Hif1-Alpha Induced Adrenomedullin Expression Supports Angiogenesis In 3d Leiomyoma Xenografts.

Cellular responses to hypoxia are critical to homeostasis. In eukaryotes, the primary regulator of angiogenesis is the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). Hormonally-stimulated human leiomyoma 3D xenografts express HIF1-α protein and undergo in vivo neovascularization. Our objective was to characterize hypoxia regulated genes and HIF-1α regulated angiogenesis in our human leiomyoma cell lines and xenograft model. Laboratory study Leiomyoma and myometrial cells exposed to normal O2 (21%) and low O2 (1%) levels and chemically induced hypoxia with cobalt chloride (CoCl2) for 6 and 24 hours; leiomyoma and myometrial cell protein; 3D leiomyoma and myometrial xenograft tissue from placebo-, estrogen- and progesterone-treated groups; Immunohistochemistry and Western blot analyses Quantitative WB analysis of myometrial cells exposed to hypoxia demonstrate a greater than 3-fold increase (3.35+0.01-fold **p<0.05) in HIF-1α expression at 24 hours in myometrial cells under hypoxic conditions. Conversely, leiomyoma cells show up regulation of HIF-1α expression (1.16+0.004-fold **p<0.01) at 6 hours and 24 hours (1.44+0.05-fold *p=0.05) under normoxic conditions. Adrenomedullin (ADM), a pro-angiogenic protein under HIF-1α transcriptional control, was upregulated in hypoxic myometrial cells at 6 (1.76+/-0.5-fold) and 24 hours (1.28+/- 0.3-fold), compared to normoxic myometrium. Leiomyoma cells show an almost 1.5-fold increased ADM expression under both normoxia and hypoxic conditions at the 6 hour time point. Exposure of myometrial cells to CoCl2 for 24 hours demonstrate a >1.5-fold increase in ADM and a >2-fold increase in the stress inducible protein, sestrin 2. After 24 hours of exposure to CoCl2, leiomyoma cells show a similar increase in ADM expression to that of myometrium at 6 hours, and a >7-fold increase in sestrin 2. IHC results for ADM expression in 3D xenografts show that the estrogen-, estrogen+progesterone and progesterone-treated groups demonstrate intense cytoplasmic/matrix staining for ADM compared to placebo treated groups, particularly under the growth-stimulatory influence of gonadal hormones. Angiogenesis is supported by hypoxia induced, HIF-1α facilitated ADM expression in 3D leiomyoma xenografts.