rHDL Infusion Research Articles

Boosting cholesterol efflux from foam cells by sequential administration of RHDL to deliver microRNA and to remove cholesterol in a triple-cell two-dimensional atherosclerosis model

Background and Aims : Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening, among others. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances towards clinical application.Methods: Here a therapeutic strategy to improve cholesterol efflux has been developed based on a two-step administration of rHDL consisting on a first dose of antagomiR-33a-loaded rHDLs to induce ABCA1 transporter overexpression, followed by a second dose of DPPC rHDLs, which remove efficiently cholesterol from foam cells. A triple-cell 2D atheroma plaque model reflecting the cellular complexity of atherosclerosis has been used to overcome the translational gap providing a suitable model to improve the efficiency of the nanoparticles in promoting cholesterol efflux.Results: The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach might be used in vivo to target more efficiently atherosclerotic lesions and improve prognosis of the disease.Conclusions: Sequential targeting of foam cells with nanoparticles including a first antagomiR-33a delivery by DPPC:CE:LPC rHDL followed by a second infusion of DPPC rHDL induces a potent cholesterol efflux from foam cells, which may overcome current technique barriers to promote clinical applications. Background and Aims : Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening, among others. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances towards clinical application. Methods: Here a therapeutic strategy to improve cholesterol efflux has been developed based on a two-step administration of rHDL consisting on a first dose of antagomiR-33a-loaded rHDLs to induce ABCA1 transporter overexpression, followed by a second dose of DPPC rHDLs, which remove efficiently cholesterol from foam cells. A triple-cell 2D atheroma plaque model reflecting the cellular complexity of atherosclerosis has been used to overcome the translational gap providing a suitable model to improve the efficiency of the nanoparticles in promoting cholesterol efflux. Results: The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach might be used in vivo to target more efficiently atherosclerotic lesions and improve prognosis of the disease. Conclusions: Sequential targeting of foam cells with nanoparticles including a first antagomiR-33a delivery by DPPC:CE:LPC rHDL followed by a second infusion of DPPC rHDL induces a potent cholesterol efflux from foam cells, which may overcome current technique barriers to promote clinical applications.

Artificial high-density lipoprotein-mimicking nanotherapeutics for the treatment of cardiovascular diseases.

Despite the ability of current efficacious low-density lipoprotein-cholesterol-lowering therapies to reduce total cardiovascular disease (CVD) risks, CVD still poses major risks for morbidity and mortality to the general population. Because of the pleiotropic endothelial protective effects of high-density lipoproteins (HDL), the direct infusion of reconstituted HDL (rHDL) products, including MDCO-216, CER001, and CSL112, have been tested in clinical trials to determine whether direct infusion of rHDL can reduce coronary events in CVD patients. In addition to these rHDL products, in the past two decades, there has been an increased focused on designing artificial HDL-mimicking nanotherapeutics to produce complementary therapeutic strategies for CVD patients beyond lowering of atherogenic lipoproteins. Although recent reviews have comprehensively discussed the developments of artificial HDL-mimicking nanoparticles as therapeutics for CVD, there has been little assessment of "plain" or "drug-free" HDL-mimicking nanoparticles as therapeutics alone. In this review, we will summarize the clinical outcomes of rHDL products, examine recent advances in other types of artificial HDL-mimicking nanotherapeutics, including polymeric nanoparticles, cyclodextrins, micelles, metal nanoparticles, and so on; and potential new approaches for future CVD interventions. Moreover, success stories, lessons, and interpretations of the utility and functionality of these HDL-mimicking nanotherapeutics will be an integral part of this article. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease.

Synergetic Effect of rHDL and LXR Agonist on Reduction of Atherosclerosis in Mice.

High-density lipoproteins (HDLs) are unique in that they play an important role in the reverse cholesterol transport process. However, reconstituted HDL (rHDL) infusions have demonstrated limited beneficial effect in clinical practice. This is perhaps a consequence of the limited cholesterol efflux abilities of atheroma macrophages due to decreased expression of cholesterol transporters in advanced atheromas and following rHDL infusion treatment. Thus, we propose that a combination therapy of rHDL and a liver X receptor (LXR) agonist could maximize the therapeutic benefit of rHDL by upregulating ATP-binding cassette transporters A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1), and enhancing cholesterol efflux to rHDL. In macrophages, rHDL downregulated the expression of ABCA1/G1 in a dose- and rHDL composition-dependent manner. Although LXR agonist, T0901317 (T1317), upregulated the expression of ABCA1 and ABCG1, the drug itself did not have any effect on cholesterol efflux (6.6 ± 0.5%) while the combination of rHDL and T1317 exhibited enhanced cholesterol efflux from [3H]-cholesterol loaded J774A.1 macrophages (23.3 ± 1.3%). Treatment with rHDL + T1317 significantly reduced the area of aortic plaque in ApoE−/− mice compared to PBS treated control animals (24.16 ± 1.42% vs. 31.59 ± 1.93%, p < 0.001), while neither rHDL nor T1317 treatment alone had a significant effect. Together, we show that rHDL paired with an LXR agonist can induce a synergetic effect in reducing atheroma burden. This synergy could lead to lower overall effective dose for both drugs, potentially overcoming the existing barriers in clinical development and renewing pharmaceutical interest in these two drug classes.

