apoA-I Mimetic Peptides Research Articles

The apoA-I mimetic peptide 4F protects apolipoprotein A-I from oxidative damage.

High density lipoprotein (HDL) is prone to modification by the oxidizing and chlorinating agent hypochlorite anion (OCl-). Oxidation of apolipoprotein (apo) A-I, the major protein in HDL, reduces ABCA-1 mediated cholesterol efflux and other protective responses to HDL. The apoA-I mimetic peptide 4F has been shown to undergo oxidation; however, the ability of the peptide to mediate cholesterol efflux remains intact. Here, we show that 4F protects apoA-I from hypochlorite-mediated oxidation. Mass spectral analysis of apoA-I shows that tyrosine residues that are prone to hypochlorite-mediated chlorination are protected in the presence of 4F. Furthermore, 4F enhances the cholesterol efflux ability of apoA-I to a greater extent than either 4F or apoA-I alone, even after hypochlorite oxidation. These observations suggest that apoA-I in lipid complexes may be protected by the presence of 4F, resulting in the preservation of its anti-inflammatory and anti-atherogenic properties. These studies also form the basis for the future studies of nanoparticles possessing both apoA-I and 4F.

Anti-tumorigenic and Platinum-Sensitizing Effects of Apolipoprotein A1 and Apolipoprotein A1 Mimetic Peptides in Ovarian Cancer.

Objective: Apolipoprotein A1 (ApoA1) is remarkably decreased in serum and ovarian tissues of ovarian cancer patients. ApoA1 and ApoA1 mimetic peptides can sequestrate pro-inflammatory phospholipids, some of which are known to activate a variety of oncogenic pathways. Besides, more intrinsic anti-tumorigenic properties, independent from interaction with lipids, have also been described for ApoA1. We aimed to disclose the effects of ApoA1 and a mimetic peptide on the malignant phenotype of ovarian cancer cells, particularly regarding cell viability, invasiveness and platinum sensitization.Methods: Cells viability was assessed by MTS assay. Extracellular matrix invasion was assessed by transwell and spheroid invasion assays. Western blotting was performed to evaluate the effect of test compounds on intracellular pathways. Sensitization assays were performed in vitro and in the biologically relevant in ovo chorioallantoic membrane model.Results: Both ApoA1 and the mimetic peptide, at a concentration of 100 μg/mL, were able to decrease the viability of SKOV3, CAOV3, and OVCAR3 cells (p < 0.05). The peptide at this concentration was not able to affect the viability of immortalized non-neoplastic ovarian cells (p > 0.05). ApoA1 decreased SKOV3 cells invasiveness at 300 μg/mL after 72 and 96 h of exposure (p < 0.05), while the ApoA1 mimetic peptide prevented cell invasion at 50 and 100 μg/mL (p < 0.01). Treatment with 100 μg/mL of ApoA1 mimetic peptide decreased Akt phosphorylation in SKOV3 cells (p < 0.01). Accordingly, treatment with increasing concentrations of the peptide sensitized SKOV3, OVCAR3 and CAOV3 cells to cisplatin. This synergistic effect was observed both in vitro and in ovo.Conclusions: These results support the role of ApoA1 and ApoA1 mimetics as suppressors of ovarian tumorigenesis and as chemo-sensitising agents.

Abstract 17268: ApoA-I Mimetic Peptide 4F Suppresses the Elevation of Pro-Inflammatory Lipid Mediators in Mouse Models of Inflammatory Bowel Disease

Introduction: Inflammatory bowel disease (IBD) has been linked to an increased prevalence of early stage vascular disease. ApoA-I mimetic peptides including 4F are potential therapeutic agents for the treatment of inflammatory diseases including atherosclerosis, and their mechanism of action appears localized to the intestine. We have reported that 4F protects against the development of disease in both the piroxicam-accelerated IL10-/- and myeloid COX2-/- mouse models of IBD. Hypothesis: We previously reported that plasma and lesion levels of oxidized products of linoleic and arachidonic acid correlate with disease in mouse models of atherosclerosis, and that 4F protects against disease in these models while inhibiting accumulation of these pro-inflammatory mediators. We thus sought to determine the complete lipid pro-inflammatory mediator profiles of both the COX2- and IL10-dependent models of IBD, while also determining the effect of 4F on the pro-inflammatory lipid profiles. Methods: We developed and validated a LC-ESI-MS/MS method for determining the levels of 40 lipid inflammatory mediators in both intestinal tissue and plasma, and we analyzed the effects of both disease and 4F upon these mediators in both IBD models. We also employed Ussing chambers to investigate ex vivo the direct effect of 4F on the clearance of pro-inflammatory lipid mediators from intestinal explants and serosal-side lipoproteins. Results: Disease in both models correlated with significantly elevated tissue and plasma levels of multiple lipid pro-inflammatory mediators, while the protective effects of 4F correlated with the significant suppression of most of these mediators. Of interest, 4F inhibited the disease dependent increase of 15HETE, 12HETE, 5HETE, 13HODE, LTB4, 6ketoPGF1α, PGF2α, and TXB2 in the COX2-/- model; and 15HETE, 12HETE, 13HODE, LTB4, and LTE4 in the IL10-/- model. Ex vivo, we showed that 4F could directly clear the pro-inflammatory mediators from inflamed intestinal explants, while also mediating their trans-intestinal efflux from serosal-side lipoproteins. Conclusions: 4F appears to protect against IBD in part by inhibiting the accumulation of pro-inflammatory lipid mediators, through a mechanism that involves the intestinal clearance of these mediators from tissue and plasma.

Role of enterocyte stearoyl-Co-A desaturase-1 in LDLR-null mice

After crossing floxed stearoyl-CoA desaturase-1 (Scd1fl/fl) mice with LDL receptor-null (ldlr-/-) mice, and then Villin Cre (VilCre) mice, enterocyte Scd1 expression in Scd1fl/fl/ldlr-/-/VilCre mice was reduced 70%. On Western diet (WD), Scd1fl/fl/ldlr-/- mice gained more weight than Scd1fl/fl/ldlr-/-/VilCre mice (P < 0.0023). On WD, jejunum levels of lysophosphatidylcholine (LysoPC) 18:1 and lysophosphatidic acid (LPA) 18:1 were significantly less in Scd1fl/fl/ldlr-/-/VilCre compared with Scd1fl/fl/ldlr-/- mice (P < 0.0004 and P < 0.026, respectively). On WD, Scd1fl/fl/ldlr-/-/VilCre mice compared with Scd1fl/fl/ldlr-/- mice had lower protein levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4), and myeloid differentiation factor-88 (MyD88) in enterocytes and plasma, and less dyslipidemia and systemic inflammation. Adding a concentrate of tomatoes transgenic for the apoA-I mimetic peptide 6F (Tg6F) to WD resulted in reduced enterocyte protein levels of LBP, CD14, TLR4, and MyD88 in Scd1fl/fl/ldlr-/- mice similar to that seen in Scd1fl/fl/ldlr-/-/VilCre mice. Adding LysoPC 18:1 to WD did not reverse the effects of enterocyte Scd1 knockdown. Adding LysoPC 18:1 (but not LysoPC 18:0) to chow induced jejunum Scd1 expression and increased dyslipidemia and plasma serum amyloid A and interleukin 6 levels in Scd1fl/fl/ldlr-/- mice, but not in Scd1fl/fl/ldlr-/-/VilCre mice. We conclude that enterocyte Scd1 is partially responsible for LysoPC 18:1- and WD-induced dyslipidemia and inflammation in ldlr-/- mice.

