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• W2013782445 abstract "Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitrostudies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivoangiogenesis. Both receptors were detected on vascular endotheliumin situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels. Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitrostudies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivoangiogenesis. Both receptors were detected on vascular endotheliumin situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels. endothelial cell basic fibroblast growth factor hyaluronan human umbilical vein endothelial cell monoclonal antibody phosphate-buffered saline bovine serum albumin fluorescence-activated cell sorting Tris-buffered saline with Tween 20 4-morpholinepropanesulfonic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol receptor for HA-mediated motility Angiogenesis, the formation of new blood vessels from a preexisting vasculature, is an essential feature of a number of important physiological processes (e.g. wound healing) and pathological conditions (e.g. diabetic eye disease and tumor growth and spread) (1Griffioen A.W. Molema G. Pharmacol. Rev. 2000; 52: 237-268PubMed Google Scholar, 2Folkman J. N. Engl. J. Med. 1995; 333: 1757-1763Crossref PubMed Scopus (2228) Google Scholar). During this process endothelial cells (EC)1 in an established vessel initially sever their normal associations with adjacent endothelial cells, migrate, and proliferate into the surrounding tissue, where they reestablish their cell-to-cell attachments to form new capillaries. Given this understanding, the interactions of endothelial cells with the extracellular matrix, and the receptors that mediate these interactions, are of critical importance to the formation of new blood vessels. Hyaluronan (HA), an important constituent of the extracellular matrix, is a glycosaminoglycan composed of repeating disaccharide units ofd-glucuronic acid andN-acetyl-d-glucosamine (3Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar). This ubiquitously distributed molecule regulates cellular events such as cell proliferation and locomotion that are required for a variety of biological processes including tumorigenesis, morphogenesis, inflammation and host response to injury (reviewed in Ref. 4Savani R.C. Bagli D.J. Harrison R.E. Turley E.A. Garg H.G. Longaker M.T. Scarless Wound Healing. Marcel Dekker, New York2000: 115-142Google Scholar). HA has also been implicated in the formation of vessels. However, its effects on in vivo angiogenesis and EC function are complex and have been reported to depend on HA concentration and molecular size (5Rooney P. Kumar S. Ponting J. Wang M. Int. J. Cancer. 1995; 60: 632-636Crossref PubMed Scopus (257) Google Scholar). High molecular weight HA (at concentrations > 100 μg/ml) inhibits EC proliferation and disrupts confluent endothelial monolayers (6West D.C. Kumar S. Exp. Cell Res. 1989; 183: 179-196Crossref PubMed Scopus (307) Google Scholar). Consistent with these findings are the observations in chick embryo limb buds that avascular regions are rich in native high molecular weight HA and that expression of this form of HA in normally vascular areas results in decreased vascularity (7Feinberg R.N. Beebe D.C. Science. 1983; 220: 1177-1179Crossref PubMed Scopus (299) Google Scholar). In contrast, low molecular weight HA stimulates EC proliferation (6West D.C. Kumar S. Exp. Cell Res. 1989; 183: 179-196Crossref PubMed Scopus (307) Google Scholar), induces in vitro endothelial tube formation (8Rahmanian M. Pertoft H. Kanda S. Christofferson R. Claesson-Welsh L. Heldin P. Exp. Cell Res. 1997; 237: 223-230Crossref PubMed Scopus (65) Google Scholar), and stimulates neovascularization in chick chorioallantoic membranes (9West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (956) Google Scholar) and cutaneous wounds (10Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledgers P. J. Invest. Dermatol. 1994; 103: 576-579Crossref PubMed Scopus (155) Google Scholar, 11Lees V.C. Fan T.-P.D. West D.C. Lab. Invest. 