Single-Cell Transcriptomics and Lineage Tracing Unveil Parallels in Lymphatic Muscle and Venous Smooth Muscle...

Lymphatic muscle cells (LMCs) are indispensable for lymphatic vessel contraction, and their aberrant recruitment or absence is associated with both primary and secondary lymphedema. Despite their critical role in lymphatic vessel function, the cellular and molecular bases that confer the unique contractile properties to LMCs are largely undefined, limiting the development of therapeutic interventions that precisely target LMCs.

We used single-cell RNA sequencing, genetic lineage tracing, whole mount immunostaining, and intravital imaging to investigate the basis for the hybrid cardiomyocyte and blood vascular smooth muscle cell (SMC) characteristics that have been described for LMCs.

Using single-cell RNA sequencing, the transcriptomes of LMCs and venous SMCs exhibited more similarities than differences, with both cell types exhibiting enrichment in overlapping molecular markers. Notably, LMCs and venous SMCs were both markedly distinct from that of arteriole SMCs. Functionally, both lymphatic vessels and blood vessels in the murine hind limb displayed pulsatile contractility, and their functions were regulated by gabapentin and nifedipine, which target the activity of voltage-gated calcium channels. Although LMCs express genes that overlap with the venous SMC transcriptome, lineage tracing demonstrates that LMCs do not originate from Myh11 (myosin heavy chain 11) lineage-derived SMCs, Nkx2.5 (NK2 homeobox 5) cardiomyocyte progenitors, or Wnt1 (Wnt family member 1) neural crest progenitors. Instead, most LMCs and SMCs in the hind limb and inguinal-axillary region originate from WT1⁺ (Wilms tumor gene 1) mesodermal progenitors from the lateral plate mesoderm. LMCs derived from WT1⁺ progenitors were critical for the maintenance of lymphatic vessel contractility.

Overall, our findings suggest that venous SMCs and LMCs derive from a related mesodermal progenitor and acquire a similar gene expression program that facilitates their contractile properties.