FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake

Abstract

Background: Therapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction. Methods: Lung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and β-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-αinduced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lungslices ex vivo and on hyaluronan uptake in vitro. Results: In vitro, FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo. We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis.

Modeling lymphangiogenesis: Pairing in vitro and in vivo metrics

“…Nuclear prospero‐related homeobox 1 (PROX‐1) protein can also identify LECs 19,20 . Other tissue sources for commercial LECs include human lung, 21,22 rat mesentery, 23 and mouse mesentery 24 . Culturing primary LECs in vitro comes with a set of limitations.…”

“…17,18 Nuclear prospero-related homeobox 1 (PROX-1) protein can also identify LECs. 19,20 Other tissue sources for commercial LECs include human lung, 21,22 rat mesentery, 23 and mouse mesentery. 24 Culturing primary LECs in vitro comes with a set strengthen the correlation between in vitro and in vivo outcomes, giving more insight into lymphangiogenesis in healthy and pathological states.…”