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 3

ORAI1 is required for normal lymphatic sprouting during development.

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ORAI1 is required for normal lymphatic sprouting during development. (A)...
(A) Immunofluorescence images of lymphatic and blood vessels in the back skins of WT and Orai1 KO embryos (E15.5). Lymphatics were stained for LYVE-1, and blood vessels were visualized by anti-CD31 staining. The graph shows the relative number of branching points (BP no.) and average distance between the branching points (BP-BP dis.). Scale bars: 100 μm. (B) The ear vasculature of 3-week-old WT and Orai1 KO mice was similarly visualized and analyzed. Scale bars: 250 μm. (C–E) Endothelial-specific deletion of Orai1 (Orai1ECKO) was induced in pregnant females by peritoneal injection of tamoxifen (1.5 mg) at E11.5 and 13.5. At E15.5, the embryos were harvested and genotyped. Lymphatics and blood vessels of the back skins of the control (CTR) embryos [Orai1+/+ Cdh5(Pac)-CreERT2 Prox1-EGFP] and Orai1ECKO embryos [Orai1fl/fl Cdh5(Pac)-CreERT2 Prox1-EGFP] were visualized by lymphatic-specific EGFP signals (C) and anti-CD31 staining (D), respectively. Scale bars: 250 μm (C), 100 μm (D). (E) Vascular analyses were performed as described above. For all studies, a total of more than 6 embryos or postnatal mice per genotype collected from at least 3 independent litters were analyzed. The dorsal midline areas of the embryos were chosen as the sites for vascular analyses. Data are expressed as SEM and SD. *P < 0.05; #P < 0.01; §P < 0.001.