Hanjoong Jo

Role of p47 phox in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II

Abstract—Hypertension caused by angiotensin II is dependent on vascular superoxide (O2·−) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2·− and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47phox, a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105±2 to 151±6 mm Hg and increased vascular O2·− formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47phox-/- mice the hypertensive response to angiotensin II infusion (122±4 mm Hg;P <0.05) was markedly blunted, and there was no increase of vascular O2·− production. In situ staining for O2·− using dihydroethidium revealed a marked increase of O2·−production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47phox-/- mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2·−, endothelial cells from WT and p47phox-/- mice were cultured. Western blotting confirmed the absence of p47phox in p47phox-/- mice. Angiotensin II increased O2·− production in endothelial cells from WT mice, but not in those from p47phox-/- mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47phox in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

Oscillatory Shear Stress Stimulates Endothelial Production of from p47phox-dependent NAD(P)H Oxidases, Leading to Monocyte Adhesion

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (± 5 dynes/cm2) increased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} compared with static conditions. In contrast, neither LS nor OS were able to induce \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.

Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47(phox)-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

NAD(P)H Oxidase-derived Hydrogen Peroxide Mediates Endothelial Nitric Oxide Production in Response to Angiotensin II*

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H2O2) increases endothelial nitric oxide (NO⋅) production. The current study is designed to determine whether endogenous levels of H2O2 are ever sufficient to stimulate NO⋅ production in intact endothelial cells. NO⋅ production was detected by a NO⋅-specific microelectrode or by an electron spin resonance spectroscopy using Fe2+-(DETC)2 as a NO⋅-specific spin trap. The addition of H2O2 to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO⋅ release. Incubation with angiotensin II (10−7 mol) elevated intracellular H2O2 levels, which were attenuated with PEG-catalase. Angiotensin II increased NO⋅ production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H2O2 in this response. In contrast, NO⋅ production evoked by either bradykinin or calcium ionophore A23187 was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO⋅ production in aortic endothelial cells from C57BL/6 mice but had no effect on NO⋅ production in endothelial cells from p47 phox−/− mice. In contrast, A23187stimulated NO⋅ production to a similar extent in endothelial cells from wild-type and p47 phox−/− mice. In summary, the present study provides direct evidence that endogenous H2O2, derived from the NAD(P)H oxidase, mediates endothelial NO⋅ production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O 2 ⨪ production from the NAD(P)H oxidase, the H2O2 stimulation of NO⋅ may facilitate peroxynitrite formation in response to this octapeptide.

Altered Shear Stress Stimulates Upregulation of Endothelial VCAM-1 and ICAM-1 in a BMP-4– and TGF-β1–Dependent Pathway

Objective—Hemodynamics has been associated with aortic valve (AV) inflammation, but the underlying mechanisms are not well understood. Here we tested the hypothesis that altered shear stress conditions stimulate the expression of cytokines and adhesion molecules in AV leaflets via a bone morphogenic protein (BMP)- and transforming growth fact (TGF)-&bgr;1–dependent pathway. Methods and Results—The ventricularis or aortic surface of porcine AV leaflets were exposed for 48 hours to unidirectional pulsatile and bidirectional oscillatory shear stresses ex vivo. Immunohistochemistry was performed to detect expressions of the 4 inflammatory markers VCAM-1, ICAM-1, BMP-4, and TGF-&bgr;1. Exposure of the aortic surface to pulsatile shear stress (altered hemodynamics), but not oscillatory shear stress, increased expression of the inflammatory markers. In contrast, neither pulsatile nor oscillatory shear stress affected expression of the inflammatory markers on the ventricularis surface. The shear stress–dependent expression of VCAM-1, ICAM-1, and BMP-4, but not TGF-&bgr;1, was significantly reduced by the BMP inhibitor noggin, whereas the TGF-&bgr;1 inhibitor SB431542 blocked BMP-4 expression on the aortic surface exposed to pulsatile shear stress. Conclusions—The results demonstrate that altered hemodynamics stimulates the expression of AV leaflet endothelial adhesion molecules in a TGF-&bgr;1– and BMP-4–dependent manner, providing some potential directions for future drug-based therapies for AV diseases.

