Temporal and Spatial Modulation of Rho GTPases during in Vitro Formation of Capillary Vascular Network
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307234200
ISSN1083-351X
AutoresIlaria Cascone, Enrico Giraudo, Francesca Caccavari, Lucia Napione, Elisa Bertotti, John G. Collard, Guido Serini, Federico Bussolino,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoEndothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of β-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs β-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis. Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of β-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs β-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis. To distribute nutrients throughout the body, vertebrates have evolved a branching blood vascular system that terminates in a network of size-invariant units, namely capillaries. The development of capillary networks characterized by typical intercapillary distances ranging from 50 to 300 μm is instrumental for optimal metabolic exchange (1.Guyton A.C. Hall J.E. Textbook of Medical Physiology, 10th Ed. W. B. Saunders Co., Philadelphia, PA2000Google Scholar). In embryo, homogeneous vascular networks form by vasculogenesis and are remodeled through angiogenesis (2.Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4791) Google Scholar). Both processes require that ECs 1The abbreviations used are: ECsendothelial cellsAbantibodyBSAbovine serum albuminFITCfluorescein isothiocyanateGFPgreen fluorescence proteinGSTglutathione S-transferasemAbmonoclonal AbMLCmyosin light chainMLC-Pphosphorylated-MLCPBSphosphate-buffered salineRBDrhotekin binding domainTRITCtetramethylrhodamine isothiocyanateVe-cadherinvascular endothelial-cadherinVEGFvascular endothelial growth factorWBDWiskott-Aldrich syndrome protein binding domainFCSfetal calf serumPBDPak 1B binding domain.1The abbreviations used are: ECsendothelial cellsAbantibodyBSAbovine serum albuminFITCfluorescein isothiocyanateGFPgreen fluorescence proteinGSTglutathione S-transferasemAbmonoclonal AbMLCmyosin light chainMLC-Pphosphorylated-MLCPBSphosphate-buffered salineRBDrhotekin binding domainTRITCtetramethylrhodamine isothiocyanateVe-cadherinvascular endothelial-cadherinVEGFvascular endothelial growth factorWBDWiskott-Aldrich syndrome protein binding domainFCSfetal calf serumPBDPak 1B binding domain. or their mesodermic precursors, angioblasts, move, proliferate, and then change their shape folding into capillaries. The ability to form networking capillary tubes is a cell autonomous property of ECs and requires permissive extracellular cues such as growth factors, autacoids, signals coming from the extracellular matrix, and physical forces (2.Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4791) Google Scholar, 3.Ingber D.E. Folkman J. Cell. 1989; 58: 803-805Abstract Full Text PDF PubMed Scopus (381) Google Scholar, 4.Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (414) Google Scholar).It is well known that culturing ECs on a tridimensional scaffold of extracellular matrix protein markedly accelerates their morphological differentiation in geometric tubular networks (5.Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 6.Grant D.S. Tashiro K. Segui-Real B. Yamada Y. Martin G.R. Kleinman H.K. 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When this process occurs on Matrigel, a natural basal membrane matrix, it is rapid (6–16 h), does not require cell proliferation, and is mainly characterized by cell motility and shape rearrangement, which imply dramatic changes in EC cytoskeletal dynamics and adhesive behavior (7.Folkman J. Haudenschild C. Nature. 