The central nervous system (CNS) microvasculature differs morphologically and functionally from that of other organs. The CNS is the dose-limiting tissue in the radiotherapeutic management of a wide variety of tumors. Alleviating or protecting against radiation-induced CNS injury would thus be of obvious advantage in cancer treatment. Among the most significant clinical manifestations or radiation toxicity in the CNS are severe alterations in the structural and functional integrity of the brain microvasculature, which may lead to abnormal glial- and endothelial-cell proliferation, fibrosis, edema, and possibly necrosis. The proposed research is based on the hypothesis that (a) the recovery/repair response of the CNS to radiation dependence on gene induction or reduction of specific proteases and inhibitors, and (b) protease/inhibitor activation regulates the astrocyte/glial-endothelial cell interaction.
Our Specific Aims are: (1) Define the mechanisms responsible for radiation-induced uPA, uPAR, and PN-I by using in vitro solo and co-cultures of human astrocytes, glial, and cerebral microvascular endothelial cells. These investigations will include determinations of transcriptional activity, mRNA stability, and active protein levels. First they will determine in vitro, the gene and protein expression of uPA, uPAR and PN-I, which can be induced by radiation in solo and co-cultures of these cells by ELISA, western blotting and northern blotting. Second, they will determine the effect of various pharmacological inhibitors that downregulate these molecules. Finally in addition to pharmacologic inhibitors that affect these molecules, they will also target these molecules by antisense approach (obtain stable transfectants for uPA/uPAR in these cell lines) or antisense sequences delivered via the recombinant adenoviral system which is already established in their laboratory. The effects of these direct and indirect reagents that regulate these molecules will also be assessed in combination with radiation to determine if they exert a synergistic or additive effect on capillary-like structure formation. (2) Determine the effects of induced or decreased gene expression of uPA, uPAR, and PN-I on the radioresponse in in vivo models, and assess the effect of the antisense stable transfectants and inhibitor specified in Specific Aim 1 on tumor angiogenesis in vivo. These investigations will first focus on determining whether the effects in the brain microvasculature correlate with the results of in vitro experiments. Next they will determine the effect of antisense stable transfectants, AEBSF and recombinant adenoviral antisense constructs for uPA/uPAR with and without irradiation, on tumor angiogenesis in vivo. The proposed studies should generate insight not only into the pathogenesis of radiation-induced CNS injury, but also into the processes necessary for successful recovery. This information in turn should suggest novel targets for therapeutic intervention.
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