Project Summary/Abstract. Lymphatic dysfunction is known to be associated with various disorders involved in both aging and inflammation, such as Alzheimer’s disease, cardiovascular decline, and arthritic progression. Throughout aging, lymphatic contractility has been shown to deteriorate related to reduced gene expression of essential pathways that mediate homeostatic contractions and cytoskeletal integrity in lymphatic muscle cells (LMCs). Similarly, we previously discovered that age-dependent cellular mechanisms of lymphatic dysfunction in the tumor necrosis factor transgenic (TNF-Tg) mouse model of chronic inflammatory arthritis are associated with progression of joint disease and the loss of joint-draining popliteal lymphatic vessel (PLV) contractions in aged mice. In these aged mice, the PLV demonstrates significant LMC atrophy at an ultrastructural level, which is proposed to drive the elimination of lymphatic function and exacerbate disease progression. As a clinical correlate, we have found that patients with active rheumatoid arthritis have fewer functional LVs and diminished lymphatic clearance on the surface of the hands. Thus, we hypothesize that inflammation mediates accelerated age-related damage to LMCs where inflamed LMCs will exhibit a comparable dysfunction in the gene pathways necessary for LMC contractility and cytoskeletal integrity. To test this hypothesis, we propose to assess alpha smooth muscle actin (αSMA)+ PLV-LMC coverage and investigate transcriptional changes by single cell RNA sequencing (scRNAseq) in young (2-month-old), aged (8-month-old), and elderly (24-month- old) WT and TNF-Tg mice. Towards this goal, we have demonstrated that αSMA+ PLV-LMC coverage is reduced in aged (8-month-old) TNF-Tg mice relative to WT littermates. Additionally, we achieved preliminary success in scRNAseq of aged LMCs to indicate the feasibility of this approach. Given the inadequate characterization of LMCs necessary for targeted research into their role in health and disease, our scRNAseq data also provides evidence that LMCs are transcriptionally distinct vascular muscle cells. Through the completion of the research aims embodied in this grant proposal, we have the opportunity to provide considerable innovation for future pre-clinical research and targeted therapeutic interventions of LMCs. In line with the applicant’s Research Training Plan, the PI will gain valuable experience primarily in bench research with additional opportunities to foster abilities in clinical trial design, medical care, academic leadership, and community outreach at the University of Rochester with a cherished history of producing successful physician- scientists. For the following PA-21-049 F30 grant submission, we propose to elucidate the mechanisms of LMC dysfunction during both aging and inflammation for the benefit of lymphatic cellular biology and our understanding of the enigmatic age-related progression of arthritis.