Abstract 250: Pulmonary Hypertension Induced by 15-HETE is Reverse by Apoai Mimetic Peptide 6f Administration

Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial occlusion leading to increased pulmonary arterial pressure. Recently, a growing body of evidence demonstrated a robust increase in oxidized lipids, including 15-hydroxyeicosatetraenoic acids (15HETE), in the lungs and plasma of PAH patients and animal models of pulmonary hypertension (PH). Recently, our group demonstrated that 15HETE induce PH. Nonetheless, the precise mechanism by which 15HETE participates in PH development remains elusive. In order to investigate this mechanism, we fed wild type mice with a diet rich in 15HETE and examine whether ApoA-I mimetic peptide can rescue PH induced by 15HETE. Methods: Wild type male mice (C57BL/6) were fed for 3 weeks for 3 weeks with regular chow (n=16-21 mice/group), 15HETE (5μg/day), 15HETE diet supplemented with either empty vector or 6F for the last week of 15HETE diet. PH development was assessed every week via serial echocardiography. Right ventricular systolic pressure (RVSP) was measured via heart catheterization. RV hypertrophy index (RV/[IVS+LV]) was measured. Lung morphology and lipid accumulation were assessed using H&amp;E and Oil red O staining. Results: Echocardiography revealed the first sign of PH as early as one week after starting 15HETE diet and a significant decrease in the pulmonary arterial acceleration time (PAAT) after 2 weeks of treatment (16.6±1.9 vs. 20.6±1.4 msec, p&lt;0.05). At the end of three weeks, mice on 15HETE diet had significantly higher RVSP (31.3±1.1 vs. 38.4±2.3 mmHg, p&lt;0.05) and RV hypertrophy index (0.26 ± 0.02 vs. 0.33 ±0.02, p&lt;0.05). This increased pressure was concomitant with a significant increase in pulmonary arteriolar thickness in mice on 15-HETE diet compared to regular diet (35.1±0.8 vs 53.4±1, p&lt;0.05). Furthermore, these WT mice did not develop atherosclerosis as there was no detectable plaque in aorta of the mice on 15HETE diet. At the end of three weeks mice treated with 6F showed a PAAT similar to control value(19±0.5 msec) concomitant with a significantly lower RVSP than mice fed with 15HETE +empty vector (33.6 ±5.7 vs 40.3±4.6, p&lt;0.05). Conclusion: Our data demonstrates that APOA-I mimetic peptide 6F is able to rescue pre-exisiting PH induced by 15-HETE diet in wild type mice.

ApoA-1 Mimetic Peptide ELK-2A2K2E Decreases Inflammatory Factor Levels Through the ABCA1-JAK2-STAT3-TTP Axis in THP-1-Derived Macrophages.

The aim of this study was to determine whether the apolipoprotein A-1 (apoA-1) mimetic peptide ELK-2A2K2E regulates inflammatory cytokine expression through activating the adenosine triphosphate-binding cassette transporter A1 (ABCA1)-janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3)-tristetraprolin (TTP) signaling pathway in THP-1 macrophage-derived foam cells. The cells were treated with the apoA-1 mimetic peptide ELK-2A2K2E at different concentrations (0, 20, 40, and 80 μg/mL) or incubated with ELK-2A2K2E (40 μg/mL) for different times (0, 6, 12, and 24 hours). Our results showed that the levels of the cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), were decreased at both concentration- and time-dependent manners. When the cells were exposed to lipopolysaccharides and actinomycin D, ELK-2A2K2E significantly decreased the mRNA stability of inflammatory cytokines at different time points (0, 30, 60, and 120 minutes) by increasing TTP expression as analyzed by real-time quantitative polymerase chain reaction. The effect of ELK-2A2K2E on TTP was obviously blocked by the inhibition of the JAK-STAT3 pathway. Furthermore, we found that ELK-2A2K2E activated the JAK-STAT3-TTP pathway through the upregulation of ABCA1 and then decreased inflammatory cytokine expression. ApoA-I mimetic peptide ELK-2A2K2E increases the degradation of TNF-α, IL-6, and MCP-1 mRNA and reduces the levels of inflammatory cytokines through activating the JAK2-STAT3-TTP signaling pathway that is dependent on the upregulation of ABCA1.

