Obesity is a serious health concern in the US and is a significant risk factor for many causes of mortality, including stroke and cardiovascular disease. Significant evidence points to an important pathogenic link between lymph barrier dysfunction and obesity. Lymphatic endothelial cells (LECs) are crucial regulators of lymphatic barrier function. Impaired LEC barrier can cause lymph leakage and lead to adipose tissue accumulation in obesity, although few pathways have been identified that regulate the barrier function of lymphatic vessels. Growing evidence supports a crucial role for LEC Ca2+ signaling pathways in regulating the function of collecting lymphatic vessels, which collect lymph from peripheral tissues. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels serve as a Ca2+ influx pathway in LECs. In preliminary studies, I determine the first recordings of TRPV4 channel activity in the intact LECs (TRPV4LEC) from mesenteric collecting lymphatic vessels (MCLVs) of mice. In addition, I show that LEC barrier is disrupted in MCLVs from LEC-specific Trpv4 knockout (Trpv4LECKO) mice, and Trpv4LECKO mice gain more weight after high-fat diet (HFD) feeding. I also observed that HFD feeding impairs the activity of LEC TRPV4 (TRPV4LEC) channels in control mice. In addition, my preliminary studies show that stimulation of mechanosensitive cation channel, Piezo1, increases the activity of TRPV4LEC channels, supporting a Piezo1LEC–TRPV4LEC signaling axis. Moreover, Piezo1LEC–TRPV4LEC signaling axis is impaired after HFD feeding, raising the possibility that impaired Piezo1LEC–TRPV4LEC signaling may lead to leaky MCLVs in obesity. Therefore, I hypothesize that 1) Piezo1LEC–TRPV4LEC signaling regulates body weight; and 2) disrupted Piezo1LEC–TRPV4LEC signaling impairs LEC barrier function, contributing to weight gain in obesity. I will test these hypotheses by executing the following specific aims: In Aim 1, I will determine whether LEC-specific TRPV4 deletion results in leaky collecting lymph vessels and increased body weight; in Aim 2, I will determine whether a loss of Piezo1LEC–TRPV4LEC signaling leads to leaky collecting lymph vessels and increased body weight; in Aim 3, I will determine whether impaired Piezo1LEC–TRPV4LEC signaling contributes to weight gain in obesemice. The proposed studies will identify a novel LEC Ca2+ signaling mechanism that maintains the barrier function of collecting lymphatic vessels and regulates body weight. This application will contribute to Dr. Yen-Lin Chen’s career development as he transitions from a postdoctoral fellow to an independent researcher. Dr. Sonkusare, a world-renowned physiologist in studying ion channels and Ca2+ signaling, will support this proposal and mentor Dr. Chen’s scientific and career development, and transition to independence during this award. To strengthen Dr. Chen’s training, the proposed plan additionally enlists a mentoring team, including Drs. Scallan, Isakson...