Laminar flow downregulates Notch activity to promote lymphatic sprouting
Dongwon Choi, … , Alex K. Wong, Young-Kwon Hong
Dongwon Choi, … , Alex K. Wong, Young-Kwon Hong
Published March 6, 2017
Citation Information: J Clin Invest. 2017;127(4):1225-1240. https://doi.org/10.1172/JCI87442.
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Research Article Vascular biology

Laminar flow downregulates Notch activity to promote lymphatic sprouting

Abstract

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow–induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow–induced lymphatic sprouting.

Authors

Dongwon Choi, Eunkyung Park, Eunson Jung, Young Jin Seong, Jaehyuk Yoo, Esak Lee, Mingu Hong, Sunju Lee, Hiroaki Ishida, James Burford, Janos Peti-Peterdi, Ralf H. Adams, Sonal Srikanth, Yousang Gwack, Christopher S. Chen, Hans J. Vogel, Chester J. Koh, Alex K. Wong, Young-Kwon Hong

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Figure 8

Overexpression of DTX3L rescues the defective lymphatic sprouting of Orai1 KO mice.

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Overexpression of DTX3L rescues the defective lymphatic sprouting of Ora...
(A and B) Lymphatic and blood vessels were visualized in control embryos (Prox1-EGFP) or Dtx3lTG transgenic embryos [Prox1-EGFP Cdh5(PAC)-CreERT2 LSL-Dtx3l]. (A) Pregnant females were injected with tamoxifen at E11.5 and E13.5. At E15.5, dermal lymphatic and blood vessels were visualized by EGFP and CD31 staining, respectively. Panels i and ii display lymphatic vessels growing into the dorsal midline region. Boxed areas are enlarged in panels iii and iv, respectively. Arrows indicate distance between front lines of lymphatic vessels growing from the lateral areas into the midline. Panels v and vi highlight the dorsal midline area. Blood vessels in the corresponding area are shown in panels vii and viii, respectively. (B) Vascular analyses were performed and graphed to show branching point numbers (BP no.) and distance between the branching points (BP-BP dis.) of lymphatic vessels (LV) and blood vessels (BV). More than 5 embryos total per genotype obtained from 3 independent litters were analyzed. The dorsal midline regions were imaged for the vascular analyses. (C and D) Ectopic expression of DTX3L rescues the lymphatic sprouting defects in Orai1 KO embryos. (C) Endothelial-specific Orai1ECKO and/or Dtx3l ectopic expression were induced in pregnant females at E11.5 and E13.5 by i.p. tamoxifen injection. Lymphatic and blood vessels were detected by LYVE-1 and CD31 staining, respectively, in the back skins of embryos with indicated genotypes at E15.5. (D) Vascular analyses were performed to determine the number of branching points (BP no.) and distance between the branching points (BP-BP dis.) of lymphatic vessels. (E) Schematic illustration showing a current working model of laminar flow–induced lymphatic sprouting. More than 4 embryos total per genotype were harvested from at least 3 independent litters and analyzed. The dorsal midline areas of the embryos were imaged for the vascular analyses. Scale bars: 100 μm (A, iii–viii; C); 1 mm (A, i, ii). *P < 0.05; #P < 0.01; §P < 0.001.