., Barrio, L.C., Bargiello, T.A., Spray, D.C., Hertzberg, endothelial cell types (Madhukar e t al., 1989; MaldonE., and Saez, J.C. (19911 Gap Junctions: New tools, new answers, new questions. Neuron 6t305-320. ado et al., 1988; van Zoelen and Tertoolen, 1991), our present in vitro findings suggest that cytokines may be Beyer, E.C.: Paul, D.L., and Goodenough, D.A. (1987) Connexin43: A prolein from rat heart homologous to a gap junction protein from widespread physiological regulators of junctional couliver. J. Cell. Riol., 205:2621-2629. pling. Beyer, E.C., Kistler, J., Paul, D.L., and Goodenough, D.A. (1989) AnWhat is the possible functional significance of our tisera directed against connexin43 peptides react with a 43kd protein localized to gap junctions in myocardium and other tissues. J. findings? First, bFGF is a potent angiogenic factor, Cell Biol., 108:595405. which induces endothelial cell migration, replication, Bradford, H. (1976) A rapid and sensitive method for the quantitation and protease production, all of which are considered to of +g quantities of proteins, using the principle of protein-dye bindbe essential components of the angiogenic process (reing. Anal. Biochem., 72:248-254. viewed by Klagsbrun and D’Amore, 1991). To this list Burgess, W.H., Dianne, C.A., Kaplow, J.,Mudd, R., Friesel, R., Zilberstein, A., Schlessinger, J., and Jaye, M. (1990)Characterization and of bFGF-modulated endothelial cell functions must now cDNA cloning of phospholipase C,-y, a major substrate for heparinalso be added junctional coupling. In our model of binding growth factor 1 (acidic fibroblast growth factorkactivated wound-induced coupling in microvascular endothelial tyrosine kinase. Mol. Cell. Biol., 10:47704777. cells (Pepper et al., 1989), the region of increased cou- Burk, R.R. (1973)A factor from a transformed cell line that affects cell migration. Proc. Natl. Acad. Sci. USA, 70:369372. pling extends several cell rows in front of, a s well a s Busso, N., Belin, D., Failly-Crepin, C.: and Vassalli, J.-D. (1986) Plasbehind, the original wound edge. Therefore, the bFGFminogen activators and their inhibitors in a human mammary cell induced increase in coupling may well represent a line (HBL-100).J. Biol. Chem., 261:9309-9315. physiological mechanism whereby cells along the lead- Cuevas, P., Carceller, F., Ortega, S., Zazo, M., Nieto, I., and GimenezGal1eg.o. G. (1991) Hvootensive activitv of fibroblast growth factor. ing front communicate both with adjacent cells as well ~cien~e,’254:1208-lii0. as with cells situated further behind. In order to form Davies, P.F., Olesen, S.-P., Clapham, D.E., Morrel, E.M., andschoen, structurally and functionally competent capillary blood F.J. (1988)Endothelial communication. Hypertension, 1I : 563-572. vessels during angiogenesis, coordinated endothelial Dennis, P A . , and Rifkin, D.B. (1990) Studies on the role of basic fibroblast growth factor in viva: Inability of neutralizing antibodies cell migration and replication as well a s maintenance to block tumor growth. J . Cell. Physiol., 144:84-98. of intercellular contacts are likely to be important. This Dugaiczyk, A,, Haron, H.A., Stone, E.M., Dennison, O.E., Rothblum, might be achieved via gap junction mediated-coordinaK.N., and Schwartz, R.J. (1983) Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphatedehydrogenase tion of cellular functions (Pepper e t al., 1989). Second, it messenger ribonucleic acid isolated from chicken muscle. Biochemhas been proposed that endothelial cell coupling may istry, 22:1605-1613. play a role in arteriolar vasodilation (Segal and Duling, El Aoumari, A,, Fromaget, C., Dupont, E., Reggio, H., Durbec, P., 1986, 1987, 1989). In this context, it has recently been Briand, J.-P., Boller, K., Kreitman, B., and Gros, D. (1990) Conservation of a cvtoolasmic carboxv-terminal domain of connexin43. a reported that systemically administered bFGF degap junctional protein in mammalian heart and brain. J. Membr. creases arterial blood pressure in a n endothelium-deBiol., 115:229-240. pendent manner (Cuevas e t al., 1991). If our findings on Folkman, J., Haudenschild, C.C., and Zetter, B.R. (1979) Long-term microvascular endothelial cells can be extrapolated to culture of caaillarv endothelial cells. Proc. Natl. Acad. Sci. USA, 7fi:m-mi. ” arteriolar endothelial cells, it could be speculated that by affecting endothelial cell coupling, systemically ad- Furie, M.B., Cramer, E.B., Naprstek, B.L., and Silverstein, S.C. (1984) Cultured endothelial cell monolayers that restrict the transendotheministered bFGF may mediate arteriolar vasodilation lial uassape of macromolecules and electrical current. J. Cell. Biol.. and thereby decrease systemic blood pressure. Finally, 98: