Over the years, numerous studies have implicated roles of many transcription factors in endothelial differentiation and function. However, many questions remain unanswered. For example, transcriptional mechanisms that regulate vascular endothelial growth factor receptors (VEGFRs) expression during angiogenesis are largely unknown. Earlier, we found that Vegfr1 transcription is rapidly induced in endothelial cells in response to angiogenic signals, mediated by fibroblast growth factor 2 and epidermal growth factor. We also found that transcription factors ETS1 and hypoxia inducible factor 2-alpha function in a combinatorial fashion to directly mediate the transcriptional induction of Vegfr1 in endothelial cells. To further understand transcription mechanisms in endothelial cells, we examined molecular mechanisms that mediate alteration of the nucleoprotein structure at the Vegfr1 chromatin domain. In particular, we were interested about the alteration of histone modification patterns. Interestingly, we found that angiogenic signal-induced transcription at the Vegfr1 locus is dependent on the function of a histone chaperone, histone cell cycle regulation defective homolog A (HIRA). Our analyses revealed that in response to angiogenic signals, HIRA mediates incorporation of acetylated histone H3 (acH3) at the Vegfr1 chromatin domain. Intriguingly, knockdown of HIRA by RNA interference (RNAi) in endothelial cells inhibits angiogenic signal-induced induction of Vegfr1 and other angiogenesis regulatory genes and also inhibits endothelial network/tube formation on matrigel. Furthermore, in a matrigel plug assay we found that depletion of HIRA also inhibits angiogenesis in vivo. Therefore, in this proposal, we will further test function of HIRA in endothelial gene regulation and postnatal/pathological angiogenesis.
Two specific aims are proposed.
Aim 1 will test the hypothesis that HIRA function is important for the induction or maintenance of the transcriptionally active nucleoprotein structure of the Vegfr1 locus. We will test whether the chromatin accessibility and transcription factor occupancy at the Vegfr1 locus as well as the subnuclear localization of the Vegfr1 locus is altered by angiogenic signals and whether HIRA is important for regulating these mechanisms. In addition, using mouse embryonic stem cell differentiation system and isolated endothelial cells, we will determine whether HIRA function differentially regulates histone acetylation, chromatin accessibility, and thereby transcription of angiogenesis regulatory genes in endothelial progenitors vs. in matured endothelial cells.
In Aim 2, we will determine HIRA function in the regulation of pathological angiogenesis. We will implement an RNAi approach for testing HIRA function in pathological angiogenesis by using a laser-induced choroidal neovascularization model and also analyzing tumor angiogenesis in a orthotopic xenograft model of breast cancer. These studies will indicate whether histone chaperones could be an important target for modulating angiogenesis.
Angiogenesis, the development of new blood vessels from existing vasculature, is a key event in many physiological processes, like organ growth and development, wound healing, and reproduction. Angiogenesis is also critical for certain pathological disorders including tumor growth/metastasis and age-related macular degeneration. Endothelial gene regulation is critical for blood vessel development and is also important during angiogenesis. Therefore, Information gleaned from these studies will contribute to our understanding of the molecular mechanisms of angiogenesis and hopefully will lead to novel therapeutic modalities for vascular regeneration and anti-angiogenesis therapy.
