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• W2007590503 abstract "Transforming growth factor-β1 (TGF-β1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-β receptors. We explored the possibility of inhibiting TGF-β- mediated events in glomerular capillary endothelial cells using a TGF-β type II receptor (TβR-II) transdominant negative mutant.A mutant TGF-β type II receptor (TβR-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat TβR-II cDNA as template. Since TβR-II and TGF-β type I receptor (TβR-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type TβR-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with TβR-IIM, and four independent clones were expanded. That the TβR-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface TβR-IIM protein was shown by affinity cross-linking with 125I-TGF-β1. In wild-type endothelial cells, TGF-β1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 ± 10% of control (n = 4). In transfected endothelial cells carrying TβR-IIM, TGF-β1 stimulated [3H]thymidine incorporation to 131 ± 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-β1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying TβR-IIM.This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with TβR-II signaling inhibits this process in glomerular capillary endothelial cells. Transforming growth factor-β1 (TGF-β1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-β receptors. We explored the possibility of inhibiting TGF-β- mediated events in glomerular capillary endothelial cells using a TGF-β type II receptor (TβR-II) transdominant negative mutant. A mutant TGF-β type II receptor (TβR-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat TβR-II cDNA as template. Since TβR-II and TGF-β type I receptor (TβR-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type TβR-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with TβR-IIM, and four independent clones were expanded. That the TβR-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface TβR-IIM protein was shown by affinity cross-linking with 125I-TGF-β1. In wild-type endothelial cells, TGF-β1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 ± 10% of control (n = 4). In transfected endothelial cells carrying TβR-IIM, TGF-β1 stimulated [3H]thymidine incorporation to 131 ± 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-β1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying TβR-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with TβR-II signaling inhibits this process in glomerular capillary endothelial cells. INTRODUCTIONAngiogenesis, the process of new blood vessel formation, is an integral part of development, wound repair, and tumor growth. The formation of capillary networks requires a complex series of cellular events, in which endothelial cells locally degrade their basement membrane, migrate into the connective tissue stroma, proliferate at the migrating tip, elongate and organize into capillary loops(1Folkman J. Klagsbrun M. Science. 1987; 235: 442-447Crossref PubMed Scopus (4020) Google Scholar). In response to angiogenic stimuli, endothelial cells in culture develop networks of capillary-like tubes.Transforming growth factor-β1 (TGF-β1) 1The abbreviations used are: TGFtransforming growth factorECMextracellular matrixPAplasminogen activatorFGFfibroblast growth factoraFGFacidic FGFbFGFbasic FGFTβR-IITGF-β type II receptorPCRpolymerase chain reactionFBSfetal bovine serumntnucleotidekbkilobase(s)MOPS3-(N-morpholino)propanesulfonic acidHBSSHanks' balanced salt solution. is a 25-kDa homodimeric polypeptide that belongs to a family of homologous multifunctional cytokines. TGF-β1 regulates diverse cellular functions including proliferation and differentiation. TGF-β1 is strongly expressed during embryogenesis (2Rappolee D.A. Brenner C.A. Schultz R. Mark D. Werb Z. Science. 1988; 241: 1823-1825Crossref PubMed Scopus (544) Google Scholar, 3Heine U. Munoz E.F. Flanders K.C. Ellingsworth L.R. Lam H.Y. Thompson N.L. Roberts A.B. Sporn M.B. J. Cell Biol. 1987; 105: 2861-2876Crossref PubMed Scopus (586) Google Scholar) and in sites undergoing intense development and morphogenesis(4Wilcox J.N. Derynck R. Mol. Cell. Biol. 1988; 8: 3415-3422Crossref PubMed Scopus (228) Google Scholar, 5Sandberg M. Vuorio T. Hirvonen H. Alitalo K. Vuorio E. Development. 