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Regulation of Angiogenesis

D. Cheresh, R. Klemke, C.A. Andrews, L.M. Collins, B. Eliceiri, A. Le, J. Leng, R. Molander, A.C. Reddy, M. Rosenfeld, S. Silletti, K. Spencer, C.M.A. Storgard, M. Storgard, D. Stupack, T.L. von Schalscha, L.M. Yeh, W. Laug*

* University of Southern California, Los Angeles, CA

CLINICAL TRIALS IN LATE-STAGE CANCER PATIENTS

Previous studies from our laboratory implicated integrins _vß₃ and _vß₅ in angiogenesis, the growth of new blood vessels. We found that antagonists of these integrins disrupt angiogenesis by causing unscheduled apoptosis in angiogenic blood vessels and that use of the antagonists can cause regression of preestablished tumors in animals. These studies led to the clinical testing of both antibody and peptide antagonists of these integrins. Specifically, a phase 1 clinical trial of Vitaxin, the humanized version of a mouse monoclonal antibody to _vß₃, in patients with late-stage cancer has been completed. The trial was conducted by J. Gutheil at the Sidney Kimmel Cancer Center, San Diego.

By definition, a phase 1 clinical trial is designed to simply examine the toxicity of a given drug. Beginning in February 1997, Vitaxin was given once a week for 6 weeks to 15 patients with late-stage cancer in a dose-escalation study. No side effects or toxic reactions were observed. However, surprisingly, 9 of the patients had either disease stabilization or clinical benefit. One patient who responded early to the treatment was allowed to continue treatment and has been taking the drug for more than 18 months. This patient has experienced steady shrinkage of tumor and no side effects. Although these results are preliminary and anecdotal, a phase 2 trial will begin soon to actually measure the potential clinical benefits of this drug.

A second phase 1 clinical trial has now been started in Europe. This trial involves the administration of a chemically modified cyclic peptide antagonist of integrins _vß₃ and _vß₅. This peptide is also being given to patients with late-stage cancer in a dose-escalation study to determine a possible maximum tolerable dose. This trial should be completed by the end of 1998.

MOLECULAR BASIS OF ANGIOGENESIS

Our goals in current studies are to gain a molecular understanding of angiogenesis and to determine the mechanism by which antagonists of integrins _vß₃ and _vß₅ disrupt this growth of new vessels. It is clear that angiogenesis depends on growth factors such as basic fibroblast growth factor and vascular endothelial cell growth factor and the ligation of integrin _vß₃ or _vß₅. These cell-surface receptors cooperate to promote intracellular signaling that potentiates survival, migration, proliferation, and differentiation of endothelial cells during the formation of new blood vessels.

We have taken a gene-delivery approach to determine some of the key signaling molecules regulated by the ligation of growth factor receptors and integrins that ultimately lead to angiogenesis. For example, retroviral delivery of a kinase inactive mutant of Src selectively disrupts angiogenesis induced by vascular endothelial cell growth factor on chick chorioallantoic membranes. In contrast, an active form of Src actually promotes angiogenesis on such membranes, which can be blocked by a selective antagonist of _vß₅ but not by one of _vß₃. Therefore, Src appears to play a critical role in a pathway of angiogenesis that depends on vascular endothelial cell growth factor and _vß₅

A second genetic approach for regulating angiogenesis is based on use of adenoviral vectors containing activated or inactivated forms of the small GTPase Ras. Gene delivery of a mutationally active form of Ras promotes growth of blood vessels on chick chorioallantoic membranes, and this growth can be blocked by the mouse monoclonal antibody to _vß₃. In addition, angiogenesis induced with either basic fibroblast growth factor or vascular endothelial cell growth factor can be disrupted by adenoviral delivery of a mutationally inactive form of Ras. These findings indicate that the Ras/MAP kinase pathway plays a key role in angiogenesis and suggest that gene-delivery strategies may be an effective means of regulating angiogenesis in vivo.

MULTIPLE ROLES FOR THROMBIN IN ANGIOGENESIS

The coagulation cascade is often dysregulated in cancer patients, but its role in the progression of cancer is not clear. In fact, tumors are often surrounded by fibrin, which is a ligand for _vß₃. We have shown that fibrin, which is generated from fibrinogen by the coagulation protease thrombin, promotes the survival of endothelial cells in vitro in a 3-dimensional extracellular matrix and that this survival depends on the expression of _vß₃. The fact that survival of endothelial cells is critical for angiogenesis in vivo prompted us to examine whether fibrin at tumor sites provides a survival signal for invading angiogenic endothelial cells.

We found that thrombin, which promotes formation of fibrin, also directly potentiates angiogenesis when added to the surface of chick chorioallantoic membranes. This effect can be duplicated with a peptide agonist of the G protein--coupled thrombin receptor. Together, these findings suggest that thrombin has at least 2 functions in tumor-induced angiogenesis: to generate a compatible extracellular matrix environment for invasion by blood vessels and to ultimately activate endothelial cells to form new blood vessels.

