Synthetic High-density Lipoprotein Research Articles

Antiinflammatory Actions of HDL

Atherosclerosis is an inflammatory disorder characterized by the accumulation of macrophages and T-lymphocytes in the arterial intima. The macrophages are derived from blood monocytes that adhere to, and transmigrate across, an activated or injured endothelial surface. The firm adhesion of monocytes to the endothelium requires expression of integrins such as CD11/CD18 on the monocyte surface and endothelial expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. Monocytes that are firmly tethered to the endothelium transmigrate into the subendothelial space in a process that is dependent on endothelial expression of chemokines such as monocyte chemotactic protein-1 (MCP-1).1 See accompanying article on page 2071 After entering the artery wall, monocytes differentiate into macrophages in processes that are mediated by macrophage colony stimulating factor (CSF) and granulocyte-macrophage CSF.2 Macrophages express a range of scavenger receptors that bind and internalize modified LDL in an unregulated manner. This leads to a progressive accumulation of lipids in the macrophages and the formation of foam cells: the hallmark cells of atherosclerotic lesions. The results of numerous epidemiological studies have established that …

Acute effects of high-density lipoproteins: biochemical basis and clinical findings

This review aims to provide an overview and an update of all therapeutic applications of synthetic high-density lipoproteins tested in animal models or in clinical trials. Starting from 1990, when plasma-derived high-density lipoproteins were administered for the first time to cholesterol-fed rabbits to evaluate a possible atherosclerosis regression, the efficacy of high-density lipoprotein therapy has been assessed in a variety of cardiovascular diseases. Synthetic high-density lipoproteins constituted by purified apolipoprotein (apo)A-I, recombinant apoA-I variants or small apoA-I-mimetic peptides complexed with phospholipids have proven their efficacy in animal models and recently also in humans. Clinical investigations on the effects of acute HDL treatment have been focused only on atherosclerosis regression. Short-term administration of the mutant apoA-IMilano provided clearcut benefit in coronary atheromas. More recently, treatment with synthetic high-density lipoproteins containing human purified apoA-I was associated with moderate activity in atheroma regression, albeit with some safety concerns at high doses. Administration of synthetic high-density lipoproteins has proven to be effective in promoting atherosclerosis regression not only in animal models, but also in humans. Applications to other areas of cardiovascular disease, such as restenosis and ischemia/reperfusion, have only, up to now, been tested in experimental models.

High-Density Lipoprotein Mimetics: Focus on Synthetic High-Density Lipoprotein

Epidemiologic studies show an inverse relation between high-density lipoprotein (HDL) cholesterol levels and coronary artery disease, and proof-of-concept experimental studies suggest that HDL and its apolipoproteins, specifically apolipoprotein (apo) A-I , have atheroprotective effects. Atheroprotective effects of HDL are attributed to its ability to remove macrophage cholesterol by stimulating reverse cholesterol transport as well as anti-inflammatory and antioxidant effects. Several different strategies are currently being pursued to exploit the vascular-protective effects of HDL. One such approach involves direct administration of synthetic reconstituted HDL made from linking phospholipid carriers with recombinant mutant apoA-I or plasma-derived wild-type apoA-I.

Anti-Inflammatory and Cardioprotective Activities of Synthetic High-Density Lipoprotein Containing Apolipoprotein A-I Mimetic Peptides

Apolipoprotein A-I (apoA-I) mimetic peptides may represent an alternative to apoA-I for large-scale production of synthetic high-density lipoproteins (sHDL) as a therapeutic agent. In this study, the cardioprotective activity of sHDL made with either L37pA peptide or its d-stereoisomer, D37pA, was compared to sHDL made with apoA-I. The peptides were reconstituted with palmitoyl-oleoyl-phosphatidylcholine, which yielded sHDL particles comparable to apoA-I sHDL in diameter, molecular weight, and alpha-helical content. Pretreatment of endothelial cells with either peptide sHDL reduced tumor necrosis factor alpha-stimulated vascular cell adhesion molecule-1 expression to the same extent as apoA-I sHDL. In an isolated rat heart model of ischemia/reperfusion (I/R) injury, L37pA and D37pA sHDL significantly reduced postischemic cardiac contractile dysfunction compared to the saline control, as indicated by a 49.7 +/- 6.4% (L37pA; P < 0.001) and 53.0 +/- 9.1% (D37pA; P < 0.001) increase of left ventricular-developed pressure (LVDP) after reperfusion and by a 45.4 +/- 3.4% (L37pA; P < 0.001) and 49.6 +/- 2.6% (D37pA; P < 0.001) decrease of creatine kinase (CK) release. These effects were similar to the 51.3 +/- 3.0% (P < 0.001) increase of LVDP and 51.3 +/- 3.0 (P < 0.001) reduction of CK release induced by apoA-I sHDL. Consistent with their cardioprotective effects, all three types of sHDL particles mediated an approximate 20% (P < 0.001) reduction of cardiac tumor necrosis factor alpha (TNFalpha) content and stimulated an approximate 35% (P < 0.05) increase in postischemic release of prostacyclin. In summary, L37pA and D37pA peptides can form sHDL particles that retain a similar level of protective activity as apoA-I sHDL on the endothelium and the heart; thus, apoA-I mimetic peptides may be useful therapeutic agents for the prevention of cardiac I/R injury.