1988; 102: 461-470PubMed Google Scholar). Moreover, TGF-β1 induces angiogenesis in vivo(6Roberts A.B. Sporn M.B. Assoian R.K. Smith J.M. Roche N.S. Wakefield L.M. Heine U.I. Liotta L.A. Falanga V. Kehrl J.H. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4167-4171Crossref PubMed Scopus (2396) Google Scholar, 7Yang E.Y. Moses H.L. J. Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (420) Google Scholar) and capillary morphogenesis in vitro(8Madri J.A. Pratt B.M. Tucker A.M. J. Cell Biol. 1988; 106: 1375-1384Crossref PubMed Scopus (450) Google Scholar). The mechanism by which TGF-β1 induces angiogenesis is not yet well defined.In the early stages of angiogenesis, proteases are required for extracellular matrix (ECM) proteolysis to facilitate endothelial cell migration(9Saksela O. Rifkin D.B. Annu. Rev. Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (714) Google Scholar). TGF-β1 induces endothelial cell secretion of plasminogen activator (PA) which activates plasmin, a protease that degrades ECM proteins(10Sato Y. Rifkin D.B. J. Cell Biol. 1989; 109: 309-315Crossref PubMed Scopus (764) Google Scholar, 11Montesano R. Pepper M.S. Möhle-Steinlein U. Risau W. Wagner E.F. Orci L. Cell. 1990; 62: 435-445Abstract Full Text PDF PubMed Scopus (370) Google Scholar). Increased production of PA has been associated with the invasive properties of cultured endothelial cells in response to angiogenic stimuli(10Sato Y. Rifkin D.B. J. Cell Biol. 1989; 109: 309-315Crossref PubMed Scopus (764) Google Scholar, 11Montesano R. Pepper M.S. Möhle-Steinlein U. Risau W. Wagner E.F. Orci L. Cell. 1990; 62: 435-445Abstract Full Text PDF PubMed Scopus (370) Google Scholar). In addition, plasmin activates latent TGF-β1(12Lyons R.M. Gentry L.E. Purchio A.F. Moses H.L. J. Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (669) Google Scholar), in an autocrine fashion. Furthermore, TGF-β1 is a potent chemoattractant for macrophages and fibroblasts (13Wahl S.M. Hunt D.A. Waterfield L.M. McCartney-Francis N. Wahl L.M. Roberts A.B. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5788-5792Crossref PubMed Scopus (1085) Google Scholar, 14Wiseman D.M. Polverini P.J. Kamp D.W. Leibovich S.J. Biochem. Biophys. Res. Commun. 1988; 157: 793-800Crossref PubMed Scopus (187) Google Scholar), which are postulated to release angiogenic peptides in vivo, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, or tumor necrosis factor-α(15Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. Nature. 1987; 329: 630-632Crossref PubMed Scopus (992) Google Scholar).Two transmembrane serine/threonine kinases, types I and II, have been identified as signal transducing TGF-β receptors. TGF-β type II receptor (TβR-II), a constitutively active kinase, directly binds TGF-β1, and this ligand binding results in the recruitment and phosphorylation of TGF-β type I receptor (TβR-I) to produce a heteromeric signaling complex(16Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2093) Google Scholar). TβR-I alone is unable to bind TGF-β1, and TβR-II is unable to signal without TβR-I(17Wrana J.L. Attisano L. Cárcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1366) Google Scholar).We explored the possibility of inhibiting TGF-β1-mediated events in renal glomerular capillary endothelial cells using a TβR-II transdominant negative mutant. A mutant TβR-II construct (TβR-IIM), lacking the cytoplasmic serine/threonine kinase domain, but with full transmembrane spanning and extracellular domains, was produced by polymerase chain reaction (PCR) using rat TβR-II cDNA (18Choi M.E. Kim E.G. Huang Q. Ballermann B.J. Kidney Int. 1993; 44: 948-958Abstract Full Text PDF PubMed Scopus (81) Google Scholar) as template. Since TβR-II and TβR-I heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type TβR-I, hence acting in a dominant negative fashion(19Chen R.H. Ebner R. Derynck R. Science. 