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WO1992021363A1 - Inhibition de proliferation de cellules musculaires lisses par la vitronectine - Google Patents

Inhibition de proliferation de cellules musculaires lisses par la vitronectine Download PDF

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Publication number
WO1992021363A1
WO1992021363A1 PCT/GB1992/000958 GB9200958W WO9221363A1 WO 1992021363 A1 WO1992021363 A1 WO 1992021363A1 GB 9200958 W GB9200958 W GB 9200958W WO 9221363 A1 WO9221363 A1 WO 9221363A1
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WO
WIPO (PCT)
Prior art keywords
vitronectin
smooth muscle
smc
pdgf
muscle cell
Prior art date
Application number
PCT/GB1992/000958
Other languages
English (en)
Inventor
Errol Wijelath
Catherine Demoliou-Mason
Sibylle Hess
Klaus Theodore Preissner
Vijay Vir Kakkar
Original Assignee
Thrombosis Research Institute
Max Planck Gesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thrombosis Research Institute, Max Planck Gesellschaft filed Critical Thrombosis Research Institute
Publication of WO1992021363A1 publication Critical patent/WO1992021363A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • This invention relates to the inhibition of smooth muscle cell proliferation and particularly to substances and compositions for use therein.
  • SMC smooth muscle cells
  • vascular smooth muscle cells have prompted research towards the identification of negative modulators that may regulate normal SMC growth in vivo.
  • Vascular SMC in culture are highly responsive to the chemotactic and mitogenic properties of platelet-derived growth factor (PDGF) and it is believed that the paracrine and autocrine activity of this factor may play a major role in SMC migration from the media into the intima and in intimal SMC proliferation in atheroma development [3,4].
  • PDGF platelet-derived growth factor
  • Recent studies [8] have shown that in atherosclerotic lesions there occurs deposition of a particular adhesion protein, namely vitronectin. Vitronectin is one example of a so-called adhesion protein.
  • Vitronectin is involved particularly in focal adhesion and cell spreading, processes which are essential for wound healing and cellular movement [5,6,7].
  • Vitronectin shares the unique recognition sequence Arg-Gly-Asp (RGD) with the group of substrate adhesion proteins such as fibrinogen, fibronectin, van Willebrand factor and laminin.
  • RGD Arg-Gly-Asp
  • a common characteristic in binding of these proteins to cell surfaces is via the integrins [2], a family of heterodimeric membrane receptors which enable cells to anchor, migrate and make contact with other cells or extracellular matrix components, processes which are important for cell growth and tissue integrity.
  • vitronectin acts as a growth inhibitory modulator of vascular smooth muscle cell proliferation.
  • vitronectin The binding and effect of vitronectin on the response of SMC to PDGF and other mitogens have been characterised.
  • the antagonistic effects of vitronectin on cell proliferation induced by a variety of known growth factors notably that contained in 2% Fetal Calf Serum (FCS) and also PDGF and insulin ⁇ like growth factor (IGF-I), have been identified.
  • FCS Fetal Calf Serum
  • IGF-I insulin ⁇ like growth factor
  • the present invention provides a method of inhibiting smooth muscle cell proliferation, especially vascular smooth muscle cell proliferation, comprising administration of an effective amount of vitronectin.
  • the invention provides vitronectin for use as an antiproliferative agent.
  • the invention also provides pharmaceutical compositions comprising vitronectin together with a pharmaceutically acceptable diluent and/or carrier.
  • vitronectin may exist in either a folded ("native" - as present circulating in plasma) or an unfolded ("extended") conformation.
  • the "extended” form is especially preferred, because of its superior antiproliferative activity.
  • vitronectin As an antiproliferative agent in accordance with the invention, therefore, it will usually be the extended form which is supplied.
  • the extended form of vitronectin is preferably multimeric, e.g. greater than the dimer and more preferably greater than the tetramer. This feature is preferred because of the superior antiproliferative activity of the higher molecular weight species.
  • vitronectin in its native form is naturally present in the body, it is within the scope of the present invention to generate the extended form of vitronectin in situ by the action of a suitable agent which affects the equilibrium of the multimeric vitronectin species, for example proteoglycans, glycosaminoglycans and similarly acting agents and/or agents which are generated upon activation of the coagulation/fibrinolysis cascade.
  • a suitable agent which affects the equilibrium of the multimeric vitronectin species, for example proteoglycans, glycosaminoglycans and similarly acting agents and/or agents which are generated upon activation of the coagulation/fibrinolysis cascade.
  • a method of inhibiting smooth muscle cell proliferation comprising generating the extended form of vitronectin in situ by administration of an agent which converts native vitronectin to its extended form.