DISRUPTION OF ANGIOGENESIS BY PEX

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 and _vß₃ are functionally associated on the surface of angiogenic blood vessels. A fragment of the metalloproteinase that makes up the C-terminal hemopexin-like domain, termed PEX, prevents binding of the enzyme to _vß₃ and blocks cell-surface collagenolytic activity. PEX blocks activity of matrix metalloproteinase 2 on chick chorioallantoic membranes and disrupts angiogenesis and tumor growth. A naturally occurring form of PEX can be detected in vivo in conjunction with expression of _vß₃ in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell _vß₃ and acts as a natural inhibitor of the activity of the metalloproteinase, thereby regulating the invasive behavior of new blood vessels.

ROLE OF ANGIOGENESIS IN ARTHRITIS

Rheumatoid arthritis is an inflammatory disease associated with neovascularization. We showed that _vß₃ on synovial endothelial cells is an effector of angiogenesis in a rabbit model of rheumatoid arthritis and that a cyclic peptide antagonist of this integrin blocks synovial angiogenesis. This blocking resulted in reduced synovial cellular infiltration, decreased pannus formation, and prevention of cartilage erosion. These results substantiate neovascularization as a central factor in arthritic disease and implicate _vß₃ as a therapeutic target in rheumatoid arthritis.

REGULATION OF CELL MIGRATION

The interaction of cells with extracellular matrix proteins or cytokines and growth factors promotes signaling events that regulate cell migration. We showed that ligation of integrin or cytokine receptors activates 2 distinct signaling pathways necessary for cell movement. One pathway involves activation of Ras/MAP kinase. Activated MAP kinase influenced the motility machinery of cells by directly phosphorylating myosin light-chain kinase, a situation that led to increased phosphorylation of myosin light chains. The second pathway involves the molecular coupling of the adaptor proteins c-crk and CAS (crk-associated substrate protein). Expression of c-crk and CAS in cells promoted a crk-CAS association and activation of the small GTPase Rac. This pathway promotes migration by inducing actin organization and membrane ruffles at the leading edge of migratory cells. Moreover, activation of these signaling pathways is associated with induction of tumor cell invasion and pulmonary metastasis in vivo.

PUBLICATIONS

Boudreau, N., Andrews, C., Srebrow, A., Ravanpay, A., Cheresh, D.A. Regulation of the angiogenic phenotype by Hox D3. J. Cell Biol. 139:257, 1997.

Brooks, P.C., Montgomery, A.M.P., Cheresh, D.A. Use of the 10-day-old chick embryo model for studying angiogenesis. Methods Mol. Biol., in press.

Brooks, P.C., Silletti, S., von Schalscha, T.L., Friedlander, M., Cheresh, D.A. Disruption of angiogenesis by PEX, a non-catalytic metalloproteinase fragment with integrin binding activity. Cell 92:391, 1998.

Cheresh, D.A. Death to a blood vessel, death to a tumor. Nature Med. 4:408, 1998.

Eliceiri, B.P., Strömblad, S., Klemke, R., Cheresh, D.A. Integrin requirement for sustained MAP kinase activity during angiogenesis. J. Cell. Biol. 140:1255, 1998.

Felding-Habermann, B., Silletti, S., Mei, F., Siu, C., Yip, P., Brooks, P.C., Cheresh, D.A., Ginsberg, M.H., Montgomery, A.M.P. A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins. J. Cell Biol. 139:1567, 1997.

Klemke, R.L., Leng, J., Molander, R., Brooks, P.C., Vuori, K., Cheresh, D.A. CAS/crk coupling serves as a "molecular switch" for induction of cell migration. J. Cell. Biol. 140:961, 1998.

Li, E., Stupack, D., Klemke, R.C., Cheresh, D.A., Nemerow, G.R. Adenovirus endocytosis and cell motility require distinct signaling pathways mediated by _v integrins. J. Virol. 7:2055, 1998.

Nicolaou, K.C., Trujillo, J.I., Jandeleit, B., Chibale, K., Rosenfeld, M., Diefenbach, B., Cheresh, D.A., Goodman, S.L. Design, synthesis, and biological evaluation of nonpeptide integrin antagonists. J. Bioorg. Med. Chem., in press.

Radar, C., Cheresh, D.A., Barbas, C.F. A phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries. Proc. Natl. Acad. Sci., U.S.A. 95:8910, 1998.

Sipkins, D.A., Cheresh, D.A., Kazemi, M.R., Bednarski, M.D., Li, K.C.P. Detection of tumor angiogenesis in vivo by _vß₃-targeted magnetic resonance imaging. Nature Med. 4:623, 1998.

van der Zee, R., Murohara, T., Passeri, J., Kearney, M., Cheresh, D.A., Isner, J.M. Reduced intimal thickening following _vß₃ blockage is associated with smooth muscle cell apoptosis. Cell Adhes. Commun., in press.