Transforming growth factor-β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo.
Bottom Line: In cells from Nox4-deficient mice, TGF-β1-induced cell proliferation, migration and tube formation were abolished.In vivo, TGF-β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice.Thus, endothelial cells are regulated by a TGF-β1 signalling pathway involving Nox4-derived ROS to promote angiogenesis.
Affiliation: Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia; O'Brien Institute, Fitzroy, Victoria, Australia.Show MeSH
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Mentions: To illustrate the functional importance of TGF-β1-induced Nox4 up-regulation, we used an adenovirus carrying RNA interference targeting human Nox4 (Adv-Nox4i) to down-regulate Nox4 in HMECs. As expected, Adv-Nox4i markedly reduced TGF-β1-stimulated Nox4 mRNA expression in HMECs (Fig.3A). Importantly, we also showed that Adv-Nox4i suppressed H2O2 production in the presence of TGF-β1 stimulation (Fig.3B). To further confirm that TGF-β1-mediated H2O2 generation was dependent on Nox4 gene expression, we isolated heart endothelial cells (MHEC) from Nox4 knockout mice (Nox4 KO) and their wild-type littermates (WT). Murine heart endothelial cells and HUVECs were found to share some similar characteristics as demonstrated in Figure S2. Both cell types exhibited cobblestone morphology, uptake of Dil-Ac-LDL and formed tubes when they were suspended in growth factor containing Matrigel. The gene expression of eNOS was also found to be similar in both cell types (Figure S2), confirming the homogeneity of the isolated MHECs. As shown in Figure3C, MHEC from WT mice also responded to TGF-β1 with an increased H2O2 production, and this response reduced in Nox4 KO mouse-derived MHEC. This confirms that Nox4 is required for TGF-β1-induced H2O2 formation in endothelial cells.
Affiliation: Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia; O'Brien Institute, Fitzroy, Victoria, Australia.