[CANCER RESEARCH 60, 2520 –2526, May 1, 2000] Anti-angiogenic Cues from Vascular Basement Membrane Collagen 1 Pablo C. Colorado, Adriana Torre, George Kamphaus, Yohei Maeshima, Helmut Hopfer, Keiko Takahashi, Ruediger Volk, Eric D. Zamborsky, Seth Herman, Pradip K. Sarkar, Mark B. Ericksen, Mohanraj Dhanabal, Michael Simons, Mark Post, Donald W. Kufe, Ralph R. Weichselbaum, Vikas P. Sukhatme, and Raghu Kalluri 2 Department of Medicine and the Cancer Center, Beth Israel Deaconess Medical Center and Harvard Medical School [P. C. C., A. T., G. K., Y. M., H. H., K. T., R. V., E. D. Z., S. H., P. K. S., M. B. E., M. D., M. S., M. P., V. P. S., R. K.], and Dana Farber Cancer Institute and Harvard Medical School [D. W. K.], Boston, Massachusetts 02215, and Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois 60637 [R. R. W.] ABSTRACT Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We spec- ulate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important anti- angiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the  1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant mol- ecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting en- dothelial cell proliferation, migration, tube formation, and Matrigel neo- vascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Addition- ally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the  1  1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for thera- peutic use. INTRODUCTION The development of new blood vessels from preexisting ones is generally referred to as angiogenesis (1). In the adult, new blood vessels arise via angiogenesis, a process critical for normal physio- logical events such as wound repair, the menstrual cycle, and endo- metrium remodeling (2). In the last three decades, considerable re- search has been conducted documenting that tumor growth and metastasis require angiogenesis (3). This process is pivotal to the survival and subsequent growth of solid tumors beyond a few cubic millimeters in size (4). Vascular basement membrane constitutes an insoluble structural wall of newly formed capillaries and undergoes several changes during tumor-induced angiogenesis (5). Initially, the membrane is degraded and disassembled but is finally reorganized to a native state around a newly formed capillary (5). Such vascular matrix changes during angiogenesis are associated with the expression of matrix proteins that can interact with vascular endothelium and provide endogenous angiogenic and anti-angiogenic signals (5). Base- ment membranes are composed of macromolecules such as type IV collagen, laminin, HSPGs, 3 fibronectin, and entactin (6). Type IV collagen is composed of six genetically distinct gene products, namely,  1 – 6 (7). The  1 and  2 isoforms are ubiquitously present in human basement membranes (8). The other four isoforms exhibit restricted distributions (9). Type IV collagen promotes cell adhesion, migration, differentiation, and growth (8). It is thought to play a crucial role in endothelial cell proliferation and behavior during the angiogenic process (5). Several studies have shown the anti-angio- genic properties associated with inhibitors of collagen metabolism, supporting the notion that basement membrane collagen synthesis and deposition are crucial for blood vessel formation and survival (10). Additionally, the COOH-terminal globular NC1 domain of type IV collagen is speculated to play an important role in the assembly of type IV collagen suprastructure, basement membrane organization, and modulation of cell behavior (11, 12). Recently, the NC1 domain of the  2 chain of type IV collagen (canstatin) was identified as an angiogenesis inhibitor (13) In the present study, we demonstrate the pivotal role of arresten, the NC1 domain of the  1 chain of type IV collagen, in modulating the function of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angio- genesis and tumor growth. MATERIALS AND METHODS Recombinant Production of Arresten in Escherichia coli. The sequence encoding arresten was amplified by PCR from the  1 NC1 (IV)/pDS vector (14) using a forward primer (5'-CGGGATCCTTCTGTTGATCACGGCTTC- 3') and a reverse primer (5'-CCCAAGCTTTGTTCTTCTCATACAGAC-3'). The resulting cDNA fragment was digested with BamHI and Hind III and ligated into predigested pET22b(+) (Novagen, Madison, WI). This placed arresten downstream of and in frame with the pelB leader sequence, allowing for periplasmic localization and expression of soluble protein. Additional vector sequence was added to the protein encoding amino acids MDIGINSD. The 3' end of the sequence was ligated in frame with the polyhistidine tag sequence. Additional vector sequence between the 3' end of the cDNA and the his tag encoded the amino acids KLAAALE. Positive clones were sequenced on both strands. Plasmid constructs encoding arresten were first transformed into E. coli HMS174 (Novagen) and then transformed into BL21 for expression (Nova- gen). Overnight bacterial culture was used to inoculate a 500-ml culture in Luria-Bertani medium. This culture was grown for 4 h until the cells reached an A 600 of 0.6. Then, protein expression was induced by addition of isopropyl- 1-thio--D-galactopyranoside to a final concentration of 1–2 mM. After a 2-h induction, cells were harvested by centrifugation at 5,000  g and lysed by resuspension in 6 M guanidine, 0.1 M NaH 2 PO 4 , and 0.01 M Tris-HCl (pH 8.0). Resuspended cells were sonicated briefly, and centrifuged at 12,000  g for 30 Received 12/22/99; accepted 3/1/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grants DK-51711 and DK-55001 (to R. K.) and R01-CA- 42596-12 (to R. W.), Deutsche Forschungsgemeinschaft Grant HO 2138/1-1 (to H. H.), a 1998 Hershey Prostate Cancer Research Award (to R. K.), a 1998 American Society of Nephrology Carl Gottschalk Research Award (to R. K.), a 1998 National Kidney Foun- dation Murray award (to R. K.), a 1998 Beth Israel Deaconess Medical Center Enterprise Award (to R. K.), and research funds from the Beth Israel Deaconess Medical Center. M. D., G. K., R. R. W., D. W. K., V. P. S., and R. K. have an equity position with Ilex Oncology, a company that is clinically developing arresten. 2 To whom requests for reprints should be addressed, at Nephrology Division, Depart- ment of Medicine, RW 563a, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 667-0445; Fax: (617) 975-5663; E-mail: rkalluri@caregroup.harvard.edu. 3 The abbreviations used are: HSPG, heparan sulfate proteoglycan; HPLC, high- performance liquid chromatography; HPEC, human prostate epithelial cell; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PCNA, proliferating cell nuclear antigen; HUVEC, human umbilical vein endothelial cell; CPAE, calf pulmo- nary arterial endothelial. 2520 Research. on February 12, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from