Lymphatic muscle cells are unique cells that undergo aging induced changes.

Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas-including LMCs-of collecting lymphatic vessels in mouse dermis at various ages. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and uncovered a pro-inflammatory microenvironment that suppresses the contractile apparatus in advanced-aged LMCs. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to preserve lymphatic vessel function as well as supporting studies to identify genetic causes of primary lymphedema currently with unknown molecular explanation.

Dissection of murine collecting lymphatic vessels for imaging, single-cell analysis, and tissue culture of lymphatic muscle cells.

In vivo and in vitro systems have the potential to provide a framework to study the organization, gene expression, and functionality of lymphatic and blood vessel smooth muscle cells in physiology and disease settings. A series of procedures are described here, including the surgical isolation of mouse collecting lymphatic vessels and blood vessels, whole-mount immunofluorescence staining of muscle cells on the vessels, and the enzymatic digestion and culture of α- smooth muscle actin+ cells from the vessels. For complete details on the use and execution of this protocol, please refer to Jones et al. (2018).