Treating the Intestine with Oral ApoA-I Mimetic Tg6F Reduces Tumor Burden in Mouse Models of Metastatic Lung Cancer

Having demonstrated that apolipoprotein A-I (apoA-I) mimetic peptides ameliorate cancer in mouse models, we sought to determine the mechanism for the anti-tumorigenic function of these peptides. CT-26 cells (colon cancer cells that implant and grow into tumors in the lungs) were injected into wild-type BALB/c mice. The day after injection, mice were either continued on chow or switched to chow containing 0.06% of a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F). After four weeks, the number of lung tumors was significantly lower in Tg6F-fed mice. Gene expression array analyses of jejunum and lung identified Notch pathway genes significantly upregulated, whereas osteopontin (Spp1) was significantly downregulated by Tg6F in both jejunum and lung. In jejunum, Tg6F increased protein levels for Notch1, Notch2, Dll1, and Dll4. In lung, Tg6F increased protein levels for Notch1 and Dll4 and decreased Spp1. Tg6F reduced oxidized phospholipid levels (E06 immunoreactivity) and reduced 25-hydroxycholesterol (25-OHC) levels, which are known to inhibit Notch1 and induce Spp1, respectively. Notch pathway promotes anti-tumorigenic patrolling monocytes, while Spp1 facilitates pro-tumorigenic myeloid derived suppressor cells (MDSCs) formation. Tg6F-fed mice had higher numbers of patrolling monocytes in jejunum and in lung (p < 0.02), and lower plasma levels of Spp1 with reduced numbers of MDSCs in jejunum and in lung (p < 0.03). We conclude that Tg6F alters levels of specific oxidized lipids and 25-OHC to modulate Notch pathways and Spp1, which alter small intestine immune cells, leading to similar changes in lung that reduce tumor burden.

Abstract 389: The Impact of Phospholipid Composition of Synthetic High-Density Lipoprotein on Its Pharmacological Activity

Objective: Synthetic high-density lipoprotein (sHDL) is a nanoparticle that can mimic biological activities of endogenous HDL such as reverse cholesterol transport (RCT) and anti-inflammatory properties. We hypothesize that differences in the fluidity of sHDL phospholipids at body temperature effect plasma stability of sHDL, particle ability to efflux cholesterol and inhibit inflammation. Methods: sHDL particles with different membrane fluidities were prepared complexing phospholipids with different fatty acid chain length and saturation (POPC, DMPC, DPPC, and DSPC) with the apoA-I mimetic peptide, PVLDLFRELLNELLEALKQKLK (22A). The ability of various sHDL compositions to efflux cholesterol, inhibit NF-kB activation and cytokine release, and cause lipid raft disruption was examined in RAW264.7 macrophages. Various sHDL were administered to mice challenged by injection of 0.05 mg/kg LPS at 10 mg/kg dose and the levels of cytokine release were measured at 2 hours post-dose. Various sHDL were dosed to normal rats at 50 mg/kg and cholesterol mobilization and pharmacokinetics were examined. Results: 22A-POPC and 22A-DMPC sHDL have relatively fluid phospholipid layer at body temperature compared to 22A-DPPC and 22A-DSPC sHDL due to lower phospholipid transition temperature. 22A-POPC and 22A-DMPC sHDL inhibited NF-κB activation and cytokine release in a concentration-dependent manner. From murine endotoxin infusion studies, 22A-DMPC displayed the significant inhibition of cytokine release. Cholesterol efflux studies demonstrated that 22A-POPC and 22A-DMPC displayed highest cholesterol efflux. Interestingly, in vivo RCT study showed that 22A-DSPC had longest plasma residence time and resulted in greatest cholesterol mobilization. Conclusions: Phospholipid composition of sHDL particles has a significant effect on its cholesterol efflux and anti-inflammatory properties, yet the effect appears to be different in vitro and in vivo . in vitro effect is driven by the ability of fluid phospholipid bilayer to bind LPS, cholesterol, and LCAT, while in vivo effect is defined by particle stability in plasma and residence time in the body.

Methotrexate-Loaded Extracellular Vesicles Functionalized with Therapeutic and Targeted Peptides for the Treatment of Glioblastoma Multiforme.

