Sigma‐1 Receptors Promote Lymphatic Endothelial Barrier Function

Abstract

The lymphatic endothelium plays important roles in lymphatic contractile activity, lymph transport and maintaining a barrier between the lymph and interstitial compartments and its dysfunction is a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma‐1 (σ1) receptors are chaperone proteins located in the endoplasmic reticulum were recently identified to promote collecting lymphatic dilation through endothelial production of nitric oxide. However, the role of σ1 in other functions of lymphatic endothelial cells are not clear. The aim of the current study was to test the hypothesis that σ1 promotes barrier function of lymphatic endothelium. We confirmed expression of σ1 in adult and juvenile human dermal lymphatic endothelial cells (HDLEC) using the ProteinSimple Western (WES) system and immunofluorescence microscopy. To test whether activation of σ1 promotes lymphatic endothelial barrier function, we applied the selective agonist PRE‐084 to cultured monolayers of HDLEC and determined the transendothelial electrical resistance (TER) as an index of barrier function using electrical cell‐substrate impedance sensing. The contribution of basal σ1 activity to HDLEC TER was also assessed either with the σ1 antagonist BD1047 (200 nM) or by silencing σ1 expression with siRNA. Because σ1 has been previously described to be located in the ER‐mitochondrial membrane interface, we also evaluated its role in endothelial bioenergetics using the Agilent Seahorse system glycolytic rate assay. Lastly, we tested the ability of PRE‐084 to counteract IL‐1β‐induced barrier dysfunction in HDLEC monolayers. The results show that the activation of σ1 receptor with PRE‐084 increases HDLEC TER in a concentration‐dependent manner, with 50, 100, 150, and 200 μM PRE‐084 eliciting significant elevations in TER (100 μM was chosen for subsequent experiments). Blockade of σ1 either with the antagonist BD‐1047 or reducing σ1 expression with siRNA resulted in decreased HDLEC TER. PRE‐084 also enhanced basal and compensatory glycolysis as well as proton efflux rate in HDLEC. Moreover, PRE‐084 partially counteracted the IL‐1β‐induced TER drop indicating its potential anti‐inflammatory impact on lymphatic endothelial cells. Collectively our results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic ATP production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.