Apolipoprotein A-IMilano/POPC complex attenuates post-ischemic ventricular dysfunction in the isolated rabbit heart

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-IMilano (apoA-IM) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-IM/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs–Henseleit buffer (10min), pretreated with either apoA-IM/POPC (0.45mg/mL) or vehicle (10min), subjected to global ischemia (30min) and reperfused for 60min. ApoA-IM/POPC (n=7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n=6) at the end of reperfusion (p<0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p<0.001), but not with apoA-IM/POPC. ApoA-IM/POPC reduced the release of creatine kinase at the end of the ischemic period (p<0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p<0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-IM was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-IM/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury.The apoA-IM/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

Molecular Mechanisms Responsible for the Anti-Inflammatory and Protective Effect of High-Density Lipoprotein on the Endothelium

In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDLs) exert several beneficial effects, including the prevention and correction of endothelial dysfunction. HDLs promote endothelium proliferation and diminish endothelial apoptosis; they play a key role in vasorelaxation by increasing the release of nitric oxide and prostacyclin through the induction of the expression and the activity of endothelial nitric oxide synthase and the coupling of cyclo-oxygenase 2 and prostacyclin synthase. In addition, HDLs affect coagulation, fibrinolysis, platelet adhesion, adhesion molecules and protease expression, and exert antioxidant activity. These effects are achieved at the gene expression level and are dependent on the activation of several intracellular signalling pathways, including PI3K/Akt, extracellular signal-regulated kinase (ERK) 1/2, protein kinase C, and p38MAPK (mitogen-activated protein kinase). The complexity of the signalling pathways modulated by HDL reflects the different effects of the components of this class of lipoproteins such as apolipoproteins or lipids on endothelial cell gene expression and the subsequent observed modulation of endothelial function. The in vivo relevance of these findings to endothelial recovery during physiological or pathological conditions remain to be addressed; nevertheless, the results of clinical studies with synthetic HDL, apolipoprotein A-I mimetics and drugs selectively affecting HDL plasma levels and biological functions that are becoming available support the importance of the correction of endothelial function by HDL.

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.

A Model Structure for the Heterodimer apoA-IMilano–apoA-II Supports Its Peculiar Susceptibility to Proteolysis

In this study, we propose a structure for the heterodimer between apolipoprotein A-IMilano and apolipoprotein A-II (apoA-IM–apoA-II) in a synthetic high-density lipoprotein (HDL) containing L-α-palmitoyloleoyl phosphatidylcholine. We applied bioinformatics/computational tools and procedures, such as molecular docking, molecular and essential dynamics, starting from published crystal structures for apolipoprotein A-I and apolipoprotein A-II. Structural and energetic analyses onto the simulated system showed that the molecular dynamics produced a stabilized synthetic HDL. The essential dynamic analysis showed a deviation from the starting belt structure. Our structural results were validated by limited proteolysis experiments on HDL from apoA-IM carriers in comparison with control HDL. The high sensitivity of apoA-IM–apoA-II to proteases was in agreement with the high root mean-square fluctuation values and the reduction in secondary structure content from molecular dynamics data. Circular dichroism on synthetic HDL containing apoA-IM–apoA-II was consistent with the α-helix content computed on the proposed model.

