p38 mitogen activated protein (MAP) kinases are widely expressed protein kinases that regulate growth and development. p38alpha is the most abundant isoform expressed in mammalian cells. Depending on cell types and the nature of stimuli, p38alpha regulates a wide range of physiological processes, such as cell proliferation, survival, and differentiation. Its pivotal role in embryogenesis is best demonstrated in p38alpha knockout (p38a-/-) mice where embryos display defective vascularization and vessel structures, suggesting a critical role of p38alpha in the development of vascular system. p38alpha knockout is lethal, thus limited information can be obtained from animal models. However, the embryonic stem (ES) cells isolated from knockout embryos (p38a-/-ES cells) are viable, which can be a valuable cell system to analyze the specific functions of p38alpha. p38alpha has been intensively studied in somatic cells, but we know little of its functions in ES cells. We have shown that p38a-/-ES cells display several altered properties from wild type ES cells, including cell adhesion, morphology and viability. The goals of this proposal are: 1. to further characterize several lines of p38a and p38a-/-ES cells and to determine the molecular mechanisms underlying the altered properties of ES cells caused by p38alpha deletion, 2. to investigate the role of p38alpha in ES differentiation, specifically focusing on how the ability of ES cells to differentiate to endothelial cells (ECs) is affected in p38a-/-ES cell, and 3. to analyze ES cell-differentiated EC function by an 3-dimendsional (3D) in vitro angiogenesis model. In this model, ECs will be cultured in a 3D collagen matrix, where they undergo a series of morphological changes to form tube-like structures, mimicking the steps of in vivo blood vessel formation. This novel assay will be used as a functional analysis to test hypothesis that ECs derived from p38a-/-ES cells may retain certain abilities to differentiate, but differentiated cells may have impaired functions to assemble into normal vessels. This study is expected to provide well characterized and genetically defined pSSalpha deficiency ES cell lines, which will be particularly useful for in-depth investigation of the roles of pSSalpha in ES cell differentiation and vascular development. The knowledge derived from this study could be valuable for the development of cell-based therapeutic approaches for diseases associated with angiogenesis and cardiovascular malfunction. ? ? ?