1980; 288: 551-556Crossref PubMed Scopus (854) Google Scholar, 13.Connolly J.O. Simpson N. Hewlett L. Hall A. Mol. Biol. Cell. 2002; 13: 2474-2485Crossref PubMed Scopus (147) Google Scholar).In the last few years it has been widely demonstrated that Rho GTPases are major regulators of cell polarization and motility. In fibroblasts, Cdc42 mediates filopodia extension and Rac1 both lamellipodia formation and membrane ruffling, and RhoA controls stress fiber formation (14.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar). Both Rac1 and RhoA also participate in the ruffling phenomena of ECs (15.Menager C. Vassy J. Doliger C. Legrand Y. 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Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar) and regulate the shape of ECs.We analyzed the temporal and spatial activation of Rac1 and RhoA during capillary vascular network formation by ECs and show that their spatially and temporally regulated activation was required for proper formation of such a network. By employing dominant negative molecules, we found that Rac1 and RhoA, respectively, regulate the early formation of adherens junction, among ECs that at the beginning self-organize into clusters and chains, and the phosphorylation level of myosin light chain (MCL) when ECs start stretching to remodel the vascular network.EXPERIMENTAL PROCEDURESReagents—Glutathione S-transferase (GST)-Pak 1B binding domain (amino acids 56–141) (GST-PBD), GST-rhotekin binding domain (GSTRBD) (amino acids 1–90), and GST-Wiskott-Aldrich syndrome protein binding domain (WBD) (amino acids 201–321) (GST-WBD) fusion proteins were produced and purified as described (27.Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (585) Google Scholar). GST-PBD, GSTRBD, and GST-WBD bind the GTP-bound form of Rac1 and Cdc42, RhoA, and Cdc42, respectively (28.Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1289) Google Scholar, 29.Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 30.Li R. Debreceni B. Jia B. Gao Y. Tigyi G. Zheng Y. J. Biol. Chem. 1999; 274: 29648-29654Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 31.Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). cDNA of N17Rac1, N17cdc42, and N19RhoA dominant negative molecules (kindly provided by G. Bockoc, University of San Diego, San Diego, CA; Dr. A. Hall, University of London, London, UK; and Dr. G. Scita, European Institute of Oncology, Milano, Italy) (32.Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar, 33.Obermeier A. Ahmed S. Manser E. Yen S.C. Hall C. Lim L. EMBO J. 1998; 17: 4328-4339Crossref PubMed Scopus (173) Google Scholar, 34.Feig L.A. Nat. Cell Biol. 1999; 1: E25-E27Crossref PubMed Scopus (340) Google Scholar) were subcloned into the BamHI/EcoRI site of Pinco retroviral vector (35.Cutrupi S. Baldanzi G. Gramaglia D. Maffe A. Schaap D. Giraudo E. van Blitterswijk W. Bussolino F. Comoglio P.M. Graziani A. EMBO J. 2000; 19: 4614-4622Crossref PubMed Google Scholar) and expressed under the control of the two long terminal repeats. Green fluorescence protein (GFP) cDNA was under the control of cytomegalovirus promoter. cDNA of RacQ61L (Rac1QL) and RhoQ63L (RhoAQL) (36.Rottner K. Hall A. Small J.V. Curr. Biol. 1999; 9: 640-648Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 37.Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar) were subcloned into the HindIII/NotI site of Pinco. GST-RBD and GST-PBD cDNAs were respectively subcloned into BamHI/EcoRI sites of pGEX2TK and BamHI/EcoRI sites of pGEX3X and expressed as fusion proteins in Escherichia coli BL21 cells. Cells carrying GST-PBD, GST-RBD, or GST-WBD were respectively lysed by sonication in 50 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 1% Triton X-100, 2 mm dithiothreitol, 5% glycerol, and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 50 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin). Lysates were purified by affinity chromatography with glutathione-coupled Sepharose 4B beads (Amersham Biosciences). For pull-down assays, beads were extensively washed six times in 50 mm Tris-HCl, pH 