Zebrafish Model for Functional Screening of Flow-Responsive Genes.
Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries.



First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow.



We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Jovana
J
Serbanovic-Canic
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Amalia
A
de Luca
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Christina
C
Warboys
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Pedro F
PF
Ferreira
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Le A
LA
Luong
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Sarah
S
Hsiao
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Ismael
I
Gauci
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Marwa
M
Mahmoud
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Shuang
S
Feng
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Celine
C
Souilhol
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Neil
N
Bowden
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
John-Paul
JP
Ashton
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Henning
H
Walczak
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
David
D
Firmin
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Rob
R
Krams
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Justin C
JC
Mason
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Dorian O
DO
Haskard
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Spencer
S
Sherwin
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Victoria
V
Ridger
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom.
Timothy J A
TJ
Chico
From the Department of Infection, Immunity and Cardiovascular Disease (J.S.-C., L.A.L., S.H., I.G., M.M., S.F., C.S., N.B., J.-P.A., V.R., T.J.A.C., P.C.E.), INSIGNEO Institute for In Silico Medicine (J.S.-C., V.R., T.J.A.C., P.C.E.), and the Bateson Centre (J.S.-C., J.-P.A., T.J.A.C., P.C.E.), University of Sheffield, United Kingdom; and Departments of Cardiovascular Science (A.d.L., C.W., J.C.M., D.O.H.), Imaging (P.F.F., D.F.), Bioengineering (R.K.), and Aeronautics (S.S.) Imperial College London, United Kingdom; and Cancer Institute, Faculty of Medical Sciences (H.W.), University College London, United Kingdom. paul.evans@sheffield.ac.uk.
Paul C
PC
Evans
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D050197:Atherosclerosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian / Q000098:blood supply; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000235:genetics*; D051379:Mice; D010641:Phenotype; D034622:RNA Interference; D012039:Regional Blood Flow; D013314:Stress, Mechanical; D013552:Swine; D059467:Transcriptome; D014162:Transfection; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
D029961:Zebrafish Proteins
Zebrafish; apoptosis; atherosclerosis; endothelial cells; shear stress