• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Jul 2, 1993; 122(2): 497–511.
PMCID: PMC2119646

Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity

Abstract

Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2- terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19- residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.

Full Text

The Full Text of this article is available as a PDF (2.8M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Asch AS, Nachman RL. Thrombospondin: phenomenology to function. Prog Hemost Thromb. 1989;9:157–176. [PubMed]
  • Asch AS, Silbiger S, Heimer E, Nachman RL. Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding. Biochem Biophys Res Commun. 1992 Feb 14;182(3):1208–1217. [PubMed]
  • Avery RL, Connor TB, Jr, Farazdaghi M. Systemic amiloride inhibits experimentally induced neovascularization. Arch Ophthalmol. 1990 Oct;108(10):1474–1476. [PubMed]
  • Bagavandoss P, Wilks JW. Specific inhibition of endothelial cell proliferation by thrombospondin. Biochem Biophys Res Commun. 1990 Jul 31;170(2):867–872. [PubMed]
  • Berman M, Winthrop S, Ausprunk D, Rose J, Langer R, Gage J. Plasminogen activator (urokinase) causes vascularization of the cornea. Invest Ophthalmol Vis Sci. 1982 Feb;22(2):191–199. [PubMed]
  • Bornstein P. Thrombospondins: structure and regulation of expression. FASEB J. 1992 Nov;6(14):3290–3299. [PubMed]
  • Bouck N. Tumor angiogenesis: the role of oncogenes and tumor suppressor genes. Cancer Cells. 1990 Jun;2(6):179–185. [PubMed]
  • Canfield AE, Schor AM, Schor SL, Grant ME. The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes. Biochem J. 1986 Apr 15;235(2):375–383. [PMC free article] [PubMed]
  • Canfield AE, Wren FE, Schor SL, Grant ME, Schor AM. Aortic endothelial cell heterogeneity in vitro. Lack of association between morphological phenotype and collagen biosynthesis. J Cell Sci. 1992 Aug;102(Pt 4):807–814. [PubMed]
  • Catimel B, Leung L, el Ghissasi H, Mercier N, McGregor J. Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region. Biochem J. 1992 May 15;284(Pt 1):231–236. [PMC free article] [PubMed]
  • Caughey GH, Raymond WW, Vanderslice P. Dog mast cell chymase: molecular cloning and characterization. Biochemistry. 1990 May 29;29(21):5166–5171. [PubMed]
  • Cheifetz S, Bassols A, Stanley K, Ohta M, Greenberger J, Massagué J. Heterodimeric transforming growth factor beta. Biological properties and interaction with three types of cell surface receptors. J Biol Chem. 1988 Aug 5;263(22):10783–10789. [PubMed]
  • Chu ML, de Wet W, Bernard M, Ding JF, Morabito M, Myers J, Williams C, Ramirez F. Human pro alpha 1(I) collagen gene structure reveals evolutionary conservation of a pattern of introns and exons. Nature. 310(5975):337–340. [PubMed]
  • Dardik R, Lahav J. Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics. Biochemistry. 1991 Sep 24;30(38):9378–9386. [PubMed]
  • Davis CG. The many faces of epidermal growth factor repeats. New Biol. 1990 May;2(5):410–419. [PubMed]
  • Dixit VM, Grant GA, Santoro SA, Frazier WA. Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin. J Biol Chem. 1984 Aug 25;259(16):10100–10105. [PubMed]
  • Eiján AM, Davel L, Oisgold-Dagá S, de Lustig ES. Modulation of tumor-induced angiogenesis by proteins of extracellular matrix. Mol Biother. 1991 Mar;3(1):38–40. [PubMed]
  • Fajardo LF, Kowalski J, Kwan HH, Prionas SD, Allison AC. The disc angiogenesis system. Lab Invest. 1988 Jun;58(6):718–724. [PubMed]
  • Fisher LW, Robey PG, Tuross N, Otsuka AS, Tepen DA, Esch FS, Shimasaki S, Termine JD. The Mr 24,000 phosphoprotein from developing bone is the NH2-terminal propeptide of the alpha 1 chain of type I collagen. J Biol Chem. 1987 Oct 5;262(28):13457–13463. [PubMed]
  • Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992 Jun 5;267(16):10931–10934. [PubMed]
  • Frazier WA. Thrombospondin: a modular adhesive glycoprotein of platelets and nucleated cells. J Cell Biol. 1987 Aug;105(2):625–632. [PMC free article] [PubMed]
  • Frazier WA. Thrombospondins. Curr Opin Cell Biol. 1991 Oct;3(5):792–799. [PubMed]
  • Galvin NJ, Vance PM, Dixit VM, Fink B, Frazier WA. Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy. J Cell Biol. 1987 May;104(5):1413–1422. [PMC free article] [PubMed]
  • Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, Bouck NP. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6624–6628. [PMC free article] [PubMed]
  • Goundis D, Reid KB. Properdin, the terminal complement components, thrombospondin and the circumsporozoite protein of malaria parasites contain similar sequence motifs. Nature. 1988 Sep 1;335(6185):82–85. [PubMed]
  • Grant DS, Tashiro K, Segui-Real B, Yamada Y, Martin GR, Kleinman HK. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell. 1989 Sep 8;58(5):933–943. [PubMed]
  • Grant DS, Kinsella JL, Fridman R, Auerbach R, Piasecki BA, Yamada Y, Zain M, Kleinman HK. Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo. J Cell Physiol. 1992 Dec;153(3):614–625. [PubMed]
  • Guo NH, Krutzsch HC, Nègre E, Vogel T, Blake DA, Roberts DD. Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3040–3044. [PMC free article] [PubMed]
  • Hennessy SW, Frazier BA, Kim DD, Deckwerth TL, Baumgartel DM, Rotwein P, Frazier WA. Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3' untranslated region. J Cell Biol. 1989 Feb;108(2):729–736. [PMC free article] [PubMed]
  • Hogg PJ, Stenflo J, Mosher DF. Thrombospondin is a slow tight-binding inhibitor of plasmin. Biochemistry. 1992 Jan 14;31(1):265–269. [PubMed]
  • Ingber D, Folkman J. Inhibition of angiogenesis through modulation of collagen metabolism. Lab Invest. 1988 Jul;59(1):44–51. [PubMed]
  • Iruela-Arispe ML, Bornstein P, Sage H. Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):5026–5030. [PMC free article] [PubMed]
  • Kakudo S, Kikuchi N, Kitadokoro K, Fujiwara T, Nakamura E, Okamoto H, Shin M, Tamaki M, Teraoka H, Tsuzuki H, et al. Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. J Biol Chem. 1992 Nov 25;267(33):23782–23788. [PubMed]
  • Klagsbrun M, D'Amore PA. Regulators of angiogenesis. Annu Rev Physiol. 1991;53:217–239. [PubMed]
  • Klar A, Baldassare M, Jessell TM. F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension. Cell. 1992 Apr 3;69(1):95–110. [PubMed]
  • Knowles DM, 2nd, Tolidjian B, Marboe C, D'Agati V, Grimes M, Chess L. Monoclonal anti-human monocyte antibodies OKM1 and OKM5 possess distinctive tissue distributions including differential reactivity with vascular endothelium. J Immunol. 1984 May;132(5):2170–2173. [PubMed]
  • Lahav J. Thrombospondin inhibits adhesion of endothelial cells. Exp Cell Res. 1988 Jul;177(1):199–204. [PubMed]
  • Laherty CD, O'Rourke K, Wolf FW, Katz R, Seldin MF, Dixit VM. Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development. J Biol Chem. 1992 Feb 15;267(5):3274–3281. [PubMed]
  • Lawler J. The structural and functional properties of thrombospondin. Blood. 1986 May;67(5):1197–1209. [PubMed]
  • Lawler J, Hynes RO. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. [PMC free article] [PubMed]
  • Lawler J, Connolly JE, Ferro P, Derick LH. Thrombin and chymotrypsin interactions with thrombospondin. Ann N Y Acad Sci. 1986;485:273–287. [PubMed]
  • Lawler J, Duquette M, Whittaker CA, Adams JC, McHenry K, DeSimone DW. Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family. J Cell Biol. 1993 Feb;120(4):1059–1067. [PMC free article] [PubMed]
