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1.  Apolipoprotein A-I Mimetics 
Current opinion in lipidology  2014;25(4):304-308.
Purpose of Review
To summarize recent publications in the field of apolipoprotein mimetics.
Recent Findings
Apolipoprotein mimetic peptides continue to show efficacy in a number of animal models of disease and demonstrate properties that make them attractive as potential therapeutic agents. A number of new apolipoprotein mimetics have been described recently. A major site of action of apolipoprotein mimetic peptides was found to be in the small intestine where they decrease the levels of pro-inflammatory bioactive lipids. A major problem related to the use of apolipoprotein mimetic peptides is their cost, particularly those that need to be generated by solid phase synthesis with chemical addition of end blocking groups. Novel approaches to apolipoprotein mimetic therapy have emerged recently that show promise in overcoming these barriers.
Despite the recent failure of therapies designed to raise HDL-cholesterol in humans, an approach to therapy using mimetics of HDL and its components continues to show promise.
PMCID: PMC4213064  PMID: 24977978
Apolipoproteins; Apolipoprotein mimetics; HDL; Atherosclerosis; Cancer
2.  Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer 
We previously reported that adding freeze-dried tomato powder from transgenic plants expressing the apolipoprotein A-I mimetic peptide 6F at 2.2% by weight to a Western diet (WD) ameliorated dyslipidemia and atherosclerosis in mice. The same dose in a human would require three cups of tomato powder three times daily. To reduce the volume, we sought a method to concentrate 6F. Remarkably, extracting the transgenic freeze-dried tomato overnight in ethyl acetate with 5% acetic acid resulted in a 37-fold reduction in the amount of transgenic tomato needed for biologic activity. In a mouse model of dyslipidemia, adding 0.06% by weight of the tomato concentrate expressing the 6F peptide (Tg6F) to a WD significantly reduced plasma total cholesterol and triglycerides (P < 0.0065). In a mouse model of colon cancer metastatic to the lungs, adding 0.06% of Tg6F, but not a control tomato concentrate (EV), to standard mouse chow reduced tumor-associated neutrophils by 94 ± 1.1% (P = 0.0052), and reduced tumor burden by two-thirds (P = 0.0371). Adding 0.06% of either EV or Tg6F by weight to standard mouse chow significantly reduced tumor burden in a mouse model of ovarian cancer; however, Tg6F was significantly more effective (35% reduction for EV vs. 53% reduction for Tg6F; P = 0.0069). Providing the same dose of tomato concentrate to humans would require only two tablespoons three times daily making this a practical approach for testing oral apoA-I mimetic therapy in the treatment of dyslipidemia and cancer.
PMCID: PMC4492730  PMID: 26171234
ApoA-I mimetic peptides; cancer; drug delivery; lipids; transgenic tomatoes
3.  Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression 
Circulation research  2003;93(12):1225-1232.
Shear stress regulates endothelial nitric oxide and superoxide (O2−·) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 μg/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress (τave) of 25 dyne/cm2 and (2) oscillating flow at τave of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL− and LDL2−. In response to oscillatory shear stress, gp91phox mRNA expression was upregulated by 2.9±0.3-fold, and its homologue, Nox4, by 3.9±0.9-fold (P<0.05, n=4), with a corresponding increase in O2−· production rate. The proportion of LDL− and LDL2− relative to static conditions increased by 67±17% and 30±7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (P<0.05, n=5). In contrast, pulsatile flow downregulated both gp91phox and Nox4 mRNA expression (by 1.8±0.2-fold and 3.0±0.12-fold, respectively), with an accompanying reduction in O2−· production, reduction in the extent of LDL modification (51±12% for LDL− and 30±7% for LDL2−), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O2−· production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.
