Trophic Effects of Platelets on Cultured Endothelial Cells are Mediated by Platelet-associated Fibroblast Growth Factor-2 (FGF-2) and Vascular Endothelial Growth Factor (VEGF)
Giuseppe Pintucci
1
The Seymour Cohn Cardiovascular Surgical Research Laboratory, Division of Cardiothoracic Surgery
,
Scott Froum
2
Division of Hematology, Cornell University Medical College, New York, USA
,
Jared Pinnell
2
Division of Hematology, Cornell University Medical College, New York, USA
,
Paolo Mignatti
1
The Seymour Cohn Cardiovascular Surgical Research Laboratory, Division of Cardiothoracic Surgery
3
Surgery and Cell Biology, New York University Medical Center, New York
,
Shahin Rafii
4
Division of Hematology and Oncology, Cornell University Medical College, New York, USA
,
David Green
2
Division of Hematology, Cornell University Medical College, New York, USA
› Author AffiliationsSupported by National Institutes of Health grants KO8CA77132-05 (D.G.) and SBIR CA80476-01 (P.M.), Department of the Army grant DAMD 17-99-1-9324 (P.M.) and the Seymour Cohn Foundation for Cardiovascular Surgery Research (G.P. and P.M.)
In addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two plateletassociated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releasates of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium.
Keywords
Platelets -
FGF -
VEGF -
MAPK
References
1
Spaet TH.
The platelet in hemostasis. Ann NY Acad Sci 1964; 115: 31-42.
3
Gimbrone Jr MA,
Aster RH,
Cotran RS,
Corkery J,
Jandl JH,
Folkman J.
Preservation of vascular integrity in organs perfused in vitro with a plateletrich medium. Nature 1969; 222: 33-6.
5
Kitchens CS,
Pendergast JF.
Human thrombocytopenia is associated with structural abnormalities of the endothelium that are ameliorated by glucocorticosteroid administration. Blood 1986; 67: 203-6.
7
D’Amore PA,
Shepro D.
Stimulation of growth and calcium influx in cultured, bovine, aortic endothelial cells by platelets and vasoactive substances. J Cell Phys 1977; 92: 177-83.
10
Pipili-Synetos E,
Papadimitriou E,
Maragoudakis ME.
Evidence that platelets promote tube formation by endothelial cells on matrigel. Br J Phar 1998; 125: 1252-7.
13
Gasic GJ,
Gasic TB,
Galanti N,
Johnson T,
Murphy S.
Platelet-tumor-cell interactions in mice. The role of platelets in the spread of malignant disease. Int J Cancer 1973; 11: 704-18.
14
Pearlstein E,
Ambrogio C,
Karpatkin S.
Effect of antiplatelet antibody on the development of pulmonary metastases following injection of CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 amelanotic melanoma tumor cells into mice. Cancer Res 1984; 44: 3884-7.
16
Frenette PS,
Johnson RC,
Hynes RO,
Wagner DD.
Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA 1995; 92: 7450-4.
19
Brunner G,
Nguyen H,
Gabrilove J,
Rifkin DB,
Wilson EL.
Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells. Blood 1993; 81: 631-8.
20
Martyre MC,
Le Bousse-Kerdiles,
Romquin N,
Chevillard S,
Praloran V,
Demory JL,
Dupriez B.
Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis. Br J Haematol 1997; 97: 441-8.
21
Möhle R,
Green D,
Moore MAS,
Nachman RL,
Rafii S.
Constitutive production and thrombin-stimulated release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997; 94: 663-8.
24
Sthoeger D,
Nardi M,
Travis S,
Karpatkin M,
Karpatkin S.
Micromethod for demonstrating increased platelet surface immunoglobulin G: findings in acute, chronic, and human immunodeficiency virus-1-related immunologic thrombocytopenias. Am J Hematol 1990; 34: 275-82.
26
Gimbrone Jr GA,
Buchanan MR.
Interactions of platelets and leukocytes with vascular endothelium: In vitro studies. Ann NY Acad Sci 1982; 401: 171-83.
27
Wartiovaara U,
Salven P,
Mikkola H,
Lassila R,
Kaukonen M,
Joensuu H,
Alitalo K,
Palotie A.
Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost 1998; 80: 171-5.
29
Zubilewicz A,
Hecquet C,
Jeanny JC,
Soubrane G,
Courtois Y,
Mascarelli F.
Two distinct signaling pathways are involved in fibroblast growth factor-2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF. Oncogene 2001; 20: 1403-13.
30
Pepper MS,
Ferrara N,
Orci L,
Montesano R.
Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro
. Biochem Biophys Res Commun 1992; 189: 824-31.
31
Goto F,
Goto K,
Weindel K,
Folkman J.
Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Invest 1993; 69: 508-17.
34
Papapetropoulos A,
Garcia-Cardena G,
Dengler TJ,
Maisopierre PC,
Yancopoulos G,
Sessa WC.
Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest 1999; 79: 213-23.
36
Florkiewicz RZ,
Sommer A.
Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc Natl Acad Sci USA 1989; 86: 3978-81.
37
Mignatti P,
Morimoto T,
Rifkin DB.
Basic fibroblast growth factor released by single, isolated cells stimulates their migration in an autocrine manner. Proc Natl Acad Sci USA 1991; 88: 11007-11.
38
Bikfalvi A,
Klein S,
Pintucci G,
Quarto N,
Mignatti P,
Rifkin DB.
Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight form. J Cell Biol 1995; 129: 233-43.
39
Moscatelli D,
Joseph-Silverstein J,
Manejias R,
Rifkin DB.
Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Natl Acad Sci USA 1987; 84: 5778-82.