7.5, containing 150 mm NaCl, 0.5% Triton X-100, 5 mm MgCl2, and 1 mm dithiothreitol and stored at -80 °C in the presence of 10% glycerol. For immunofluorescence studies of localization of Rac1-GTP, RhoA-GTP, and Cdc42-GTP, GST-PBD and GST-WBD were eluted with 50 mm Tris-HCl, pH 8.0, containing 100 mm NaCl, 2 mm dithiothreitol, 5% glycerol, 20 mm reduced glutathione, and protease inhibitors. GST-RBD was eluted with the same buffer containing 50 mm NaCl. GST fusion proteins were stored at -80 °C.All indicated monoclonal (mAb) and polyclonal antibody (Ab) were provided by Pharmingen (BD Biosciences) (mAb anti-Rac1, fluorescein isothiocyanate (FITC), and tetramethylrhodamine isothiocyanate (TRITC) conjugated-secondary Abs) and by Santa Cruz Biotechnology (Santa Cruz, CA) (mAb and Ab anti-RhoA, Ab anti-Cdc42, mAb and Ab anti-GST, mAb anti-β-catenin, Ab anti-α-catenin, Ab anti-IQGAP1, mAb anti-β-tubulin). mAbs anti-phosphorylated-MLC (MLC-P) and anti-vascular endothelial (Ve)-cadherin were obtained from Dr. S. Komatsu (University of Massachusetts, Worcester, MA) and Prof. E. Dejana (Institute of Molecular Oncology, Milan, Italy), respectively. Other products were purchased from Sigma.Cell Culture—Human dermal capillary ECs (Biotech, BD Biosciences) were cultured according to the manufacturer's instructions. EC infection was performed as described previously (38.Primo L. Roca C. Ferrandi C. Lanfrancone L. Bussolino F. Oncogene. 2000; 19: 3632-3641Crossref PubMed Scopus (24) Google Scholar). Briefly, N17Rac1, N17Cdc42, N19RhoA, RhoAQL, and Rac1QL cDNAs subcloned into Pinco vector were transiently transfected into Phoenix packaging cells, and the supernatants were used to infect ECs. As control of infection, GFP analysis was performed both through fluorescence microscope and fluorescence-activated cell sorting (FACS Advantage SE, Biotech, BD Biosciences, San Jose, CA) giving 85–90% of positive ECs. The expression of transgene molecules was evaluated by Western blot analysis and always gave an increase of proteins recognized by specific mAbs anti-Rac1, anti-RhoA, and Ab anti-Cdc42 or as compared with ECs infected with vector alone (not shown). Infection conditions per se were without effect on EC morphology and on cell cycle analyzed by propidium iodide fluorescence by FACS flow cytometer.In Vitro Angiogenesis—To analyze in vitro angiogenesis, glass coverslips (2 cm2 growth area) were coated with 0.25 ml of Matrigel (8.8 mg/ml; Biotech, BD Biosciences), which was immediately aspirated, and the remaining film was allowed to solidify. ECs (125 cells/mm2) were incubated at 37 °Cina5%CO2 humidified atmosphere for 14 h on glass coverslips and observed with an inverted photomicroscope (model DM IRB HC; Leica Microsystems, Solms, Germany). Phase contrast snap photographs and 12–14-h long movies (one frame every 5 min) were taken with a cooled digital CCD Hamamatsu ORCA camera (Hamamatsu Photonics Italy, Milano, Italy), recorded, and analyzed with ImageProPlus 4.0 imaging software to count the number of capillaries/mm2 (Media Cybernetics, Carlsbad, CA) (39.Roca C. Primo L. Valdembri D. Cividalli A. Declerck P. Carmeliet P. Gabriele P. Bussolino F. Cancer Res. 2003; 63: 1500-1507PubMed Google Scholar).In the sprouting assay capillary ECs carrying vector alone or N17Rac1 or N19RhoA were suspended at a density of 4 cells/μl in culture medium containing 20% of Methocel stock (6 g of carboxymethylcellulose in 500 ml of M199) in M199 containing 20% FCS, 0.1 mg/ml porcine heparin. Eight hundred cells were seeded into non-adherent round-bottomed 96-well plates (Falcon, BD Biosciences), and cultured overnight at 37 °C (5% CO2, 100% humidity). The spheroids were harvested by gently pipetting them and were centrifuged at 300 × g for 15 min. The EC spheroids were suspended in 200 μl of M199 medium containing 40% FCS, 1.2% (v/w) methylcellulose