PMCID: PMC4433384  PMID: 14593003
shear stress; NADPH oxidase; LDL oxidation; Nox4; gp91phox
5.  Monocyte recruitment to endothelial cells in response to oscillatory shear stress 
Leukocyte recruitment to endothelial cells is a critical event in inflammatory responses. The spatial, temporal gradients of shear stress, topology, and outcome of cellular interactions that underlie these responses have so far been inferred from static imaging of tissue sections or studies of statically cultured cells. In this report, we developed micro-electromechanical systems (MEMS) sensors, comparable to a single endothelial cell (EC) in size, to link real-time shear stress with monocyte/EC binding kinetics in a complex flow environment, simulating the moving and unsteady separation point at the arterial bifurcation with high spatial and temporal resolution. In response to oscillatory shear stress (τ) at ± 2.6 dyn/cm2 at a time-averaged shear stress (τave) = 0 and 0.5 Hz, individual monocytes displayed unique to-and-fro trajectories undergoing rolling, binding, and dissociation with other monocyte, followed by solid adhesion on EC. Our study quantified individual monocyte/EC binding kinetics in terms of displacement and velocity profiles. Oscillatory flow induces up-regulation of adhesion molecules and cytokines to mediate monocyte/EC interactions over a dynamic range of shear stress ± 2.6 dyn/cm2 (P= 0.50, n= 10).—Hsiai, T. K., Cho, S. K., Wong, P. K., Ing, M., Salazar, A., Sevanian, A., Navab, M., Demer, L. L., Ho, C.-M. Monocyte recruitment to endothelial cells in response to oscillatory shear stress. FASEB J. 17, 1648–1657 (2003)
PMCID: PMC4108745  PMID: 12958171
micro-electromechanical systems (MEMS); cell tracking velocimetry; shear stress sensors; endothelial cells; monocytes
6.  Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS 
Exposure to airborne particulate pollutants is intimately linked to vascular oxidative stress and inflammatory responses with clinical relevance to atherosclerosis. Particulate matter (PM) has been reported to induce endothelial dysfunction and atherosclerosis. Here, we tested whether ambient ultrafine particles (UFP, diameter < 200 nm) modulate eNOS activity in terms of nitric oxide (NO) production via protein S-glutathionylation. Treatment of human aortic endothelial cells (HAEC) with UFP significantly reduced NO production. UFP-mediated reduction in NO production was restored in the presence of JNK inhibitor (SP600125), NADPH oxidase inhibitor (Apocynin), anti-oxidant (N-acetyl cysteine), and superoxide dismutase mimetics (Tempol and MnTMPyP). UFP exposure increased the GSSG/GSH ratio and eNOS S-glutathionylation, whereas over-expression of Glutaredoxin-1 (to inhibit S-glutathionylation) restored UFP-mediated reduction in NO production by nearly 80%. Thus, our findings suggest that eNOS S-glutathionylation is a potential mechanism underlying ambient UFP-induced reduction of NO production.
PMCID: PMC3743434  PMID: 23751346
Ultrafine Particles/UFP; Oxidative Stress; eNOS; S-Glutathionylation; Endothelial dysfunction; Air Pollution
7.  High-Density Lipoprotein and 4F Peptide Reduce Systemic Inflammation by Modulating Intestinal Oxidized Lipid Metabolism 
Oxidized phospholipids are found in the vasculature of animal models of atherosclerosis, in human atherosclerotic lesions, and in other inflammatory diseases. Oxidized phospholipids cause vascular and nonvascular cells to initiate an inflammatory reaction. Metabolites of arachidonic acid, such as 12-hydroxyeicosatetraenoic acid, can mimic some of the inflammatory properties of oxidized phospholipids. In vitro and in vivo normal high-density lipoprotein (HDL), normal apolipoprotein A-I, and apolipoprotein A-I mimetic peptides, each likely acting in a different manner, prevent the inflammatory reaction characteristic of atherosclerosis, and this is associated with decreased levels of oxidized lipids in tissues and cells. HDL from animal models of atherosclerosis or from humans with atherosclerosis or from humans or animals with other chronic inflammatory diseases does not prevent the inflammatory reaction characteristic of atherosclerosis and may even enhance the inflammatory reaction. In mice and perhaps humans, ≈30% of the steady-state plasma HDL-cholesterol pool is derived from the small intestine. The metabolism of phospholipids by gut bacteria has been recently implicated in atherosclerosis in both mice and humans. Studies with apolipoprotein A-I mimetic peptides suggest that the small intestine is a major tissue regulating systemic inflammation in mouse models of atherosclerosis and may be important for determining the functionality of HDL.
PMCID: PMC3597084  PMID: 23077141
apolipoprotein A-I; apolipoprotein A-I mimetic peptides; high-density lipoprotein; hydroxyeicosatetraenoic acid; hydroxyoctadecadienoic acid; lipoproteins; oxidized lipids; small intestine
8.  HDL mimetics inhibit tumor development in both induced and spontaneous mouse models of colon cancer 
Molecular Cancer Therapeutics  2012;11(6):1311-1319.
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.
