? PROJECT 1, Molecular Genetic Alterations Contributing to CCM Pathogenesis We have previously shown that CCM pathogenesis follows a two-hit mutation mechanism where the inherited, constitutional CCM gene mutation is augmented by a somatic mutation occurring in the remaining wild-type copy gene. We have further shown that this second hit occurs in the endothelial cells (ECs) of the lesion. But it is unknown whether this mutation is the sole or even main cause of the progressive shift of the CCM from a single dilated vessel to the mature, multi-cavernous, hemorrhagic lesion. We hypothesize that CCM-deficient ECs exert a dominant, non-cell autonomous effect on CCM pathogenesis, recruiting non-mutant cells into the developing lesion. We will test this hypothesis by crossing the multicolor Cre-reporter, R26R-Confetti, into a floxed Ccm mutant line to visually trace the lineage of the mutant ECs in the developing lesion. In parallel, working with the Dr. Awad?s Mouse Phenotyping and Human Tissue Core, we will analyze the micro-dissected endothelial cells from human CCMs to determine the anatomic location of the somatically mutated cells in the different caverns of the lesion. These data will complement the murine lineage tracing studies to determine whether the original somatic mutation is required in all regions of the growing CCM lesion. Finally, based on information gained from Drs. Ginsberg?s and Kahn?s studies of signaling aberrations associated with loss of CCM proteins, we will determine the significance of other signaling pathways (Wnt/beta-catenin, ROS, NOTCH, KLF2, others) in CCM lesion pathogenesis. We will employ pharmacological inhibitors of these signaling pathways with our Ccm1 and Ccm3 mouse models, and working with Dr. Awad?s Core, determine the effects of inhibition of these pathways on lesion size, number, biomarkers of maturation, and the clinically important phenotype of hemorrhage. These combined analyses will identify the molecular and signaling aberrations that are critical for lesion maturation, providing new hypotheses for lesion pathogenesis, and identifying new targets for CCM therapy.
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