The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.
Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis.



A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs.



NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Ralf P
RP
Brandes
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Sabine
S
Harenkamp
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Christoph
C
Schürmann
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Ivana
I
Josipovic
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Beliza
B
Rashid
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Flavia
F
Rezende
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Oliver
O
Löwe
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Franziska
F
Moll
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Jeremy
J
Epah
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Jeanette
J
Eresch
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Arnab
A
Nayak
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Irakli
I
Kopaliani
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Cornelia
C
Penski
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Michel
M
Mittelbronn
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.).
Norbert
N
Weissmann
From the Institute for Cardiovascular Physiology (R.P.B., S.H., C.S., I.J., B.R., F.R., O.L., F.M., J.E., K.S.), Pharmazentrum Frankfurt (J.E.), Institute for Biochemistry II (A.N.), and Neurological Institute (Edinger Institute) (C.P., M.M.), Goethe University, Frankfurt, Germany; Department of Physiology, Medical Faculty, TU Dresden, Dresden, Germany (I.K.); Justus-Liebig Universität Giessen, Giessen, Germany (N.W.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany (R.P.B., C.S., I.J., F.R., O.L., F.M., K.S.). Schroeder@vrc.uni-frankfurt.de.
Katrin
K
Schröder
D000072197:ADAM10 Protein / Q000378:metabolism; D000072198:ADAM17 Protein / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000201:enzymology*; D005838:Genotype; D006614:Hindlimb; D007511:Ischemia / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D008565:Membrane Proteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply*; D009247:NADH, NADPH Oxidoreductases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000172:deficiency / Q000235:genetics; D018919:Neovascularization, Physiologic; D018384:Oxidative Stress; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism; D012039:Regional Blood Flow; D015861:Retinal Neovascularization / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D015398:Signal Transduction; D013997:Time Factors
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D051880:Receptors, Notch; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D053829:Amyloid Precursor Protein Secretases; D000072197:ADAM10 Protein; C491234:Adam10 protein, mouse; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse
ADAM17 protein; Notch; NoxO1 protein, mouse; angiogenesis effect; dibenzazepine; reactive oxygen species