Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)−/− mice, relative to apoE−/− mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE−/− mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE−/− mice is directly associated with altered production of macrophage proinflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE−/− mice is mediated by a shift in the balance of NO and ONOO− (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.
apolipoprotein E (apoE); insulin receptor substrate-1 (IRS-1); liver; macrophage; paraoxonase; paraoxonase 2 (PON2)
Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.
atherosclerosis; obesity; genetics
To utilize proteomic analysis to identify protein biomarkers associated with pro-inflammatory HDL in patients with active rheumatoid arthritis.
Liquid chromatography-mass spectrometry (LC-MS) was used to analyze proteins associated with immunoaffinity purified HDL from plasma of two sets of RA patients carrying distinct HDL (anti- or pro-) inflammatory properties. Proteins were fractionated by Offgel electrophoresis and analyzed by LC-MS/MS equipped with a high capacity high performance liquid chromatography chip (HPLC-Chip) incorporating C18 reverse phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate select HDL-associated proteins in a second RA cohort.
Seventy-eight proteins were identified in the HDL complexes. Twelve proteins were significantly increased in RA patients with pro-inflammatory HDL compared to RA patients with anti-inflammatory HDL. These proteins included acute phase proteins, including apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, C9). Four of the proteins associated with HDL were validated in a second RA cohort.
Pro-inflammatory HDL in patients with RA contains a significantly altered proteome including increased amounts of acute phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation from active RA with resultant loss of its anti-inflammatory function. The characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CVD in RA patients.
The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE−/− background, deletion of LXRα, but not LXRβ, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRβ on the ApoE−/− background led to an even more severe cholesterol accumulation phenotype compared to LXRα−/−ApoE−/− mice, indicating that LXRβ does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα−/−ApoE−/− and LXRβ−/−ApoE−/− mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα−/−ApoE−/− mice. Mechanistically, this defect was linked to a specific requirement for LXRα−/− in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice.
atherosclerosis; nuclear receptor; cholesterol metabolism; apoliporotein
Recent studies suggest that 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 report, 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 pro-inflammatory 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 APCmin/+ 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.
HDL; Mimetic Peptides; Colon Cancer; LPA; Cancer Therapeutics
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion.
The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides.
Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of 1O2 generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.
The threat of emerging and re-emerging viruses underscores the need to develop broad-spectrum antivirals. LJ001 is a non-cytotoxic, membrane-targeted, broad-spectrum antiviral previously reported to inhibit the entry of many lipid-enveloped viruses. Here, we delineate the molecular mechanism that underlies LJ001's antiviral activity. LJ001 generates singlet oxygen (1O2) in the membrane bilayer; 1O2-mediated lipid oxidation results in changes to the biophysical properties of the viral membrane that negatively impacts its ability to undergo virus-cell fusion. These changes are not apparent on LJ001-treated cellular membranes due to their repair by cellular lipid biosynthesis. Thus, we generated a new class of membrane-targeted broad-spectrum antivirals with improved photochemical, photophysical, and pharmacokinetic properties leading to encouraging in vivo efficacy against a lethal emerging pathogen. This study provides a mechanistic paradigm for the development of membrane-targeting broad-spectrum antivirals that target the biophysical process underlying virus-cell fusion and that exploit the difference between inert viral membranes and their biogenic cellular counterparts.
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 proinflammatory molecule, depending on the context and environment. Based on a number of recent studies, it appears that the anti- or proinflammatory 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.
ApoA-I mimetic peptides; High-density lipoprotein; Inflammation; Oxidative stress
Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and the bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAEC) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression.
Methods and Results
Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the MAPK signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally-modified LDL. HAEC in which PON2 was silenced by siRNA showed increased pro-inflammatory and UPR responses when treated with 3OC12-HSL or Ox-PAPC.
3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules such as 3OC12-HSL contribute to the pro-atherogenic effects of chronic infection. The anti-atherogenic effects of PON2 include destruction of quorum sensing molecules.