Despite promising in vitro evidence for effective glioblastoma treatment, most drugs are hindered from entering the central nervous system because of the presence of the blood-brain barrier (BBB). Thus, successful modification of drug delivery and novel therapeutic strategies are needed to overcome this obstacle. Extracellular vesicles (EVs), cell-derived membrane-encapsulated structures with diameters ranging from 50 to 1000 nm, have been explored as the drug delivery system to deliver their cargo to the brain tissue. Moreover, tumor targeting and selective drug delivery has been facilitated by engineering their parent cells to secrete modified EVs. However, the method suffers from many shortcomings including poor repeatability and complex and time-consuming operations. In this context, we present an easy-to-adapt and highly versatile methodology to modify EVs with an engineered peptide capable of recognition and eradication of glioma. On the basis of molecular recognition between phospholipids on EV lipid bilayer membranes and ApoA-I mimetic peptides, we have developed methotrexate (MTX)-loaded EVs functionalized with therapeutic [Lys-Leu-Ala (KLA)] and targeted [low-density lipoprotein (LDL)] peptides. In vitro experiments demonstrated that EVs decorated with LDL or KLA-LDL could obviously ameliorate their uptake by human primary glioma cell line U87 and permeation into three-dimensional glioma spheroids in contrast to blank EVs, and consequently, the treatment outcome of the payload is improved. Both ex vivo and in vivo imaging experiments revealed that peptide LDL could obviously promote EV extravasation across the BBB and distribution in the glioma site. Furthermore, compared with the mice administrated with MTX and MTX@EVs, MTX@EVs-KLA-LDL-treated mice showed the longest median survival period. In conclusion, functionalizing with the peptide onto EV surfaces may provide a substantial advancement in the application of EVs for selective target binding as well as therapeutic effects for brain tumor treatment.

Beneficial Effects of High-Density Lipoproteins on Acquired von Willebrand Syndrome in Aortic Valve Stenosis.

Infusions of apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins (HDL), result in aortic valve stenosis (AVS) regression in experimental models. Severe AVS can be complicated by acquired von Willebrand syndrome, a haemorrhagic disorder associated with loss of high-molecular-weight von Willebrand factor (vWF) multimers (HMWM), the latter being a consequence of increased shear stress and enhanced vWF-cleaving protease (ADAMTS-13) activity. Although antithrombotic actions of HDL have been described, its effects on ADAMTS-13 and vWF in AVS are unknown. We assessed ADAMTS-13 activity in plasma derived from a rabbit model of AVS (n = 29) as well as in plasma collected from 64 patients with severe AVS (age 65.0 ± 10.4 years, 44 males) undergoing aortic valve replacement (AVR). In both human and rabbit AVS plasma, ADAMTS-13 activity was higher than that in controls (p < 0.05). Accordingly, AVS patients had less HMWM than controls (66.3 ± 27.2% vs. 97.2 ± 24.1%, p < 0.0001). Both ADAMTS-13 activity and HMWM correlated significantly with aortic transvalvular gradients, thereby showing opposing correlations (r = 0.3, p = 0.018 and r = -0.4, p = 0.003, respectively). Administration of an apoA-I mimetic peptide reduced ADAMTS-13 activity in AVS rabbits as compared with the placebo group (2.0 ± 0.5 RFU/sec vs. 3.8 ± 0.4 RFU/sec, p < 0.05). Similarly, a negative correlation was found between ADAMTS-13 activity and HDL cholesterol levels in patients with AVS (r = -0.3, p = 0.045). Our data indicate that HDL levels are associated with reduced ADAMTS-13 activity and increased HMWM. HDL-based therapies may reduce the haematologic abnormalities of the acquired von Willebrand syndrome in AVS.

