Chapter 8 ANGIOSTATIN Generation, Structure and Function of the Isoforms Jennifer A. Doll and Gerald A. Soff Northwestern University Feinberg School of Medicine, Department of Medicine, Division oj Hernatology/Oncology and the Robert H. Lurie Comprehensive Cancer Center, Chicago, ZL 1. INTRODUCTION: DISCOVERY AND FUNCTION OF ANGIOSTATIN The discovery of angiostatin in 1994 provided a major advance in the field of angiogenesis research. As Folkman first proposed in 1971, angiogenesis, the growth of new vessels from the pre-existing vasculature, is required for tumors to grow beyond a few millimeters in diameter and for tumor metastasis'. With a few exceptions (wound healing and reproductive cycles), the vasculature in the adult is maintained in a quiescent state by a net balance of angiogenic inducers and inhibitors secreted into the tissue Tumors shift this balance to favor vessel growth, by increasing inducer levels, decreasing inhibitor levels, or most often, a combination of Tumor angiogenesis involves many processes, including increased vascular permeability, endothelial cell activation, proliferation, migration and tube formation as well as matrix degradation. Designing therapies targeting any of these steps would inhibit angiogenesis, and thus inhibit tumor growth. Therefore, much research has been devoted to developing such agents for use in cancer therapy. Numerous inducers and inhibitors of angiogenesis have now been identified, both endogenous and exogenous. It is of interest to note that the endogenous regulators, both inducers and inhibitors, span extremely diverse groups of molecules, including growth factors and cytokines, proteins and enzymes, protein cleavage products, enzyme inhibitors, carbohydrates, lipids, hormones and Most of the naturally occurring angiogenesis inhibitors, such as thrombospondin-1 and pigment epithelium- 176 CYTOKINES AND CANCER derived factor, have a wide variety of cellular activities and affect multiple cell types6,'. Few inhibitors have been identified which have high specificity to activated endothelial cell suppression. Angiostatin was the first natural inhibitor discovered and one of the few to show high selectivity for the endothelial cells lining the blood Angiostatin's discovery stemmed from research into several observations that had perplexed clinicians and researchers alike for many years. First, as described in some case reports as well as in animal tumor models, rapid growth of distant metastases has been observed following the removal of the primary tumor (reviewed in 8). The second observation is that a secondary tumor can be suppressed by the presence of a different primary tumor at a distant location (reviewed in 8). From these observations and their knowledge of tumor angiogenesis, Folkman developed the hypothesis that some tumors, while able to stimulate angiogenesis within their own capillary beds, produce angiogenesis inhibitors which enter the circulation and suppress angiogenesis in metastatic foci8. They tested this hypothesis using a variant of the murine Lewis lung carcinoma (LLC) cell line with a low metastatic potential (LLC-LM)~. They resected the primary subcutaneous LLC-LM tumors 14 days after implantation in mice and compared metastatic growth to sham operated mice in which the primary tumor was left intact8. Mice with resected primary tumors had 10-fold more metastatic growth compared to sham-operated mice, suggesting that the primary tumor had been inhibiting the growth of metastases8. In addition, corneal neovascularization toward an implanted pellet containing basic fibroblast growth factor (bFGF), a potent angiogenesis inducer, was inhibited in mice with intact primary tumors but not in mice with resected tumors, indicating that a circulating factor was indeed inhibiting angiogenesis8 From more than 100 liters of urine collected from mice bearing LLC-LM tumors, O'Reilly and colleagues isolated a 38 kDa murine protein, which they named angiostatin8. By sequence analysis, they determined that this protein was an internal fragment of plasminogen (PLG), beginning with amino acid 98 (initial sequence of 98-102: valine-tyrosine-leucine-serine- glutamic acid) and with a C-terminus at approximately amino acid 4408. This fragment included the first four of five loop structures, called kringle domains, in the PLG protein (Figure l)8. The angiostatin produced in the mice was dependent on the presence of the Lewis lung tumor8. However, at this time, it was not known if the tumor itself was producing the angiostatin or if the tumor was producing protein(s) that could generate angiostatin or that could block inhibitors of PLG activators8. To generate human angiostatin for study, O'Reilly and colleagues digested human PLG with elastase, as it was