Elevated cyclic stretch alters matrix remodeling in aortic valve cusps: implications for degenerative aortic valve disease

Matrix metalloproteinases (MMPs) and cathepsins are proteolytic enzymes that are upregulated in diseased aortic valve cusps. The objective of this study was to investigate whether elevated cyclic stretch causes an increased expression and activity of these proteolytic enzymes in the valve cusp. Circumferentially oriented fresh porcine aortic valve cusp sections were stretched to 10% (physiological), 15% (pathological), and 20% (hyperpathological) in a tensile stretch bioreactor for 24 and 48 h. The expression and activity of MMP-1, MMP-2, MMP-9, tissue inhibitor of MMP-1, and cathepsin L, S, and K were quantified and compared with fresh controls. Cell proliferation and apoptosis were also analyzed. As a result, at 10% physiological stretch, the expression and activity of remodeling enzymes were comparable with fresh controls. At 15% stretch, the expression of MMP-1, -2, -9 and cathepsin S and K were upregulated, whereas the expression of cathepsin L was downregulated compared with controls. A similar trend was observed at 20% stretch, but the magnitudes of upregulation and downregulation of the expression were less than those observed at 15%. In addition, there were significantly higher cell proliferation and apoptosis at 20% stretch compared with those of other treatment groups. In conclusion, elevated mechanical stretch on aortic valve cusps may detrimentally alter the proteolytic enzyme expression and activity in valve cells. This may trigger a cascade of events leading to an accelerated valve degeneration and disease progression.

Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences. Influence of Shear Stress

Objective—The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood. Methods and Results—Monolayers of porcine aortic endothelial cells (PAECs) or porcine aortic valve endothelial cells (PAVECs) were exposed to 20 dyne/cm2 steady laminar shear stress for 48 hours, with static cultures serving as controls. Multiple microarray comparisons were made using RNA from sheared and control batches of both cell types. More than 400 genes were significantly differentially expressed in each comparison group. The resulting profiles were validated at the transcription and protein level and expression patterns confirmed in vivo by immunohistochemistry. PAVECs were found to be less intrinsically inflammatory than PAECs, but both cell types expressed similar antioxidant and antiinflammatory genes in response to shear stress. PAVECs expressed more genes associated with chondrogenesis, whereas PAECs expressed osteogenic genes, and shear stress had a protective effect against calcification. Conclusions—Transcriptional differences between PAVECs and PAECs highlight the valvular endothelial cell as a distinct organ system and suggest more attention needs to be given to valvular cells to further our understanding of similarities and differences between valvular and vascular pathology.

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.

Mechanisms of Cell Signaling by Nitric Oxide and Peroxynitrite: From Mitochondria to MAP Kinases

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Bone Morphogenic Protein Antagonists Are Coexpressed With Bone Morphogenic Protein 4 in Endothelial Cells Exposed to Unstable Flow In Vitro in Mouse Aortas and in Human Coronary Arteries. Role of Bone Morphogenic Protein Antagonists in Inflammation and Atherosclerosis

Background— Exposure to disturbed flow, including oscillatory shear stress, stimulates endothelial cells (ECs) to produce bone morphogenic protein (BMP) 4, which in turn activates inflammation, a critical atherogenic step. BMP activity is regulated by the level of BMP antagonists. Until now it was not known whether shear also regulates the expression of BMP antagonists and whether they play a role in EC pathophysiology. Methods and Results— BMP antagonists follistatin, noggin, and matrix Gla protein were expressed in cultured bovine and human arterial ECs. Surprisingly, oscillatory shear stress increased expression of the BMP antagonists in ECs, whereas unidirectional laminar shear decreased such expression. Immunohistochemical studies with mouse aortas showed data consistent with in vitro findings: Only ECs in the lesser curvature exposed to disturbed flow, but not those in the greater curvature and straight arterial regions exposed to undisturbed flow, showed coexpression of BMP4 and the BMP antagonists. Similarly, in human coronary arteries, expression of BMP4 and BMP antagonists in ECs positively correlated with the severity of atherosclerosis. Monocyte adhesion induced by oscillatory shear stress was inhibited by knockdown of BMP4 or treatment with recombinant follistatin or noggin, whereas it was increased by knockdown of follistatin and/or noggin. Conclusions— The present results suggest that ECs coexpress BMP antagonists along with BMP4 in an attempt to minimize the inflammatory response by oscillatory shear stress as part of a negative feedback mechanism. The balance between the agonist, BMP4, and its antagonists may play an important role in the overall control of inflammation and atherosclerosis.