Abstract 21376: Apo A-I Mimetic Peptides Inhibit IBD In Multiple Mouse Models

Introduction: Inflammatory bowel disease (IBD) has been linked to an increased prevalence of early stage vascular disease. ApoA-I mimetic peptides including 4F and Tg6F (6F expressed in tomatoes) are potential therapeutic agents for the treatment of inflammatory diseases. We previously reported that 4F protects against the development of IBD in a COX2-deficient and cholate-dependent mouse model of Crohn’s disease. Hypothesis: We sought to determine, a) the mechanism by which 4F inhibits IBD, b) whether 4F is protective in additional mouse models of IBD, and c) whether Tg6F could also protect against IBD. Methods: We examined the effects of cholate on changes in barrier permeability and translocation of LPS into portal vein plasma, in COX2-deficient and wild-type mice. We studied the effects of both COX2 and 4F on the pro-inflammatory response of LPS-stimulated macrophages. We investigated the effects of 4F in the IL10 -/- + piroxicam model of colitis. Finally, we examined the protective effects of Tg6F in myeloid conditional COX2 KO mice that were challenged with cholate. Results: Cholate increased both barrier permeability and the translocation of LPS into portal vein plasma equally in both COX2-deficient and wild-type mice, while only COX2-deficient mice including myeloid COX2-deficient mice subsequently developed IBD. Bone marrow derived macrophages lacking COX2 expressed 10-15 fold higher pro-inflammatory cytokines including IL1-β compared to WT macrophages, when stimulated with LPS. Both pre-treatment and co-treatment of these macrophages with 4F significantly reduced the expression of pro-inflammatory cytokines, returning them to the level of WT. 4F significantly inhibited the development of colitis in IL10 -/- mice challenged with the COX inhibitor piroxicam, as determined by colon length, colitis score, and H&amp;E score. Furthermore, Tg6F significantly protected against the development of IBD in myeloid COX2 KO mice challenged with cholate, as determined by gross pathology and H&amp;E score. Conclusions: 4F protects against IBD in the COX2-deficient mouse model, in part, by inhibiting the pro-inflammatory phenotype of COX2-deficient macrophages. Our findings suggest a novel therapeutic role for ApoA-I mimetic peptides including Tg6F in IBD.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch’s Membrane and Causes Its Structural Remodeling

ABSTRACTPurpose: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch’s membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch’s membrane.Methods: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch’s membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch’s membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection.Results: All injected eyes showed a dose-dependent reduction of Bruch’s membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch’s membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets.Conclusion: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch’s membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

Synthetic high-density lipoprotein nanodisks for targeted withalongolide delivery to adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and has a 5-year survival rate of <35%. ACC cells require cholesterol for steroid hormone production, and this requirement is met via expression on the cell surface of a high level of SRB1, responsible for the uptake of high-density lipoproteins (HDLs), which carry and transport cholesterol in vivo. Here, we describe how this natural lipid carrier function of SRB1 can be utilized to improve the tumor-targeted delivery of a novel natural product derivative – withalongolide A 4,19,27-triacetate (WGA-TA) – which has shown potent antitumor efficacy, but poor aqueous solubility. Our strategy was to use synthetic HDL (sHDL) nanodisks, which are effective in tumor-targeted delivery due to their smallness, long circulation half-life, documented safety, and ability to bind to SRB1. In this study, we prepared sHDL nanodisks using an optimized phospholipid composition combined with ApoA1 mimetic peptide (22A), which has previously been tested in clinical trials, to load WGA-TA. Following optimization, WGA-TA nanodisks showed drug encapsulation efficiency of 78%, a narrow particle size distribution (9.81±0.41 nm), discoid shape, and sustained drug release in phosphate buffered saline. WGA-TA-sHDL nanodisks exhibited higher cytotoxicity in the ACC cell line H295R half maximal inhibitory concentration ([IC50] 0.26±0.045 μM) than free WGA-TA (IC50 0.492±0.115 μM, P<0.05). Fluorescent dye-loaded sHDL nanodisks efficiently accumulated in H295R adrenal carcinoma xenografts 24 hours following dosing. Moreover, daily intraperitoneal administration of 7 mg/kg WGA-TA-loaded sHDL nanodisks significantly inhibited tumor growth during 21-day administration to H295R xenograft-bearing mice compared to placebo (P<0.01). Collectively, these results suggest that WGA-TA-loaded nanodisks may represent a novel and beneficial therapeutic strategy for the treatment of ACC.

