A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids - PubMed (original) (raw)

A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids

Sangderk Lee et al. Free Radic Biol Med. 2009.

Abstract

Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cells (HAECs). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species. We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.

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Figures

FIG. 1

FIG. 1. NOX4 complex is an important source of intracellular reactive oxygen species (ROS) formation in response to Ox-PAPC

(A–C) HAECs were transfected with scrambled (sc), NOX4, p22phox, and rac1-specific siRNAs (NOX4si, p22si, rac1si) for gene silencing. After 2 days, protein levels of NOX4, p22phox and rac1 were determined. (D) To examine the efficiency of siRNA transfection in HAECs, the scrambled- (20nM) and fluorescently-labeled siRNA (GLO-siRNA, 20nM) were transfected into HAECs using Lipofectamine 2000 reagent. After 24hr incubation, the presence of fluorescently-labeled GLO-siRNA in the cells was confirmed by detecting fluorescence intensity (514nm wavelength for excitation and 560–615nm wavelength for detection, resolution:100x). (E) After 2 days of siRNA transfection, intracellular ROS formation in response to Ox-PAPC (50ug/ml, Ox50) or media (C) were measured by DCF assay as described in the method section. The image shown is the relative fluorescence intensities at 0, 20min points of Ox-PAPC treatment. (F) Quantification of the DCF signal by measuring mean fluorescence intensities of 10 randomly chosen cell area from the images in (D). Values are represented as mean ± SEM, *** p<0.001; n

FIG. 2

FIG. 2. NOX4 and p22phox gene silencing downregulated induction of pro-inflammatory and sterol synthetic genes by Ox-PAPC but not redox- and UPR-regulating genes

(A) HAECs were transfected with scrambled (sc) or NOX4-specific siRNA (NOX4 si) as described in the method section. After 2 days of cell growth, cells were treated Ox-PAPC (50μg/ml, Ox50) or media (C) for 4 hrs, and relative mRNA levels of proinflammatory and sterol synthetic genes (IL-8 MCP-1, LDLR) were determined by QRT-PCR. (B) Transcriptional levels of other NOX subtypes were measured by QRT-PCR to examine the presence of compensatory up-regulations of other NOX subtypes by NOX4 silencing in HAEC. (C) HAECs were transfected with scrambled (sc) or siRNA to p22phox (p22 si), and after 2 days the expression of IL-8 by Ox-PAPC (50ug/ml) was measured by QRT-PCR. (D) HAECs were transfected with NOX4 siRNA and treated with Ox-PAPC as in (A), and the transcriptional levels of HO-1 and ATF3 were determined by QRT-PCR. Values are mean ± SEMs of triplicate PCR reactions; *** p<0.001.

FIG. 3

FIG. 3. A role for NOX4 component rac1 in Ox-PAPC effects on IL-8 expression

(A) Ox-PAPC causes rac1 recruitment to the cell membrane. HAECs were treated with Ox-PAPC (50ug/ml, Ox50) for the indicated times in M199 containing 1% FBS. The membrane fractions were prepared and the rac1 content in the fractions was determined by Western blotting. The same blot was reprobed with caveolin-1 antibody for normalization. This Western blot was representative of at least 3 repeated experiments with similar results. (B) Rac1 siRNA inhibits IL-8 induction. HAECs were transected with scrambled (sc) and siRNA to rac1 (rac1 si), and after 2 days of cell growth, the IL-8 induction by Ox-PAPC (50ug/ml, Ox50, 4hr) were measured by QRT-PCR. Values are mean ± SEMs of triplicate PCR reactions; ** p<0.01.

FIG. 4

FIG. 4. Vascular endothelial growth factor receptor 2 (VEGFR2) mediates rac1 recruitment induced by Ox-PAPC in HAEC

(A) HAECs were pretreated with SU1498 (10uM) for 1hr and co-treated with Ox-PAPC (50ug/ml) for an additional 4hrs. The rac1 content in the membrane fractions was determined as in Figure 3A. The relative band densities normalized by caveolin-1 content in the fractions were shown as a graph. (B) HAECs were transfected with scrambled (sc) or VEGFR2-specfic siRNA (VEGFR2si) as described in the method section. After 2 days, the relative mRNA levels of VEGFR2 in the cells were determined by QRT-PCR. Values in (B) are mean ± SEMs of triplicate PCR reactions; *** p<0.001. (C) After 2 days of siRNA transfection, cells were treated with Ox-PAPC (50ug/ml, Ox50) for 4 hrs and the rac1 content in the membrane fractions were determined as in (A). A representative of at least 3 repeated experiments with similar results is shown.

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