PROJECT SUMMARY Lymphatic vessels (LVs) 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 and immune cells into the lymphatic system through initial LVs and collecting LVs. The initial and collecting LVs have different structures and roles in maintaining lymphatic drainage. The initial LVs have permeable cell-cell junctions and are ready to uptake interstitial fluid and cells; by contrast, the collecting LVs are less permeable, so that the collecting LVs can transport ‘lymph’ to lymph nodes without leaking. Impaired lymphatic drainage contributes to LV-related human diseases, such as lymphedema, immune dysfunction, fibrosis, obesity, cancer, Alzheimer’s disease, etc. While little is known about why LVs become dysfunctional, clinical studies reveal that inflammation is one of the leading contributors to the lymphatic dysfunction. Mechanisms of how inflammation affects both initial and collecting LVs making them dysfunctional are unclear, because in our current experimental models, including animal models, we often cannot decouple multifactorial inflammatory factors in the initial and collecting LVs. 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, LV permeability, and LV interactions with surrounding cells. However, these previous models have not created 3D lymphatics with LEC junctions enabling controlled drainage, valve formation, or physiological inflammatory response. Also, they have not considered the distinct roles of initial and collecting LVs. In this proposal, we will establish a bioengineered in vitro 3D lymphatic vascular system, including both initial and collecting LVs, and determine how inflammatory conditions make the initial and collecting LVs dysfunctional. In Aim 1, we will establish collecting LV-on-chip, considering (i) tightened cell-cell junctions, (ii) luminal valves, and (iii) mural cell coverage (Aim 1.1). Next, we will combine the collecting LVs with the initial LVs and demonstrate fluid and immune cell transport through these LVs in the normal condition (Aim 1.2). In Aim 2, we will determine the effects of inflammatory conditions by using inflammatory cytokines that are relevant to lymphatic dysfunction on the initial and collecting LVs and overall lymphatic drainage (Aim 2.1). Next, we will identify new targets to reverse the lymphatic dysfunction by screening pathways of lymphatic vascular mechanotransduction (Aim 2.2). We will then validate the mechanisms and targets in an in vivo skin inflammation model (Aim 2.3). In summary, we will develop a new bioengineered model of 3D lymphatic vascular system and fluid/immune cell transport through the initial and collecting LVs and p...