Abstract 132: Glomerular Podocyte Damage is Mitigated by Normal HDL and ApoA1

We and others have shown that chronic kidney disease (CKD) disrupts the normally beneficial effects of apoAI/HDL on many different cell types. Proteinuria which characterizes CKD is a hallmark of glomerular podocyte injury and a powerful risk factor for cardiovascular disease (CVD). Although the glomerular filtration barrier normally limits direct contact of lipoproteins with podocytes, disruption in the filtration barrier promotes such interactions. We therefore investigated the effects of normal HDL and ApoAI on injured podocytes and compared these effects to HDL isolated from subjects with CKD. Primary podocytes were isolated from wild type mice. The cells were exposed to puromycin (PAN, 100ug/ml) with and without HDL isolated from controls with normal kidney function, individuals with CKD, normal ApoA1 (EMD Millipore Company), and ApoA-I mimetic peptide L-4F. We assessed podocyte viability and proliferation (colorimetrically), cellular production of superoxide (ROS)(HPLC), membrane lipid rafts, phosphorylated caveolin-1 (Cav-1)(western blot). PAN significantly altered ROS production, cell viability, proliferation. ApoA1 or HDL Cont , but not HDL CKD , significantly improved each of these responses. In addition, L-4F increased podocyte viability (0.60±0.04 PAN vs 0.65±0.04 PAN+L-4F) and HDL Cont , but not HDL CKD , normalized the significant increase in PAN-induced Cav-1 phosphorylation (0.65±0.04 vs 0.81±0.05 vs 0.70±0.09, respectively). We conclude that podocyte damage including ROS production, cellular membrane and functions can be mitigated by normal HDL or its primary lipoprotein, ApoA1, findings that parallel beneficial functions of HDL/ApoA1 on other cell types. By contrast, HDL of subjects with CKD do not provide similar benefit.

Abstract 564: Structure-Function Studies of ApoA-I Mimetic Peptides for ABCA1-Dependent Cholesterol Efflux and HDL Formation

To examine the mechanism of action of ApoA-I mimetics, we designed 4 peptides with a variable number of E, L, K and A residues and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. They were named based on a unique physical property: N=ELK-neutral (EKLKELLEKLLEKLKELL), H=ELK-hydrophobic (EKLLELLKKLLELLKELL), P=ELK-positive (EKLKALLEKLKAKLKELL), Neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed that H and P were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment, all peptides showed greater than 40% helicity. In DMPC vesicle solubilization assay, we observed following order: P&gt;N&gt;H&gt;&gt;Neg. By non-denaturing gel electrophoresis, N, H, P formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC. However, Neg did not form any detectable particle. A mixture of natural lipids (PC:SM:LysoPC:PE:PS:PI; mol% 55:12:5:10:8:10), intended to simulate ABCA1 lipid membrane microdomain, yielded similar results . Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: H&gt;N&gt;&gt;P, whereas Neg peptide was inactive. All-atom MD simulation of N carried out on a 12–nm disc containing POPC:cholest (200:20), 26 peptides, and initially placed in belt-like configuration as head-to-tail dimers, the starting structure was maintained for at least 3 μ-sec, indicated a strong preference for this configuration. However, when N was initially oriented in a picket-fence configuration, it distorted to a belt-like pattern within 1 μ-sec. For Neg, both starting configurations were much less stable with dimers losing their connectivity and monomers migrating to the top and bottom of the disc, leaving large hydrophobic patches of acyl chains exposed. Cross-linking studies were consistent with the ability of H and N to form dimers to stabilize the HDL structure, and thereby, were also consistent with simulation. Together, we show that a net neutral charge, a relatively large hydrophobic moment (0.78), and a broad hydrophobic face (180 degrees) are optimum features for an apoA-I mimetic peptide to promote cholesterol efflux and to stabilize a nascent discoidal HDL structure.

