Malignant astrocytomas demonstrate a prominent angiogenic response that promotes the highly invasive and proliferative phenotype of these tumors. Tumors typically synthesize both anti- and pro-angiogenic molecules; however, when the balance shifts toward pro-angiogenic molecules tumors exhibit very aggressive behavior. Intact thrombospondin (TSP)-1 and 2 and peptides derived from the type 1 repeat domain of TSP-1 and 2 have been shown to act as anti-angiogenic molecules. However, the amino terminal domain of TSP-1 and 2 may have a different function than the type 1 repeat domain, as recent in vitro data from other investigators indicates that the amino-terminal domain of TSP-1 can be pro-angiogenic. In this regard, we have preliminary data indicating that an amino-terminal domain fragment of TSP-1 and 2 is proteolytically generated in vivo in malignant astrocytomas but is minimally detected in the normal brain. The mechanism by which the amino-terminal domain of TSP-1 and 2 can promote angiogenesis is unclear. TSP-1 and 2 complex with matrix metalloproteinases (MMP)-2 and 9 and the complex is internalized by the LDL receptor-related protein (LRP), reducing the activity of MMP-2 and 9. TSP-1 and 2 bind to LRP through their amino terminal domain. We hypothesize that the in vivo amino-terminal fragments of TSP-1 and 2 compete with intact TSP-1 or 2 (complexed with MMP-2 or 9) for binding to and internalization by LRP. The latter competition should result in increased MMP-2 and 9 activity in these tumors, promoting angiogenesis and tumor invasion.
The aims of this project are as follows. 1) Characterize the proteolytic fragments of TSP-1 and 2 that are found in malignant astrocytomas, and create rec-proteins corresponding to these in vivo fragments. 2) Test the angiogenic modulatory effect of rec-TSP- 1 and 2 fragments in capillary tube formation assays and the corneal model of angiogenesis, and determine whether the amino-terminal fragments of TSP-1 and 2 inhibit LRP internalization of intact TSP-1or 2 complexed with MMP-2 or 9. 3) Determine the biologic role of host or stromal cell-derived TSP-2 in malignant gliomas, and the effect on the angiogenic phenotype of stable tumor cell over-expression of TSP-2 fragments identified in patient biopsies in aim 1. Mouse malignant glioma cell clones will be injected intracerebrally into the TSP-2 knockout mouse and the wild-type mouse, and studies to analyze tumor angiogenesis and proliferation performed.
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