  • the known vitronectin structure may vary by substitution, deletion or addition at one or more amino acid residues, so long as it retains essentially the same biological activity.
  • Vitronectin for use in accordance with the invention may be provided in any suitable form appropriate to the protocol of administration and/or the needs of a patient.
  • compositions comprising vitronectin together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Suitable carriers/diluents are well known in the art and include saline or other sterile aqueous media, optionally including additional components such as buffer salts and preservatives.
  • vitronectin for administration may be provided in lyophilised or freeze dried solid forms.
  • Administration may be via any suitable protocol.
  • administration is by intravenous injection or infusion, and may be systemic or topical.
  • vitronectin as an antiproliferative agent is in such an amount as to give the desired effective result of inhibiting SMC proliferation at the intended site.
  • a quantity which constitutes an "effective" amount may depend upon various parameters, such as body weight of the patient, degree of inhibition required, intended site of activity, all of which will be well understood and appreciated by persons skilled in the art.
  • an amount of vitronectin will be administered which gives a concentration in plasma of from about 1 to about 100 mg ml" - , more preferably from about 1 to about 10 mg ml" - .
  • the present invention provides vitronectin for use in kits and assays, as well as such kits and assays, for example for screening individuals for those with a high risk of atherosclerosis. This may be achieved for example by exploiting the inhibitory effect of vitronectin on PDGF- induced SMC proliferation and observing the antagonistic effect of blood components, e.g. fibrinogen, on the degree of cell proliferation inhibition by vitronectin. (Such blood components bind vitronectin, thereby preventing it from performing its inhibitory function. )
  • blood components e.g. fibrinogen
  • a further example of the use of the present invention as a diagnostic tool is in the in vitro testing for whether a patient will react to antiproliferative treatment with vitronectin.
  • Figure la illustrates the inhibitory effect of vitronectin on vascular SMC proliferation induced by various growth factors
  • Figure lb illustrates the anti-mitogenic effect of vitronectin on PDGF-stimulated SMC when pre-added and post-added at various times;
  • Figure lc illustrates the differences in SMC antiproliferative activity of the native and extended forms of vitronectin
  • Figure 2a shows the dose-response profile of vitronectin on PDGF-induced DNA synthesis
  • Figure 2b shows the electrophoresis profiles of native and extended vitronectin in the presence or absence of SDS
  • Figure 2c is a plot of total binding of radioisotope-labelled vitronectin (native and extended forms) to SMC monolayers;
  • Figure 2d illustrates the inhibition of binding of radioisotope-labelled vitronectin (extended form) to SMC in suspension by various peptides
  • Figure 3a illustrates vitronectin inhibition of PDGF-induced DNA synthesis in human vascular SMC
  • Figure 3b shows a Northern blot illustrating the absence of vitronectin inhibition of PDGF-induced mRNA expression of the c-fos proto-oncogene.
  • Vascular SMC were isolated from sections of aortic or vein tissue removed during surgery and transferred into DMEM supplemented with 100 IU ml" * ⁇ penicillin, lOO ⁇ g ml" 1 streptomycin sulphate, 100 ⁇ g ml" l neomycin and 2.5 ⁇ g ml" • ⁇ fungizone (Gibco). Dissected medial tissue was finely minced, rinsed and digested under constant agitation (18-20 h, 37 ⁇ C) in DMEM 15 mM Hepes (Flow), 10% FCS with 0.1% collagenase Type II (Worthington) and 0.05% elastase Type I (Sigma).
  • Figure 1(b) illustrates SMC stimulation with 10 ng ml" 1 PDGF-BB in serum-free medium in the presence of 0.1 uM vitronectin (VN) added at the indicated times before (pre-addition) or after (post-addition) PDGF.
  • VN vitronectin
  • vitronectin had no effect on mitogen- induction of DNA synthesis. This indicates that the response to vitronectin may depend on the differential expression of vitronectin receptor(s) and/or of other cellular components) with cell growth.
  • vitronectin Although the concentration of native (i.e. folded) vitronectin in serum is high, serum-vitronectin is not anti-mitogenic suggesting that it is in an inactive state or associated with other serum components that may neutralize its anti-mitogenic activity. Only the extended (i.e. unfolded) form of vitronectin which is considered to be similar to that generated upon activation of the coagulation/fibrinolysis cascade or to that associated with extracellular matrix (ECM) components [9,10], appeared to have the preferential conformational state required for the transmission of this inhibitory effect. Purified human "native" vitronectin which is believed to be in the folded conformation [11] and constitutes the major form present in plasma [1], was considerably less active.
  • ECM extracellular matrix