1993; 260: 1335-1338Crossref PubMed Scopus (356) Google Scholar, 20Wieser R. Attisano L. Wrana J.L. Massagué J. Mol. Cell. Biol. 1993; 13: 7239-7247Crossref PubMed Google Scholar, 21Brand T. MacLellan W.R. Schneider M.D. J. Biol. Chem. 1993; 268: 11500-11503Abstract Full Text PDF PubMed Google Scholar). When the transdominant negative mutant construct was stably expressed in glomerular capillary endothelial cells, capillary morphogenesis and associated apoptosis were entirely blocked in these cells.EXPERIMENTAL PROCEDURESCell CultureGlomerular capillary endothelial cells were isolated from bovine kidney cortex as described previously(22Ballermann B.J. Am. J. Physiol. 1989; 256: C182-C189Crossref PubMed Google Scholar), with the following modifications. After collagenase digestion, the cells were plated at low density on gelatin-coated plates, in RPMI 1640 medium containing 15% fetal bovine serum (FBS) to which 8 ng/ml acidic fibroblast growth factor (aFGF) (R & D Systems), 0.1 μg/ml heparin, and 5 units/ml penicillin, and 5 μg/ml streptomycin were added. The aFGF was used to stimulate endothelial cell proliferation, and the heparin both to increase the affinity of aFGF for endothelial cell FGF receptors and to inhibit mesangial cell growth. Colonies of endothelial cells were subjected to two rounds of cloning to establish cell lines free of contaminating mesangial cells. Once the cells were established in culture, they were maintained in RPMI 1640 with 15% FBS and 5 units/ml penicillin, and 5 μg/ml streptomycin. Cells between passages 5 and 15 were used for experiments described herein. That the cells are endothelial cells was documented for each isolation by labeling with fluorescent acetylated low density lipoprotein (Biomedical Technologies Inc.).To induce capillary tube formation, cells grown to confluence were placed in RPMI medium containing 0.5% FBS, in the presence or absence of exogenous 2 ng/ml TGF-β1 (Collaborative Biomedical Products). For transmission electron microscopy, the cells were fixed in 3% glutaraldehyde in phosphate-buffered saline, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in epon directly on the culture dish, in order to preserve morphology of the capillary tubes. Thin sections were cut, stained with uranyl acetate and lead citrate, and subjected to electron microscopy (Paragon Biotech Inc., Baltimore, MD). In experiments with neutralizing antibody to TGF-β1, cells grown to confluence were placed in RPMI medium containing 0.5% FBS in the presence or absence of 10 ng/ml turkey anti-human TGF-β1 IgG (Collaborative Biomedical Products).Mutant TβR-II ConstructA truncated TGF-β type II receptor construct (TβR-IIM), lacking the cytoplasmic serine/threonine kinase domain, but with full transmembrane spanning and extracellular domains, was generated by PCR using a rat TβR-II cDNA as the template (Fig. 1). Primer sequences were as follows: sense primer 5′-GTTAAGGCTAGCGACGGGGGCTGCCATG-3′; antisense primer 5′-GGCGGTCGACTAGACACGGTAACAGTAGAAG-3′; and contained the sequences for the restriction enzymes NheI and SalI, respectively (underlined), for directional cloning, and a stop codon in the antisense primer. Each 100-μl PCR reaction mixture contained 1 ng of template DNA, 0.25 μM primers, 0.05 mM dNTPs, 0.75 mM MgCl2, 1 × PCR buffer II (Perkin-Elmer), and 2 units of Taq polymerase (Perkin-Elmer). Amplification consisted of initial denaturation at 95°C for 1 min, followed by 25 cycles (15 s at 95°C, 15 s at 50°C, and 15 s at 72°C) in GeneAmp PCR System 9600 (Perkin-Elmer). This reaction product was gel-purified and cloned with NheI and SalI into pMAMneo (CLONTECH), a glucocorticoid-inducible mammalian expression vector. That the clone contained correct directionality and in-frame sequences of the PCR product were verified by restriction mapping with EcoRI, BamHI, and HindIII, and sequencing by dideoxy chain termination technique using Sequenase 2.0 (U. S. Biochemical).Stable Transfection of Glomerular Capillary Endothelial CellsTo generate clones that stably expressed TβR-IIM, glomerular capillary endothelial cells were transfected by using Lipofectin (Life Technologies, Inc.) as follows. Cells grown to approximately 50% confluency on 6-well plates were washed with RPMI, then incubated with 1-5 μg of DNA (TβR-IIM ligated in pMAMneo) in RPMI