Research Article: Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

Date Published: February 1, 2016

Publisher: Public Library of Science

Author(s): Ji Hwan Min, Chul Hee Lee, Yong Woo Ji, Areum Yeo, Hyemi Noh, Insil Song, Eung Kweon Kim, Hyung Keun Lee, John A Chiorini.


By 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).

C57BL/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.

DE 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.

Dll4/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.

Partial Text

Dry eye (DE) is a highly prevalent ocular inflammatory disorder affecting millions of people worldwide. However, disparities in the definition, diagnostic criteria, and treatment guidelines of the condition suggest that DE is a complicated heterogeneous disease involving many different pathophysiologic mechanisms.[1, 2] Although most DE patients complain of discomfort on the ocular surface area, the lacrimal gland (LG) is a major target organ of DE pathogenesis for both non-Sjögren DE and Sjögren syndrome.[3, 4] Inflammatory cytokines, inflammatory cells, and matrix proteases were upregulated after DE stress in human and mouse LGs. [5–7] Despite the importance of LGs and inflammation in DE pathophysiology, the exact mechanisms underlying increased inflammation in LGs affected by DE remain unknown.

The novel findings of this study are as follows: (1) Expression levels of the Dll4/Notch signaling pathway and lymphangiogenesis are significantly upregulated in DE-induced LGs. (2) HIF-1α is important for Dll4/Notch induced lymphangiogenesis in LGs of DE. (3)Lastly, the suppression of lymphangiogenesis significantly increases CD45+ cell infiltration in DE-induced LGs. A schematic figure summarizing the results of this study is shown in Fig 6.