PMCID: PMC3374063  PMID: 22416044
HDL; Mimetic Peptides; Colon Cancer; LPA; Cancer Therapeutics
9.  Dysfunctional High-Density Lipoprotein and the Potential of Apolipoprotein A-1 Mimetic Peptides to Normalize the Composition and Function of Lipoproteins 
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.
PMCID: PMC3625624  PMID: 21628835
ApoA-I mimetic peptides; High-density lipoprotein; Inflammation; Oxidative stress
10.  Anti-inflammatory and Antioxidant Properties of HDLs Are Impaired in Type 2 Diabetes 
Diabetes  2011;60(10):2617-2623.
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.
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.
PMCID: PMC3178289  PMID: 21852676
11.  Effects of lipid-probe interactions in biochemical fluorometric methods that assess HDL redox activity 
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.
PMCID: PMC3409024  PMID: 22768920
12.  D-4F, an apoA-I mimetic peptide, inhibits proliferation and tumorigenicity of epithelial ovarian cancer cells by upregulating the antioxidant enzyme MnSOD 
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.
PMCID: PMC3248802  PMID: 21425255
MnSOD; apolipoprotein A-I; mimetic peptides; oxidative stress; animal models; epithelial ovarian cancer
13.  Vasculitis, Atherosclerosis, and Altered HDL Composition in Heme-Oxygenase-1-Knockout Mice 
To elucidate roles of heme oxygenase-1 (HO-1) in cardiovascular system, we have analyzed one-year-old HO-1-knockout mice. Homozygous HO-1-knockout mice had severe aortitis and coronary arteritis with mononuclear cellular infiltration and fatty streak formation even on a standard chow diet. Levels of plasma total cholesterol and HDL were similar among the three genotypes. However, homozygous HO-1-knockout mice had lower body weight and plasma triglyceride. HO-1-deficiency resulted in alteration of the composition of HDL. The ratio of apolipoprotein AI to AII in HO-1-knockout mice was reduced about 10-fold as compared to wild-type mice. In addition, paraoxonase, an enzyme against oxidative stress, was reduced less than 50% in HO-1-knockout mice. The knockout mice also exhibited significant elevation of plasma lipid hydroperoxides. This study using aged HO-1-knockout mice strengthened the idea that HO-1 functions to suppress systemic inflammation in artery wall and prevents plasma lipid peroxidation.
PMCID: PMC3296294  PMID: 22518297
14.  In vitro stimulation of HDL anti-inflammatory activity and inhibition of LDL pro-inflammatory activity in the plasma of patients with end-stage renal disease by an apoA-1 mimetic peptide 
Kidney International  2009;76(4):437-444.
Features of end-stage renal disease such as oxidative stress, inflammation, hypertension, and dyslipidemia are associated with accelerated atherosclerosis and increased risk of death from cardiovascular disease. By inhibiting the formation and increasing the disposal of oxidized lipids, HDL exerts potent antioxidant and anti-inflammatory actions. Given that apolipoproteinA-1 can limit atherosclerosis, we hypothesized that an apolipoproteinA-1 mimetic peptide, 4F, may reduce the proinflammatory properties of LDL and enhance the anti-inflammatory properties of HDL in uremic plasma. To test this, plasma from each of 12 stable hemodialysis patients and age-matched control subjects was incubated with 4F or vehicle. The isolated HDL and LDL fractions were added to cultured human aortic endothelial cells to quantify monocyte chemotactic activity, thus measuring their pro- or anti-inflammatory index. The LDL from the hemodialysis patients was more pro-inflammatory and their HDL was less anti-inflammatory than those of the control subjects. Pre-incubation of the plasma from the hemodialysis patients with 4F decreased LDL pro-inflammatory activity and enhanced HDL anti-inflammatory activity. Whether 4F or other apolipoproteinA-1 mimetic peptides will have any therapeutic benefit in end-stage renal disease will have to be examined directly in clinical studies.