Functionalization of reconstituted hdl for theranostic in cardiovascular disease

Background and Aims: ApoA-1 containing HDL lipoproteins are the major cholesterol acceptors from extra-hepatic tissues. In normal condition, their role in reverse cholesterol transport (RCT) favours lipid homeostasis. Furthermore, they are one of the key effectors in atheroprotection when dyslipidemia occurs and accordingly they have been widely studied1. Nowadays, in vitro reconstituted-HDL (rHDL) can be adequately produced and applied in biomedicine research. Referred to the cardiovascular field, it has been demonstrated that treatments based on rHDL are beneficial both in vitro and in animal studies. However, despite promising, rHDL infusions have failed to reach the prespecified efficacy endpoints when mortality benefit has been tested in clinical trials2. Therefore, with the aim to overcome this handicap, we have set up a methodology to “functionalize” rHDL with a microRNA in order to improve their therapeutic outcomes.

Role of High-Density Lipoproteins in Cholesterol Homeostasis and Glycemic Control.

Role of High-Density Lipoproteins in Cholesterol Homeostasis and Glycemic Control.

Abstract 169: Lipoproteins and their Modified Forms Regulate Smooth Muscle Cell Calcification

Background: Vascular calcification (VC), alongside atherogenic lipoprotein profiles, have been correlated with poor cardiovascular outcome, however there is a paucity of literature exploring the relationship between the two regarding VC progression. We therefore aim to examine the roles of lipoprotein species and their oxidised forms on both medial and intimal VC. Methods: Human aortic smooth muscle cells (HAoSMC) were pre-treated with 200 μg/ml high (HDL), low (LDL) and very low (VLDL) density lipoproteins for 24 hours before treating cells with a calcification medium (CM; Ca 2.7mM, PO 4 2.0 mM). Cells were harvested using an alizarin red (ARS) calcification assay, or at various time points for qPCR analysis. In parallel, in vivo studies using apolipoprotein E knock-out mice were fed an atherogenic diet for 40 weeks and received reconstituted HDL (rHDL) infusions containing apoA-I (20mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine during the final 4 weeks of the study. Tissues were harvested and stained for plaque assessment (H&amp;E) and calcium deposits (ARS). Results: Pre-treatment of HAoSMC with rHDL inhibited calcification (43.9%, p&lt;0.001), whereas ox-rHDL removed its protective effects. Likewise, ox-LDL (77.1%, p&lt;0.05) also upregulated calcium deposition and interestingly ox-VLDL significantly decreased calcification (70.5%, p&lt;0.05) with their native counterparts having no effects. PCR measures of calcification markers Runx2, RANKL and alkaline phosphatase show a time-dependent increase in expression as calcification occurs. In animal studies, no change in weight gain, cholesterol or triglyceride levels were observed with treatment. In addition, rHDL infusions did not alter plaque size however ARS staining of the brachiocephalic artery demonstrated a significant reduction (6.58%, p&lt;0.05) in calcification present in the atherosclerotic plaque. Conclusions: This study is the first to demonstrate the effects of lipoproteins on VC in vitro and the effects of rHDL on in vivo VC. Somewhat in accordance to the roles of lipoproteins in atherosclerosis, HDL and ox-VLDL show a reduction of calcification, where-as ox-LDL enhances calcification of HAoSMC.

Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with an approximately two-fold elevated risk of cardiovascular (CV)-related mortality. Patients with RA present with systemic inflammation including raised circulating myeloid cells, but fail to display traditional CV risk-factors, particularly dyslipidaemia. We aimed to explore if increased circulating myeloid cells is associated with impaired atherosclerotic lesion regression or altered progression in RA. Using flow cytometry, we noted prominent monocytosis, neutrophilia, and thrombocytosis in two mouse models of RA. This was due to enhanced proliferation of the haematopoietic stem and progenitor cells (HSPCs) in the bone marrow and the spleen. HSPCs expansion was associated with an increase in the cholesterol content, due to a down-regulation of cholesterol efflux genes, Apoe, Abca1, and Abcg1. The HSPCs also had enhanced expression of key myeloid promoting growth factor receptors. Systemic inflammation was found to cause defective cellular cholesterol metabolism. Increased myeloid cells in mice with RA were associated with a significant impairment in lesion regression, even though cholesterol levels were equivalent to non-arthritic mice. Lesions from arthritic mice exhibited a less stable phenotype as demonstrated by increased immune cell infiltration, lipid accumulation, and decreased collagen formation. In a progression model, we noted monocytosis, enhanced monocytes recruitment to lesions, and increased plaque macrophages. This was reversed with administration of reconstituted high-density lipoprotein (rHDL). Furthermore, RA patients have expanded CD16+ monocyte subsets and a down-regulation of ABCA1 and ABCG1. Rheumatoid arthritis impairs atherosclerotic regression and alters progression, which is associated with an expansion of myeloid cells and disturbed cellular cholesterol handling, independent of plasma cholesterol levels. Infusion of rHDL prevented enhanced myelopoiesis and monocyte entry into lesions. Targeting cellular cholesterol defects in people with RA, even if plasma cholesterol is within the normal range, may limit vascular disease.

VEGFR2 is activated by high‐density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia

High-density lipoproteins augment hypoxia-induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high-density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2-neutralizing antibody attenuated rHDL-induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia-driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2-neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL-infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2-neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.-Cannizzo, C. M., Adonopulos, A. A., Solly, E. L., Ridiandries, A., Vanags, L. Z., Mulangala, J., Yuen, S. C. G., Tsatralis, T., Henriquez, R., Robertson, S., Nicholls, S. J., Di Bartolo, B. A., Ng, M. K. C., Lam, Y. T., Bursill, C. A., Tan, J. T. M. VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

Beneficial Effects of Reconstituted High-Density Lipoprotein (rHDL) on Circulating CD34+ Cells in Patients after an Acute Coronary Syndrome.

BackgroundHigh-density lipoproteins (HDL) favorably affect endothelial progenitor cells (EPC). Circulating progenitor cell level and function are impaired in patients with acute coronary syndrome (ACS). This study investigates the short-term effects of reconstituted HDL (rHDL) on circulating progenitor cells in patients with ACS.Methods and FindingsThe study population consisted of 33 patients with recent ACS: 20 patients from the ERASE trial (randomized to receive 4 weekly intravenous infusions of CSL-111 40 mg/kg or placebo) and 13 additional patients recruited as controls using the same enrolment criteria. Blood was collected from 16 rHDL (CSL-111)-treated patients and 17 controls at baseline and at 6–7 weeks (i.e. 2–3 weeks after the fourth infusion of CSL-111 in ERASE). CD34+ and CD34+/kinase insert domain receptor (KDR+) progenitor cell counts were analyzed by flow cytometry. We found preserved CD34+ cell counts in CSL-111-treated subjects at follow-up (change of 1.6%), while the number of CD34+ cells was reduced (-32.9%) in controls (p = 0.017 between groups). The level of circulating SDF-1 (stromal cell-derived factor-1), a chemokine involved in progenitor cell recruitment, increased significantly (change of 21.5%) in controls, while it remained unchanged in CSL-111-treated patients (p = 0.031 between groups). In vitro exposure to CSL-111 of early EPC isolated from healthy volunteers significantly increased CD34+ cells, reduced early EPC apoptosis and enhanced their migration capacity towards SDF-1.ConclusionsThe relative increase in circulating CD34+ cells and the low SDF-1 levels observed following rHDL infusions in ACS patients point towards a role of rHDL in cardiovascular repair mechanisms.

Reconstituted high-density lipoproteins promote wound repair and blood flow recovery in response to ischemia in aged mice.