known to liberate kringle Angiostatin 177 containing fragments, including a fragment comprised of kringle domains 1- 3, isolated kringle 4 and mini-PLG, which is kringle 5 attached to the plasmin catalyhc domain". From the elastase digestion, they isolated a fragment of approximately 40 kl3as. This fragment included the first three Figure I. Structure of Human Plasminogen and Angiostatin K1-4. A) Plasminogen, the zyrnogen form of plasmin, contains five conserved kringle domains (K1 - KS), as well as the protease domain. The triangle indicates where plasminogen activators (uPA, tPA) cleave plasminogen to yield the active serine protease plasmin (picture thanks to M. Llinas and co-workers, Carnegie Mellon University, Pittsburgh, PA). B) Angiostatin as originally described by O'Reilly et aL8, consists of the first four of the five kringle domains of plasminogen. kringle domains of human PLG, with an N-terminus of amino acid 97 or 99 of the human PLG protein, a region corresponding to the murine angiostatin8. The purified elastase-generated human angiostatin specifically 178 CYTOKINES AND CANCER inhibited endothelial cell proliferation in vitro and inhibited neovascularization in the in vivo chick chorioallantoic membrane (CAM) assay8. In the LLC-LM model, following removal of the primary tumor, systemic treatment with the elastase-generated human angiostatin suppressed metastases8. Elastase-generated human angiostatin also inhibited bFGF and vascular endothelial growth factor (VEGF)-induced endothelial cell migration and tube formation in a collagen matrix system". By careful histologic analysis, this group found that dormancy of micro metastases in mice with intact LLC tumors was due to a balance between proliferation and apoptosis of the tumor cells12. When the primary tumor was removed, and thus the circulating angiostatin also removed, angiogenesis ensued and apoptosis was significantly decreased, allowing for expansion of the metastatic foci12. A later study showed that the elastase-generated human angiostatin could also inhibit primary tumor growth of human prostate, breast and colon cancer cells in subcutaneous mouse models treated with a systemic dose of 50 mgkg twice daily9. Consistent with their findings in the LLC model, this suppression was also due to an increase in apoptotic rate while the proliferation rate remained unchanged9. In another study, Folkman and colleagues generated an expression vector to produce recombinant murine angiostatin, and the purified protein encompassed the first four kringle domains with an amino acid sequence as follows: AspZOthrough Ser32-Ser-Arg97through Gly4,, 13 . This recombinant angiostatin was significantly larger than the in vivo generated angiostatin, at 52 kDa versus 38 kDa13. The N-terminal addition of 14 amino acids contributed to this increase but could not account for the total difference in size; therefore, the remaining difference was thought to be due to glycosylation differenced3. The size difference did not affect activity, and in fact, the recombinantly generated angiostatin was more potent than the elastase-generated human angiostatin against endothelial proliferation in vitro and inhibited LLC-LM subcutaneous primary tumor growth in vivo13. They further showed that when T241 fibrosarcoma tumor cells were transfected with an angiostatin expression vector and implanted in mice, primary and metastatic tumor growth were both inhibited14. Subsequently, other researchers isolated angiostatin and angiostatin-like proteins. These related proteins, or angiostatin isoforms, had differing NH2- and COOH-termini of PLG and varied mainly in their kringle domain content. The differences in structure and in anti-endothelial cell and anti- tumor activity are discussed in detail below. Overall, studies by several groups, including our own, confirmed that angiostatin isoforms inhibit endothelial cell proliferation, migration and tube formation induced by a variety of angiogenesis inducers in ~itro"-'~and also inhibit vessel formation Angiostatin 179 in vivo in the corneal pocket assay, in the embryonic body model and in the aortic ring m~del'~"~"~.The anti-tumor activity of angiostatin isoforms has now been demonstrated against a variety of tumor types in mouse models as well, including hemangioendotheliomaZ0,glioma21~22, liver can~er~~-~',lung can~er~~'~',ovarian cancerz8, colorectal ~ance?~and breast ~ancer'~>~O.As angiogenesis is important in physiologic and pathophysiologic processes in addition to cancer, angiostatin isoforms have been investigated in other diseases. Potential therapeutic benefits have also been observed in models of corneal wound healing3', collagen-induced arthritis32133and endometri~sis~~. In contrast to angiostatin, the parent PLG protein does not