  • Maione TE, Gray GS, Petro J, Hunt AJ, Donner AL, Bauer SI, Carson HF, Sharpe RJ. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 1990 Jan 5;247(4938):77–79. [PubMed]
  • Maragoudakis ME, Sarmonika M, Panoutsacopoulou M. Inhibition of basement membrane biosynthesis prevents angiogenesis. J Pharmacol Exp Ther. 1988 Feb;244(2):729–733. [PubMed]
  • Mosher DF. Physiology of thrombospondin. Annu Rev Med. 1990;41:85–97. [PubMed]
  • Müller G, Behrens J, Nussbaumer U, Böhlen P, Birchmeier W. Inhibitory action of transforming growth factor beta on endothelial cells. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5600–5604. [PMC free article] [PubMed]
  • Murphy-Ullrich JE, Hök M. Thrombospondin modulates focal adhesions in endothelial cells. J Cell Biol. 1989 Sep;109(3):1309–1319. [PMC free article] [PubMed]
  • Murphy-Ullrich JE, Mosher DF. Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation. J Cell Biol. 1987 Oct;105(4):1603–1611. [PMC free article] [PubMed]
  • Murphy-Ullrich JE, Lightner VA, Aukhil I, Yan YZ, Erickson HP, Hök M. Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin. J Cell Biol. 1991 Nov;115(4):1127–1136. [PMC free article] [PubMed]
  • Murphy-Ullrich JE, Schultz-Cherry S, Hök M. Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell. 1992 Feb;3(2):181–188. [PMC free article] [PubMed]
  • Myoken Y, Kan M, Sato GH, McKeehan WL, Sato JD. Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites. Exp Cell Res. 1990 Dec;191(2):299–304. [PubMed]
  • O'Shea KS, Dixit VM. Unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo. J Cell Biol. 1988 Dec;107(6 Pt 2):2737–2748. [PMC free article] [PubMed]
  • Oldberg A, Antonsson P, Lindblom K, Heinegård D. COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins. J Biol Chem. 1992 Nov 5;267(31):22346–22350. [PubMed]
  • Paglia L, Wilczek J, de Leon LD, Martin GR, Hörlein D, Müller P. Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides. Biochemistry. 1979 Oct 30;18(22):5030–5034. [PubMed]
  • Pepper MS, Belin D, Montesano R, Orci L, Vassalli JD. Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J Cell Biol. 1990 Aug;111(2):743–755. [PMC free article] [PubMed]
  • Plouët J, Gospodarowicz D. Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells. J Cell Physiol. 1989 Nov;141(2):392–399. [PubMed]
  • Polverini PJ, Cotran RS, Sholley MM. Endothelial proliferation in the delayed hypersensitivity reaction: an autoradiographic study. J Immunol. 1977 Feb;118(2):529–532. [PubMed]
  • Polverini PJ, Bouck NP, Rastinejad F. Assay and purification of naturally occurring inhibitor of angiogenesis. Methods Enzymol. 1991;198:440–450. [PubMed]
  • Postlethwaite AE, Keski-Oja J, Moses HL, Kang AH. Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta. J Exp Med. 1987 Jan 1;165(1):251–256. [PMC free article] [PubMed]
  • Prater CA, Plotkin J, Jaye D, Frazier WA. The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J Cell Biol. 1991 Mar;112(5):1031–1040. [PMC free article] [PubMed]
  • Rastinejad F, Polverini PJ, Bouck NP. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell. 1989 Feb 10;56(3):345–355. [PubMed]
  • Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S. Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell. 1991 Nov 15;67(4):687–699. [PubMed]
  • Rich KA, George FW, 4th, Law JL, Martin WJ. Cell-adhesive motif in region II of malarial circumsporozoite protein. Science. 1990 Sep 28;249(4976):1574–1577. [PubMed]
  • Roberts AB, Sporn MB. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta. Am Rev Respir Dis. 1989 Oct;140(4):1126–1128. [PubMed]