Recombinant apolipoprotein A-IMilano for the treatment of cardiovascular diseases

Apolipoprotein A-I(Milano) (apoA-I(M)) is a natural variant of apoA-I characterized by a cysteine for arginine substitution at position 173 of the primary sequence. ApoA-I(M) carriers have much less atherosclerosis than expected from their very low plasma high-density lipoprotein (HDL) cholesterol levels, suggesting that the variant might be protective. Synthetic HDL (sHDL) made with a recombinant form of the dimeric A-I(M) (A-I(M)/A-I(M)) and phospholipids given in single or multiple injections is effective in inducing the regression of atherosclerotic plaques, preventing arterial restenosis, and limiting cardiac dysfunction after ischemia/reperfusion injury. In a phase II trial in patients with acute coronary syndromes, a short-term treatment with A-I(M)/A-I(M) sHDL caused a remarkable reduction of atheroma burden. Although at early stages of drug development, A-I(M)/A-I(M) sHDL holds vast promise for the treatment of a variety of cardiovascular diseases in humans.

Therapeutic use of the high-density lipoprotein protein and peptides

High-density lipoprotein (HDL) therapy is a novel and emerging area of therapeutic development in the cardiovascular field. It attempts to supplement and improve the vascular benefit exerted by other agents that are active on lipid metabolism, for example, hypolipidaemic drugs. Furthermore, it takes advantage of the novel techniques of coronary evaluation. A number of reports have examined the potential therapeutic properties of the synthetic HDLs prepared by complexing recombinant apolipoprotein (apo) A-IMilano, a variant form of native apoA-I, with phospholipids. The availability of synthetic HDL complexes containing recombinant apoA-IMilano has opened up a new era of therapeutic management for coronary disease. HDL formulations of recombinant apoA-IMilano–phospholipid complexes have clearly shown rapid regression of a focal carotid atheroma as well as powerful protection from myocardial infarction in a rabbit model. In a pilot study, ETC-216 showed a significant reduction in coronary plaque burden after five weekly treatments, assessed by intravascular ultrasound in patients with acute coronary syndrome. Other therapeutic options of HDL therapy have recently became available.

High Density Lipoprotein Administration: A New Therapeutic Modality for the Treatment of Cardiovascular Diseases

Apolipoprotein A-I (apoA-I) is a 243 amino-acid cysteine deficient protein, containing 10 amphipathic domains, that allow its binding to a lipid-rich surface. It is the major protein component of high-density lipoproteins (HDL). Each HDL contains 2 or 3 copies of apoA-I plus various combinations of additional apolipoproteins (e.g. apoE, apoA-IV, apoA-II), lipid transport proteins (e.g. CETP and PLTP), and surface acting enzymes (e.g. paraoxonase, LCAT, PAF). This heterogenous and dynamic class of lipoproteins mainly functions in reverse cholesterol transport, a process resulting in the net removal of excess cholesterol from tissues, to be transported to the liver for excretion. The apolipoprotein AIMilano (A-IMilano) is a rare and naturally occurring variant form of the native apoA-I. The A-IMilano variant was a chance discovery in an individual with very low HDL-cholesterol (7-10 mg/dL) compared to the normal human levels of 40-50 mg/dL. This clinical observation dates back 30 years, and was accompanied by similar findings in two of the probands children as well as in his father. Surprisingly, these carriers appeared to be remarkably free from any vascular abnormalities. Extensive studies led to the identification of the mutation, a Cysteine for Arginine substitution at position 173, i.e. the first description of a human apolipoprotein mutation associated to altered lipoprotein composition and metabolism. This led to an extensive evaluation of the population of Limone sul Garda for the presence of the mutation and the cardiovascular disease status of the population, confirming the earlier suggestion that this might be a protective variant. In the past 25 years some important additional insights were provided. Particularly, it could be shown that the HDL containing AIMilano is endowed with a remarkably enhanced capacity to remove cholesterol from tissues. "HDL therapy" is a novel and emerging area of therapeutic development in the cardiovascular field. It attempts to supplement and improve the vascular benefit exerted by other agents active on lipid metabolism, ie hypolipidemic drugs. Further, it takes advantage on novel techniques of coronary evaluation. A number or reports have examined the potential therapeutic properties of synthetic HDL prepared by complexing recombinant apo A-IMilano with phospholipids. The availability of synthetic HDL complexes containing recombinant apoA-IMilano has opened a new era of therapeutic management of coronary disease. Prototype HDL formulations of recombinant apoA-IMilano/phospholipid complexes have used dipalmitoylphosphatidylcholine (DPPC) or 1-palmitoyl-2-oleyl phosphatidylcholine (POPC) as phospholipid component. HDL containing apoAIMilano/ DPPC have clearly shown rapid regression of a focal carotid atheroma in a rabbit model. The mechanism of this very rapid effect appears to be associated with the cholesterol removing capacity and extended serum half-life of HDL containing A-IMilano. In a pilot study, ETC-216 (apoA-IMilano/POPC) similarly showed significant reduction of coronary plaque burden after five weekly treatments as assessed by intravascular ultrasound (IVUS) in patients with acute coronary syndromes. It is likely that additional safety, efficacy and dose regimen studies in a larger patient population will need to be performed to fully evaluate the potential of this agent as an "HDL therapy" product candidate. "HDL therapy" such as ETC- 216 is being evaluated for use with the current standard of care for patients with acute coronary syndromes, including the use of statins. Keywords: synthetic hdl, apolipoprotein a-iMilano, cardiovascular disease, atherosclerosis, ivus, recombinant proteins, regression, animal models