BackgroundThe average population age is increasing and the incidence of age-related vascular complications is rising in parallel. Impaired wound healing and disordered ischemia-mediated angiogenesis are key contributors to age-impaired vascular complications that can lead to amputation. High-density lipoproteins (HDL) have vasculo-protective properties and augment ischemia-driven angiogenesis in young animals. We aimed to determine the effect of reconstituted HDL (rHDL) on aged mice in a murine wound healing model and the hindlimb ischemia (HLI) model.MethodsMurine wound healing model—24-month-old aged mice received topical application of rHDL (50 μg/wound/day) or PBS (vehicle control) for 10 days following wounding. Murine HLI model—Femoral artery ligation was performed on 24-month-old mice. Mice received rHDL (40 mg/kg) or PBS, intravenously, on alternate days, 1 week pre-surgery and up to 21 days post ligation. For both models, blood flow perfusion was determined using laser Doppler perfusion imaging. Mice were sacrificed at 10 (wound healing) or 21 (HLI) days post-surgery and tissues were collected for histological and gene analyses.ResultsDaily topical application of rHDL increased the rate of wound closure by Day 7 post-wounding (25 %, p < 0.05). Wound blood perfusion, a marker of angiogenesis, was elevated in rHDL treated wounds (Days 4–10 by 22–25 %, p < 0.05). In addition, rHDL increased wound capillary density by 52.6 %. In the HLI model, rHDL infusions augmented blood flow recovery in ischemic limbs (Day 18 by 50 % and Day 21 by 88 %, p < 0.05) and prevented tissue necrosis and toe loss. Assessment of capillary density in ischemic hindlimb sections found a 90 % increase in rHDL infused animals. In vitro studies in fibroblasts isolated from aged mice found that incubation with rHDL was able to significantly increase the key pro-angiogenic mediator vascular endothelial growth factor (VEGF) protein (25 %, p < 0.05).ConclusionrHDL can promote wound healing and wound angiogenesis, and blood flow recovery in response to ischemia in aged mice. Mechanistically, this is likely to be via an increase in VEGF. This highlights a potential role for HDL in the therapeutic modulation of age-impaired vascular complications.

Disordered haematopoiesis and athero-thrombosis.

Atherosclerosis, the major underlying cause of cardiovascular disease, is characterized by a lipid-driven infiltration of inflammatory cells in large and medium arteries. Increased production and activation of monocytes, neutrophils, and platelets, driven by hypercholesterolaemia and defective high-density lipoproteins-mediated cholesterol efflux, tissue necrosis and cytokine production after myocardial infarction, or metabolic abnormalities associated with diabetes, contribute to atherogenesis and athero-thrombosis. This suggests that in addition to traditional approaches of low-density lipoproteins lowering and anti-platelet drugs, therapies directed at abnormal haematopoiesis, including anti-inflammatory agents, drugs that suppress myelopoiesis, and excessive platelet production, rHDL infusions and anti-obesity and anti-diabetic agents, may help to prevent athero-thrombosis.

Abstract 17001: Reconstituted High-density Lipoprotein (CSL-111) Infusion Improves Post-ischemic Heart Function Through Modulating the Acute Inflammatory Response and Angiogenesis

Aims: High-density lipoprotein (HDL) has multiple actions which may benefit post-ischemic heart function. We have shown in patients with type 2 diabetes that a single infusion of reconstituted HDL (rHDL, CSL-111) reduces inflammation1 including the number of circulating monocytes, neutrophils and lymphocytes. Whether such effects may contribute to improved heart function after ischemia and reperfusion is unknown. This study aimed to determine the effect of HDL on post-ischemic inflammatory response and heart function in insulin resistant mice. Methods and Results: A left coronary artery ligation model was used to study acute ischemia (30min) and reperfusion (I/R) in mice fed a chow (CD) or high fat diet (HFD) for 8 weeks. A single intravenous bolus of rHDL (CSL-111; 80mg/kg) or placebo (saline) was delivered at the time of reperfusion. At 15 days post I/R, rHDL increased ejection fraction from 40±3% to 48±2% in both diet groups (p=0.004). rHDL also increased stroke volume (p=0.002) and cardiac output in both diet groups (p=0.015). Histological analysis revealed that rHDL increased the myocardial capillary density by 22±4% and 15±4% in CD and HFD mice respectively (p=0.0007; p=0.042). However, rHDL did not alter cardiomyocyte hypertrophy. Interestingly, rHDL infusion modulated acute post-ischemic inflammatory response by decreasing the total number of circulating leucocytes (p=0.030) and neutrophils (p=0.005) in both diet groups 1 day following I/R. However, rHDL reduced the number of circulating monocytes (p=0.044) and B lymphocytes (p=0.009) only in HFD mice. In both diet groups, at 3 days post-I/R, rHDL reduced recruitment of leukocytes into the heart including monocytes (p=0.038, p=0.014), neutrophils (p=0.005, P=0.020), T lymphocytes (p=0.044, p=0.018) and B lymphocytes (p=0.043, p=0.010). Moreover, rHDL decreased the proportion of B lymphocytes recruited into the heart in HFD mice only (p=0.040), highlighting its strong anti-inflammatory effect particularly in the insulin-resistant state. Conclusion: A single infusion of rHDL at time of reperfusion improves post-ischemic heart function through modulation of angiogenesis and acute inflammatory response in insulin-resistant mice.