1995; 73: 259-266PubMed Google Scholar). HA appears to exert its biological effects through binding interactions with specific cell-associated receptors (12Sherman L. Sleeman J. Herrlich P. Ponta H. Curr. Opin. Cell Biol. 1994; 6: 726-733Crossref PubMed Scopus (377) Google Scholar). A number of HA-binding proteins have been identified, and two molecularly distinct cell-surface receptors for HA have been characterized, namely CD44 and RHAMM (for receptor for hyaluronan-mediated motility) (12Sherman L. Sleeman J. Herrlich P. Ponta H. Curr. Opin. Cell Biol. 1994; 6: 726-733Crossref PubMed Scopus (377) Google Scholar, 13Entwistle J. Hall C.L. Turley E.A. J. Cell. Biochem. 1996; 61: 569-577Crossref PubMed Scopus (420) Google Scholar, 14Naor D. Slonov R.V. Ish-Shalom D. Woude G.F.V. Klein G. Advances in Cancer Research. 71. Academic Press, San Diego1997: 241-319Google Scholar, 15Sneath R.J.S. Mangham D.C. J. Clin. Pathol. (Lond.). 1998; 51: 191-200Crossref Scopus (244) Google Scholar). Although several other binding interactions for CD44 and RHAMM have been reported (14Naor D. Slonov R.V. Ish-Shalom D. Woude G.F.V. Klein G. Advances in Cancer Research. 71. Academic Press, San Diego1997: 241-319Google Scholar, 16Yang B. Hall C.L. Yang B.L. Savani R.C. Turley E.A. J. Cell. Biochem. 1994; 56: 455-468Crossref PubMed Scopus (35) Google Scholar), currently the interaction with HA appears to be the one most likely to directly activate intracellular signals required to stimulate processes relevant to angiogenesis. Specifically, preliminary in vitro studies have suggested potential roles for these two molecules in aspects of HA-dependent endothelial function (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar, 18Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 19Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). However, the relative contributions of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. In this paper, we therefore investigated the contribution of the HA-binding receptors, RHAMM and CD44, to EC functions and to angiogenesis in vivo. Both receptors were noted to be present on vascular endothelium in situ and on the surface of cultured EC. Using blocking antibodies specific to each receptor, we show that CD44 is the major determinant of EC adhesion to HA and EC proliferation, whereas RHAMM regulates EC migration through the basement membrane substrate Matrigel. Further, although antibodies against each receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked bFGF-induced neovascularization in mice. Together, these data provide evidence for the involvement endothelial HA-binding receptors in specific endothelial cell functions and angiogenesis and suggest that they may represent new targets for anti-angiogenic therapy. Human endothelial cells (HUVEC) and a murine endothelial cell line (H5V), were cultured as described previously (20Christofidou-Solomidou M. Nakada M. Williams J. Muller W. DeLisser H. J. Immunol. 1997; 158: 4872-4878PubMed Google Scholar). All reagents and chemicals were obtained from Sigma unless otherwise specified. The following antibodies were used: antibody R36, a rabbit polyclonal anti-serum raised against amino acids 585–605 encoded in the complete murine RHAMM cDNA derived from the RHAMM gene structure (21Feiber C. Plug R. Sleeman J. Dall P. Ponta H. Hoffman M. Gene (Amst.). 1999; 226: 41-50Crossref PubMed Scopus (25) Google Scholar), which binds bovine aortic smooth muscle cells (22Savani R.C. Wang C. Yang B. Zhang S. Kinsella M.G. Wight T.N. Stern R. Nance D.M. Turley E.A. J. Clin. Invest. 1995; 95: 1158-1168Crossref PubMed Scopus (172) Google Scholar) and rat macrophages (23Savani R.C. Khalil N. Turley E.A. Proc. West. Pharmacol. Soc. 1995; 38: 131-136PubMed Google Scholar); mAb J-173, a murine antibody against human CD44 (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar); mAb KM81 against murine CD44 (24Miyake K. Medina K.L. Hayashi S. Ono S. Hamaoka T. Kincade P.W. J. Exp. Med. 1990; 171: 477-488Crossref PubMed Scopus (530) Google Scholar, 25Miyake K. Underhill C.B. Lesley J. Kincade P.W. J. Exp. Med. 1990; 172: 69-75Crossref PubMed Scopus (556) Google Scholar); and mAb 390 and 4G6 against murine and human PECAM-1, respectively (20Christofidou-Solomidou M. Nakada M. Williams J. Muller W. DeLisser H. J. Immunol. 1997; 158: 4872-4878PubMed Google Scholar). For all studies involving R36, protein G-Sepharose-purified antibody was used. Cells were washed in phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde for 10 min, and then permeabilized with ice-cold 0.5% Nonidet P-40 for 1 min. After washing, cells were stained by immunofluorescent staining using the appropriate antibody as described previously (26DeLisser H.M. Chilkotowsky J. Yan H.