Nox2 Deficiency Reduces Cartilage Damage and Ectopic Bone Formation in an Experimental Model for Osteoarthritis

Osteoarthritis (OA) is a destructive disease of the joint with age and obesity being its most important risk factors. Around 50% of OA patients suffer from inflammation of the synovial joint capsule, which is characterized by increased abundance and activation of synovial macrophages that produce reactive oxygen species (ROS) via NADPH-oxidase 2 (NOX2). Both ROS and high blood levels of low-density lipoprotein (LDL) are implicated in OA pathophysiology, which may interact to form oxidized LDL (oxLDL) and thereby promote disease. Therefore, targeting NOX2 could be a viable treatment strategy for OA. Collagenase-induced OA (CiOA) was used to compare pathology between wild-type (WT) and Nox2 knockout (Nox2−/−) C57Bl/6 mice. Mice were either fed a standard diet or Western diet (WD) to study a possible interaction between NOX2-derived ROS and LDL. Synovial inflammation, cartilage damage and ectopic bone size were assessed on histology. Extracellular ROS production by macrophages was measured in vitro using the Amplex Red assay. Nox2−/− macrophages produced basal levels of ROS but were unable to increase ROS production in response to the alarmin S100A8 or the phorbol ester PMA. Interestingly, Nox2 deficiency reduced cartilage damage, synovial lining thickness and ectopic bone size, whereas these disease parameters were not affected by WD-feeding. These results suggest that NOX2-derived ROS are involved in CiOA development.

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Accumulation of LDL/ox-LDL in the necrotic region participates in osteonecrosis of the femoral head: a pathological and in vitro study

Lipids in Health and Disease ◽

10.1186/s12944-021-01601-x ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Xin-Yuan Wang ◽

Tian-Le Ma ◽

Kang-Ning Chen ◽

Zhi-Ying Pang ◽

Hao Wang ◽

...

Keyword(s):

Femoral Head ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

In Vitro Study ◽

Long Bone ◽

Blood Levels ◽

Necrotic Region

Abstract Background Osteonecrosis of the femoral head (ONFH) is a common but intractable disease that appears to involve lipid metabolic disorders. Although numerous studies have demonstrated that high blood levels of low-density lipoprotein (LDL) are closely associated with ONFH, there is limited evidence to explain the pathological role of LDL. Pathological and in vitro studies were performed to investigate the role of disordered metabolism of LDL and oxidized LDL (ox-LDL) in the femoral head in the pathology of ONFH. Methods Nineteen femoral head specimens from patients with ONFH were obtained for immunohistochemistry analysis. Murine long-bone osteocyte Y4 cells were used to study the effects of LDL/ox-LDL on cell viability, apoptosis, and metabolism process of LDL/ox-LDL in osteocytes in normoxic and hypoxic environments. Results In the pathological specimens, marked accumulation of LDL/ox-LDL was observed in osteocytes/lacunae of necrotic regions compared with healthy regions. In vitro studies showed that ox-LDL, rather than LDL, reduced the viability and enhanced apoptosis of osteocytes. Pathological sections indicated that the accumulation of ox-LDL was significantly associated with impaired blood supply. Exposure to a hypoxic environment appeared to be a key factor leading to LDL/ox-LDL accumulation by enhancing internalisation and oxidation of LDL in osteocytes. Conclusions The accumulation of LDL/ox-LDL in the necrotic region may contribute to the pathology of ONFH. These findings could provide new insights into the prevention and treatment of ONFH.

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Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.1.68-75.2005 ◽

2005 ◽

Vol 12 (1) ◽

pp. 68-75 ◽

Cited By ~ 27

Author(s):

Gabriel Virella ◽

M. Brooks Derrick ◽

Virginia Pate ◽

Charlyne Chassereau ◽

Suzanne R. Thorpe ◽

...

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Gas Chromatography Mass Spectrometry ◽

Low Density ◽

Modified Ldl ◽

Capture Assay ◽

Carboxymethyl Lysine

ABSTRACT Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N ε(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.

