Anti-Angiogenics

We have demonstrated the existence of two cytokine-dependent angiogenic pathways defined by their dependency on distinct vascular cell integrins (Friedlander, et. al, 1995). Using in vivo ocular models we demonstrated that angiogenesis in vivo can be regulated by distinct, cytokine driven pathways and that specific integrin antagonists inhibit these pathways. Furthermore, we demonstrated, for the first time, direct therapeutic application of this mechanistic distinction by inhibiting ocular neovascularization stimulated by two cytokines known to be one of, if not the, "vasoformative" factor responsible for angiogenesis associated with retinal ischemia (the leading cause of blindness in Americans under the age of 65). Recently (Stromblad, et. al., 2002) we have studied the role of p53 in facilitating angiogenesis mediated by these distinct pathways and demonstrate that mice lacking p53 are refractory to this treatment, indicating that neovascularization in normal mice depends on alpha v integrin-mediated suppression of p53. Blockade of alpha v during neovascularization results in an induction of p21CIP1 in wild type and, surprisingly, in p53 null retinas, demonstrating that alpha v -integrin ligation regulates p21CIP1 levels in a p53-independent manner. Our findings demonstrate that p53 and alpha v-integrin act in concert in the control of retinal neovascularization.

We have demonstrated that human ocular neovascular tissue selectively expresses either alpha v beta 3 or alpha v beta 3 and alpha v beta 5 depending on whether the tissue is from retinal or choroidal neovascular diseases, respectively, and that cyclic-RGD peptide antagonists of both alpha v beta 3 and alpha v beta 5 inhibit retinal angiogenesis (Friedlander, et. al., 1996) After defining the presence of at least 2 integrin-mediated pathways of angiogenesis, we decided to evaluate human pathological tissue specimens obtained from patients with either retinal (e.g., diabetic retinopathy) or choroidal (e.g., macular degeneration) neovascularization. In this series of experiments we were able to extend the concept of two distinct integrin-mediated pathways of angiogenesis outlined in our Science paper to clinical relevance. Ocular tissues taken from patients with ischemic retinal neovascular disease (e.g., diabetes) were found to selectively express both alpha v beta 3 and alpha v beta 5 integrins while tissue from patients with degenerative sub-retinal neovascular disease (e.g., macular degeneration) preferentially up-regulated alpha v beta 3. These observations not only serve to reinforce the clinical concept that retinal and sub-retinal neovascularization represent distinct ocular pathologies, but also has profound implications for therapeutic approaches to treating such diseases. To test the therapeutic application of this concept, we have used peptide and non-peptidic integrin antagonists to inhibit naturally occurring retinal neovascularization in a neonatal mouse model.

We have demonstrated that a naturally occurring form of the carboxy-terminal, non-catalytic domain of matrix metalloproteinase-2 (PEX) can be detected in vivo in conjunction with expression of alpha v beta 3 during developmental retinal neovascularization (Brooks, et. al., 1998). We have also used the newborn mouse retinal vascular model to further explore the in vivo relevance of a previous in vitro observation. Our collaborators, David Cheresh and Peter Brooks, had observed that matrix metalloproteinase-2 (MMP-2) and the integrin alpha v beta 3 co-localized in angiogenic blood vessels of the chick chorioallantoic membrane (CAM). They also observed that a non-catalytic carboxy terminal domain of MMP-2 ("PEX") binds specifically to alpha v beta 3, inhibiting, in a dose-dependent fashion the binding of proteolytically-active MMP-2., resulting in the inhibition of angiogenesis in CAM and mouse tumor models. We decided to examine angiogenic corneas and newborn mouse retinas undergoing active retinal vasoproliferation for the presence of MMP-2 and PEX domain. Both tissues contained active forms of MMP-2 as determined by gelatinase assays. Furthermore, we observed, using western blot analysis, that the PEX domain was present in newborn mouse retinas, its highest levels correlating with the time at which active vessel migration decreases and vessel maturation occurs. When we quantified the levels of PEX present in the mouse retinas, we found that the levels were comparable to that used to inhibit tumor- or cytokine-stimulated angiogenesis in model systems. Thus, a proteolytic fragment of a naturally occurring enzyme is generated during physiological angiogenesis and may serve to auto regulate the angiogenic process itself.