Molecular mechanisms responsible for the antiinflammatory and protective effect of HDL on the endothelium

In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDL) exert several beneficial effects, including the prevention and correction of endothelial dysfunction. HDL promote endothelium proliferation and diminish endothelial apoptosis; they play a key role in vasorelaxation by increasing the release of nitric oxide and prostacyclin through the induction of the expression and the activity of endothelial nitric oxide synthase and the coupling of cyclooxygenase 2 and prostacyclin synthase. In addition, HDL affect coagulation, fibrinolysis, platelet adhesion, adhesion molecules, and protease expression, and they exert antioxidant activity. These effects are achieved at the gene expression level and are dependent on the activation of several intracellular signaling pathways, including PI3K/Akt, ERK1/2, PKC, and p38MAPK. The complexity of the signaling pathways modulated by HDL reflects the different effects of the components of this class of lipoproteins such as apolipoproteins or lipids on endothelial cell gene expression and the subsequent modulation of endothelial function observed. The in vivo relevance of these findings to endothelial recovery during physiological or pathological conditions remains to be addressed; nevertheless, the results of clinical studies with synthetic HDL, ApoA-I mimetics, and drugs that are becoming available that selectively affect HDL plasma levels and biological functions support the importance of the correction of endothelial function by HDL.

Apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complex (ETC-216) protects the in vivo rabbit heart from regional ischemia-reperfusion injury.

Ex vivo studies demonstrated that a synthetic high-density lipoprotein (HDL) comprised of a complex of recombinant apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine protects the isolated rabbit heart from reperfusion injury. Therefore, we sought to determine whether a pharmaceutical preparation of this complex, ETC-216, was cardioprotective in an in vivo model of left anterior descending artery (LAD) occlusion and reperfusion. Initially, ETC-216 (100 mg/kg) was tested in acute (one-treatment) and chronic (two-treatment) i.v. administrations. ETC-216-treated rabbits developed smaller infarcts expressed as percentage of area at risk (p <0.01) compared with vehicle treatments. No differences were noted between chronic and acute administration. Therefore, ETC-216 (10, 3, or 1 mg/kg) or equivalent vehicle volumes were acutely infused. Compared with vehicle, ETC-216 reduced infarct size as a percentage of the area at risk at 10 (p <0.0005) and 3 mg/kg (p <0.05). No significant differences occurred at 1 mg/kg. To determine whether ETC-216 could protect the heart after initiation of ischemia, the synthetic HDL (10 mg/kg) was infused intravenously beginning 5 min before the end of 30 min of LAD occlusion. Infarct size as percentage of the area at risk was 31.6 +/- 3.0 (ETC-216) versus 49.5 +/- 2.5 (vehicle) (p <0.001), and as percentage of left ventricle was 19.7 +/- 1.6 (ETC-216) versus 34.1 +/- 2.3 (vehicle) (p <0.0005). Electron microscopy demonstrated that ETC-216 prevented irreversible cardiac damage as assessed by mitochondrial granulation and sarcomere contraction band formation. These findings suggest ETC-216 reduces reperfusion injury and may have utility for coronary artery revascularization procedures.

Synthetic high-density lipoproteins exert cardioprotective effects in myocardial ischemia/reperfusion injury.

