An Osteopontin–NADPH Oxidase Signaling Cascade Promotes Pro–Matrix Metalloproteinase 9 Activation in Aortic Mesenchymal Cells

Mukhopadhyay P, Rajesh M, Bátkai S, Yoshihiro K, Haskó G, Liaudet L, Szabó C, Pacher P. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol 296: H1466 -H1483, 2009. First published March 13, 2009 doi:10.1152/ajpheart.00795.2008.-Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22phox , xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX-or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit. heart failure; apoptosis; inducible nitric oxide synthase; hemodynamics BECAUSE OF ITS unmatched efficacy and broad-spectrum effect, doxorubicin (DOX; Adriamycin), an anthracycline antibiotic, continues to be a widely used chemotherapeutic agent to treat a variety of cancers (55) despite its potential to elicit serious dose-dependent cardiotoxicity often leading to degenerative cardiomyopathy...

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“…Sections were air dried and hydrated with PBS. Sections were then incubated with 5 M DHE at 37°C for 30 min as previously described (21).…”

Section: Methodsmentioning

confidence: 99%

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Mukhopadhyay

Rajesh

Bátkai

et al. 2009

American Journal of Physiology-Heart and Circulatory Physiology

324

270

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“…These authors suggested that the presence of both osteopontin and thrombin were likely to occur in cancer, inflammation, and wound healing. Subsequently, multiple authors have shown activity of the thrombin-cleaved NH 2 -terminal domain in mediating integrin binding and cell adhesion (5,6,8,9). However, to date, very little is known of the activity and fate of the corresponding COOHterminal domain.…”

Section: Introductionmentioning

confidence: 99%

Thrombin-Cleaved COOH-Terminal Osteopontin Peptide Binds with Cyclophilin C to CD147 in Murine Breast Cancer Cells

Mi¹,

Oliver

Guo³

et al. 2007

Cancer Research

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Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombincleaved osteopontin COOH-terminal fragment. [Cancer Res 2007;67(9):4088-97]

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“…Preclinical models should be studied for consequences of vitamin D deficiency on vascular inflammation, matrix remodeling, and arterial oxidative stress-and the contributions of the renin-angiotensin-aldosterone axis to the beneficial vascular effects of adequate vitamin D nutrition. Histomorphometric and biochemical techniques that spatially resolve arterial tunic inflammation and oxidative stress (42) should be used to assess vascular effects of vitamin D insufficiency. Moreover, Cre-Lox technology (43) could be applied to evaluate how cell-autonomous VDR signaling in VSMC, macrophages, T cells, and endothelial cells contributes to vascular physiology and pathobiology in murine models.…”

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confidence: 99%

Calciotropic Hormones and Arterial Physiology

Towler¹

2007

Journal of the American Society of Nephrology

Self Cite

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V ascular calcification increasingly afflicts our aging and dysmetabolic population (1). In the past decade, vascular calcification has emerged as an actively regulated form of calcified tissue metabolism, resembling mineralization of endochondral and membranous bone (1). Demer and colleagues (2) were the first to provide molecular insights into disease pathobiology. In 1993, they identified expression of the powerful bone morphogen, BMP2, in calcium-laden type Vb atherosclerotic plaques. Detailed studies have confirmed contributions of osteogenic BMP2 and Wnt signaling cascades not only in atherosclerotic disease (2) but also during medial artery calcification of diabetes (3) and calcific aortic stenosis (4). Executive cell types that are familiar to bone biologists are seen in calcifying macrovascular specimens, controlling calcified matrix remodeling via paracrine signals that include osteopontin, matrix Gla protein, and inorganic pyrophosphate (1,3,5). Ectopic skeletal morphogens divert aortic mesoangioblasts, mural pericytes, and valve myofibroblasts to osteogenic fates (1,3,6). Ectopic vascular expression of pro-osteogenic morphogens is entrained to physiologic stimuli that promote calcification (1,3). Inflammation, mechanical shear, oxidative stress, hyperphosphatemia, and elastinolysis provide stimuli that promote osteogenic matrix remodeling and compromise vascular defenses that limit calcium deposition (1,3).In ESRD, a "perfect storm" of metabolic, mechanical, and inflammatory insults challenges vascular health, leading to cardiovascular mortality rates that approach 20% per annum (7). Perturbations in parathyroid hormone (PTH) production that compromise normal bone formation increase the risk for vascular calcium load (8,9). Indeed, in patients stage 5 chronic kidney disease, low-turnover osteoporosis with inappropriately normal PTH levels is a strong risk factor for vascular calcium accumulation (9). Vascular calcification contributes to vascular stiffening-abnormal aortofemoral Windkessel physiology that generates systolic hypertension, increases myocardial workload, and compromises distal tissue perfusion (10).In this issue of JASN, London et al. (11) again advance our understanding of human arterial physiology and calciotropic hormones, highlighting the importance of adequate vitamin D nutrition to maintenance of vascular structure and function in ESRD (8). As expected (10), age and hypertension strongly contribute to risk for reduced arterial compliance, evidenced by reduced brachial artery (BA) distensibility, increased aortic pulse wave velocity (PWV), and diminished flow-mediated dilation (FMD). As a sensitive index of perturbed intimal-medial signaling, reduced FMD identifies and confirms dysfunction in arterial physiology (10). The BA distensibility relationship also is important; this assessment of arterial compliance is not altered significantly by blood's rheologic properties and, like PWV, quantifies how vascular material and geometric properties integrate to determine arterial stiffnes...

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An Osteopontin–NADPH Oxidase Signaling Cascade Promotes Pro–Matrix Metalloproteinase 9 Activation in Aortic Mesenchymal Cells

Cited by 107 publications

References 26 publications

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Thrombin-Cleaved COOH-Terminal Osteopontin Peptide Binds with Cyclophilin C to CD147 in Murine Breast Cancer Cells

Calciotropic Hormones and Arterial Physiology

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