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 2

ORAI1 is essential for the laminar flow–induced NOTCH1 suppression in LECs.

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ORAI1 is essential for the laminar flow–induced NOTCH1 suppression in LE...
(A and B) Activation of Ca2+ mobilization in LECs by laminar flow: Fluo-4–loaded LECs were pretreated with PBS (CTR) or SKF-96365 (SKF), an inhibitor of store-operated Ca2+ entry (SOCE), for 10 minutes, and subjected to laminar flow (2 dyn/cm2). Calcium signals were captured by a time-lapse microscope (A). Relative signal intensity was plotted in the graph (B). (C) NICD1 protein level in LECs and BECs that were transfected with control siRNA (siCTR) or ORAI1 siRNA (siORAI1) for 24 hours, then subjected to laminar flow (2 dyn/cm2). The vertical line marks a boundary between 2 different areas in the same gel. Blots presented are derived from replicate samples run on parallel gels. (D) qRT-PCR data showing the mRNA level of NRARP, HEY1, and HEY2 in ORAI1-inhibited LECs and BECs. Statistical comparison was made between control siRNA and ORAI1 siRNA samples at each time point to assess the reversal of the laminar flow–mediated suppression of the genes. (E) Effect of ORAI1 knockdown on the flow-induced sprouting of LECs and BECs. Cells were transfected with control or ORAI1 siRNA for 24 hours, exposed to laminar flow (2 dyn/cm2) for 24 hours, stained with a CellTracker dye, aggregated in methylcellulose polymers, and embedded in Matrigel. After 24 hours, spheroid images (>20) were taken and relative sprout numbers were quantified by NIH ImageJ. Scale bars: 50 μm. Data are expressed as SEM and SD of a representative of 3 independent experiments. *P < 0.05; #P < 0.01; §P < 0.001; t test.