ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.
BACKGROUND
Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown.


OBJECTIVE
We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation.


RESULTS
Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs fromOrai1knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient inOrai1,Klf2, orKlf4showed a significantly reduced lymphatic density and impaired lymphatic development.


CONCLUSIONS
Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Dongwon
D
Choi
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Eunkyung
E
Park
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Eunson
E
Jung
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Young Jin
YJ
Seong
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Mingu
M
Hong
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Sunju
S
Lee
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
James
J
Burford
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Georgina
G
Gyarmati
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Janos
J
Peti-Peterdi
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Sonal
S
Srikanth
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Yousang
Y
Gwack
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Chester J
CJ
Koh
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Evgenii
E
Boriushkin
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Anne
A
Hamik
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.). alex.wong@med.usc.edu young.hong@usc.edu.
Alex K
AK
Wong
From the Section of Plastic and Reconstructive Surgery, Department of Surgery (D.C., E.P., E.J., Y.J.S., M.H., S.L., A.K.W., Y.-K.H.), Department of Biochemistry and Molecular Biology (D.C., E.P., E.J., Y.J.S., M.H., S.L., Y.-K.H.), Norris Comprehensive Cancer Center, and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute (J.B., G.G., J.P.-P.), Keck School of Medicine, University of Southern California, Los Angeles; Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA (S.S., Y.G.); Department of Pediatric Urology, Texas Children's Hospital, Baylor College of Medicine, Houston (C.J.K.); Division of Cardiovascular Medicine, Department of Medicine, Stony Brook University, NY (E.B., A.H.); and Northport Veterans Affairs Medical Center, NY (A.H.).
Young-Kwon
YK
Hong
D000818:Animals; D049109:Cell Proliferation; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004729:Endothelium, Lymphatic / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D004730:Endothelium, Vascular / Q000378:metabolism; D005786:Gene Expression Regulation; D005838:Genotype; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D042583:Lymphangiogenesis; D040542:Mechanotransduction, Cellular; D018345:Mice, Knockout; D000071740:ORAI1 Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics / Q000378:metabolism
C493965:Cdkn1c protein, mouse; D050761:Cyclin-Dependent Kinase Inhibitor p57; C000715467:KLF4 protein, human; C498314:Klf2 protein, mouse; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; C467480:vascular endothelial growth factor A, mouse; C467483:vascular endothelial growth factor C, mouse; C496366:Fgfr3 protein, mouse; D051498:Receptor, Fibroblast Growth Factor, Type 3
calcium channel; capillary; cell proliferation; lymphatic vessels; vascular endothelial growth factor A