We have shown that a recombinant form of a carboxyl-terminal fragment of TrpRS is a potent antagonist of VEGF-induced angiogenesis in a mouse model, and of naturally occurring retinal angiogenesis in the neonatal mouse. (Otani, et. al., 2002). Recent work suggests that human tyrosyl- and tryptophanyl-tRNA synthetases (TrpRS) link protein synthesis to signal transduction pathways including angiogenesis. We have been studying the anti-angiogenic activity of tryptophanyl-tRNA synthetase (TrpRS) fragments. In normal human cells TrpRS exists as a full length form and a truncated (mini-TrpRS) one in which an amino-terminal domain is deleted due to alternative splicing of the pre-mRNA. This latter form is preferentially synthesized in cells exposed to interferon-g. Further truncation of mini-TrpRS results in a 42 kD form (T2) that is the most potent of the angiostatic forms of TrpRS evaluated to date. We have used recombinant and cell-based delivery forms of T2 to inhibit angiogenesis in several in vitro and animal models of neovascularization. In each system, T2 was a very potent angiostatic in a dose-dependent fashion. These results suggest that fragments of TrpRS, as naturally occurring and potentially non-immunogenic anti-angiogenics, can be used for the treatment of neovascular eye diseases. We are currently pursuing studies to identify the receptor to which T2 TrpRS binds. We are also using gene therapy approaches (targeted nanoparticles, viral- and cell-based vectors) to deliver a secreted form of T2 TrpRS to treat animal, and ultimately human, neovascular retinal diseases.

Relevant Publications:

Scheppke, L., Aguilar, E., Gariano, R. F., Jacobson, R., Hood, J., Doukas, J., Cao, J., Noronha, G., Yee, S., Weis, S., Martin, M., B., Soll, R., Cheresh, D. A., and Friedlander, M. (2008). Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits. Journal of Clinical Investigation. 118(6): 2337-46. PMCID: PMC2381746.

Dorrell, M., Aguilar, E., Scheppke, L., Barnett, F., and Friedlander, M. (2007). Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc. Natl. Acad. Sci. 104:967-972.

Dorrell, M., Aguilar, E., Schepke, L., Barnett, F., and M. Friedlander. (2007). Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc. Natl. Acad. 104(3):967-72.

Banin, E., Dorrell, M.I., Aguilar, E., Ritter, M.R.,Aderman, C.M., Smith, A.C.H., Friedlander, J., and M. Friedlander. (2006). T2-TrpRS inhibits pre-retinal neovascularization and enhances physiological vascular regrowth in oxygen-induced retinopathy as assessed by a new method of quantification. Invest. Ophthal. Vis. Sci., 47(5):2125-34.

Barnett, F.H., Scharer-Schusz Wood, M.M., Yu, X., Wagner, T.E. and Friedlander, M. (2004). Intra-arterial delivery of endostatin gene to brain tumors prolongs survival and alters tumor vessel ultrastructure. Gene Therapy, 11:1283-1289.

Otani, A., Kinder, K., Ewalt, K., Otero, F., Schimmel, P. and Friedlander, M. (2002). Bone marrow derived stem cells cells target retinal astrocytes and have pro- or anti-angiogenic activity. Nature Medicine 8:1004-1010.

Otani, A., Slike, B., Dorrell, M. I., Hood, J., Kinder, K., Ewalt, K., Cheresh, D.A., Schimmel, P. and Friedlander, M. (2002). A fragment of human TrpRS as a potent antagonist of ocular angiogenesis. Proceedings National Academy of Sciences, 99:178-183.

Strömblad, S, Fotedar, A., Brickner, H., Theesfeld, C., Aguilar de Diaz, E., Friedlander, M. and Cheresh, D.A. (2002). Loss of p53 compensates for alpha v function in retinal neovascularization. J Biol Chem. 277(16):13371-4.

Wakasugi, K., Slike, B., Ewalt, K., Hood, J., Otani, A., Ewalt, K.L., Friedlander, M., Cheresh, D.A., and Schimmel, P. (2002). A human aminoacyl-tRNA synthetase as a regulator of angiogenesis. Proceedings National Academy of Sciences, 99:173-177.

Brooks, P.C., Siletti, S., von Schalscha, T.L., Friedlander, M. and Cheresh, D.A. (1998). Disruption of angiogenesis by PEX, a non-catalytic metalloproteinase fragment with integrin binding activity. Cell 92:391-400.

Friedlander, M., Theesfeld, C.L., Sugita, M., Fruttiger, M., Thomas, M.A., Chang, S. and Cheresh, D.A. (1996). Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. (U.S.A.) 93:9764-9769.

Friedlander, M., Brooks, P., Shaffer, R., Kincaid, C., Varner, J., Cheresh, D. (1995). Two pathways of angiogenesis defined by homologous alpha v integrins. Science, 270:1500-1502.