Regulation of Collecting Lymphatic Vessel Contractile Function by TRPV4 Channels.

BACKGROUND: Dysregulation of TRPV4 (transient receptor potential vanilloid type 4)-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 Trpv4(fx/fx) mouse strain and the Cre-lines Prox1-CreER(T2) and LysM-Cre we assessed the role of TRPV4 channels in lymphatic endothelial cells and peri-lymphatic myeloid cells, respectively. Confocal microscopy and extensive functional experimentation on isolated and pressurized lymphatics, including measurements of intracellular calcium activity, were used to validate our single-cell RNA sequencing findings and to elucidate the underlying mechanisms. Clinical significance was assessed using biopsies from patients with breast cancer-related lymphedema. RESULTS: We characterized the single-cell transcriptome of collecting lymphatic vessels and surrounding tissues. Trpv4 was highly enriched in lymphatic endothelial cells and in a subset of Lyve1+ (lymphatic vessel endothelial hyaluronan receptor 1) macrophages displaying a tissue-resident profile. In clinical samples, breast cancer-related lymphedema was associated with increased infiltration of macrophages coexpressing LYVE1 and TRPV4. Pharmacological activation of TRPV4 channels led to contractile dysregulation in isolated collecting lymphatics. The response was multiphasic, including initial vasospasm and subsequent vasodilation and inhibition of contractions, which was associated with the activation of TXA2Rs (thromboxane A2 receptors) in lymphatic muscle cells by secreted prostanoids from TRPV4+ myeloid cells, and increased nitric oxide (and perhaps other vasodilatory prostanoids) from lymphatic endothelial cells. The TXA2R-mediated vasospasm resulted from increased mobilization of calcium from intracellular stores through inositol trisphosphate 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 lymphatic muscle cells or lymphatic endothelial cells. These findings highlight potentially important roles of TRPV4 channels in lymphatic dysfunction associated with inflammation, including secondary lymphedema.