with or without 30 ng/ml vascular endothelial growth factor (VEGF)-A165 (R&D Systems, Minneapolis, MN), and mixed with an equal volume of diluted collagen solution (7 volumes of collagen from rat tail, 1 volume of 10× M199, 1 volume of 0.1 n NaOH, and 1 volume of 0.2 m Hepes, pH 7.3) (39.Roca C. Primo L. Valdembri D. Cividalli A. Declerck P. Carmeliet P. Gabriele P. Bussolino F. Cancer Res. 2003; 63: 1500-1507PubMed Google Scholar). After 24 h spheroid photographs were taken with digital CCD Hamamatsu ORCA camera linked to an inverted photomicroscope (model DM IRB HC, Leica).Biochemical Assays during in Vitro Angiogenesis—Capillary ECs, ECs carrying vector alone, N17Rac1, or N19RhoA (9 × 106) were seeded on 100-mm Petri dishes coated with 9 ml of Matrigel, which was immediately aspirated. Cells were incubated at 37 °C for the indicated times, and after washing with cold PBS, pH 7.4, cells were treated with 8 ml of MatriSperse Cell Recovery Solution (Biotech, BD Biosciences) at 4 °C for 2 h. After complete release from the gel, ECs were centrifuged at 500 × g for 5 min at 4 °C.To analyze the GTP-bound form of Rho GTPases, cells were lysed for 20 min on ice in 50 mm Tris, pH 7.4, 2 mm MgCl2, 100 mm NaCl, 1% Nonidet P-40, 10% glycerol supplemented with protease inhibitors. Lysates were centrifuged for 5 min at 13,000 × g, and equal amounts of proteins (500 μg) were incubated with 30 μg of GST-RBD, GST-PBD, or GST-WBD G-Sepharose beads coupled for 60 min at 4 °C. At the end of incubation, beads were washed with the same buffer, and solubilized proteins were resolved on SDS-PAGE (12%), transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA), probed with mAbs anti-RhoA, anti-Rac1, or Ab anti-Cdc42, and detected by enhanced chemiluminescence techniques (Amersham Biosciences). Total cell lysates were treated in the same conditions to evaluate the total amounts of RhoA, Rac1, and Cdc42.To detect phosphorylated MLC (MLC-P), ECs were washed twice with cold PBS containing 1 mm NaOV4 and then immediately boiled in 1 ml of 20 mm Tris, 22 mm glycine, 10 mm dithiothreitol, 1% SDS. Proteins were separated by SDS-PAGE (12%) and immunoblotted with mAb anti-MLC-P.To analyze Ve-cadherin-catenin complexes, ECs were washed twice with Ca2+- and Mg2+-containing PBS and lysed in 10 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl2, pH 7.5, 1% Nonidet P-40, 1% Triton X-100, and protease inhibitors for 20 min on ice. Lysates were centrifuged for 15 min at 14,000 × g. The supernatants were incubated with protein G-Sepharose (Amersham Biosciences) and a mAb anti-β-catenin for 2 h at 4 °C. Then beads were washed with lysis buffer. Pellets were solved in SDS buffer (1% Triton X-100, 1% Nonidet P-40, 0.5% SDS in Ca2+ -and Mg2+-containing PBS) and boiled for 5 min. Solubilized proteins were separated by SDS-PAGE (8%) and transferred onto nitrocellulose (Bio-Rad). Nitrocellulose was blocked with 10% BSA in Ca2+- and Mg2+-containing PBS and immunoblotted with mAbs anti-Ve-cadherin or anti-β-catenin, or Abs anti-α-catenin, or anti-IQGAP1. Cell apoptosis was evaluated by Annexin V staining as described (40.Strasly M. Cavallo F. Geuna M. Mitola S. Colombo M.P. Forni G. Bussolino F. J. Immunol. 2001; 166: 3890-3899Crossref PubMed Scopus (144) Google Scholar). Where shown, densitometry was performed with Phoretix 1D software (Nonlinear USA Inc., Durham, NC).Adhesion Assay—96-Well plates were coated with 1.28 μg/μl Matrigel for 3 h at 37 °C, washed, and blocked with 3% BSA for 2 h at 37 °C. 1 × 104 capillary ECs carrying vector alone or N17Rac1 or N19RhoA were plated to adhere for 30 min. Attached cells were fixed and stained by crystal violet, and the absorbance was read at 540 nm in microtiter plate spectrophotometer (HT6 7000 Bio Assay Reader, PerkinElmer Life Sciences).Motility Assay—24-Well transwell chambers (Falcon, BD Bioscience Biotech) containing