Regulation of Collecting Lymphatic Vessel Contractile Function by TRPV4 Channels
Background: Dysregulation of TRPV4-mediated signaling has been associated with inflammation and tissue fibrosis, both of which are key features in the pathophysiology of lymphatic system diseases; however, the expression and functional roles of lymphatic TRPV4 channels remain largely unexplored.  Methods: We generated a single-cell RNA sequencing dataset from microdissected mouse collecting lymphatic vessels to characterize the expression of Trpv4. Using a novel Trpv4fx/fx mouse strain and the Cre-lines Prox1-CreERT2 and LysM-Cre we assessed the role of TRPV4 channels in lymphatic endothelial cells (LECs) and peri-lymphatic myeloid cells, respectively. Confocal microscopy and extensive functional experimentation on isolated and pressurized lymphatics, including measurements of intracellular calcium activity, were employed to validate our scRNAseq findings and to elucidate the underlying mechanisms. Clinical significance was assessed using biopsies from breast cancer-related lymphedema (BCRL) patients.  Results: We characterized the single-cell transcriptome of collecting lymphatic vessels and surrounding tissues. Trpv4 was highly enriched in LECs and in a subset of Lyve1+ macrophages displaying a tissue-resident profile. In clinical samples, BCRL was associated with increased infiltration of macrophages co-expressing LYVE1 and TRPV4. Pharmacological activation of TRPV4 channels led to contractile dysregulation in isolated collecting lymphatics. The response was multi-phasic, including initial vasospasm and subsequent vasodilation and inhibition of contractions, which was associated with the activation of thromboxane A2 receptors (TXA2Rs) in lymphatic muscle cells (LMCs) by secreted prostanoids from TRPV4+ myeloid cells, and increased nitric oxide (and perhaps other vasodilatory prostanoids) from LECs. The TXA2R-mediated vasospasm resulted from increased mobilization of calcium from intracellular stores through IP3 receptors and store-operated calcium entry.  Conclusions: Our results uncovered a novel mechanism of lymphatic contractile dysregulation mediated by the crosstalk between TRPV4-expressing myeloid cells, including LYVE1+ macrophages, and LMCs or LECs. These findings highlight potentially important roles of TRPV4 channels in lymphatic dysfunction associated with inflammation, including secondary lymphedema. Overall design: Collecting lymphatic vessels and surrounding tissues were microdissected from male and female C57BL/6J (N=4 for each sex). These mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 10-12 weeks of age and utilized for experiments after a 2-week acclimation period. For each sample, single-cell suspensions were prepared from lymphatic tissues pooled from 4 different anatomical regions, i.e., popliteal, inguinal axillary, superficial cervical, and mesentery.