Small molecule inhibitors of collagen-induced angiogenesis

Small molecule inhibitors of collagen-induced angiogenesis

Abstracts cleaved from the cellular membrane and potentially bind to endothelial cells, however, a receptor for this process has not been identified...

60KB Sizes 0 Downloads 18 Views

Abstracts

cleaved from the cellular membrane and potentially bind to endothelial cells, however, a receptor for this process has not been identified. Our lab, in collaboration with Bouck and colleagues, identified CD36, a transmembrane glycoprotein on capillary endothelial cells, as a receptor that mediates antiangiogenic activity. Our laboratory has also identified a circulating protein, histidine-rich glycoprotein (HRGP) that shares significant homology with CD36. Through in vitro binding, in vivo cellular migration and tube formation assays, we show that CD36 is indeed the endothelial cell receptor for vasculostatin and that HRGP serves as a soluble “decoy” receptor and thereby inhibits the antiangiogenic activity of BAI1. Tumor growth in mice was dramatically increased when HRGP was introduced into tumor cells that contain vasculostatin when compared to tumors with vasculostatin alone. This result shows that HRGP is an important modulator of BAI1 within the tumor environment and the BAI1-CD36-HRGP interactions potentially can be exploited in future tumor therapies.

S17

endothelial cells, fibrillar collagen induced widespread capillary tube formation by 12 h. In contrast, tube formation was reduced by ∼ 70% only SMI 496, α β1 integrin A-domain inhibitor, and by function blocking antiα2β1, but not α1β1 integrin antibodies. By flow cytometry, endothelial cells bound fluorescein-type I collagen, an interaction specifically inhibited (∼ 65%) by SMI 496. Moreover, endothelial cells cultured on collagen and phalloidin-stained for filamentous actin were well-spread with abundant stress fibers, but SMI 496 caused cytoskeletal collapse, and delayed endothelial monolayer wound healing in vitro. SMI activities were examined on zebrafish (Danio rerio) embryos expressing green fluorescent protein under control of the vascular endothelial growth factor receptor 2 promoter. SMI 496, but not a control SMI interfered with angiogenesis by reversibly inhibiting sprouting from the axial vessels. Thus, an inhibitor of the integrin β1I-domain disrupts endothelial cell–collagen interactions and collageninduced capillary morphogenesis and may represent a novel therapy to combat pathological angiogenesis in vivo.

doi:10.1016/j.matbio.2008.09.247 doi:10.1016/j.matbio.2008.09.249

33 Chondrostatin inhibits angiogenesis

35 MMP14 and TIMP regulation of angiogenesis in aortic ring cultures

Zhepeng Wanga, Jennifer Bryana, Brian J. Ellb, Allan C. Rapraegerb, Linda Sandella a Washington University, United States b University of Wisconsin, United States

Alfred C. Aplina, Eric Fogelb, Roberto F. Nicosiaa,b Department of Pathology, University of Washington, Seattle, WA, United States b VA Medical Center, Seattle, WA, United States

Cartilage is antiangiogenic and resistant to tumor invasion. There has been a good deal of research aimed at isolating inhibitors from cartilage, but the mechanism for the lack of vascularity of cartilage is still unknown. Chondrostatin (chon) is a fragment from type II procollagen. It is removed from procollagen prior to formation of collagen fiber in ECM. Chon contains an RGD sequence that is conserved across species, indicating potential biological function. We showed previously that the integrins αvβ3 and αvβ5 mediate cell adhesion to chon. Three different assays were performed to determine effects of chon on angiogenesis. In the tube formation assay, recombinant chon, but not the mutated chon, inhibited HUVEC cell tube formation in a dose-dependent manner: 0.1 μM of chon suppress tube formation by 50% of the control. In the aortic ring assay, chon, but not the mutated chon, inhibited FGF-stimulated microvessel outgrowth and the inhibition is dose-dependent. In the mouse corneal assay, chon, but not the mutated chon, inhibited vascularization as seen in the fluorescence-labeled mouse cornea. These results suggested that chon is an angiogenesis inhibitor that is dependent on the RGD motif. Since microvascular endothelial cells, which are recruited by tumors, have become an important target in tumor therapy, chon may be a potential antitumor agent. In addition, chon is liberated from the collagen molecule during biosynthesis, reaching the highest level during cartilage formation. As we show it inhibits angiogenesis, chon is an excellent candidate for the molecular mechanism by which cartilage is avascular.

The purpose of this study was to define the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in vascular regression following angiogenesis. Formation of new blood vessels in collagen–gel cultures of rat and mouse aortic rings is associated with collagen lysis and production of MMPs including membrane type 1-MMP (MMP14). By immunohistochemistry, high levels of MMP14 are detected at the tips of sprouting neovessels. Inhibition of MMP14 with specific antibodies blocks angiogenic sprouting in the aortic ring assay. Antiangiogenic effects are also obtained with recombinant TIMP-2, -3, and -4 which block the activity of MMP14 but not with TIMP-1, a poor inhibitor of MMP14. All four TIMPs are expressed in collagen gel cultures during angiogenesis, with increasing levels during the vascular regression phase. Interestingly, TIMPs and anti-MMP14 antibodies stabilize the neovasculature and allow vessels to survive longer than untreated controls if administered after the angiogenic growth phase. Conversely, vascular regression is accelerated in aortic cultures from TIMP-1 and TIMP-2 deficient mice. TIMPs with antiMMP14 activity are more potent than TIMP-1 in preventing vascular regression. The vascular survival effect of these TIMPs is associated with complete inhibition of collagen lysis whereas TIMP-1 has no anti-collagenolytic effect. These results indicate that MMP14 plays a critical role in both angiogenesis and vascular regression and demonstrate that TIMPs with antiMMP14 activity have opposite effects on angiogenesis depending on the stage of the angiogenic process.

doi:10.1016/j.matbio.2008.09.248

doi:10.1016/j.matbio.2008.09.250

34 Small molecule inhibitors of collagen-induced angiogenesis

36 ECM-ligand functionalized fibrillar peptides

Kari Haburskya, Renato V. Iozzob, Kevin Turnera, Michelle Burrowsb, Sung-wook Choic, Soni Basrac, Joel S. Bennettd, William F. DeGradoc, James D. San Antonioa a Department of Med, Thom Jeff U, Phil, PA, United States b Department of Path, Anat, Cell Biol, Thom Jeff U, Phil, PA, United States c Departments of Chem, Biochem and Biophys, U PA, Phil, PA, United States d Department of Med, U PA, Phil, PA, United States

Jangwook P. Jung, Joel H. Collier Department of Surgery, University of Chicago, Chicago, IL 60626, United States

Type I collagen induced angiogenesis requires ligation of collagens GFPGER sequence to the endothelial cell α2β1 integrin. We investigated the effects of anti-α2β 1 integrin small molecule inhibitors (SMI) designed to disrupt integrin α2I- or β1I-domain function on angiogenesis. In control

a

Regeneration of epithelial and endothelial cell monolayers after wounding is a complex sequence of events regulated by cell–matrix interactions, soluble growth factors, and the mechanics of the ECM. A major challenge for studying the biology of these processes is creating experimentally controllable microenvironments where a multitude of these factors may be independently and precisely adjusted. Towards this end, self-assembling multi-component biomaterials are increasingly seen as an attractive route. Here we report co-assembling fibril-forming peptides capable of forming hydrogels displaying fibronectin- and laminin-derived sequences. We