Human high-density lipoproteins (HDLs) protect the heart against ischemia/reperfusion injury. In the present study, the cardioprotective effects of synthetic high-density lipoproteins (sHDLs) made of phosphatidylcholine and apolipoprotein A-I were investigated in isolated rat hearts, which underwent a 20-min low-flow ischemia followed by a 30-min reperfusion. The administration of sHDL during the 10 min immediately before ischemia caused a rapid, dose-dependent improvement of postischemic cardiac function: at the maximum dose (2.0 mg of sHDL protein/ml), left ventricular developed pressure (LVDP) recovered to 71.0 +/- 3.2 versus 40.5 +/- 3.8 mm Hg in saline-treated hearts, and coronary perfusion pressure (CPP) increased to 100.3 +/- 6.2 versus 132.0 +/- 9.0 mm Hg. The preservation of postischemic cardiac function was associated with a dose-dependent reduction of creatine kinase release into the coronary effluent. sHDLs administered in the first 10 min postischemia also exerted a significant, dose-dependent improvement of postischemic LVDP, CPP, and creatine kinase release, but the cardioprotective effect was less than when sHDLs were given preischemia. The preservation of postischemic cardiac function by sHDL was mediated through a reduction of cardiac tumor necrosis factor-alpha content and an enhanced cardiac production of prostaglandin E2 and I2. The present experimental data indicate that sHDLs may provide a novel therapeutic approach to clinical conditions in which myocardial ischemia/reperfusion occurs, such as acute coronary syndromes, cardiac surgery, or revascularization procedures.

Recombinant apolipoprotein A-I[Formula: See Text]: a novel agent for the induction of regression of atherosclerotic plaques

Apolipoprotein (apo) A-I, because of its anti-atherogenic properties, provides a potentially powerful approach to the management of vascular diseases. In the clinic, patients with low high density lipoproteins (HDL)/apoA-I are at dramatically increased risk of coronary disease, the opposite being true for individuals with high HDL. Drug studies, e.g., the VA-HIT trial with gemfibrozil, clearly associated a reduced risk of events with raised HDL-cholesterolemia. A number of animal studies have shown that the infusion of apoA-I containing synthetic HDL can inhibit atherosclerosis progression in experimental animals, being also able to stimulate reverse cholesterol transport in humans. Recently, high interest has been devoted to a molecular variant of apoA-I, apoA-I[Formula: See Text] (apoA-I[Formula: See Text]), characterized by a Cys for Arg substitution and formation of apoA-I[Formula: See Text]/A-I[Formula: See Text] dimers. These latter are characterized by a prolonged permanence in plasma and a more effective cholesterol removing function, which may offer an improved approach to the therapeutic management of arterial disease. Aside from a number of clinical studies on human apoA-I[Formula: See Text] carriers, all indicating a clear protection from cardiovascular disease in spite of markedly reduced HDL levels, animal investigations have provided definite indication as to the potential of apoA-I[Formula: See Text] infusion to directly reduce the extent of atherosclerotic plaques. In addition to the well known powerful cholesterol effluxing capacity of apoA-I[Formula: See Text], fibrinolytic properties and possibly antioxidant/vasodilator mechanisms seem to be in play. Ongoing clinical studies will provide final indication as to the potential of this new therapeutic approach.

C05 High density lipoproteins protect the heart against ischemia-reperfusion injury

A high plasma concentration of high density lipoproteins (HDL) is associated with a decreased risk for cardiovascular disease, an effect commonly attributed to the central role of HDL in reverse cholesterol transport, the process by which excess cholesterol in the arterial wall is transported to the liver for excretion. Nevertheless, HDL exert additional cellular effects, like stimulation of prostacyclin release, that may well contribute to the protection of the arterial wall in conditions not associated with increased lipid deposition. This prompted us to investigate the direct protective effect of HDL against postischemic myocardial dysfunction in an isolated heart model. Rat hearts were perfused at a constant flow of 15 mllmin for 30 min; a moderate ischemia was induced by reducing the flow to 1 mllmin for 20 min and was followed by a 30 min reperfusion at 15 ml/min. Buffer-perfused hearts showed a clear-cut reduction of left ventricular function with a substantial increase of coronary resistance, associated with a consistent release of CK in the perfusates. The perfusion of the hearts with human HDL during the last 10 min of pre-ischemia caused a dosedependent protection against ischemia-reperfusion damage, as shown by an almost complete recovery of lefl ventricular developed pressure, a minimal increase of coronary perfusion pressure and a blunted CK release. Synthetic HDL containing lecithin and apoA-I were also effective in preventing ischemiareperfusion injury, whereas lipid-free apoA-I and lecithin liposomes were totally ineffective. These findings indicate that HDL exert a direct protective effect against ischemia-reperfusion damage, providing support to the therapeutic use of synthetic HDL in a variety of clinical conditions characterized by myocardial stunning, e.g., thrombolytic therapy, angioplasty or cardiac surgery.