The apolipoprotein A-I mimetic peptide, D-4F, alleviates ox-LDL-induced oxidative stress and promotes endothelial repair through the eNOS/HO-1 pathway

Apolipoprotein A-I (apoA-I) mimetic peptide exerts many anti-atherogenic properties. However, the underlying mechanisms related to the endothelial protective effects remain elusive. In this study, the apoA-I mimetic peptide, D-4F, was used. Proliferation assay, wound healing, and transwell migration experiments showed that D-4F improved the impaired endothelial proliferation and migration resulting from ox-LDL. Endothelial adhesion molecules expression and monocyte adhesion assay demonstrated that D-4F inhibited endothelial inflammation. Caspase-3 activation and TUNEL stain indicated that D-4F reduced endothelial cell apoptosis. A pivotal anti-oxidant enzyme, heme oxygenase-1 (HO-1) was upregulated by D-4F. The Akt/AMPK/eNOS pathways were involved in the expression of HO-1 induced by D-4F. Moreover, the anti-oxidation, pro-proliferation, and pro-migration capacities of D-4F were diminished by the inhibitors of both eNOS (L-NAME) and HO-1 (Znpp). Additionally, downregulation of ATP-binding cassette transporter A1 (ABCA1) by siRNA abolished the activation of Akt, AMPK and eNOS, and reduced the upregulation of HO-1 triggered by D-4F. Furthermore, D-4F promoted the reendothelialization of injured intima in carotid artery injury model of C57BL/6J mice in vivo. In summary, these findings suggested that D-4F might be a powerful candidate in the protection of endothelial cells and the prevention of cardiovascular disease (CVD).

Influence of route of administration and lipidation of apolipoprotein A-I peptide on pharmacokinetics and cholesterol mobilization

apoA-I, apoA-I mimetic peptides, and their lipid complexes or reconstituted high-density lipoprotein (HDL) have been studied as treatments for various pathologies. However, consensus is lacking about the best method for administration, by intravenous (IV) or intraperitoneal (IP) routes, and formulation, as an HDL particle or in a lipid-free form. The objective of this study was to systematically examine peptide plasma levels, cholesterol mobilization, and lipoprotein remodeling in vivo following administration of lipid-free apoA-I peptide (22A) or phospholipid reconstituted 22A-sHDL by IV and IP routes. The mean circulation half-life was longer for 22A-sHDL (T1/2 = 6.27 h) than for free 22A (T1/2 = 3.81 h). The percentage of 22A absorbed by the vascular compartment after the IP dosing was ∼50% for both 22A and 22A-sHDL. The strongest pharmacologic response came from IV injection of 22A-sHDL, specifically a 5.3-fold transient increase in plasma-free cholesterol (FC) level compared with 1.3- and 1.8-fold FC increases for 22A-IV and 22A-sHDL-IP groups. Addition of either 22A or 22A-sHDL to rat plasma caused lipoprotein remodeling and appearance of a lipid-poor apoA-I. Hence, both the route of administration and the formulation of apoA-I peptide significantly affect its pharmacokinetics and pharmacodynamics.

ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer.

Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.

Apolipoprotein Mimetic Peptides as Modulators of Lipoprotein Function.

Apolipoprotein (apo)A-I and apoE are the two protein components that have been extensively investigated for their anti-atherogenic properties. Both apolipoproteins possess amphipathic helical structures, responsible for the solubilization of lipids. While apoA-I possesses class A amphipathic helical structures, apoE possesses a 59 residue long amphipathic helical domain linked to a four helix bundle containing the Arg-rich, 10 residue receptor binding domain. An 18 residue model peptide (18A) was designed to mimic the amphipathic helical domains of apoA-I. This and several analogs were able solubilize phospholipids and, when administered into animal models of atherosclerosis, were able to inhibit lesion formation without any effect on plasma cholesterol levels. These analogs were subsequently termed as apoA-I mimetic peptides. When this peptide (18A) was covalently linked to the Arg-rich receptor binding domain of apoE, the resulting peptide Ac-hE18A-NH2, in which hE refers to the 141-150 Arg-rich region of apoE, dramatically reduced plasma cholesterol in several dyslipidemic animal models, resulting in the reduction of lesion formation. This and several other analogs which were able to dramatically decrease plasma cholesterol, analogous to apoE, were termed as apoE mimetic peptides. These observations developed the field of apolipoprotein mimetic peptides which are involved in interacting with lipoproteins and modulating their function. The present review describes progress made in this field which have culminated in clinical trials in humans for both the apoA-I and apoE mimetic peptides.

Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol.