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Arnab Chattopadhyay,
Mohamad Navab,
Greg Hough,
Feng Gao, David Meriwether,
Victor Grijalva,
James R Springstead,
Mayakonda N Palgunachari,
Ryan Namiri-Kalantari,
Feng Su,
Brian J Van Lenten,
Alan C Wagner,
G M Anantharamaiah,
Robin Farias-Eisner,
Srinivasa T Reddy,
Alan M Fogelman
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ABSTRACT: Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western Diet (WD) at 2.2% by weight, and fed to LDLR-/- mice at 45 mg/kg/day 6F. After 13 weeks, percent aorta with lesions was 4.1+/-4, 3.3+/-2.4, and 1.9+/-1.4 for WD, WD+EV, and WD+6F, respectively (WD+6F vs. WD, p=0.0134; WD+6F vs. WD+EV, p=0.0386; WD+EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD+6F mice; p<0.0295. HDL-cholesterol and paroxonase-1 activity (PON) were higher in WD+6F mice (p=0.0055, p=0.0254, respectively), but not in WD+EV mice. Plasma SAA, total cholesterol, triglycerides, LPA and 15-HETE levels positively correlated with lesions (p<0.0001); HDL-cholesterol and PON were inversely correlated (p<0.0001). After feeding WD+6F, i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared to WD+EV (p<0.0469); iii) small intestine LPA 18:2 positively correlated with percent aorta with lesions (p<0.0178). These data suggest that 6F acts in the small intestine and provide a novel approach to oral apoA-I mimetic therapy.
The Journal of Lipid Research 02/2013; · 5.56 Impact Factor
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ABSTRACT: Paraoxonase 2 deficiency (PON2-def) alters mitochondrial function and exacerbates the development of atherosclerosis in mice. PON2 overexpression protects against ER stress in cell culture. In this paper, we examined the role of PON2 in the unexplored link between ER stress and mitochondrial dysfunction and tested whether restoration of PON2 in macrophages is sufficient to reduce aggravated atherosclerosis in PON2-def/apoE(-/-) mice on a Western diet. ER stress response genes, intracellular calcium levels, and apoptotic nuclei were significantly elevated in PON2-def/apoE(-/-) macrophages compared to apoE(-/-) macrophages in response to ER stressors, but not at the basal level. In contrast, PON2-def/apoE(-/-) macrophages exhibited greater mitochondrial stress at the basal level, which was further worsened in response to ER stressors. There was no difference in ER stress response genes and apoptotic nuclei between apoE(-/-) and PON2-def/apoE(-/-) macrophages when pretreated with xestospongin (which blocks the release of calcium from ER) suggesting that PON2 modulates cell survival and ER stress by maintaining calcium homeostasis. Treatment with a mitochondrial calcium uptake inhibitor, RU360, attenuated ER stressor mediated mitochondrial dysfunction in PON2-def/apoE(-/-) macrophages. CHOP expression (ER stress marker) and apoptotic nuclei were significantly higher in aortic lesions of PON2-def/apoE(-/-) mice compared to apoE(-/-) mice fed a Western diet. Restoration of PON2 in macrophages reduced ER stress, mitochondrial dysfunction and apoptosis in response to ER stressors. Furthermore, restoration of PON2 in macrophages reduced lesional apoptosis and atherosclerosis in PON2-def/apoE(-/-) mice on a Western diet. Our data suggest that macrophage PON2 modulates mechanisms that link ER stress, mitochondrial dysfunction and the development of atherosclerosis.
Molecular Genetics and Metabolism 07/2012; · 3.19 Impact Factor
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ABSTRACT: Fluorescence-based cell-free assays offer an attractive alternative to current cell-based assays for measuring the redox activity of High-Density Lipoprotein (HDL). We have recently developed a biochemical assay that assesses the effect of HDL on the oxidation rate of dihydrorhodamine 123 (DHR), reflected by increasing fluorescence over time. However, an immediate reduction in the fluorescence signal is observed after addition of HDL to DHR, due to fluorescence quenching from lipid-probe interactions. Understanding this process is important for interpretation of the results of all fluorescence-based cell-free assays that measure oxidative properties of lipids.
We determined the effect of quenchers (proteins or lipids) on the fluorescence signal of two fluorescence-based cell-free assays: the rhodamine 123 (RHD)-based assay, and a previously described assay based on dichlorodihydrofluorescein (DCF) in patients with systemic inflammation or atherosclerosis versus healthy subjects.