Scavenger Receptor BI Promotes High Density Lipoprotein-mediated Cellular Cholesterol Efflux

Scavenger receptor BI (SR-BI) binds high density lipoproteins (HDL) with high affinity and mediates the selective uptake of HDL cholesteryl ester. We examined the potential role of SR-BI in mediating cellular cholesterol efflux. In Chinese hamster ovary cells stably transfected with murine SR-BI, overexpression of SR-BI resulted in a 3-4-fold stimulation of initial cholesterol efflux rates. Efflux rates correlated with SR-BI expression in cells and HDL concentration in the medium. When incubated with synthetic cholesterol-free HDL, SR-BI-transfected cells showed approximately 3-fold increases in initial rates of efflux compared with control cells, indicating that SR-BI expression enhances net cholesterol efflux mediated by discoidal HDL. In six different cell types, including cultured macrophages, the rate of efflux of cholesterol mediated by HDL or serum was well correlated with cellular SR-BI expression level. In addition, in situ hybridization experiments revealed that SR-BI mRNA was expressed in the thickened intima of atheromatous aorta of apolipoprotein E knockout mice. Thus, SR-BI is an authentic HDL receptor mediating cellular cholesterol efflux. SR-BI may facilitate the initial steps of HDL-mediated cholesterol efflux in the arterial wall as well as later steps of reverse cholesterol transport involving uptake of HDL cholesterol in the liver.

Structure-function relationship in recombinant A-I M/A-I M

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-IMilano (apoA-IM) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-IM/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs–Henseleit buffer (10 min), pretreated with either apoA-IM/POPC (0.45 mg/mL) or vehicle (10 min), subjected to global ischemia (30 min) and reperfused for 60 min. ApoA-IM/POPC (n = 7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n = 6) at the end of reperfusion (p < 0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p < 0.001), but not with apoA-IM/POPC. ApoA-IM/POPC reduced the release of creatine kinase at the end of the ischemic period (p < 0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p < 0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-IM was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-IM/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury.The apoA-IM/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

Comparative acyl specificities for transfer and selective uptake of high density lipoprotein cholesteryl esters

This study compares the specificities of selective uptake and transfer mediated by plasma cholesteryl ester transfer protein (CETP) for various species of cholesteryl esters in high density lipoproteins (HDL). [3H]Cholesterol was esterified with a series of variable chain length saturated acids and a series of variably unsaturated 18-carbon acids. These were incorporated into synthetic HDL particles along with 125I-labeled apoA-I as a tracer of HDL particles and [14C]cholesteryl oleate as an internal standard for normalization between preparations. Selective uptake by Y1-BS1 mouse adrenal cortical tumor cells was most extensively studied, but uptake by human HepG2 hepatoma cells and fibroblasts of human, rat, and rabbit origin were also examined. Acyl chain specificities for selective uptake and for CETP-mediated transfer were conversely related; selective uptake by all cell types decreased with increasing acyl chain length and increased with the extent of unsaturation of C18 chains. In contrast, CETP-mediated transfer increased with acyl chain length, and decreased with unsaturation of C18 chains. The specificities of human and rabbit CETP were also compared, and were found to differ little. Associated experiments showed that HDL-associated triglycerides, traced by [3H]glyceryl trioleyl ether, were selectively taken up but at a lesser rate than cholesteryl esters. The mechanism of this uptake appears to be the same as for selective uptake of cholesteryl esters.

Synthetic high density lipoprotein particles. Application to studies of the apoprotein specificity for selective uptake of cholesterol esters.

Particles closely resembling rat high density lipoproteins (HDL) in terms of equilibrium density profile and particle size were prepared by sonication of apoA-I with a microemulsion made with egg lecithin and cholesterol oleate. These particles, like authentic HDL, allowed selective uptake of their cholesterol ester moieties by cultured cells without parallel uptake of the particle itself. That uptake was saturable and competed by HDL. In rats, the plasma decay kinetics and sites of uptake of a cholesteryl ether tracer were similar whether that tracer was incorporated into synthetic or authentic HDL. Synthetic particles containing other apoproteins were made by generally the same method, but using in place of apoA-I either a mixture of rat apoCs or apoE that was either competent or reductively methylated to prevent interaction with the B/E receptor. These particles, of lower density and larger Stokes radius than those made with apoA-I, also allowed selective uptake of cholesterol esters, albeit with a lower degree of selectivity than in the case of apoA-I. Thus a specific apoprotein component in the subject lipoprotein particle is not required for selective uptake. However, selective uptake was shown to be a function of particle density or size, and part of the difference in rates of selective uptake from the particles made with various apoproteins was explained by their differences in density or size.