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Oxidative stress augments pulmonary hypertension in chronically hypoxic mice overexpressing the oxidized LDL receptor

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00169.2012 ◽

2013 ◽

Vol 305 (2) ◽

pp. H155-H162 ◽

Cited By ~ 24

Author(s):

Sayoko Ogura ◽

Tatsuo Shimosawa ◽

ShengYu Mu ◽

Takashi Sonobe ◽

Fumiko Kawakami-Mori ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Vascular Remodeling ◽

Chronic Hypoxia ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Ros Generation ◽

Low Density ◽

Ros Production ◽

Oxidized Low Density Lipoprotein

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

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Vascular responses and ion exchange in diabetes: Authors' reply

Clinical Science ◽

10.1042/cs0820339a ◽

1992 ◽

Vol 82 (3) ◽

pp. 339-339

Author(s):

J. M. Ritter ◽

G. C. Viberti

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Large Body ◽

Plasma Lipid ◽

Density Lipoprotein ◽

High Plasma ◽

Diabetic Patients ◽

Vascular Sensitivity

1. Na+/Li+ countertransport is not a gold standard, or indeed any other kind of standard. It is a measure of the activity of one particular cation exchanger. 2. There is a large body of literature regarding the effects of oxidized low-density lipoprotein (LDL) in experimental animals and in vitro. Whether abnormal oxidized LDL or one of many other possible mechanisms underlies the inverse relationship that we observed between vascular sensitivity in vivo to nitroprusside or carbachol with erythrocyte Na+/Li+ countertransport in diabetic patients remains to be seen. 3. We caution against post hoc subgroup analysis (smokers versus non-smokers, low versus high plasma lipid levels, etc.) in studies of this size.

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Gemfibrozil metabolite inhibits in vitro low-density lipoprotein (LDL) oxidation and diminishes cytotoxicity induced by oxidized LDL

Metabolism ◽

10.1016/s0026-0495(00)80012-8 ◽

2000 ◽

Vol 49 (4) ◽

pp. 479-485 ◽

Cited By ~ 4

Author(s):

Mitsunobu Kawamura ◽

Shigeru Miyazaki ◽

Tamio Teramoto ◽

Keiko Ashidate ◽

Hisako Thoda ◽

...

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Ldl Oxidation ◽

Low Density

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Oxidized low density lipoprotein inhibits the migration of aortic endothelial cells in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.120.4.1011 ◽

1993 ◽

Vol 120 (4) ◽

pp. 1011-1019 ◽

Cited By ~ 78

Author(s):

G Murugesan ◽

G M Chisolm ◽

P L Fox

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Lipid Hydroperoxide ◽

Density Lipoprotein ◽

Low Density ◽

Dependent Manner ◽

Oxidized Low Density Lipoprotein ◽

Inhibitor Molecule ◽

Healing Response

Endothelial cell (EC) migration is a critical and initiating event in the formation of new blood vessels and in the repair of injured vessels. Compelling evidence suggests that oxidized low density lipoprotein (LDL) is present in atherosclerotic lesions, but its role in lesion formation has not been defined. We have examined the role of oxidized LDL in regulating the wound-healing response of vascular EC in vitro. Confluent cultures of bovine aortic EC were "wounded" with a razor, and migration was measured after 18 to 24 h as the number of cells moving into the wounded area and the mean distance of cells from the wound edge. Oxidized LDL markedly reduced migration in a concentration- and oxidation-dependent manner. Native LDL or oxidized LDL with a thiobarbituric acid (TBA) reactivity < 5 nmol malondialdehyde equivalents/mg cholesterol was not inhibitory; however, oxidized LDL with a TBA reactivity of 8-12 inhibited migration by 75-100%. Inhibition was half-maximal at 250-300 micrograms cholesterol/ml and nearly complete at 350-400 micrograms/ml. The antimigratory activity was not due to cell death since it was completely reversed 16 h after removal of the lipoprotein. The inhibitor molecule was shown to be a lipid; organic solvent extracts of oxidized LDL inhibited migration to nearly the same extent as the intact particle. When LDL was variably oxidized by dialysis against FeSO4 or CuSO4, or by UV irradiation, the inhibitory activity correlated with TBA reactivity and total lipid peroxides, but not with electrophoretic mobility or fluorescence (360 ex/430 em). This indicates that a lipid hydroperoxide may be the active species. These results suggest the possibility that oxidized LDL may limit the healing response of the endothelium after injury.

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Scavenger Receptors Class A-I/II and CD36 Are the Principal Receptors Responsible for the Uptake of Modified Low Density Lipoprotein Leading to Lipid Loading in Macrophages

Journal of Biological Chemistry ◽

10.1074/jbc.m209649200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49982-49988 ◽

Cited By ~ 613

Author(s):

Vidya V. Kunjathoor ◽

Maria Febbraio ◽

Eugene A. Podrez ◽

Kathryn J. Moore ◽

Lorna Andersson ◽

...

Keyword(s):

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Gas Chromatography Mass Spectrometry ◽

Scavenger Receptors ◽

Low Density ◽

Lipid Uptake ◽

Modified Ldl ◽

Ldl Uptake

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptakein vitroand atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75–90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

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The Ability of Different Ketohexoses to Alter Apo-A-I Structure and Function In Vitro and to Induce Hepatosteatosis, Oxidative Stress, and Impaired Plasma Lipid Profile in Hyperlipidemic Zebrafish

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/3124364 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Dhananjay Yadav ◽

Suk-Jeong Kim ◽

Myung Ae Bae ◽

Jae-Ryong Kim ◽

Kyung-Hyun Cho

Keyword(s):

Zebrafish Embryo ◽

Antioxidant Activities ◽

Cell Model ◽

Oxidized Ldl ◽

Plasma Lipid ◽

Density Lipoprotein ◽

Ldl Oxidation ◽

Zebrafish Model ◽

Oil Red O

In the current study, we have tested the nonenzymatic glycation activities of ketohexoses, such as tagatose and psicose. Although tagatose-treated apoA-I (t-A-I) and psicose-treated apoA-I (p-A-I) exerted more inhibitory activity you cupric ion-mediated low-density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL) phagocytosis into macrophage than fructose-treated apoA-I (f-A-I). In the lipid-free state, t-A-I and f-A-I showed more multimerized band without crosslinking. Since t-A-I lost its phospholipid binding ability, the rHDL formation was not as successful as f-A-I. However, injecting t-A-I showed more antioxidant activities in zebrafish embryo under the presence of oxLDL. Three weeks of consumption of fructose (50% of wt in Tetrabit/4% cholesterol) showed a 14% elevation of serum triacylglycerol (TG), while tagatose-administered group showed 30% reduction in serum TG compared to high cholesterol control. Fructose-fed group showed the biggest area of Oil Red O staining with the intensity as strong as the HCD control. However, tagatose-consumed group showed much lesser Oil Red O-stained area with the reduction of lipid accumulation. In conclusion, although tagatose treatment caused modification of apoA-I, the functional loss was not as much severe as the fructose treatment in macrophage cell model, zebrafish embryo, and hypercholesterolemic zebrafish model.

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Levels of Oxidized LDL, Estrogens, and Progesterone in Placenta Tissues and Serum Paraoxonase Activity in Preeclampsia

Mediators of Inflammation ◽

10.1155/2013/862982 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 19

Author(s):

Şerefden Açıkgöz ◽

Ülkü Özmen Bayar ◽

Murat Can ◽

Berrak Güven ◽

Görkem Mungan ◽

...

Keyword(s):

Pregnant Women ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Trophoblast Invasion ◽

Pon1 Activity ◽

Serum Paraoxonase ◽

The Relationship ◽

Serum Pon1 Activity

In vitro literature studies have suggested that atherosclerotic oxidized low density lipoprotein (OxLDL) inhibits trophoblast invasion. The objective of this study was to determine the levels of OxLDL and to examine the relationship between antioxidative estradiol, estriol, and prooxidative progestin in normal and preeclamptic placental tissues and measure the serum activity of antioxidative paraoxonase (PON1). The study included 30 preeclamptic and 32 normal pregnant women. OxLDL was determined with ELISA, estradiol, unconjugated estriol, and progesterone that were determined with chemiluminescence method in placental tissues. Serum PON1 activity was determined with spectrophotometric method. Levels of OxLDL (), estriol (), estradiol (), and progesterone () were lower in the placental tissues of preeclamptic group compared to the normal pregnant women. Serum PON1 activity was higher in preeclamptic group () and preeclamptic group without intrauterine growth restriction () compared to normal pregnant women. Tissue estriol of preeclamptic group without/with IUGR (, ) was lower than the normal group. Results of our study suggest that the events leading to fetoplacental insufficiency lead to a reduction in the levels of estriol limit deposition of OxLDL in placental tissues. The serum PON1 activity is probably important in the inhibition of OxLDL in preeclampsia.

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Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Biochemical Journal ◽

10.1042/bj3220317 ◽

1997 ◽

Vol 322 (1) ◽

pp. 317-325 ◽

Cited By ~ 192

Author(s):

Jesús R. REQUENA ◽

Min Xin FU ◽

Mahtab U. AHMED ◽

Alicia J. JENKINS ◽

Timothy J. LYONS ◽

...

Keyword(s):

Oxidative Stress ◽

Gas Chromatography ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Thiobarbituric Acid ◽

Low Density ◽

Lysine Residues

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(Nε-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(Nε-lysino)propane (LML)] and lysine-HNE [3-(Nε-lysino)-4-hydroxynonan-1-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70Ő80% of total lysine loss during the reaction with MDA. LM and LML (0.002Ő0.12mmol/mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

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