PMCID: PMC3280585  PMID: 19471321
anti-inflammatory agents; atherosclerosis; cardiovascular disease; inflammation; lipid disorders; oxidative stress
15.  Salutary Effects of Hemodialysis on Low-Density Lipoprotein Proinflammatory and High-Density Lipoprotein Anti-inflammatory Properties in Patient With End-Stage Renal Disease 
End-stage renal disease (ESRD) causes oxidative stress, inflammation, low-density lipoprotein (LDL) oxidation, high-density lipoprotein (HDL) deficiency and accelerated atherosclerosis. Uptake of oxidized LDL by macrophages results in foam cell and plaque formation. HDL mitigates atherosclerosis via reverse cholesterol transport and inhibition of LDL oxidation. ESRD heightens LDL inflammatory activity and suppresses HDL anti-inflammatory activity. The effect of hemodialysis on the LDL and HDL inflammatory properties is unknown. By removing the potential pro-oxidant/proinflammatory uremic toxins, dialysis may attenuate LDL inflammatory and HDL anti-inflammatory properties. Conversely, exposure to dialyzer membrane and tubing and influx of impurities from dialysate can intensify LDL and HDL inflammatory activities. This study examined the effect of hemodialysis on LDL and HDL inflammatory activities. Plasma samples were obtained from 12 normal control and 26 ESRD patients before and after hemodialysis with (16 patients) or without (10 patients) heparinization. HDL and LDL were isolated and tested for monocyte chemotactic activity in cultured endothelial cells. ESRD patients had increased LDL chemotactic activity, reduced HDL anti-inflammatory activity, paraoxonase and glutathione peroxidase levels, and elevated plasma IL-6 before dialysis. Hemodialysis partially improved LDL inflammatory and HDL anti-inflammatory activities and enhanced patients’ HDL ability to suppress their LDL inflammatory activity. The salutary effect on LDL inflammatory activity was significantly greater in patients dialyzed with than those without heparin. ESRD heightens LDL inflammatory and impairs HDL anti-inflammatory activities. Hemodialysis partially improves LDL and HDL inflammatory activities. The salutary effects of hemodialysis are in part mediated by heparin, which is known to possess lipolytic and antioxidant properties.
PMCID: PMC3276406  PMID: 21830637
inflammation; cardiovascular; drugs; lipids; kidney; atherosclerosis
16.  L-5F, an apolipoprotein A-I mimetic, inhibits tumor angiogenesis by suppressing VEGF/basic FGF signaling pathways†‡ 
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.
PMCID: PMC3248743  PMID: 21283904
17.  Apolipoprotein A-I Mimetic Peptides Prevent Atherosclerosis Development and Reduce Plaque Inflammation in a Murine Model of Diabetes 
Diabetes  2010;59(12):3223-3228.
To determine the effect of the apolipoprotein A-I (ApoA-I) mimetic peptide, D-4F, on atherosclerosis development in a pre-existing diabetic condition.
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.
PMCID: PMC2992786  PMID: 20826564
18.  Amelioration of nephropathy with apoA-1 mimetic peptide in apoE-deficient mice 
Nephrology Dialysis Transplantation  2010;25(11):3525-3534.
Background. There is mounting evidence that dyslipidaemia may contribute to development and progression of renal disease. For instance, hyperlipidaemia in apolipoprotein E-deficient (apoE−/−) mice is associated with glomerular inflammation, mesangial expansion and foam cell formation. ApoA-1 mimetic peptides are potent antioxidant and anti-inflammatory compounds which are highly effective in ameliorating atherosclerosis and inflammation in experimental animals. Given the central role of oxidative stress and inflammation in progression of renal disease, we hypothesized that apoA-1 mimetic peptide, D-4F, may attenuate renal lesions in apoE−/− mice.
Methods. Twenty-five-month-old female apoE−/− mice were treated with D-4F (300 µg/mL in drinking water) or placebo for 6 weeks. Kidneys were harvested and examined for histological and biochemical characteristics.
Results. Compared with the control mice, apoE−/− mice showed significant proteinuria, tubulo-interstitial inflammation, mesangial expansion, foam cell formation and up-regulation of oxidative [NAD(P)H oxidase subunits] and inflammatory [NF-κB, MCP-1, PAI-1 and COX-2] pathways. D-4F administration lowered proteinuria, improved renal histology and reversed up-regulation of inflammatory and oxidative pathways with only minimal changes in plasma lipid levels.
Conclusions. The apoE−/− mice develop proteinuria and glomerular and tubulo-interstitial injury which are associated with up-regulation of oxidative and inflammatory mediators in the kidney and are ameliorated by the administration of apoA-1 mimetic peptide. These observations point to the role of oxidative stress and inflammation in the pathogenesis of renal disease in hyperlipidaemic animals and perhaps humans.