-C. Daise M.L. Buck C.A. Albelda S.M. J. Cell Biol. 1994; 124: 195-203Crossref PubMed Scopus (99) Google Scholar). Cells were viewed on a Zeiss phase-epifluorescent microscope using a 80× fluorescence lens and photographed with TMAX film at 3200 ASA. Confocal microscopic images were obtained using a computer-interfaced, laser-scanning microscope (Leica TCS 4D) in the Confocal Core Facility at the Children's Hospital of Philadelphia. Simultaneous wavelength scanning allowed superimposition of fluorescent labeling with fluorescein isothiocyanate and Texas Red fluorophores at wavelengths of 488 nm and 568 nm respectively. Laser power was fixed at 75% for all image acquisition. Image output was at 1024 × 1024 pixels. Endothelial cells were treated with various anti-human or anti-murine PECAM-1 mAbs for 1 h at 4 °C. The primary antibody was then removed, the cells washed with PBS, and a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-mouse or anti-rat secondary antibody (Cappell) was added for 30 min at 4 °C. After washing in PBS, flow cytometry was performed using an Ortho Cytofluorograph 50H cell sorter equipped with a 2150 data handling system (Ortho Instruments, Westwood, MA). HUVEC, cultured in T-75 flasks, were washed with Ca2+/Mg2+-free PBS and then incubated at 37 °C/5% CO2 for 15 min with 3 ml of 1 mmEDTA in Ca2+/Mg2+-free PBS. Cells were then scraped with a rubber policeman. The resulting lysates were transferred to 15-ml tubes, centrifuged for 10 min at 1000 rpm, washed once with cold PBS, and then centrifuged. The residual pellets were sonicated several times in 1-s bursts on ice in 5 ml of membrane preparation buffer (0.25 m sucrose, 10 mm HEPES, 0.05 mm, 0.5 mm 1,4-dithiothreitol, pH 7.1) followed by centrifugation at 1000 rpm for 10 min. After the supernatants were removed, the tubes were ultracentrifuged at 40,5000 rpm for 45 min and the pellet resuspended in 125 μl of membrane preparation buffer. Samples were stored at −80 °C. HUVEC, cultured in T-75 flasks, were washed with cold Ca2+/Mg2+-free PBS. The cells were then incubated for 10 min with 500 μl of lysis buffer (25 mmTris-HCl, 0.15 m NaCl, 1.0 mm EDTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and Sigma protease inhibitor at 1:20 dilution). Cells were then scraped, transferred to microcentrifuge tubes, centrifuged at 14000 rpm for 10 min, and the resulting supernatant collected and stored at −80 °C. HUVEC cultured in T-75 flasks were washed with Ca2+/Mg2+-free PBS. The cells were then harvested by scraping the flasks with 1 ml of PBS and the resulting lysates transferred to microcentrifuge tubes that were centrifuged at 3000 rpm for 5 min. The pellets were washed twice with ice-cold PBS and then resuspended in 1 ml of fresh Buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 1.5 mmMgCl2, 0.2% Igepal, 1.0 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride), incubated for 5 min on ice with occasional stirring, and then centrifuged at 300 rpm for 5 min. Nuclear extracts were then released by resuspending the pellets in Buffer B (20 mm Hepes, 420 mm NaCl, 0.1 mm EDTA, 1.5 MgCl, 25% glycerol, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 1.0 μg/liter leupeptin and pepstatin) and incubating the tubes on ice for 10 min with intermittent gentle shaking. Nuclear extracts were stored at −80 °C. Western blot analysis was performed using the NOVEX NuPAGE electrophoresis system (Invitrogen, Carlsbad, CA) with 1 mm 4–15% BisTris gels according to manufacturer's instructions. Briefly, 10 μg of lysate was loaded to each well and gels were run at 200 V at 4 °C for 50 min in NuPAGE MOPS SDS running buffer under reducing conditions. Proteins were transferred to nitrocellulose membrane at 30 V for 60 min at room temperature. The membrane was then blocked for 1 h at room temperature with 5% nonfat dry milk in Tween/Tris-buffered saline (TTBS) (100 mm Tris base, 1.5 m NaCl adjusted to pH 7.4 with 0.1% Tween 20). The primary antibody was then applied overnight at 4 °C. On the following day, the membrane was washed with TTBS four times, for 10 min each time. A horseradish peroxidase conjugated goat anti-rabbit secondary antibody was applied for 1 h at room temperature. Following this, the membrane was washed with TTBS followed by two 15-min washes with TBS. The blots were developed using a chemiluminescence system (Amersham Pharmacia Biotech). HUVEC were cultured for 24 h in 96-well plates and the number of viable cells determined using a commercially available non-radioactive colorimetric assay according to the manufacturer's instructions (Cell Titer 96® AQueous non-radioactive cell proliferation assay, Promega, Madison WI). HA (as Healon) at a concentration of 0.2 mg/ml in water containing N-hydroxysulfosuccinimide at 0.184 mg/ml, was mixed with an equal volume of 1-ethyl-3-(dimethyaminopropyl)carbodiimide HCl (1.23 mg/ml). 