Atherosclerosis; Chronic Infection; Inflammation; Oxidative Stress; Unfolded Protein Response
TNFα may change from a stimulator of reversible activation of endothelial cells (ECs) to a killer when combined with cycloheximide (CHX). The means by which endothelial cells are destined to either the survival or the apoptotic pathways are not fully understood. We investigated the role of p38 MAPK and protein phosphatase 2A (PP2A) activation and their regulation of 4E-BP1 stability in ECs to determine whether this pathway contributes to apoptosis induced by TNFα and CHX.
Methods and Results
Apoptosis was induced in human umbilical vein ECs (HUVECs) by treating them with a combination of TNFα and cycloheximide (CHX) [TNFα/CHX]. Activation of p38 MAPK was increased in HUVECs undergoing apoptosis, which was associated with degradation of eIF4E regulator, 4E-BP1, in a p38 MAPK-dependent manner. CHX attenuated a TNFα-stimulated increase in the expression and activity of PP2A. Silencing PP2A expression with siRNA transfection mimicked CHX-sensitization, increasing HUVEC apoptosis with TNFα stimulation, suggesting a protective role for PP2A in the apoptotic process.
Our data suggest i) TNFα stimulates PP2A and that HUVECs elude apoptosis by PP2A-dependent de-phosphorylation of p38 MAPK and ii) CHX-induced inhibition of PP2A leads to maintenance of p38 activity and degradation of 4E-BP1, resulting in enhanced TNFα-induced apoptosis.
apoptosis; endothelial cell signaling; TNFα; initiation factor 4E-binding protein; protein phosphatase 2A; p38 MAPK
In mice, 4F, an apolipoprotein A-I mimetic peptide that restores HDL function, prevents diabetes-induced atherosclerosis. We sought to determine whether HDL function is impaired in type 2 diabetic (T2D) patients and whether 4F treatment improves HDL function in T2D patient plasma in vitro.
RESEARCH DESIGN AND METHODS
HDL anti-inflammatory function was determined in 93 T2D patients and 31 control subjects as the ability of test HDLs to inhibit LDL-induced monocyte chemotactic activity in human aortic endothelial cell monolayers. The HDL antioxidant properties were measured using a cell-free assay that uses dichlorofluorescein diacetate. Oxidized fatty acids in HDLs were measured by liquid chromatography–tandem mass spectrometry. In subgroups of patients and control subjects, the HDL inflammatory index was repeated after incubation with L-4F.
The HDL inflammatory index was 1.42 ± 0.29 in T2D patients and 0.70 ± 0.19 in control subjects (P < 0.001). The cell-free assay was impaired in T2D patients compared with control subjects (2.03 ± 1.35 vs. 1.60 ± 0.80, P < 0.05), and also HDL intrinsic oxidation (cell-free assay without LDL) was higher in T2D patients (1,708 ± 739 vs. 1,233 ± 601 relative fluorescence units, P < 0.001). All measured oxidized fatty acids were significantly higher in the HDLs of T2D patients. There was a significant correlation between the cell-free assay values and the content of oxidized fatty acids in HDL fractions. L-4F treatment restored the HDL inflammatory index in diabetic plasma samples (from 1.26 ± 0.17 to 0.71 ± 0.11, P < 0.001) and marginally affected it in healthy subjects (from 0.81 ± 0.16 to 0.66 ± 0.10, P < 0.05).
In patients with T2D, the content of oxidized fatty acids is increased and the anti-inflammatory and antioxidant activities of HDLs are impaired.
Reverse cholesterol transport (RCT) is a major antiatherogenic function of high density lipoprotein (HDL). In the current work, the authors evaluated whether the RCT capacity of HDL from rheumatoid arthritis (RA) patients is impaired when compared to healthy controls.
HDL was isolated from 40 patients with RA and 40 age and sex matched healthy controls. Assays of cholesterol efflux, HDL’s antioxidant function and paraoxanase-1 (PON-1) activity were performed as described previously. Plasma myeloperoxidase (MPO) activity was assessed by a commercially available assay.
Mean cholesterol efflux capacity of HDL was not significantly different between RA patients (40.2%±11.1%) and controls (39.5%±8.9%); p=0.75. However, HDL from RA patients with high disease activity measured by a disease activity score using 28 joint count (DAS28>5.1), had significantly decreased ability to promote cholesterol efflux compared to HDL from patients with very low disease activity/clinical remission (DAS28<2.6). Significant correlations were noted between cholesterol efflux and the DAS28 (r=−0.39, p=0.01) and erythrocyte sedimentation rate, (r=−0.41, p=0.0009). Higher plasma MPO activity was associated with worse HDL function (r=0.41/p=0.009 (antioxidant capacity); r=0.35, p=0.03 (efflux)). HDL’s ability to promote cholesterol efflux was modestly but significantly correlated with its antioxidant function (r=−0.34, p=0.03).