We found lipid-probe interactions between the non-fluorescent substrate and the lipid, which affect the observed rate of change of fluorescence after addition of lipids to DHR and DCFH. These interactions depended on: sample collection and storage, types and concentrations of lipid and fluorescent probe, method of HDL isolation, diluents and matrices, and pH. The RHD-based assay yielded reproducible measurements despite fluorescence quenching, while the DCF-based assay displayed more experimental variability. Furthermore, the lipid-probe interactions varied according to the setting of systemic inflammation when using apolipoprotein (apo) B-depleted plasma. However, under fixed conditions the rhodamine assay could reliably detect similar mean relative differences in the redox activity of HDL samples between different groups of patients using either purified HDL or apo-B depleted plasma.
Lipid-probe interactions should be considered when interpreting the results of fluorescence assays for measuring lipid oxidative state. Ideally, samples should be freshly obtained and purified HDL should be utilized rather than Apo B-depleted serum. Assay variability can be reduced by strict standardization of conditions (particularly sample collection, storage, lipid isolation method). Data comparisons between different studies similarly require strict standardization of conditions between studies and this caveat must be considered when using these assays to study the role of HDL function in the development of atherosclerosis in vivo.
Lipids in Health and Disease 07/2012; 11:87. · 2.17 Impact Factor
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ABSTRACT: Recent studies suggest that high-density lipoprotein (HDL) levels are inversely related to colon cancer risk. HDL mimetics constructed from a number of peptides and proteins with varying structures possess anti-inflammatory and antioxidant properties reminiscent of HDL. In this article, we examined whether HDL mimetics, L-4F (an apolipoprotein A-I mimetic peptide) and G* (an apolipoprotein J mimetic peptide) affect tumor growth and development in mouse models of colon cancer. HDL mimetics reduced viability and proliferation of CT26 cells, a mouse colon adenocarcinoma cell line, and decreased CT26 cell-mediated tumor burden in BALB/c mice when administered subcutaneously or orally. Plasma levels of lysophosphatidic acid (LPA), a serum biomarker for colon cancer, were significantly reduced in mice that received HDL mimetics, suggesting that binding and removal of proinflammatory lipids is a potential mechanism for the inhibition of tumor development by HDL mimetics. Furthermore, L-4F significantly reduced size and number of polyps in APC(min/+) mice, a mouse model for human familial adenomatous polyposis, suggesting that HDL mimetics are effective in inhibiting the development of both induced and spontaneous cancers of the colon. Our results, for the first time, identify HDL mimetics as a novel therapeutic strategy for the treatment of colon cancer.
Molecular Cancer Therapeutics 03/2012; 11(6):1311-9. · 5.23 Impact Factor
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ABSTRACT: Most current assays of HDL functional properties are cell-based. We have developed a fluorometric biochemical assay based on the oxidation of dihydrorhodamine 123 (DHR) by HDL. This cell-free assay assesses the intrinsic ability of HDL to be oxidized by measuring increasing fluorescence due to DHR oxidation over time. The assay distinguishes the oxidative potential of HDL taken from different persons, and the results are reproducible. Direct comparison of this measurement correlated well with results obtained using a validated cell-based assay (r(2) = 0.62, P < 0.001). The assay can be scaled from a 96-well format to a 384-well format and, therefore, is suitable for high-throughput implementation. This new fluorometric method offers an inexpensive, accurate, and rapid means for determining the oxidative properties of HDL that is applicable to large-scale clinical studies.
The Journal of Lipid Research 09/2011; 52(12):2341-51. · 5.56 Impact Factor
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David Meriwether,
Satoshi Imaizumi,
Victor Grijalva,
Greg Hough,
Ladan Vakili,
G M Anantharamaiah,
Robin Farias-Eisner,
Mohamad Navab,
Alan M Fogelman,
Srinivasa T Reddy,
Ishaiahu Shechter
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ABSTRACT: The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.