100 μl of the resulting solution was then added to each well of a 96-well plate for 2 h at room temperature, followed by washing of the wells with PBS. In other experiments 100 μl of Matrigel (25 μg/ml; Collaborative Research, Bedford, MA) was added to the well and allowed to dry overnight at 37 °C. The wells with HA or Matrigel coupled to the plate were then blocked with 2% BSA and washed with PBS. EC (20,000 cells) labeled overnight with 3[H]thymidine and resuspended in serum-free media containing BSA or antibody were added to the wells and incubated for 30 min at 37 °C in 5% CO2. Following incubation, lysis buffer (0.5% Triton X-100 and 1% SDS in PBS) was added to each and the radioactivity counts of the lysate determined. Matrigel-coated transwell inserts (Costar; 8-mm pore filter) were prepared by twice adding 100 μl of Matrigel (250 μg/ml) to the transwell and allowing the Matrigel to dry at 37 °C in a non-humidified oven for 24 h. EC (100,000 cells) labeled overnight with 3[H]thymidine and resuspended in media (with 10% serum) containing BSA or antibody were added to the transwells and incubated for 8 h at 37 °C in 5% CO2. The EC pass through the pores of the filter and adhere on the lower surface of the filter (27Ito Y. Iwamoto Y. Tanaka K. Okuyama K. Sugioka Y. Int. J. Cancer. 1996; 67: 148-152Crossref PubMed Scopus (35) Google Scholar). After incubation with BSA or antibody, the wells were removed and washed, and the top surface of the filter wiped with a cotton swab. The filters were then carefully cut out, placed in scintillation fluid, and counted in a β-counter. For each antibody condition, migration was expressed as percentage of BSA control. In vitro tube formation was studied using previously described procedures (28Zhou Z. Christofidou-Solomidou M. Garlanda C. DeLisser H.M. Angiogenesis. 1999; 3: 181-188Crossref PubMed Scopus (67) Google Scholar). Matrigel was diluted with cold serum-free medium to 10 mg/ml. 50 μl of the solution were added to each well of a 96-well plate and allowed to form a gel at 37 °C for 30 min. HUVEC (150,00 cells/ml) were initially incubated for 15 min with IgG or antibody in complete medium. Two hundred μl of the cell/antibody suspension (30,000 cells) were then subsequently added to each well and incubated for 6–8 h at 37 °C in 5% CO2. Under these conditions EC form delicate networks of tubes that are detectable within 2–3 h and are fully developed after 8–12 h. After incubation with IgG or antibody, the wells were washed and the Matrigel and its endothelial tubes fixed with 3% paraformaldehyde. The total tube length per well was determined by computer-assisted image analysis with the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD). This model has been extensively characterized by Passaniti et al.(29Passaniti A. Taylor R.M. Pili R. Guo Y. Long P.V. Haney J.A. Pauly R.R. Grant D.S. Martin G.R. Lab. Invest. 1992; 67: 519-528PubMed Google Scholar). Briefly C57Bl/6 mice were injected subcutaneously with 0.5 ml of Matrigel supplemented with bFGF (500 ng/ml) to induce the growth of vessels into the gel. There were three experimental groups: IgG, antiCD44 (KM81), and anti-RHAMM (R36). For each, the following amounts of IgG or antibody were added to the Matrigel plugs: IgG and KM81, 200 μg; R36, 500 μg. Animals were then injected with IgG (200 μg), KM81 (200 μg), or R36 (400 μg) daily via the intraperitoneal route for 5 days. The dosing of the antibodies was based on previous studies (28Zhou Z. Christofidou-Solomidou M. Garlanda C. DeLisser H.M. Angiogenesis. 1999; 3: 181-188Crossref PubMed Scopus (67) Google Scholar) and reflected concentrations of each antibody that result in saturable binding to murine EC by FACS analysis (data not shown). After 5 days, the animals were sacrificed and the gels processed for hemoglobin analysis. Differences among groups were analyzed using one-way analysis of variance. When statistically significant differences were found (p < 0.05), individual comparisons were made using the Bonferroni/Dunn test. Although CD44 is present on endothelial cells (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar, 18Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), the endothelial expression of RHAMM, the other well defined cell surface HA-binding receptor, has not been fully characterized (19Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Endothelial surface expression of these receptors was therefore assessed initially by immunofluorescence staining of human skin (neonatal foreskin) using the J-173 and R36 antibodies against CD44 (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar) and RHAMM (22Savani R.C. Wang C. Yang B. Zhang S. Kinsella M.G. Wight T.N. Stern R. Nance D.M. Turley E.A. J. Clin. Invest. 1995; 95: 1158-1168Crossref PubMed Scopus (172) Google Scholar), respectively. Antibodies against RHAMM and CD44 not only stained the smooth muscle of the dermis and the vessel wall, but both were also noted to bind to the vascular endothelium as identified by PECAM-1 (Fig. 1,A–F). Surface expression of RHAMM and CD44 on cultured HUVEC was subsequently demonstrated with these antibodies by confocal immunofluorescence microscopy (Fig. 1, G–I) and by FACS analysis (Fig. 2A). The presence of RHAMM on the cell surface of HUVEC was further confirmed by immunoblotting with anti-RHAMM antibody that demonstrated the presence of RHAMM (∼80 kDa) in membrane, cytoplasmic, and nuclear fractions of HUVEC cell lysates (Fig. 2B). Further, the binding of biotinylated HA to HUVEC was inhibited by the either anti-CD44 or anti-RHAMM antibody (Fig. 2C).Figure 2Expression of HA-binding receptors on EC and their binding of biotinylated HA. A, the expression of PECAM-1, RHAMM, and CD44 on HUVEC and H5V was assessed by FACS analysis. The following antibodies were used: anti-human PECAM-1 (4G6), anti-murine PECAM-1 (390), anti-human CD44 (J-173), anti-murine CD44 (KM81), and anti-RHAMM (R36). Filled and unfilled tracings represent the background staining and staining for the antibodies, respectively. The HUVEC and H5V lines were recognized by the species-specific PECAM-1 antibodies (a andd). Antibody J-173 bound to HUVEC (b), whereas KM81 bound to the H5V line (e). R36 bound to both cell lines (c and f). B, endothelial cell lysates were partitioned into nuclear (N), cytoplasmic (C), and membrane (M) fractions and then blotted with anti-RHAMM antibody. RHAMM (∼80 kDa) was detected in all three fractions. C, the binding in solution of biotinylated HA to HUVEC in the absence or presence of anti-RHAMM or anti-CD44 antibody was determined by FACS analysis. The binding of biotinylated HA to HUVEC, as assessed by the mean fluorescence intensity, was reduced by both antibodies. Data are representative of two experiments in which similar results were obtained.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A number of important aspects of EC function relevant to angiogenesis including cell proliferation, migration, and tube formation are regulated in part by endothelial adhesive interactions with the ECM (30Brooks P.C. Strömblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar, 31Eliceiri B.P. Klemke R. Strömblad S. Cheresh D.A. J. Cell Biol. 1998; 140: 1255-1263Crossref PubMed Scopus (361) Google Scholar, 32Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1214) Google Scholar). We therefore investigated the effect of anti-RHAMM and anti-CD44 antibodies on the adhesion of cultured EC to plastic surfaces coated with HA. Although both antibodies inhibited the binding of biotinylated HA to EC in solution (Fig. 2C), antibody against CD44 (20 μg/ml), but not RHAMM (300 μg/ml), inhibited EC adhesion to HA immobilized on plastic (Fig.3A). This suggests that EC adhesion to HA is largely mediated by CD44, and not RHAMM. Antibody against CD44 (J-173) has been shown previously to inhibit EC proliferation (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar, 18Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To determine if antagonism of RHAMM also had a similar effect, HUVEC were cultured for 24 h in the presence of control (anti-MHC1), anti-CD44 (J-173), or anti-RHAMM (R36) antibodies (all antibody concentrations = 100 μg/ml). Compared with cells cultured in media alone, J-173 