and 5-10 μl of Lipofectin suspension for 5 h at 37°C in a 5% CO2 atmosphere. Control cells were incubated with pMAMneo vector (not containing TβR-IIM) and Lipofectin. Following a 5-h incubation, medium containing 20% FBS in RPMI was added to each well to make a final concentration of 10% FBS, and incubated further for 48 h. Then the medium was changed to 10% FBS in RPMI (no antibiotics) and incubated for another 24 h. To select for stable transfectants, cells were treated with 400 μg/ml Geneticin (Life Technologies, Inc.) in RPMI medium containing 15% FBS, and the medium was changed every 2-3 days. Clones emerged at approximately 14 days after lipofection. Stably transfected clones were subcloned using ring cylinders, expanded, and maintained in RPMI medium containing 15% FBS, 200 μg/ml Geneticin, 5 units/ml penicillin, and 5 μg/ml streptomycin. Four independent, stably transfected clones containing TβR-IIM and 4 clones containing empty vector were expanded. Non-transfected glomerular endothelial cells similarly treated with Lipofectin served as additional controls.Solution Hybridization/RNase ProtectionRNase protection analysis was done using the RPA II kit (Ambion) according to the manufacturer's instructions. The 32P-labeled antisense RNA probe was prepared from the linearized plasmid containing a fragment of the rat TβR-II cDNA using T7 RNA polymerase, yielding a probe 488 nucleotides (nt) long. 20 μg of total RNA from wild-type and transfected cells were hybridized with the 32P-labeled probe. Hybridization was for 16-18 h at 42°C in 50% formamide, 5 × SSPE, 0.1 M Tris, pH 7.4, and 50 μg/ml salmon sperm DNA. The samples were then digested with RNase A/T1, and resolved on a 6% acrylamide, 7.7 M urea sequencing gel. A sample of 32P-labeled 1-kb ladder DNA was loaded in adjacent lanes as the molecular size marker.Northern Blot AnalysisTotal RNA from cells grown in the absence or presence of 1 μM dexamethasone (Sigma) was isolated by lysis with TRI reagent (Molecular Research Center, Inc.) according to the manufacturer's instructions, and size fractionated (30 μg/lane) on a 1% agarose, 2% formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.2. Messenger RNA was transferred to a nylon membrane (Nytran, Schleicher & Schuell) and UV linked to the membrane. The blot was prehybridized at 65°C using 1% bovine serum albumin (Sigma), 7% SDS, 0.5 M phosphate buffer, 1 mM EDTA, pH 8.0, and 100 μg/ml heat-denatured salmon sperm DNA for 2 h, hybridized in the same solution containing the appropriate 32P-labeled cDNA at 65°C overnight, followed by two 30-min washes at 65°C with 0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer, 1 mM EDTA, pH 8.0, then four 15-min washes with 1% SDS, 40 mM phosphate buffer, 1 mM EDTA, pH 8.0, at 65°C. The membrane was then exposed to Kodak X-AR 5 film for 25-48 h. The TβR-II probe is a 2.8-kb rat TβR-II full-length cDNA (17Wrana J.L. Attisano L. Cárcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1366) Google Scholar) which was labeled with [32P]dCTP using random primer labeling system (Life Technologies, Inc.).Covalent Labeling of TGF-β ReceptorsCells on 100-mm plates (Corning) were washed twice with cold 40 mM HEPES, pH 7.4, in Hanks' balanced salt solution (HBSS), then incubated in binding assay buffer (HBSS, 40 mM HEPES, pH 7.4, and 1 mM bacitracin) with 400 pM125I-TGF-β1 (DuPont) in the presence or absence of 100 nM unlabeled TGF-β1 (Collaborative Biomedical Products), at room temperature for 90 min. The cells were then washed twice with 40 mM HEPES, pH 7.4, in HBSS, followed by incubation for 30 min at 4°C with covalent cross-linking reagent disuccinimidyl suberate (Pierce) in dimethyl sulfoxide at a final concentration of 0.3 mM in 40 mM HEPES, pH 7.4, HBSS. The cross-linking reaction was quenched by washing three times with cold 250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA. The cells were lysed with 100 μl of 1% Triton X-100, 10 mM Tris, pH 7.4, 1 mM EDTA, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 10 μg/ml pepstatin, and subjected to centrifugation at 13,000 × g for 30 min to remove particulate matter. Sample loading buffer (sucrose, 0.01% bromphenol blue, 2%β-mercaptoethanol, 5 mM EDTA) was added (1:1, v/v) and boiled for 5 min, followed by 10% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Brilliant Blue (Bio-Rad) to visualize equivalence in protein loading, and destained prior to autoradiography.