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 1

Laminar flow selectively suppresses NOTCH1 activity in LECs.

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Laminar flow selectively suppresses NOTCH1 activity in LECs. (A and B) R...
(A and B) Real-time quantitative reverse transcription PCR (qRT-PCR) data showing the mRNA levels of KLF2 in LECs and BECs (A) and those of Notch target genes in LECs (B) in response to steady laminar flow (2 dyn/cm2). Expression of the Notch target genes in BECs by laminar flow is shown in Supplemental Figure 1C. (C) Protein levels of NICD1 in LECs and BECs in response to laminar flow (2 dyn/cm2). The intensity of the Western blotting bands is quantitated in Supplemental Figure 1E. A monoclonal anti-NOTCH1 antibody that specifically detects the cleaved form of NOTCH1 at Val1744 was used. Blots presented are derived from replicate samples run on parallel gels. (D) Luciferase assay showing the flow-mediated suppression of Notch activity in LECs. LECs were transfected with a Notch activity reporter (pGa981-6) (60) and exposed to laminar flow (2 dyn/cm2) for 24 or 48 hours before measurement of luciferase activity. (E) Spheroid-based sprouting assays. Cells were exposed or not exposed to laminar flow (2 dyn/cm2) for 24 hours, stained with a CellTracker dye, aggregated in methylcellulose polymers, and then embedded in Matrigel. After 24 hours, images of more than 20 spheroids were taken for analyses, and relative sprout numbers were quantified in the graph. (F) Biomimetic sprouting assay. Intraluminal laminar flow (5 dyn/cm2) was applied onto a layer of LECs lining the inner wall of the vascular mimetic channels made in collagen gel. Scale bars: 100 μm. Relative sprout length and number were graphed. Each experiment was independently performed at least 3 times with consistent results. Data are expressed as SEM and SD from 1 representative data set. *P < 0.05; #P < 0.01; §P < 0.001; t test.