The cholesterol efflux capacity of HDL is impaired in RA patients with high disease activity and is correlated with systemic inflammation and HDL’s antioxidant capacity. Attenuation of HDL function, independent of HDL cholesterol levels, may suggest a mechanism by which active RA contributes to increased cardiovascular (CV) risk.
To determine whether altering the dietary content of ω-6 (n-6) and ω-3 (n-3) polyunsaturated fatty acids affects the growth of androgen-sensitive prostate cancer xenografts, tumor membrane fatty acid composition, and tumor cyclooxygenase-2 and prostaglandin E2 (PGE2) levels.
Individually caged male severe combined immunodeficiency mice were fed isocaloric 20% kcal fat diets with the fat derived either primarily from n-6 fatty acids (n-6 group) or with the fat consisting of n-6 and n-3 fatty acids in a ratio of 1:1 (n-3 group), and injected s.c. with Los Angeles Prostate Cancer 4 (LAPC-4) cells. Tumor volumes and mouse weights were measured weekly, caloric intake was measured 3 days per week, and tumors and serum were harvested at 8 weeks postinjection.
Tumor growth rates, final tumor volumes, and serum prostate-specific antigen levels were reduced in the n-3 group relative to the n-6 group. The n-3 group tumors had decreased proliferation (Ki67 staining) and increased apoptosis (terminal nucleotidyl transferase –mediated nick end labeling staining). In vitro proliferation of LAPC-4 cells in medium containing n-3 group serum was reduced by 22% relative to LAPC-4 cells cultured in medium containing serum from the n-6 group. The n-6/n-3 fatty acid ratios in serum and tumor membranes were lower in the n-3 group relative to the n-6 group. In addition, n-3 group tumors had decreased cyclooxygenase-2 protein and mRNA levels, an 83% reduction in PGE2 levels, and decreased vascular endothelial growth factor expression.
These results provide a sound basis for clinical trials evaluating the effect of dietary n-3 fatty acids from fish oil on tumor PGE2 and membrane fatty acid composition, and serum and tumor biomarkers of progression in men with prostate cancer.
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.
Cancer and atherosclerosis are major causes of death in western societies. Deregulated cell death is common to both diseases, with significant contribution of inflammatory processes and oxidative stress. These two form a vicious cycle and regulate cell death pathways in either direction. This raises interest in antioxidative systems. The human enzymes paraoxonase-2 (PON2) and PON3 are intracellular enzymes with established antioxidative effects and protective functions against atherosclerosis. Underlying molecular mechanisms, however, remained elusive until recently. Novel findings revealed that both enzymes locate to mitochondrial membranes where they interact with coenzyme Q10 and diminish oxidative stress. As a result, ROS-triggered mitochondrial apoptosis and cell death are reduced. From a cardiovascular standpoint, this is beneficial given that enhanced loss of vascular cells and macrophage death forms the basis for atherosclerotic plaque development. However, the same function has now been shown to raise chemotherapeutic resistance in several cancer cells. Intriguingly, PON2 as well as PON3 are frequently found upregulated in tumor samples. Here we review studies reporting PON2/PON3 deregulations in cancer, summarize most recent findings on their anti-oxidative and antiapoptotic mechanisms, and discuss how this could be used in putative future therapies to target atherosclerosis and cancer.
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.
MnSOD; apolipoprotein A-I; mimetic peptides; oxidative stress; animal models; epithelial ovarian cancer
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.
To determine the effect of the apolipoprotein A-I (ApoA-I) mimetic peptide, D-4F, on atherosclerosis development in a pre-existing diabetic condition.
RESEARCH DESIGN AND METHODS
We induced hyperglycemia in 6-week-old apoE−/− female mice using streptozotocin. Half of the diabetic apoE−/− mice received D-4F in drinking water. Ten weeks later, plasma lipids, glucose, insulin levels, atherosclerotic lesions, and lesion macrophage content were measured.