[3H]Thymidine Incorporation104 cells were plated in 24-well dishes and incubated in medium containing 15% FBS and grown to subconfluence. Medium was then changed to serum-free RPMI for 24 h, followed by incubation in 0.5% FBS in the presence or absence of TGF-β1 (2 ng/ml). After 45 h, the medium was removed, and cells were exposed for 3 h to 1 μCi/ml [3H]thymidine in RPMI 1640 medium containing 2% bovine platelet-poor plasma derived serum, at 37°C. The cells were washed three times with RPMI, and then extracted three times with ice-cold 6% trichloroacetic acid, followed by solubilization in 1 N NaOH and counted in a Packard Liquid scintillation counter. For determination of the time course of [3H]thymidine incorporation, similar methods were utilized and the cells were extracted at the various time points, with 3-h [3H]thymidine exposure prior to each period.Genomic DNA Isolation and AnalysisCells were plated on 150-mm plates, and grown to confluence in medium containing 15% FBS, then incubated in the presence or absence of 1 μM dexamethasone for 24 h. The medium was then changed to 0.5% FBS and incubated in the presence or absence of TGF-β1 (2 ng/ml) at 37°C for 5 days. Genomic DNA isolation was performed using Puregene (Gentra) according to the manufacturer's instructions. Briefly, cells were lysed directly on the plate by removing the culture medium and adding the cell lysis solution, followed by incubation with RNase A, then protein precipitation solution. The samples were centrifuged at 2,000 × g for 10 min and supernatant transferred to new tubes. Precipitated DNA was resuspended in DNA hydration solution, and quantitated by UV spectrophotometer. 20 μg of DNA was analyzed with a 1.5% agarose gel electrophoresis.RESULTSExpression of TβR-IIMmRNATo demonstrate that the transfected glomerular endothelial cells expressed TβR-IIM mRNA, RNase protection assay was performed using antisense RNA prepared from the rat TβR-II cDNA with some flanking vector sequence. The rat TβR-II probe contained 488 nt of authentic rat TβR-II sequence, which included only 211 nt of TβR-IIM, and predicted to hybridize fully with rat (but not bovine) TβR-II mRNA. Based on the size of the TβR-II cDNA probe, the expected size of the protected fragment produced from transfected rat TβR-IIM cDNA sequence was 211 nt. As shown in Fig. 2A, hybridization with the rat TβR-II antisense riboprobe protected a fragment 211 nt in length from RNase digestion in transfected cells. No protected fragment was seen in the wild-type bovine glomerular endothelial cells or mock transfected cells with vector alone. Expression of TβR-IIM mRNA in glomerular endothelial cells was also demonstrated by RT-PCR utilizing the sense and antisense primers to rat cDNA sequence used in producing the mutant construct (data not shown), and four independent transfected clones expressing TβR-IIM mRNA were propagated for all subsequent experiments.Figure 2:Expression of TβR-II mRNA in wild-type and transfected glomerular endothelial cells. A, RNase protection assay. Total RNA from wild-type and transfected glomerular endothelial cells, treated with 1 μM dexamethasone, was hybridized in solution with a 32P-labeled rat TβR-II cDNA probe followed by RNase digestion. As expected, no protected fragment was observed in the wild-type cells (lane 1). In the transfected cells (lane 2), a 211-nt long protected fragment was seen, demonstrating that mRNA for rat TβR-IIM was expressed. Controls represent 32P-labeled rat TβR-II cDNA probe hybridized with yeast total RNA, followed by digestion with RNase (lane 3), or without RNase treatment (lane 4). No protected fragment was seen with RNase treatment (lane 3). The undigested probe was 488 nt long (lane 4). Molecular size was determined by 32P-labeled 1-kb ladder DNA (not shown). B, Northern analysis. Total RNA (30 μg/lane) from wild-type glomerular endothelial cells incubated in