but not R36 significantly inhibited EC proliferation (Fig. 3C), suggesting that engagement of CD44, but not RHAMM, transduces signals that trigger the proliferation of these cells. HA has been implicated in cell motility, including endothelial cell migration (17Trochon V. Mabilat C. Bertrand P. Legrand Y. Smadja-Joffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Crossref PubMed Scopus (195) Google Scholar). We investigated the effect of anti-RHAMM antibody on the ability of single endothelial cells to migrate through polycarbonate filters coated with Matrigel. Antibody against RHAMM, but not CD44, inhibited migration through Matrigel-coated filters in a dose-dependent manner (data shown for 100 μg/ml antibody concentration) (Fig. 3B). This inhibition is unlikely to be due to antagonism of cell-matrix adhesion, as neither antibody decreased the adhesion of EC to Matrigel-coated surfaces (Fig.3A), indicating that there are other constituents of Matrigel that allow for EC adhesion to this substrate independent of HA. These data are consistent with a role for RHAMM in the migration of EC through the matrix of the basement membrane. The differentiation and organization of EC into vascular tubes is a critical step in the process of angiogenesis, which has been reproduced in vitro by a number of models (33Mousa S.A. Mousa S.A. Angiogenesis Inhibitors and Stimulators: Potential Therapeutic Implications. 20. Lances Bioscience, Georgetown, TX2000: 1-12Google Scholar). Using a model of endothelial tube formation on Matrigel, we noted that both anti-RHAMM and anti-CD44 antibodies inhibited tube formation by HUVEC on this substrate in a dose-dependent manner (Fig.4, A and B). Tube formation in the presence of control antibody (anti-MHC1) did not inhibit tube formation (data not shown). Of note, when tube formation was allowed to occur in the presence of both antibodies at concentrations that were individually not inhibitory (anti-CD44 = 5 μg/ml; anti-RHAMM = 50 μg/ml), the inhibition of tube formation was additive (Fig. 4C), suggesting that, with respect to this process, RHAMM and CD44 may act in a cooperative or synergistic fashion. The finding that various aspects of EC function required for angiogenesis were inhibited by antagonism of RHAMM or CD44 activity suggested that these two HA binding receptors might also be involved in the formation of vessels in vivo. To investigate this hypothesis, we studied the effect of anti-RHAMM (R36) and anti-murine CD44 (KM81) antibodies in a model of murine angiogenesis in which vessels form over 5 days within subcutaneously implanted Matrigel plugs containing bFGF. Non-immune IgG or antibody was incorporated in the plug and administered daily (days 0–4) via the intraperitoneal route (see “Experimental Procedures” for details on dosing). Both antibodies bound murine EC as determined by FACS analysis (Fig. 2A), and KM81 has been shown previously to be a functionally active CD44 blocking antibody (24Miyake K. Medina K.L. Hayashi S. Ono S. Hamaoka T. Kincade P.W. J. Exp. Med. 1990; 171: 477-488Crossref PubMed Scopus (530) Google Scholar, 25Miyake K. Underhill C.B. Lesley J. Kincade P.W. J. Exp. Med. 1990; 172: 69-75Crossref PubMed Scopus (556) Google Scholar). Compared with animals treated with KM81 or control animals treated with non-immune rabbit IgG, R36 inhibited the angiogenic response to bFGF (Fig. 5, A–D). As assessed by the hemoglobin concentration in the recovered Matrigel plugs, R36 significantly reduced the vascularization of the plugs whereas the effect of the KM81 was similar to non-immune IgG (Fig.5E). To investigate the involvement of the HA receptors, RHAMM and CD44, in blood vessel formation, the endothelial expression of these proteins was determined and the effects of antibodies against RHAMM and CD44 on EC function and in vivo angiogenesis w" @default.
• W2013782445 created "2016-06-24" @default.
• W2013782445 creator A5009942851 @default.
• W2013782445 creator A5047815710 @default.
• W2013782445 creator A5050338024 @default.
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• W2013782445 date "2001-09-01" @default.
• W2013782445 modified "2023-09-26" @default.
• W2013782445 title "Differential Involvement of the Hyaluronan (HA) Receptors CD44 and Receptor for HA-mediated Motility in Endothelial Cell Function and Angiogenesis" @default.
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