in the preliminary data in the last application showing the impact of HoxAS on vascular stability and growth. This work was done using endothelioma cells (bEND and EOMA) in cutaneous tissue.2 Further, we have now extended these studies to examine the impact of restoring HoxAS on growth of transformed endothelial cells in the mouse brain. A manuscript has just been submitted and these data are presented in Section 3.1b (Figs. 4- 7). The purpose of the work was to provide proof-of-concept that proliferative vascular tissue in the brain could be controlled by manipulation of HoxAS. Progress was hampered in part because our first choice of endothelioma cell type (bEND) did not reproducibly form lesions in vivo. The murine EOMA endothelioma cell line formed lesions similar to those induced by the bEND cells, but was phenotypically stable. We subsequently showed that restoring HoxAS could also limit growth of the EOMA tumors in the brain, and this was accompanied by a reduction in the HoxAS target Hifla and an increase in the anti-angiogenic TSP-2 gene. In addition we are currently conducting a clinical study to screen a large number of samples from the AVM tissue bank to document the prevalence of HoxAS dysregulation in a wide spectrum of AVM cases;this is part of Aim1. We previously showed preliminary ISH data (Fig.1) and have now performed this analysis on 7 AVM patient samples. A challenge which has slowed these studies is that our OCT frozen tissue sections are difficult to section due to the complex angio-architecture, thus making quantitative or semi-quantitative analysis by ISH difficult. Moreover, there is currently only one commercially available antibody against HoxAS, and while we have attempted to use this for staining cultured cells and also attempted different antigen retrieval and processing protocols for tissue samples, we have not been successful. I have also consulted with other investigators studying HoxAS who have not been able to apply this antibody for staining. We have now successfully used this for Western blot of tissue lysates (see Fig.8) and can apply this to human BAVM tissue specimens to confirm that HoxAS protein as well as mRNA is reduced. Nonetheless, we have now included new data to show that we can detect changes in HoxAS protein by performing Western blot on tissue lysates (Sec. 3.1, Fig.8) to confirm that changes in HoxAS mRNA are reflected at the level of protein. Importantly, to more accurately quantitate differences in HoxAS expression, we have now adapted a tissue micro-dissection technique that we developed for cutaneous hemangioma tissue to perform real time PCR analysis. We microscopically removed as much of the adjacent connective tissue and extract RNA from the remaining tissue, which is enriched in aberrant vessels. Real time PCR on this tissue has confirmed our ISH results: HoxAS expression is lacking in the vascular nidus of the AVM (Fig.1). We anticipate that we can now complete these experiments within the next several months as additional samples become available from Core C. All reviewers raised concerns regarding the number of different models proposed, the lack of justification for these models, and the paucity of preliminary data to support their use or their relevance to the human disease. Dr. Young has addressed the use of our model systems in the General Introduction. Moreover, the aims in Project 3 have now been revised and simplified to use only one general model?murine angiogenesis and vascular dysplasia?which has been well characterized by our group. 1. To first investigate whether HoxAS can impact cerebral angiogenesis, we will use focal VEGF stimulation using AAV-VEGF direct injection into the brain of transgenic mice which selectively expresses HoxAS in PHS 398/2590 (Rev.11/07) Page 266 Continuation Format Page
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