Investigating the Effect of a Single Infusion of Reconstituted High-Density Lipoprotein in Patients with Symptomatic Carotid Plaques

BackgroundElevation of plasma high-density lipoprotein (HDL) cholesterol concentration reduces cardiovascular mortality and morbidity. HDLs have been shown to possess acute anti-inflammatory, antioxidant, and antithrombotic properties. We hypothesize that HDL therapy can acutely alter local and systemic manifestations of plaque instability.MethodsForty patients with early symptomatic carotid disease were randomized to either receive reconstituted HDL (rHDL) 40 mg/kg (n = 20) or placebo (n = 20). Carotid endarterectomies were performed 24 hr later. Plaques were obtained intraoperatively and used for measurement of thrombomodulatory genes expression. Plasma samples were collected before the infusion, 24 and 48 hr later to measure changes in systemic markers of plaque instability.ResultsNo significant differences were noted in thrombomodulatory genes expression between the 2 groups. Systemic levels of tissue factor, matrix metalloproteinase 9 (MMP-9), and monocyte chemotactic factor-1 (MCP-1) were significantly reduced in the rHDL group. However, the effects on MMP-9 and MCP-1 were abolished in the immediate postoperative period. Although rHDL did not affect plasma interleukin-6 levels 24 hr following the infusion, it prevented the significant postoperative elevation seen in the placebo group.ConclusionsA single infusion of rHDL can acutely alter plasma biomarkers associated with plaque instability and cardiovascular morbidity.

ApoA-1 infusion reduces arterial cholesterol and myocardial lesions in a rat model of cardiac dysfunction and insulin resistance

ObjectiveLow plasma high-density lipoprotein cholesterol (HDL-C) concentration is associated with the metabolic syndrome (MetS) and increased prevalence of cardiovascular disease (CVD). Animal and human studies report infusion of apolipoprotein A-1 (apoA-1) can reduce endothelial dysfunction, and/or induce regression of atherosclerosis. However, the direct mechanisms underlying the vascular benefits of either apoA-1 or HDL-C remain unclear. In this study, we assessed the ability of reconstituted HDL (rHDL) to improve vascular complications of MetS, including left ventricular (LV)-hypertrophy, arterial cholesterol deposition and myocardial lesion development. Methods and resultsObese insulin resistant (IR) JCR:LA-cp rats were infused with rHDL (0.4mg/kg) over 3 days before assessing cardiac function (Echocardiography) at days 7 and 50 post-infusion, as well as haematoxylin and eosin staining of myocardial lesions at day 50. Acute ex vivo arterial cholesterol deposition was assessed with acute infusion of rHDL ex-vivo. Infusion of rHDL partially corrected abnormal diastolic compliance (18%; *p<0.05) and improved parameters of cardiac function in IR rats. Further, acute rHDL infusion in carotid vessels reduced remnant lipoprotein associated-cholesterol deposition (30–86%; **p<0.01) ex vivo in IR and male Wistar rats and reduced (41%; *p<0.05) the frequency of early-stage myocardial lesions in IR rats. ConclusionShort-term infusion of rHDL may beneficially reduce chronic vascular sequelae of MetS, including temporary improvement in LV-dysfunction, acute reduction of acute arterial cholesterol deposition and the development of early-stage myocardial lesions in the JCR:LA-cp rat.

HDL and ABC Transporters Reduce the Proliferation of Hematopoietic Stem and Progenitor Cells, with Anti-atherogenic Consequences

Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high fat, high cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and HDL, respectively and are targets of LXR transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common beta subunit of the IL-3/GM-CSF receptor. In Western type diet fed Apoe-/- mice, monocytosis and neutrophilia also develop in association with proliferation and expansion of hematopoietic stem, multipotential progenitor and myeloid progenitor cell populations in the bone marrow and spleen. Apoe is highly expressed in long term hematopoietic stem cells and to a lesser extent in progenitor populations, and acts in a cell autonomous fashion to control myeloid proliferation, monocytosis and accumulation of monocytes in atherosclerotic lesions. Treatment of Apoe-/- mice with infusions of reconstituted HDL and/or LXR activator resulted in normalization of HSPC proliferation, monocytosis and neutrophilia. Together, the findings suggest that ApoE acts in concert with the ATP binding cassette transporters ABCA1 and ABCG1 to control HSPC and myeloid proliferation, monocytosis and atherogenesis. LXR activators and rHDL infusions exert anti-atherogenic effects by controlling HSPC and myeloid proliferation.

