Lymphatic Vessel Pumping during Inflammation
Summary
Arkansans as well as hundreds of millions of people worldwide suffer from chronic inflammatory diseases associated with lymphatic pathologies such as lymphedema. Lymphatic vessels provide an essential conduit for the transport of fluid and cells back to circulation, thereby maintaining tissue integrity. Disruption of lymphatic vessels or lymphatic flow either from surgery, injury, or infection enhances chronic pathology. However, how dysfunctional lymphatics exacerbate inflammation is poorly understood. Therefore, our goal is to determine the cellular and molecular mechanisms by which chronic inflammation regulates lymphatic function, so that lymphatic pathologies can be targeted in the clinic to treat chronic inflammatory diseases. Importantly, the emerging trend in infection and oncology is the utilization of immunotherapies and immune modulators with a rapidly growing market that could be harnessed to treat lymphatic diseases.
Lymphatic vessels are composed of two cell types: lymphatic endothelial cells (LECs) that line vessels and lymphatic muscle cells (LMCs) that function by inducing series of coordinated contractions to pump fluid and inflammatory cells out of tissues. Lymphatic function and intrinsic pumping critically relies on the complementary actions of LECs and LMCs. If either is disrupted, defective dilated lymphatics lead to edematous swelling further exacerbating inflammation. Dilated lymphatic vessels are associated with elevated IFNg and a robust STAT1 genomic signature. Therefore, we hypothesize pro-inflammatory mediators like IFNg activate STAT1 to impair lymphatic function by decreasing pumping, thereby preventing inflammatory cell exit from tissues and promoting chronic inflammation. While specific markers provide the ability to identify and study LECs, unfortunately there is not a specific marker identifying LMCs apart from other vascular smooth muscle cells, thus limiting the experimental tools available to study the effects of inflammatory mediators on LMCs. As a result, it is not clear whether inflammatory mediators are impacting LECs or LMCs to disrupt lymphatic pumping. The lack of a specific LMC marker is a knowledge gap and severe limitation in lymphatics research.
Aim 1. Define lymphatic muscle cell molecular marker to delineate from lymphatic endothelial cells. Lymphatic vessels from rat and mouse mesentery will be isolated, digested and FACS-sorted to separate LMCs and LECs. We will perform TMT multiplex quantitative proteomics on sorted cell populations to identify proteins distinguishing LMCs from LECs. Arterial smooth muscle cells will provide controls for comparison. Studies will be initiated in rats due to the increased amount of biomaterial, while experiments in mice which are the most commonly used animal model for laboratory research, are necessary for commercialization.
Aim 2. Determine the functional consequence of manipulating IFNg/STAT1 signaling on lymphatic pumping and contraction. Mice will be infected with Leishmania parasites to induce inflammation, IFNg/STAT1 activation, and dilated lymphatics. Lymphatic vessels connecting the footpad infection site to lymph node will be analyzed ex vivo for contractile function. Additionally, in vivo lymphatic flow will be imaged following Evans blue injection. Popliteal lymphatics from naïve mice will serve as controls. The contribution of IFNg and STAT1 on contractile function and lymphatic flow will be assessed following STAT1 pharmacological inhibition or IFNg antibody neutralization, respectively.