Abstract
During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed] [Google Scholar]
- Bavisotto L. M., Schwartz S. M., Heimark R. L. Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors. J Cell Physiol. 1990 Apr;143(1):39–51. doi: 10.1002/jcp.1041430106. [DOI] [PubMed] [Google Scholar]
- Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
- Bouck N. Tumor angiogenesis: the role of oncogenes and tumor suppressor genes. Cancer Cells. 1990 Jun;2(6):179–185. [PubMed] [Google Scholar]
- Brown P. D., Levy A. T., Margulies I. M., Liotta L. A., Stetler-Stevenson W. G. Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res. 1990 Oct 1;50(19):6184–6191. [PubMed] [Google Scholar]
- Dell'Era P., Presta M., Ragnotti G. Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells. Exp Cell Res. 1991 Feb;192(2):505–510. doi: 10.1016/0014-4827(91)90070-b. [DOI] [PubMed] [Google Scholar]
- Fafeur V., Jiang Z. P., Böhlen P. Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells. J Cell Physiol. 1991 Nov;149(2):277–283. doi: 10.1002/jcp.1041490214. [DOI] [PubMed] [Google Scholar]
- Felder C. C., Ma A. L., Liotta L. A., Kohn E. C. The antiproliferative and antimetastatic compound L651582 inhibits muscarinic acetylcholine receptor-stimulated calcium influx and arachidonic acid release. J Pharmacol Exp Ther. 1991 Jun;257(3):967–971. [PubMed] [Google Scholar]
- Felder C. C., MacArthur L., Ma A. L., Gusovsky F., Kohn E. C. Tumor-suppressor function of muscarinic acetylcholine receptors is associated with activation of receptor-operated calcium influx. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1706–1710. doi: 10.1073/pnas.90.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Folkman J., Klagsbrun M. Angiogenic factors. Science. 1987 Jan 23;235(4787):442–447. doi: 10.1126/science.2432664. [DOI] [PubMed] [Google Scholar]
- Folkman J., Shing Y. Angiogenesis. J Biol Chem. 1992 Jun 5;267(16):10931–10934. [PubMed] [Google Scholar]
- Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol. 1992 Apr;3(2):65–71. [PubMed] [Google Scholar]
- Fotsis T., Pepper M., Adlercreutz H., Fleischmann G., Hase T., Montesano R., Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2690–2694. doi: 10.1073/pnas.90.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gospodarowicz D. Biological activities of fibroblast growth factors. Ann N Y Acad Sci. 1991;638:1–8. doi: 10.1111/j.1749-6632.1991.tb49012.x. [DOI] [PubMed] [Google Scholar]
- Gusovsky F., Lueders J. E., Kohn E. C., Felder C. C. Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-gamma. An alternative mechanism for cholinergic-induced phosphoinositide breakdown. J Biol Chem. 1993 Apr 15;268(11):7768–7772. [PubMed] [Google Scholar]
- Huang A. J., Manning J. E., Bandak T. M., Ratau M. C., Hanser K. R., Silverstein S. C. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J Cell Biol. 1993 Mar;120(6):1371–1380. doi: 10.1083/jcb.120.6.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jaye M., Schlessinger J., Dionne C. A. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992 Jun 10;1135(2):185–199. doi: 10.1016/0167-4889(92)90136-y. [DOI] [PubMed] [Google Scholar]
- Kohn E. C., Felder C. C., Jacobs W., Holmes K. A., Day A., Freer R., Liotta L. A. Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI. Cancer Res. 1994 Feb 15;54(4):935–942. [PubMed] [Google Scholar]
- Kohn E. C., Jacobs W., Kim Y. S., Alessandro R., Stetler-Stevenson W. G., Liotta L. A. Calcium influx modulates expression of matrix metalloproteinase-2 (72-kDa type IV collagenase, gelatinase A). J Biol Chem. 1994 Aug 26;269(34):21505–21511. [PubMed] [Google Scholar]
- Kohn E. C., Liotta L. A. L651582: a novel antiproliferative and antimetastasis agent. J Natl Cancer Inst. 1990 Jan 3;82(1):54–60. doi: 10.1093/jnci/82.1.54. [DOI] [PubMed] [Google Scholar]
- Kohn E. C., Liotta L. A., Schiffmann E. Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells. Biochem Biophys Res Commun. 1990 Jan 30;166(2):757–764. doi: 10.1016/0006-291x(90)90874-m. [DOI] [PubMed] [Google Scholar]
- Kohn E. C., Sandeen M. A., Liotta L. A. In vivo efficacy of a novel inhibitor of selected signal transduction pathways including calcium, arachidonate, and inositol phosphates. Cancer Res. 1992 Jun 1;52(11):3208–3212. [PubMed] [Google Scholar]
- Kubota Y., Kleinman H. K., Martin G. R., Lawley T. J. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol. 1988 Oct;107(4):1589–1598. doi: 10.1083/jcb.107.4.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
- Meldolesi J., Clementi E., Fasolato C., Zacchetti D., Pozzan T. Ca2+ influx following receptor activation. Trends Pharmacol Sci. 1991 Aug;12(8):289–292. doi: 10.1016/0165-6147(91)90577-f. [DOI] [PubMed] [Google Scholar]
- Minniti C. P., Kohn E. C., Grubb J. H., Sly W. S., Oh Y., Müller H. L., Rosenfeld R. G., Helman L. J. The insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor mediates IGF-II-induced motility in human rhabdomyosarcoma cells. J Biol Chem. 1992 May 5;267(13):9000–9004. [PubMed] [Google Scholar]
- Montesano R., Pepper M. S., Belin D., Vassalli J. D., Orci L. Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases. J Cell Physiol. 1988 Mar;134(3):460–466. doi: 10.1002/jcp.1041340318. [DOI] [PubMed] [Google Scholar]
- Moody T. W., Cuttitta F. Growth factor and peptide receptors in small cell lung cancer. Life Sci. 1993;52(14):1161–1173. doi: 10.1016/0024-3205(93)90098-n. [DOI] [PubMed] [Google Scholar]
- Presta M., Maier J. A., Ragnotti G. The mitogenic signaling pathway but not the plasminogen activator-inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells. J Cell Biol. 1989 Oct;109(4 Pt 1):1877–1884. doi: 10.1083/jcb.109.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rifkin D. B., Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol. 1989 Jul;109(1):1–6. doi: 10.1083/jcb.109.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Savarese D. M., Russell J. T., Fatatis A., Liotta L. A. Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility. J Biol Chem. 1992 Oct 25;267(30):21928–21935. [PubMed] [Google Scholar]
- Takigawa M., Nishida Y., Suzuki F., Kishi J., Yamashita K., Hayakawa T. Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2). Biochem Biophys Res Commun. 1990 Sep 28;171(3):1264–1271. doi: 10.1016/0006-291x(90)90822-5. [DOI] [PubMed] [Google Scholar]
- Taylor S., Folkman J. Protamine is an inhibitor of angiogenesis. Nature. 1982 May 27;297(5864):307–312. doi: 10.1038/297307a0. [DOI] [PubMed] [Google Scholar]
- Yamamoto N., Matsutani S., Yoshitake Y., Nishikawa K. Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells. Histochemistry. 1991;96(6):479–485. doi: 10.1007/BF00267073. [DOI] [PubMed] [Google Scholar]
- Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]