  • Robson KJ, Hall JR, Jennings MW, Harris TJ, Marsh K, Newbold CI, Tate VE, Weatherall DJ. A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature. 1988 Sep 1;335(6185):79–82. [PubMed]
  • Ryan MC, Sandell LJ. Differential expression of a cysteine-rich domain in the amino-terminal propeptide of type II (cartilage) procollagen by alternative splicing of mRNA. J Biol Chem. 1990 Jun 25;265(18):10334–10339. [PubMed]
  • Sage H, Bornstein P. Endothelial cells from umbilical vein and a hemangioendothelioma secrete basement membrane largely to the exclusion of interstitial procollagens. Arteriosclerosis. 1982 Jan-Feb;2(1):27–36. [PubMed]
  • Sakamoto N, Iwahana M, Tanaka NG, Osada Y. Inhibition of angiogenesis and tumor growth by a synthetic laminin peptide, CDPGYIGSR-NH2. Cancer Res. 1991 Feb 1;51(3):903–906. [PubMed]
  • Salvesen G, Enghild JJ. An unusual specificity in the activation of neutrophil serine proteinase zymogens. Biochemistry. 1990 Jun 5;29(22):5304–5308. [PubMed]
  • Silverstein RL, Leung LL, Nachman RL. Thrombospondin: a versatile multifunctional glycoprotein. Arteriosclerosis. 1986 May-Jun;6(3):245–253. [PubMed]
  • Smith KF, Nolan KF, Reid KB, Perkins SJ. Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat. Biochemistry. 1991 Aug 13;30(32):8000–8008. [PubMed]
  • Smith KF, Harrison RA, Perkins SJ. Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering. Biochemistry. 1992 Jan 28;31(3):754–764. [PubMed]
  • Sottile J, Selegue J, Mosher DF. Synthesis of truncated amino-terminal trimers of thrombospondin. Biochemistry. 1991 Jul 2;30(26):6556–6562. [PubMed]
  • Sun X, Skorstengaard K, Mosher DF. Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin. J Cell Biol. 1992 Aug;118(3):693–701. [PMC free article] [PubMed]
  • Swerlick RA, Lee KH, Wick TM, Lawley TJ. Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J Immunol. 1992 Jan 1;148(1):78–83. [PubMed]
  • Taraboletti G, Roberts D, Liotta LA, Giavazzi R. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J Cell Biol. 1990 Aug;111(2):765–772. [PMC free article] [PubMed]
  • Taraboletti G, Belotti D, Giavazzi R. Thrombospondin modulates basic fibroblast growth factor activities on endothelial cells. EXS. 1992;61:210–213. [PubMed]
  • Tuszynski GP, Rothman VL, Deutch AH, Hamilton BK, Eyal J. Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins. J Cell Biol. 1992 Jan;116(1):209–217. [PMC free article] [PubMed]
  • Tuszynski GP, Rothman VL, Papale M, Hamilton BK, Eyal J. Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain. J Cell Biol. 1993 Jan;120(2):513–521. [PMC free article] [PubMed]
  • Vuorio E, de Crombrugghe B. The family of collagen genes. Annu Rev Biochem. 1990;59:837–872. [PubMed]
  • Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5788–5792. [PMC free article] [PubMed]
  • Wiestner M, Krieg T, Hörlein D, Glanville RW, Fietzek P, Müller PK. Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures. J Biol Chem. 1979 Aug 10;254(15):7016–7023. [PubMed]
  • Woltering EA, Barrie R, O'Dorisio TM, Arce D, Ure T, Cramer A, Holmes D, Robertson J, Fassler J. Somatostatin analogues inhibit angiogenesis in the chick chorioallantoic membrane. J Surg Res. 1991 Mar;50(3):245–251. [PubMed]
  • Wu CH, Donovan CB, Wu GY. Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides. J Biol Chem. 1986 Aug 15;261(23):10482–10484. [PubMed]
  • Yang EY, Moses HL. Transforming growth factor beta 1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane. J Cell Biol. 1990 Aug;111(2):731–741. [PMC free article] [PubMed]
  • Zetter BR. Assay of capillary endothelial cell migration. Methods Enzymol. 1987;147:135–144. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • Compound
    Compound
    PubChem Compound links
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...