PMCID: PMC2980997  PMID: 20488818
atherosclerosis; chronic kidney disease; hyperlipidaemia; inflammation; oxidative stress
19.  Mitogen-Activated Protein Kinase Phosphatase-1 Deficiency Decreases Atherosclerosis in Apolipoprotein E Null Mice by Reducing Monocyte Chemoattractant Protein-1 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.
PMCID: PMC3037189  PMID: 20619710
mitogen-activated protein kinase phosphatase-1; atherosclerosis; monocyte chemoattractant protein-1; monocytes
20.  HIV-1 infected patients with suppressed plasma viremia on treatment have pro-inflammatory HDL 
The role of pro-inflammatory lipids in systemic immune activation in HIV infection remains largely unknown. We hypothesized that HIV-1-infected persons on antiretroviral therapy would have pro-inflammatory high density lipoprotein (HDL), and that an apoA-1 mimetic peptide might reverse the inflammatory properties of HDL in these persons.
Plasma was obtained from 10 HIV-1-infected individuals on combination antiretroviral therapy with suppressed viremia and was incubated with the apoA-I mimetic peptide L-4F or sham-treated prior to isolation of HDL. The HDL that was isolated from each sample was tested for its ability to inhibit LDL-induced MCP-1 production in cultures of human aortic endothelial cells.
We found in a small pilot study of HIV-1-infected individuals with suppressed viremia on combination antiretroviral therapy that oxidative stress and inflammation in HIV-1 are associated with a marked reduction of HDL antioxidant/anti-inflammatory activities. In vitro, these abnormalities were significantly improved by treatment with the apoA-1 mimetic peptide, 4F.
These preliminary observations suggest that the anti-inflammatory properties of HDL are defective in HIV-1-infected persons despite treatment that is considered to be virologically successful.
PMCID: PMC3049748  PMID: 21345230
21.  L-4F Differentially Alters Plasma Levels of Oxidized Fatty Acids Resulting in more Anti-Inflammatory HDL in Mice 
Drug metabolism letters  2010;4(3):139-148.
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.
PMCID: PMC3037264  PMID: 20642447
HETE; HODE; HPODE; EET; apoA-I mimetic peptides; L-4F; arachidonic acid metabolism
22.  Structure and Function of HDL Mimetics 
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.
PMCID: PMC2860541  PMID: 19608977
23.  L-4F Alters Hyperlipidemic (but not Normal) Mouse Plasma to Reduce Platelet Aggregation 
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.
PMCID: PMC2818809  PMID: 19965777
Platelets; apoA-I mimetic peptides; L-4F; Arachidonic acid metabolism; apoE null mice
24.  Paraoxonase 2 Deficiency Alters Mitochondrial Function and Exacerbates the Development of Atherosclerosis 
Antioxidants & Redox Signaling  2011;14(3):341-351.
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.
PMCID: PMC3011913  PMID: 20578959
25.  Ambient Particulate Pollutants in the Ultrafine Range Promote Early Atherosclerosis and Systemic Oxidative Stress 
Circulation research  2008;102(5):589-596.
Air pollution is associated with significant adverse health effects, including increased cardiovascular morbidity and mortality. Exposure to particulate matter with an aerodynamic diameter of <2.5 μm (PM2.5) increases ischemic cardiovascular events and promotes atherosclerosis. Moreover, there is increasing evidence that the smallest pollutant particles pose the greatest danger because of their high content of organic chemicals and prooxidative potential. To test this hypothesis, we compared the proatherogenic effects of ambient particles of <0.18 μm (ultrafine particles) with particles of <2.5 μm in genetically susceptible (apolipoprotein E–deficient) mice. These animals were exposed to concentrated ultrafine particles, concentrated particles of <2.5 μm, or filtered air in a mobile animal facility close to a Los Angeles freeway. Ultrafine particle–exposed mice exhibited significantly larger early atherosclerotic lesions than mice exposed to PM2.5 or filtered air. Exposure to ultrafine particles also resulted in an inhibition of the antiinflammatory capacity of plasma high-density lipoprotein and greater systemic oxidative stress as evidenced by a significant increase in hepatic malondialdehyde levels and upregulation of Nrf2-regulated antioxidant genes. We conclude that ultrafine particles concentrate the proatherogenic effects of ambient PM and may constitute a significant cardiovascular risk factor.
PMCID: PMC3014059  PMID: 18202315
air pollution; ultrafine particles; atherosclerosis; oxidative stress; HDL

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