David Meriwether

A novel approach to oral apoA-I mimetic therapy

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 LDL receptor-null (LDLR−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the 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 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (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 with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.

A Biochemical Fluorometric Method for Assessing the Oxidative Properties of HDL

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 (r2 = 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.

Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid

We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR−/−) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR−/− mice and causes changes in small intestine gene expression. Confirmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i) levels of LPA in the small intestine were similar to those induced by feeding WD; ii) gene expression changes in the small intestine mimicked WD-mediated changes; and iii) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDL-cholesterol levels, and the fast-performance liquid chromatography lipoprotein profile mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i) WD-mediated systemic inflammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii) Tg6F reduces WD-mediated systemic inflammation and dyslipidemia by preventing WD-induced increases in LPA levels in the small intestine.

A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

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 LJ001s 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 LJ001s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001s 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.

HDL Mimetics Inhibit Tumor Development in Both Induced and Spontaneous Mouse Models of Colon Cancer

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 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. Mol Cancer Ther; 11(6); 1311–9. ©2012 AACR.

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.

Apolipoprotein A-I Mimetic Peptide 4F Rescues Pulmonary Hypertension by Inducing MicroRNA-193-3p

Background— Pulmonary arterial hypertension is a chronic lung disease associated with severe pulmonary vascular changes. A pathogenic role of oxidized lipids such as hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids is well established in vascular disease. Apolipoprotein A-I mimetic peptides, including 4F, have been reported to reduce levels of these oxidized lipids and improve vascular disease. However, the role of oxidized lipids in the progression of pulmonary arterial hypertension and the therapeutic action of 4F in pulmonary arterial hypertension are not well established. Methods and Results— We studied 2 different rodent models of pulmonary hypertension (PH): a monocrotaline rat model and a hypoxia mouse model. Plasma levels of hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids were significantly elevated in PH. 4F treatment reduced these levels and rescued preexisting PH in both models. MicroRNA analysis revealed that microRNA-193-3p (miR193) was significantly downregulated in the lung tissue and serum from both patients with pulmonary arterial hypertension and rodents with PH. In vivo miR193 overexpression in the lungs rescued preexisting PH and resulted in downregulation of lipoxygenases and insulin-like growth factor-1 receptor. 4F restored PH-induced miR193 expression via transcription factor retinoid X receptor &agr;. Conclusions— These studies establish the importance of microRNAs as downstream effectors of an apolipoprotein A-I mimetic peptide in the rescue of PH and suggest that treatment with apolipoprotein A-I mimetic peptides or miR193 may have therapeutic value.

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.

Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR−/−) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR−/− mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.

Effects of lipid-probe interactions in biochemical fluorometric methods that assess HDL redox activity.

BackgroundFluorescence-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.MethodsWe 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.ResultsWe 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.ConclusionsLipid-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.