CETP expression reverses the reconstituted HDL-induced increase in VLDL

Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity.

Reconstituted High-Density Lipoprotein Shortens Cardiac Repolarization

We hypothesize that increasing high-density lipoprotein cholesterol (HDL-C) shortens cardiac repolarization. HDL-C is inversely associated with sudden death. The relation between HDL-C and repolarization of the heart is unexplored. HDL-C was elevated with reconstituted high-density lipoprotein (rHDL). Cardiac repolarization was studied by recording cardiac transmembrane potentials with the patch clamp technique from isolated rabbit cardiomyocytes that were superfused with rHDL. Infusions with rHDL (40 mg/kg body weight) were performed in dyslipidemic patients and healthy volunteers. Electrocardiograms were recorded to assess cardiac repolarization before and 24 h after infusion with rHDL. rHDL as well as purified human apolipoprotein AI shortened repolarization of isolated rabbit cardiomyocytes by ∼25% (p < 0.05). rHDL infusion shortened the heart rate-corrected QT interval on surface electrocardiograms in all participants (p < 0.001). rHDL shortens cardiac repolarization. These data provide evidence for a novel mechanism of HDL infusion that may contribute to reduction of sudden cardiac death.

Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein

Infusion of reconstituted HDL (rHDL) leads to changes in HDL metabolism as well as to an increased capacity of plasma to support cholesterol efflux providing an opportunity to investigate mechanisms linking cholesterol efflux to changes in plasma HDL. Patient plasmas after infusion of rHDL were tested ex vivo for their capacity to stimulate cholesterol efflux. Reconstituted HDL enhanced mobilization of cholesterol from tissues in vivo as shown by rising HDL cholesterol concentrations over the infusion period. Infusion of rHDL in vivo led to increased cholesterol efflux ex vivo; surprisingly, removing apoB-containing lipoproteins while preserving all HDL subfractions eliminated this increase. Infusion of rHDL led to the remodelling of plasma HDL; however, the capacity of plasma to support cholesterol efflux did not correlate with changes in the concentrations of any of HDL subfractions. Unmodified rHDL accounted for only a proportion of the increment in cholesterol efflux capacity. Furthermore, studies using HeLa and BHK cells overexpressing ABCA1, ABCG1, and SR-B1 showed that the contribution of these cellular mediators of cholesterol efflux to the enhanced capacity of plasma for the efflux was minimal. Enhanced cholesterol efflux from tissues requires the presence of apoB-containing lipoproteins and may involve enhanced flow of cholesterol through multiple components of the reverse cholesterol transport pathway rather than being determined by a specific HDL subfraction.

Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.

Predicting the Yield of Cascade Screening for Familial Hypercholesterolaemia

Method: A three-year pilot cascade screening service wasundertakenat theLipidDisordersClinicatRoyalPerth Hospital.We assessed the effectiveness of cascade screening and treatment with cholesterol lowering medication. The number of non-fatal myocardial infarctions (MI) prevented as a result of treatment with statin was estimated using two methods. Results: 274 cases of FH were identified (1.13 relatives/index case); detection rates were higher with larger pedigrees. Participation and satisfaction rates were high; 88% of patients surveyed were highly satisfied with the program. Plasma LDL-cholesterol levels in statin naive patients fell by 42%andby a further 9% in those already on a statin. Preliminary estimates suggest that from22 andup to 111MIswereprevented for every 100 cases of FH treated with statin. Conclusion: Cascade screening is an effective method for identifying new cases of FH in the Australian setting. Treatment of FH patients with statin therapy leads to significant reductions in LDL-cholesterol and prevention of non-fatal MI. This reduction in coronary events is likely to generate cost savings. However, these savings need to be weighed against costs of screening and treatment, and this balance needs to be subjected to a detailed economic evaluation. doi:10.1016/j.hlc.2011.05.137 Conclusions: TNP-470 produces a robust reduction in tumour growth but is associated with a significant loss of bodyweight, which may limit its clinical application. High dose rHDL infusions had no effect on tumour mass in the murine LLC model. Further studies are needed in other tumour models. doi:10.1016/j.hlc.2011.05.138