polycarbonate filters with 8-mm pores were used. The underside of the filters were coated with 1.28 μg/μl Matrigel for 1 h at 37 °C and saturated in 3% BSA for 1 h at 37 °C. Two hundred microliters of cell suspension containing 3 × 104 capillary ECs carrying vector alone or N17Rac1 or N19RhoA were plated in the upper chambers of transwell apparatus, and M199 supplemented with 10% FCS was added to the lower chamber. Cells were incubated for 3 h at 37 °C in a 95% air, 5% CO2 atmosphere. After washing with PBS, the cells migrated to the bottom side of the transwell membrane were fixed with 3.7% glutaraldehyde, and cells on the upper side of the filter were carefully removed by scraping with the edge of a cotton swab. Migrated cells were stained with crystal violet, and the absorbance was read at 540 nm in microtiter plate spectrophotometer. Absorbance values were linear from 1 × 102 to 2 × 104 cells.Gel Contraction—Gel contraction assay was performed as described previously (41.Kolodney M.S. Wysolmerski R.B. J. Cell Biol. 1992; 117: 73-82Crossref PubMed Scopus (403) Google Scholar). Capillary ECs (200 cells/mm2) carrying vector alone or N17Rac1 or N19RhoA were plated as detailed above. Cells were incubated at 37 °Cina5%CO2 humidified atmosphere until they spread. In some experiments 100 nm cytochalasin D or 5 μm Y-27632 (Calbiochem) were added from the beginning of incubation. To control the numbers of cell spread on Matrigel, ECs were recovered by using of MatriSperse Cell Recovery Solution and counted. To initiate Matrigel contraction, polymerized gels were gently released from the underlying culture dish. The degree of Matrigel contraction was determined after 14 h by recording pictures with a digital camera (½ inch CCD, JVC) connected to a Stereomicroscope Olympus SZX9 (Olympus Europe, Hamburg, Germany). The gel area was calculated by Image Pro Plus 4x software.Cell Dissociation Assay—Confluent cultured ECs were treated with 0.01% trypsin in Hepes-buffered saline (1 mm CaCl2, 1.3 m NaCl, 50 mm KCl, 3.3 mm Na2HPO4-7H2O, 55 mm glucose, 100 mm Hepes, pH 7.4) (TC treatment) or in Hepes-buffered saline without Ca2+ supplemented with 1 mm EGTA (TE treatment) for 15 min at 37 °C and dissociated through 10 times pipetting. The extent of dissociation cells was represented by the index NTC/NTE, where NTC and NTE are the number of clusters and single cells, respectively (42.Takeichi M. J. Cell Biol. 1977; 75: 464-474Crossref PubMed Scopus (470) Google Scholar).Immunofluorescence—Cells cultured on glass coverslips coated with Matrigel were fixed for 30 min in 4% paraformaldehyde in PBS, pH 7.5, quenched with 20 mm NH4Cl for 30 min, permeabilized with 0.1% saponin in PBS for 30 min on ice, and rinsed twice in PBS. Blocking incubations were performed in PBS containing 10% goat serum and 0.25% fish skin gelatin (1 h, at room temperature). GST-PBD, GSTRBD, or GST-WBD (30 μg) were diluted in 10% of blocking solution and incubated for 1 h at room temperature or overnight at 4 °C. Cells were extensively washed with PBS and then incubated for 30 min at 37 °C with a mAb anti-GST. After washes they were incubated with the secondary FITC-conjugated Ab. In order to exclude interferences of cell fixation on the GTP-bound state of Rho GTPases, pull-down assay was performed on cells fixed, permeabilized, and isolated from Matrigel as detailed above.Fixed and permeabilized cells were also incubated with mAbs antiVe-cadherin, anti-β-catenin, anti-MLC-P, and anti-GST or Abs anti-RhoA or anti-Rac1, and then visualized with secondary TRITC-conjugated and FITC-conjugated Abs.For F-actin staining, cells were incubated for 30 min at 37 °C with FITC-conjugated phalloidin. ECs infected with a modified Pinco vector without GFP were used for double staining experiments.RESULTSMorphological Events of Capillary EC Morphogenesis—Time-lapse video microscopy