Diabetic apoE−/− mice developed ∼300% more lesion area, marked dyslipidemia, increased glucose levels, and reduced plasma insulin levels when compared with nondiabetic apoE−/− mice. Atherosclerotic lesions were significantly reduced in the D-4F–treated diabetic apoE−/− mice in whole aorta (1.11 ± 0.73 vs. 0.58 ± 0.44, percentage of whole aorta, P < 0.01) and in aortic roots (36,038 ± 18,467 μm2/section vs. 17,998 ± 12,491 μm2/section, P < 0.01) when compared with diabetic apoE−/− mice that did not receive D-4F. Macrophage content in atherosclerotic lesions from D-4F–treated diabetic apoE−/− mice was significantly reduced when compared with nontreated animals (78.03 ± 26.1 vs. 29.6 ± 15.2 P < 0.001, percentage of whole plaque). There were no differences in glucose, insulin, total cholesterol, HDL cholesterol, and triglyceride levels between the two groups. Arachidonic acid, PGE2, PGD2, 15-HETE, 12-HETE, and 13-HODE concentrations were significantly increased in the liver tissue of diabetic apoE−/− mice compared with nondiabetic apoE−/− mice and significantly reduced by D-4F treatment.
Our results suggest that oral D-4F can prevent atherosclerosis development in pre-existing diabetic mice and this is associated with a reduction in hepatic arachidonic acid and oxidized fatty acid levels.
We previously reported that mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is necessary for oxidized phospholipids to induce monocyte chemoattractant protein-1 (MCP-1) secretion by human aortic endothelial cells. We also reported that inhibition of tyrosine phosphatases including MKP-1 ameliorated atherosclerotic lesions in mouse models of atherosclerosis.
This study was conducted to further investigate the specific role of MKP-1 in atherogenesis.
Methods and Results
We generated MKP-1−/−/apoE−/− double-knockout mice. At 24 weeks of age, the size, macrophage and dendritic cell content of atherosclerotic lesions of the aortic root were significantly lower (~-41% for lesions and macropahges, and ~-78% for dendritic cells) in MKP-1−/−/apoE−/− mice when compared with apoE−/− mice. Total cholesterol (−18.4%, p=0.045) and very low-density lipoprotein (VLDL)/ low-density lipoprotein (LDL) (-20.0%, p=0.052) cholesterol levels were decreased in MKP-1−/−/apoE−/− mice. Serum from MKP-1−/−/apoE−/− mice contained significantly lower levels of MCP-1 and possessed significantly reduced capability to induce monocyte migration in vitro. Moreover, peritoneal macrophages isolated from MKP-1−/−/apoE−/− mice produced significantly lower levels of MCP-1 when compared to peritoneal macrophages from apoE−/− mice. Furthermore, MKP-1−/−/apoE−/− mice had significantly reduced serum hydroxyeicosatetraenoic acids (HETEs) levels, which have been reported to induce MCP-1 levels.
Our results demonstrate that MKP-1 deficiency significantly decreases atherosclerotic lesion development in mice, in part, by affecting MCP-1 levels in the circulation and MCP-1 production by macrophages. MKP-1 may serve as a potential therapeutic target for the treatment of atherosclerotic disease.
mitogen-activated protein kinase phosphatase-1; atherosclerosis; monocyte chemoattractant protein-1; monocytes
Vascular networks within a living organism are complex, multi-dimensional, and challenging to image capture. Radio-angiographic studies in live animals require a high level of infrastructure and technical investment in order to administer costly perfusion mediums whose signals metabolize and degrade relatively rapidly, diminishing within a few hours or days. Additionally, live animal specimens must not be subject to long duration scans, which can cause high levels of radiation exposure to the specimen, limiting the quality of images that can be captured. Lastly, despite technological advances in live-animal specimen imaging, it is quite difficult to minimize or prevent movement of a live animal, which can cause motion artifacts in the final data output. It is demonstrated here that through the use of postmortem perfusion protocols of radiopaque silicone polymer mediums and ex-vivo organ harvest, it is possible to acquire a high level of vascular signal in preclinical specimens through the use of micro-computed tomographic (microCT) imaging. Additionally, utilizing high-order rendering algorithms, it is possible to further derive vessel morphometrics for qualitative and quantitative analysis.