  • PDGF-BB (10 ng ml" - ) 1200 ⁇ 100
  • the minimum concentration of cRGD that could be used without causing the cells to detach within the time course required to measure DNA synthesis was 1.0 ⁇ M. It could not be evaluated, therefore, whether the cRGD peptide is exerting its antagonistic effect by acting as a competitive inhibitor as in the case of cell adhesion to vitronectin [13].
  • the growth inhibitory effect of vitronectin may be transmitted via secondary interactions through other domains in addition to the RGD site, the kinetics of which may differ.
  • vitronectin Since none of the other adhesive proteins tested, including fibronectin, collagen or fibrinogen were able to inhibit PDGF-induced DNA synthesis in confluent, quiescent SMC, it would suggest that the anti-mitogenic activity of vitronectin is transmitted via receptor(s) that recognize, in addition to the RGD site, specific regions of the vitronectin molecule such as the heparin binding domain.
  • Figure 2(a) illustrates the dose-dependent inhibition of PDGF-BB-induced DNA synthesis in SMC by vitronectin.
  • SMC were stimulated with 10 ng ml" - PDGF-BB in the presence of the indicated concentrations of vitronectin.
  • PDGF-induced [ 3 H]thymidine uptake in the absence of vitronectin was 1000 +_ 110 dpm.
  • Vitronectin alone had no effect on basal DNA synthesis.
  • the dose-response profile of vitronectin (0.1 nM- 0.1 ⁇ M) was biphasic, as shown in Figure 2(a).
  • This biphasic response may reflect concentration-dependent differences in the anti-mitogenic or adhesive properties of the conformational states of oligomeric species formed as a result of exposure of epitopes in the extended form of vitronectin in agrement with the conformational lability/flexibility of this molecule [15,16].
  • Figure 2(c) illustrates the time-dependent differential association of extended (•) and native (o) [ 125 I]vitronectin 3.5-6.0 nM with SMC monolayers at 37 ⁇ C. While native [ 125 1]vitronectin did not demonstrate appreciable binding, extended (multimeric) [ 125 i]vitronectin binding to cell surface receptors was considerably higher and appeared to reach steady state within 2 h. Since approximately 50% of extended [ i25 1]vitronectin binding to SMC monolayer was due to binding to ECM, the effects of cRGD and heparin on vitronectin binding to cell surface were assessed with cells in suspension.
  • Figure 2(d) shows the inhibition of [ 125 I]vitronectin (extended form) (6.0 nM) binding to SMC in suspension at 37 ⁇ C in the absence (A) or presence (B) of 100-fold molar excess unlabelled vitronectin; 1 ⁇ m cRGD (C) or 10 ⁇ g ml" *• unfractionated heparin (Sigma) (D).
  • FIG. 2(d) shows, one hundred-fold excess of unlabelled vitronectin suppressed [ 12 I]vitronectin binding to SMC surface by more than 60%, indicating the specificity of the vitronectin-SMC interactions. Furthermore, [ 125 I]vitronectin binding was suppressed by 34% using the cRGD peptide (1 ⁇ M) or by 50% using unfractionated heparin (10 ⁇ g ml" * - ), suggesting the involvement of both the RGD and heparin binding domains in the interactions of vitronectin with SMC cell surface receptors.
  • the inhibition of [- 2 - I]vitronectin binding by heparin may be due to heparin binding and changes in the vitronectin conformation and also the monomer-oligomer equilibrium which may effect the concentration of active vitronectin species.
  • Our preliminary studies have shown that the two molecules may neutralize each other's anti-mitogenic activity.
  • vitronectin and heparin may be of importance in the regulation of cell growth, especially since heparin has been shown to inhibit mitogen and, in particular, PDGF-induced SMC proliferation [19,20] .
  • vitronectin inhibits dose-dependent PDGF-induction of DNA synthesis in human vascular smooth muscle cells without affecting c-fos mRNA expression.
  • Figure 3(b) shows a Northern blot of equal amounts (20 ⁇ g) of total cellular RNA hybridized with a 2 kb c-fos probe or 0.53 kb G6PD probe.
  • Vitronectin was able to inhibit DNA synthesis over a range of • concentrations of PDGF-BB (0.1-100 ng ml" x ), and at all concentrations the extent of inhibition did not vary (Fig. 3a). This effect was not due to binding and depletion of PDGF since vitronectin did not inhibit the induction of mRNA expression of the c-fos proto-oncogene by PDGF (Fig. 3b). These data also indicate that vitronectin may inhibit growth by affecting mechanisms other than those involved in the expression of early biochemical events induced by growth factors.
  • Vitronectin may prevent growth factor- induced medial-SMC proliferation either directly via integrin/proteoglycan receptor activation or indirectly by preventing access of growth factors and chemo- attractants to SMC as well as cell migration via inhibition of ECM degradation by proteases.
  • Generation of the extended form of circulating vitronectin at sites of vascular injury upon activation of the coagulation/fibrinolysis cascade may be of physiological significance to limit the early proliferative stages of atheroma development or in intimal hyperplasia, since both of these events involve vascular SMC migration and proliferation.