The Journal of Lipid Research 07/2011; 52(10):1795-809. · 5.56 Impact Factor
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Satoshi Imaizumi,
Mohamad Navab,
Cecilia Morgantini,
Christina Charles-Schoeman,
Feng Su,
Feng Gao,
Murray Kwon,
Ekambaram Ganapathy, David Meriwether,
Robin Farias-Eisner,
Alan M Fogelman,
Srinivasa T Reddy
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ABSTRACT: Although high-density lipoprotein-cholesterol (HDL-C) levels in large epidemiological studies are inversely related to the risk of coronary heart disease (CHD), increasing the level of circulating HDL-C does not necessarily decrease the risk of CHD events, CHD deaths, or mortality. HDL can act as an anti- or a pro-inflammatory molecule, depending on the context and environment. Based on a number of recent studies, it appears that the anti- or pro-inflammatory nature of HDL may be a more sensitive indicator of the presence or absence of atherosclerosis than HDL-C levels. The HDL proteome has been suggested to be a marker, and perhaps a mediator, of CHD. Apolipoprotein A-1 (apoA-I), the major protein in HDL is a selective target for oxidation by myeloperoxidase, which results in impaired HDL function. Improving HDL function through modification of its lipid and/or protein content maybe a therapeutic target for the treatment of CHD and many inflammatory disorders. HDL/apoA-I mimetic peptides may have the ability to modify the lipid and protein content of HDL and convert dysfunctional HDL to functional HDL. This review focuses on recent studies of dysfunctional HDL in animal models and human disease, and the potential of apoA-I mimetic peptides to normalize the composition and function of lipoproteins.
Circulation Journal 06/2011; 75(7):1533-8. · 3.77 Impact Factor
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Ekambaram Ganapathy,
Feng Su, David Meriwether,
Asokan Devarajan,
Victor Grijalva,
Feng Gao,
Arnab Chattopadhyay,
G M Anantharamaiah,
Mohamad Navab,
Alan M Fogelman,
Srinivasa T Reddy,
Robin Farias-Eisner
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ABSTRACT: We recently reported that apoA-I and apoA-I mimetic peptides prevent the development of flank tumors in immunocompetent C57BL/6J mice. To delineate the mechanism(s) of action of apoA-I mimetic peptides in tumor development, we examined the effect of D-4F (an apoA-I mimetic peptide) on the antioxidant status and on the gene expression and function of antioxidant enzymes in ID8 cells (a mouse epithelial ovarian cancer cell line) and in a mouse model. We demonstrate that D-4F treatment significantly reduces the viability and proliferation of ID8 cells, with a concomitant improvement of the antioxidant status of ID8 cells as measured by lipid peroxidation, protein carbonyl, superoxide anion, and hydrogen peroxide levels. D-4F treatment induces MnSOD (but not CuZnSOD) mRNA, protein, and activity. Inhibition of MnSOD in ID8 cells using shRNA vectors abrogates the inhibitory effects of D-4F on ID8 cell viability and proliferation. Moreover, tumor development from ID8 cells carrying shRNA for MnSOD were unaffected by D-4F treatment. Our results suggest that the inhibitory effects of D-4F on ID8 cell proliferation and tumor development are mediated, at least in part, by the induced expression and activity of MnSOD.
International Journal of Cancer 03/2011; 130(5):1071-81. · 5.44 Impact Factor
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Feng Gao,
Sergio X Vasquez,
Feng Su,
Svetlana Roberts,
Neha Shah,
Victor Grijalva,
Satoshi Imaizumi,
Arnab Chattopadhyay,
Ekambaram Ganapathy, David Meriwether,
Brad Johnston,
G M Anantharamaiah,
Mohamad Navab,
Alan M Fogelman,
Srinivasa T Reddy,
Robin Farias-Eisner
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ABSTRACT: We recently reported that apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides inhibit tumor growth and improve survival in a mouse model of ovarian cancer. The current study was designed to examine whether inhibition of angiogenesis is one of the mechanisms for the observed anti-tumorigenic effects. The apoA-I mimetic peptide L-5F had no affect on proliferation and cell viability of human umbilical vascular endothelial cells (HUVECs) in the basal state; however, treatment with L-5F at 1, 3, and 10 μg ml(-1), dose-dependently inhibited both vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced proliferation, cell viability, migration, invasion and tube formation in HUVECs. L-5F inhibited VEGF- and bFGF-induced activation of their corresponding receptors, VEGFR2 and FGFR1, as well as downstream signaling pathways, including Akt and ERK1/2. MicroCT scanning and immunohistochemistry staining demonstrated that daily injection of L-5F (10 mg kg(-1)) decreased both the quantity and size of tumor vessels in mice. L-5F treated mice showed significantly reduced levels of VEGF in both tumor tissue and the circulation, which is consistent with in vitro data showing that L-5F inhibited production and secretion of VEGF from mouse and human ovarian cell lines in the absence and presence of exogenously added lysophosphatidic acid, a potent tumor promoter. In conclusion, our data that L-5F inhibits angiogenesis suggests that apoA-I mimetic peptides may serve as novel anti-angiogenesis agents for the treatment of angiogenesis-associated diseases, including cancer.
Integrative Biology 02/2011; 3(4):479-89. · 4.51 Impact Factor