over a period of 14 h shows that in vitro vascular network formation by capillary ECs follows specific and peculiar morphological phases (Fig. 1). During the first 30 min, ECs randomly plated on Matrigel spread, moved, and started to form small and seldom interconnected clusters. After 1 h, ECs were completely spread and clusters increased in size and highly connecting among them. These changes became more evident after 2 h, when discrete Matrigel areas were empty and surrounded by EC islets or chains. Four hours after plating, endothelial chains begun stretching and ECs appeared very elongated. This process continued for another 2 h and was accompanied by a reduction in size of cell clusters and the delineation of polygonal spaces whose dimensions were similar to those observed at the end of the process. During the last 8 h EC chains became progressively thinner, and by 14 h these chains gave rise to EC cords interconnected with nodes that originated from the reduction in size of clusters and their compression.Activation and Cellular Localization of Rho GTPases during Capillary EC Morphogenesis—Because cell motility largely depends on the control of the actin cytoskeletal dynamics by the Rho GTPase family (14.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar), we studied the time courses of RhoA, Rac1, and Cdc42 activation and their intracellular localization in ECs during vascular network formation Matrigel. To analyze the enzyme GTP-bound state of Rac1, RhoA, and Cdc42 in capillary EC lysates (27.Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (585) Google Scholar), pull-down experiments were performed by using fusion proteins between GST and the Cdc42/Rac interactive binding domain of human PAK1B (GST-PBD), or the Rho binding domain of the Rho effector rhotekin (GSTRBD), or the Cdc42/Rac interactive binding domain of the Wiskott-Aldrich syndrome protein (GST-WBD), which binds specifically to active Cdc42 (31.Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar).When ECs were put in suspension the amount of RhoA-GTP was very low. Thirty min after plating ECs on Matrigel, RhoAGTP markedly increased and remained elevated up to 1 h (Fig. 2A). After 2 and 4 h, RhoA-GTP decreased, but it was again detectable at 6 h, reaching its maximum at 10 h and subsequently declining again (Fig. 2A). Rac1-GTP was detected in suspended cells and increased 2 h after plating, remaining elevated up to 4 h (Fig. 3A). Then Rac-GTP progressively decreased and was lower than the basal level after 14 h. The amount of Cdc42-GTP slightly increased in adhering cells as compared with suspended cells but did not change over the time of network formation, including the early step when Rac1 was activated (Fig. 3C). This observation suggested that modulation of Cdc42 activation was not essential in our experimental model.Fig. 2Activation of RhoA during capillary EC morphogenesis.A, RhoAGTP amount was evaluated by pull-down experiments with GST-RBD along the capillary network formation. At the indicated stage of the morphogenetic process on Petri dishes (100 mm diameter; 9 × 106 cells), ECs were dispersed with MatriSperse Cell recovery solution, lysed, and incubated with GST-RBD. Proteins were separated by SDS-PAGE (12%) and then analyzed for bound RhoA molecules by Western blotting. An aliquot of lysate was used for analyzing the total amounts of RhoA. The time courses from 30 min to 4 h and from 6 to 14 h have been performed in separated experiments. Densitometric analysis of three independent experiments is shown as mean ± S.D. Bars correspond to time points of the pull-down experiment. Data were analyzed by oneway analysis of variance (p < 0.0002) and Student-Newman-Keuls test (* indicates p < 0.05 versus suspended cells). B, subcellular localization of RhoA-GTP after 30 min from the
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