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  • Health & Medical Sciences (AREA)
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Abstract

La vitronectine, une protéine d'adhésion, agit comme un modulateur inhibiteur de croissance par rapport à la prolifération de cellules musculaires lisses, un phénomène dont on sait qu'il provoque l'athérosclérose. L'invention se rapporte à un procédé d'inhibition de la prolifération de cellules musculaires lisses, consistant à administrer de la vitronectine, ainsi qu'à la vitronectine utilisée à de telles fins et des compositions pharmaceutiques contenant cette protéine. De préférence, la vitronectine est sous sa forme allongée.
PCT/GB1992/000958 1991-05-28 1992-05-27 Inhibition de proliferation de cellules musculaires lisses par la vitronectine WO1992021363A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9111439.7 1991-05-28
GB919111439A GB9111439D0 (en) 1991-05-28 1991-05-28 Inhibition of vascular smooth muscle cell proliferation

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WO1992021363A1 true WO1992021363A1 (fr) 1992-12-10

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EP (1) EP0586451A1 (fr)
AU (1) AU1763792A (fr)
GB (2) GB9111439D0 (fr)
IE (1) IE921697A1 (fr)
WO (1) WO1992021363A1 (fr)
ZA (1) ZA923901B (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5545569A (en) * 1993-05-13 1996-08-13 Neorx Corporation Prevention and treatment of pathologies associated with abnormally proliferative smooth muscle cells
US6171609B1 (en) 1995-02-15 2001-01-09 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US6268390B1 (en) * 1991-09-27 2001-07-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
US7445628B2 (en) 1995-06-07 2008-11-04 Cook Incorporated Method of treating a patient with a coated implantable medical device
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006014190A1 (de) 2006-03-24 2007-09-27 Henkel Kgaa Hochfeste schlagschälfeste Klebstoffe

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0292663A1 (fr) * 1987-05-25 1988-11-30 Research Development Corporation Of Japan Méthode de production de vitronectine
EP0341006A2 (fr) * 1988-05-03 1989-11-08 The Board Of Trustees Of The Leland Stanford Junior University Inhibiteur de la croissance des cellules des muscles lisses
EP0410006A1 (fr) * 1989-02-13 1991-01-30 Nisshin Flour Milling Co., Ltd. Collyre de traitement de blessures de l'epithelium de la cornee

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0292663A1 (fr) * 1987-05-25 1988-11-30 Research Development Corporation Of Japan Méthode de production de vitronectine
EP0341006A2 (fr) * 1988-05-03 1989-11-08 The Board Of Trustees Of The Leland Stanford Junior University Inhibiteur de la croissance des cellules des muscles lisses
EP0410006A1 (fr) * 1989-02-13 1991-01-30 Nisshin Flour Milling Co., Ltd. Collyre de traitement de blessures de l'epithelium de la cornee

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Journal of cellular Physiology, vol. 124, no. 1, July 1985, J.J. CASTELLOT et al.: "Effect of heparin on vascular smooth muscle cells. I. Cell metabolism", pages 21-28, see page 21, left-hand column, paragraph 2 - right-hand column, paragraph 2; page 23, right-hand column, paragraph 2 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6268390B1 (en) * 1991-09-27 2001-07-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US5545569A (en) * 1993-05-13 1996-08-13 Neorx Corporation Prevention and treatment of pathologies associated with abnormally proliferative smooth muscle cells
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
US6171609B1 (en) 1995-02-15 2001-01-09 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US7445628B2 (en) 1995-06-07 2008-11-04 Cook Incorporated Method of treating a patient with a coated implantable medical device
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method

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Publication number Publication date
GB9120748D0 (en) 1991-11-13
AU1763792A (en) 1993-01-08
GB9111439D0 (en) 1991-07-17
EP0586451A1 (fr) 1994-03-16
ZA923901B (en) 1993-03-08
IE921697A1 (en) 1992-12-02

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