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Transforming growth factor-β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo.

Peshavariya HM, Chan EC, Liu GS, Jiang F, Dusting GJ - J. Cell. Mol. Med. (2014)

Bottom Line: TGF-β1-stimulated Nox4 expression and ROS formation in endothelial cells.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia; O'Brien Institute, Fitzroy, Victoria, Australia.

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Transforming growth factor-β1 (TGF-β1) regulates Nox4-derived H2O2 in endothelial cells. Treatment of HMECs with Adv-Nox4i suppressed the stimulatory effects of TGF-β1 (10 ng/ml; A) on Nox4 gene expression and (B) H2O2 generation. (C) TGF-β1 (10 ng/ml)-induced H2O2 generation was abolished in MHEC derived from Nox4 knockout (Nox4 KO) mice. All data are mean ± SEM from three to six experiments, *P < 0.05 from control without treatment; †P < 0.05 from Adv-GFP or WT-treated cells in the presence of TGF-β1 (10 ng/ml).
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fig03: Transforming growth factor-β1 (TGF-β1) regulates Nox4-derived H2O2 in endothelial cells. Treatment of HMECs with Adv-Nox4i suppressed the stimulatory effects of TGF-β1 (10 ng/ml; A) on Nox4 gene expression and (B) H2O2 generation. (C) TGF-β1 (10 ng/ml)-induced H2O2 generation was abolished in MHEC derived from Nox4 knockout (Nox4 KO) mice. All data are mean ± SEM from three to six experiments, *P < 0.05 from control without treatment; †P < 0.05 from Adv-GFP or WT-treated cells in the presence of TGF-β1 (10 ng/ml).

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.


Transforming growth factor-β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo.

Peshavariya HM, Chan EC, Liu GS, Jiang F, Dusting GJ - J. Cell. Mol. Med. (2014)

Transforming growth factor-β1 (TGF-β1) regulates Nox4-derived H2O2 in endothelial cells. Treatment of HMECs with Adv-Nox4i suppressed the stimulatory effects of TGF-β1 (10 ng/ml; A) on Nox4 gene expression and (B) H2O2 generation. (C) TGF-β1 (10 ng/ml)-induced H2O2 generation was abolished in MHEC derived from Nox4 knockout (Nox4 KO) mice. All data are mean ± SEM from three to six experiments, *P < 0.05 from control without treatment; †P < 0.05 from Adv-GFP or WT-treated cells in the presence of TGF-β1 (10 ng/ml).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508156&req=5

fig03: Transforming growth factor-β1 (TGF-β1) regulates Nox4-derived H2O2 in endothelial cells. Treatment of HMECs with Adv-Nox4i suppressed the stimulatory effects of TGF-β1 (10 ng/ml; A) on Nox4 gene expression and (B) H2O2 generation. (C) TGF-β1 (10 ng/ml)-induced H2O2 generation was abolished in MHEC derived from Nox4 knockout (Nox4 KO) mice. All data are mean ± SEM from three to six experiments, *P < 0.05 from control without treatment; †P < 0.05 from Adv-GFP or WT-treated cells in the presence of TGF-β1 (10 ng/ml).
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.

Bottom Line: TGF-β1-stimulated Nox4 expression and ROS formation in endothelial cells.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia; O'Brien Institute, Fitzroy, Victoria, Australia.

Show MeSH
Related in: MedlinePlus