PROJECT SUMMARY Lymphatic vessel (LV) differentiation, development, and morphogenesis are central in maintaining fluid homeostasis, regulating host immunity, and transporting dietary fat and neuronal waste. All these functions are governed by lymphatic drainage, a transport of interstitial fluid into the lymphatic system through the initial LVs and collecting LVs. The initial LVs show permeable button-like junction morphology and are ready to uptake interstitial fluid; by contrast, the collecting LVs are less permeable with zipper-like junction structure, so that the collecting LVs transport ‘lymph’ to lymph nodes without leaking. Impaired lymphatic drainage contributes to many human diseases, such as lymphedema, immune dysfunction, fibrosis, obesity, cancer, and Alzheimer’s disease. While little is known about why LVs become dysfunctional, clinical studies reveal that inflammation is one of the leading contributors to the lymphatic dysfunction. Although dysfunctional collecting LVs has been extensively studied, how inflammation impacts initial LV development and morphogenesis is unclear, because in our current experimental models, including animal models, we often cannot decouple multifactorial inflammatory factors in the lymphatic endothelium. Since two-dimensional cell culture has failed to recapitulate three-dimensional (3D) tissue architecture of lymphatics, researchers have developed 3D in vitro models of LVs, demonstrating lymphatic sprouting, lymphatic network formation, and LV interactions with other cells. However, these previous models have not created 3D lymphatic structure with specialized LEC junction development enabling controlled fluid drainage through the button-like junctions and physiological inflammatory response. In this proposal, we will use a bioengineered in vitro 3D lymphatic vascular system, exhibiting button-like junction morphogenesis of the LVs and fluid drainage to understand the regulation of LEC junction and drainage by focusing on ROCK1/2 and integrin α5 signaling. In Aim 1, we will examine the roles of ROCKs in LEC junction and drainage. Next, we will scrutinize the mechanisms of ROCKs-mediated junction zippering in LECs. In Aim 2, we will study integrin α5 mediated regulation of LEC junction and lymphatic drainage. We will then determine signal transduction through ROCKs and integrin α5 and evaluate therapeutic efficacy of targeting ROCKs and integrin α5 in lymphatic dysfunction and inflammation models in vivo. In summary, we will use a bioengineered model of 3D lymphatic vessels and fluid transport to provide an understanding of lymphatic drainage in normal and inflammatory conditions.