the absence (lane 1) or presence (lane 2) of 1 μM dexamethasone was subjected to Northern blot hybridization with a 32P-labeled rat TβR-II cDNA probe. Only a 5.5-kb mRNA was detected, corresponding to wild-type TβR-II. Lanes 3 and 4 represent total RNA from transfected endothelial cells incubated in the absence or presence of 1 μM dexamethasone, respectively. Both the 5.5- and a 1.8-kb mRNA are observed, reflecting wild-type TβR-II and TβR-IIM, respectively. The 1.8-kb mRNA was strongly induced by dexamethasone (lane 4). Also, a modest induction of the 5.5-kb wild-type TβR-II mRNA was seen with dexamethasone (lanes 2 and 4). Similar densities for the 18 S signals indicate approximate equivalence of RNA loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Northern blot analysis of total RNA isolated from wild-type and transfected glomerular endothelial cells, probed with TβR-II cDNA, showed a 5.5-kb band in all cells (Fig. 2B), corresponding to wild-type TβR-II. A new 1.8-kb band, corresponding to TβR-IIM, was observed only in cells transfected with TβR-IIM, and not present in wild-type or mock transfected cells, and was strongly induced by dexamethasone. Also, a modest induction of the 5.5-kb wild-type TβR-II mRNA was seen with dexamethasone.Cell Surface Expression of TβR-IIMAffinity cross-linking with 125I-TGF-β1 in wild-type glomerular endothelial cells detected two distinct bands with molecular masses of approximately 89 and 70 kDa corresponding with TβR-II and TβR-I, respectively (Fig. 3). Only in the transfected glomerular endothelial cells expressing TβR-IIM, two labeled bands of approximately 48 and 36 kDa were observed, both induced by dexamethasone. Labeling of TβR-I in transfected glomerular endothelial cells appeared diminished in three separate experiments.Figure 3:Affinity cross-linking of 125I-labeled TGF-β1 to cell surface receptors. Lanes 1 and 2 represent wild-type glomerular endothelial cells cross-linked in the presence or absence of unlabeled TGF-β1, respectively. Lanes 3 and 4 represent transfected glomerular endothelial cells in the presence or absence of unlabeled TGF-β1, respectively. Specifically labeled bands are observed at approximately 89 kDa and approximately 70 kDa corresponding to wild-type TβR-II and TβR-I, respectively. Bands of approximately 48 and 36 kDa are seen only in the transfected glomerular endothelial cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of TGF-β1 on Endothelial Cell [3H]Thymidine IncorporationTreatment of wild-type glomerular endothelial cells in culture for 48 h with exogenous TGF-β1 significantly inhibited [3H]thymidine incorporation to 63 ± 10% of control (Fig. 4A). Inhibition of [3H]thymidine incorporation was similarly observed in empty vector transfected cells treated with TGF-β1. In contrast, treatment of transfected cells carrying TβR-IIM with exogenous TGF-β1 stimulated [3H]thymidine incorporation to 131 ± 9% of control (∗, p < 0.005, Student's t test, n = 4). The time course of [3H]thymidine incorporation in transfected cells grown in 0.5% FBS and in the presence or absence of TGF-β1 (2 ng/ml) is shown in Fig. 4B. The results are mean values of triplicate determinations ± S.E. Exogenous TGF-β1 stimulated [3H]thymidine incorporation significantly above the basal rate in 0.5% FBS at 36 and 48 h (p < 0.005, analysis of variance).Figure 4:Effect of TGF-β1 on [3H]thymidine incorporation. A, cells were incubated in the presence or absence of TGF-β1 (2 ng/ml) for 45 h, followed by [3H]thymidine incorporation into the cells for 3 h. Lane 1, TGF-β1 inhibited [3H]thymidine incorporation in wild-type glomerular endothelial cells to 63 ± 10%. Lane 2, in transfected glomerular endothelial cells carrying TβR-IIM, TGF-β1 stimulated [3H]thymidine incorporation to 131 ± 9% (∗, p < 0.005, Student's t test, n = 4). B, transfected glomerular endothelial cells were incubated in 0.5% FBS and in the presence (•) or absence (Ε) of TGF-β1 (2 ng/ml), and [3H]thymidine incorporation was determined at various time points. Data represent means of triplicate determinations ± S.E. (p < 0.005, analysis of variance). A second experiment gave essentially the same