Abstract
PurposeBy using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs).MethodsC57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45+ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition.ResultsDE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45+ cell infiltration in LGs. Likewise, CD45+ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice.ConclusionsDll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.
Highlights
Dry eye (DE) is a highly prevalent ocular inflammatory disorder affecting millions of people worldwide
Inhibition of lymphangiogenesis by delta-like ligand 4 (Dll4)/Notch suppression resulted in increased CD45+ cell infiltration in lacrimal glands (LGs)
Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs
Summary
Dry eye (DE) is a highly prevalent ocular inflammatory disorder affecting millions of people worldwide. [5,6,7] Despite the importance of LGs and inflammation in DE pathophysiology, the exact mechanisms underlying increased inflammation in LGs affected by DE remain unknown. [10] Similar to DE induced cornea, we found an increase of lymphatic vessels (LVs) in the LGs of a DE-induced mouse model.[11] By using immunofluorescence staining as well as immunoblot, upregulation of a well-known marker related to LV formation, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), was observed in LGs after DE stress.[11] the underlying molecular mechanism for lymphatics growth in LGs and their functional role in the development of DE pathology have not yet been investigated
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