To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants. L-4F differentially alters plasma levels of oxidized fatty acids in mice and the resistance of C3H/HeJ mice to atherosclerosis may be mediated by a reduced reaction of this strain to these potent lipid oxidants.
HETE; HODE; HPODE; EET; apoA-I mimetic peptides; L-4F; arachidonic acid metabolism
The role of myeloid cell cyclooxygenase-2 (COX-2) in the progression of atherosclerosis has not been clearly defined.
Methods and Results
We investigated the role of COX-2 expressed in the myeloid lineage in the development of atherosclerosis using a myeloid-specific COX-2−/− (COX-2−M/−M) mouse on a hyperlipidemic apoE−/− background (COX-2−M/−M/apoE−/−). Myeloid COX-2 depletion resulted in significant attenuation of acute inflammation corresponding with decreased PGE2 levels in an air pouch model. COX-2 depletion in myeloid cells did not influence development of atherosclerosis in COX-2−M/−M/apoE−/− when compared to apoE−/− littermates on either chow or western diets. The unanticipated lack of contribution of myeloid COX-2 to the development atherosclerosis is not due to altered maintenance, differentiation, or mobilization of myeloid and lymphoid populations. Moreover, myeloid COX-2 depletion resulted in unaltered serum prostanoid levels and cellular composition of atherosclerotic lesions of COX-2−M/−M/apoE−/− mice.
Our results suggest that COX-2 expression in myeloid cells, including macrophages, does not influence the development of atherosclerosis in mice.
Atherosclerosis; Cyclooxygenase-2; Myeloid cells; Inflammation; Prostanoids
HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have anti-inflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents.
Hyperlipidemia is associated with platelet hyper-reactivity. We hypothesized that L-4F, an apoA-I mimetic peptide, would inhibit platelet aggregation in hyperlipidemic mice.
Methods and Results
Injecting L-4F into apoE null and LDL receptor null mice resulted in a significant reduction in platelet aggregation in response to agonists but there was no reduction in platelet aggregation after injection of L-4F into wild-type (WT) mice. Consistent with these results, injection of L-4F into apoE null mice prolonged bleeding time but not in WT mice. Incubating L-4F in vitro with apoE null platelet rich plasma also resulted in decreased platelet aggregation. However, incubating washed platelets from either apoE null or WT mice with L-4F did not alter aggregation. Compared to wild-type mice, unstimulated platelets from apoE null mice contained significantly more 12-HETE, thromboxane A2 (TXA2), prostaglandins D2 (PGD2) and E2 (PGE2). In response to agonists, platelets from L-4F treated apoE null mice formed significantly less TXA2, PGD2 PGE2, and 12-HETE.
By binding plasma oxidized lipids that cause platelet hyper-reactivity in hyperlipidemic mice, L-4F decreases platelet aggregation.
Platelets; apoA-I mimetic peptides; L-4F; Arachidonic acid metabolism; apoE null mice
Increased production of reactive oxygen species (ROS) as a result of decreased activities of mitochondrial electron transport chain (ETC) complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. Our previous studies established that paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. The aim of the present study was to determine the mechanism by which PON2 modulates ROS production. In this report, we demonstrate that PON2-def mice on the hyperlipidemic apolipoprotein E−/− background (PON2-def/apolipoprotein E−/−) develop exacerbated atherosclerotic lesions with enhanced mitochondrial oxidative stress. We show that PON2 protein is localized to the inner mitochondrial membrane, where it is found associated with respiratory complex III. Employing surface-plasmon-resonance, we demonstrate that PON2 binds with high affinity to coenzyme Q10, an important component of the ETC. Enhanced mitochondrial oxidative stress in PON2-def mice was accompanied by significantly reduced ETC complex I + III activities, oxygen consumption, and adenosine triphosphate levels in PON2-def mice. In contrast, overexpression of PON2 effectively protected mitochondria from antimycin- or oligomycin-mediated mitochondrial dysfunction. Our results illustrate that the antiatherogenic effects of PON2 are, in part, mediated by the role of PON2 in mitochondrial function. Antioxid. Redox Signal. 14, 341–351.