results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Serum Deprivation and TGF-β1 on Capillary MorphogenesisIn cultured wild-type glomerular endothelial cells, with serum deprivation in the presence or absence of dexamethasone pretreatment, many of the cells detached from their substratum while remaining cells organized into capillary-like structures (Fig. 5A). Furthermore, treatment with exogenous TGF-β1, in the presence or absence of dexamethasone, also induced cell detachment and formation of capillary-like structures (Fig. 5B). These events were accelerated by approximately 48 h when compared to those cells under serum deprivation alone. Both cell detachment and formation of capillary-like structures were observed with serum deprivation in mock transfected cells carrying empty vector. In contrast, cell detachment and formation of capillary-like structures were uniformly and entirely absent in transfected cells carrying TβR-IIM treated either with serum deprivation (Fig. 5C) or exogenous TGF-β1 (Fig. 5D). When examined with a high power phase-contrast objective, after 5 days of culture the cellular cords formed by wild-type glomerular endothelial cells appeared as tubes that contained a central translucent lumen-like space along their length (Fig. 6A), similar to the in vitro angiogenesis models described by Ingber and Folkman (23Ingber D.E. Folkman J. J. Cell Biol. 1989; 109: 317-330Crossref PubMed Scopus (719) Google Scholar) and Montesano et al.(11Montesano R. Pepper M.S. Möhle-Steinlein U. Risau W. Wagner E.F. Orci L. Cell. 1990; 62: 435-445Abstract Full Text PDF PubMed Scopus (370) Google Scholar). Lumen formation was confirmed by transmission electron microscopy, which revealed groups of endothelial cells that were joined by interdigitated cell processes and enclosed a central lumenal space, as shown in Fig. 6B. Amorphous material within the lumen, also previously described by Ingber and Folkman(23Ingber D.E. Folkman J. J. Cell Biol. 1989; 109: 317-330Crossref PubMed Scopus (719) Google Scholar), likely represents matrix and debris. Clathrin-coated pits and vesicles, as well as cell junctional complex are also observed.Figure 5:Effect of serum deprivation and exogenous TGF-β1 on capillary morphogenesis. Formation of capillary-like structures were observed in wild-type glomerular endothelial cells incubated in 0.5% FBS medium, in the absence (panel A) or presence (panel B) of exogenous TGF-β1 (2 ng/ml). Whereas, capillary-like structures were not observed in transfected glomerular endothelial cells carrying TβR-IIM, incubated in 0.5% FBS medium, both in the absence (panel C) or presence (panel D) of exogenous TGF-β1 (2 ng/ml).View Large Image" @default.
• W2007590503 created "2016-06-24" @default.
• W2007590503 creator A5020032238 @default.
• W2007590503 creator A5060749319 @default.
• W2007590503 date "1995-09-01" @default.
• W2007590503 modified "2023-10-10" @default.
• W2007590503 title "Inhibition of Capillary Morphogenesis and Associated Apoptosis by Dominant Negative Mutant Transforming Growth Factor-β Receptors" @default.
• W2007590503 cites W1494055834 @default.
• W2007590503 cites W1608169825 @default.
• W2007590503 cites W1641644248 @default.
• W2007590503 cites W1867003241 @default.
• W2007590503 cites W1967850071 @default.
• W2007590503 cites W1973893754 @default.
• W2007590503 cites W1975729483 @default.
• W2007590503 cites W1980235826 @default.
• W2007590503 cites W1987059068 @default.
• W2007590503 cites W1992172901 @default.
• W2007590503 cites W2002621910 @default.
• W2007590503 cites W2003598961 @default.
• W2007590503 cites W2009414873 @default.
• W2007590503 cites W2021263701 @default.
• W2007590503 cites W2023464504 @default.
• W2007590503 cites W2023502130 @default.
• W2007590503 cites W2028514452 @default.
• W2007590503 cites W2029545106 @default.
• W2007590503 cites W2041708400 @default.
• W2007590503 cites W2050251876 @default.
• W2007590503 cites W2062199188 @default.
• W2007590503 cites W2072670533 @default.
• W2007590503 cites W2075404842 @default.
• W2007590503 cites W2081948388 @default.
• W2007590503 cites W2083549154 @default.
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