Human Lecithin:cholesterol Acyltransferase Research Articles

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Abstract 16132: Cardiometabolic Risk Factors and Non-Culprit Plaque Characteristics in Patients With STEMI in REAL-TIMI 63B

Introduction: Greater non-calcified plaque volume (NCPV) measured by coronary CT angiography (CCTA) is associated with higher risk of future myocardial infarction in stable patients. However, NCPV and associations with clinical characteristics in patients presenting with a first acute STEMI are not well described. Methods: The REAL-TIMI 63B trial randomized 593 patients with no prior MI presenting with acute STEMI to intravenous recombinant human lecithin-cholesterol acyltransferase (MEDI6012) or placebo; a subset were to undergo CCTA 48-72h after STEMI. Global NCPV, low-attenuation PV (LAPV), and calcified PV (CPV) were measured by a centralized core laboratory blinded to treatment arm. Revascularized segments were excluded from coronary plaque quantification. Results: 188 patients had a CCTA performed at baseline. Of these, 80.3% had NCPV and 77.4% had LAPV outside of the revascularized segments. Median NCPV was 28.4mm 3 [IQR 1.4-92.1], LAPV 3.8mm 3 [0.1-14.9], and CPV 92.4mm 3 [22.6-201.8]. Among the characteristics examined, male vs. female sex (51.3 mm 3 [3.47-100.3] vs 5.6 mm 3 [0.0-17.3]; p=0.0003), BMI >25 kg/m 2 (overweight or obese) (41.20 mm 3 [2.01-104.50] vs 10.98 mm 3 (0.0-61.5); p=0.01) and diabetes (82.6 mm 3 [59.0-123.4] vs 24.2 mm 3 [1.21-85.2]; p=0.038) were associated with greater NCPV (Figure). Conclusions: Among patients with no prior MI undergoing primary PCI for STEMI, we found that most patients had NCPV and LAPV outside the culprit lesion. Male sex and markers of cardiometabolic disease including higher BMI and diabetes were associated with a greater burden of NCPV. These observations underscore the need for intensive medical therapies that may stabilize vulnerable plaque in patients presenting with acute myocardial infarction.

Abstract P182: Recombinant Human Lecithin-cholesterol Acyltransferase Treatment In Patients With ASCVD Increases HDL Through Increases In Cholesterol Ester Content.

Lecithin-cholesterol acyltransferase (LCAT) mediates the esterification of free cholesterol (FC) to cholesteryl ester (CE) in HDL and, therefore, plays a critical role in reverse cholesterol transport. MEDI6012, a recombinant human LCAT (rhLCAT) increases HDL-C. Our goal was to interrogate the pathways regulating the increase in HDL-C and effects of rhLCAT on apoB metabolism. Methods: We enrolled five subjects (4 Man, mean age 67) with stable ASCVD into a Phase II, placebo controlled, double-blind, randomized cross-over study to determine the effects of two IV doses of MEDI6012, administered 48-hrs apart, versus placebo, on plasma lipids and lipoproteins. Stable isotope kinetic studies with D2-Leu, 13C-Phe, D2-Glycerol were performed to examine the metabolism of apoB100, ApoA1, ApoA2 and triglyceride. Results: As expected two doses of IV MEDI6012 increased total cholesterol and HDL-C levels significantly (Table1). We did not observe significant changes in other measured lipids or lipoproteins. The significant increase in HDL-C was 34.9±10.3 (p=0.002) and due to an increase in the amount of CE (33±8.9 p=0.001); there was no change in free cholesterol (1.9±1.9). We found no changes in levels of sterols (i.e. Lathosterol) between the two periods. Preliminary in vivo kinetic studies of HDL metabolism in 3 subjects, showed no changes in the mean fractional clearance rate (FCR) or production rate (PR) of apoA1 between placebo (0.3±0.2pool/day, 1.3±0.5 mg/kg/day) and rhLCAT treatment (0.28±0.1pools/day, 1.3±0.3). There were no changes in ApoA2 FCR and PR after rhLCAT administration. Complete data for ApoA1, ApoA2 and ApoB100 and TG kinetics will be presented. Conclusions: In subjects with ASCVD, treatment with rhLCAT increased HDL-C mainly by increasing HDL CE; there were no changes in the FCR or PR of HDL ApoA1 or ApoA2. Together, these results suggest that rhLCAT treatment is associated with increased steady-state transport of CE by HDL.

Correction of Familial LCAT Deficiency by AAV-hLCAT Prevents Renal Injury and Atherosclerosis in Hamsters-Brief Report.

[Figure: see text].

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

Hepatic Levels of DHA-Containing Phospholipids Instruct SREBP1-Mediated Synthesis and Systemic Delivery of Polyunsaturated Fatty Acids.

SummaryPolyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs.

Low Plasma Lecithin: Cholesterol Acyltransferase (LCAT) Concentration Predicts Chronic Kidney Disease.

Low high-density lipoprotein-cholesterol (HDL-c) is the most remarkable lipid trait both in mild-to-moderate chronic kidney disease (CKD) patients as well as in advanced renal disease stages, and we have previously shown that reduced lecithin:cholesterol acyltransferase (LCAT) concentration is a major determinant of the low HDL phenotype. In the present study, we test the hypothesis that reduced LCAT concentration in CKD contributes to the progression of renal damage. The study includes two cohorts of subjects selected from the PLIC study: a cohort of 164 patients with CKD (NefroPLIC cohort) and a cohort of 164 subjects selected from the PLIC participants with a basal estimated glomerular filtration rate (eGFR) > 60 mL/min/1.73 m2 (PLIC cohort). When the NefroPLIC patients were categorized according to the LCAT concentration, patients in the 1st tertile showed the highest event rate at follow-up with an event hazard ratio significantly higher compared to the 3rd LCAT tertile. Moreover, in the PLIC cohort, subjects in the 1st LCAT tertile showed a significantly faster impairment of kidney function compared to subjects in the 3rd LCAT tertile. Serum from subjects in the 1st LCAT tertile promoted a higher reactive oxygen species (ROS) production in renal cells compared to serum from subjects in the third LCAT tertile, and this effect was contrasted by pre-incubation with recombinant human LCAT (rhLCAT). The present study shows that reduced plasma LCAT concentration predicts CKD progression over time in patients with renal dysfunction, and, even more striking, it predicts the impairment of kidney function in the general population.

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL‐C levels, low plasma cholesterol esterification, and the formation of Lipoprotein‐X (Lp‐X), an abnormal cholesterol‐rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp‐X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp‐X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp‐X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT‐deficient (KO), wild type (WT), and LCAT‐transgenic (Tg) mice by gavaging mice with alpha‐naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT‐Tg mice, however, prevented the formation of Lp‐X and other plasma lipid abnormalities in WT and LCAT‐KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp‐X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp‐X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp‐X in other diseases besides FLD.

Recombinant LCAT (Lecithin:Cholesterol Acyltransferase) Rescues Defective HDL (High-Density Lipoprotein)-Mediated Endothelial Protection in Acute Coronary Syndrome.

Objective- Aim of this study was to evaluate changes in LCAT (lecithin:cholesterol acyltransferase) concentration and activity in patients with an acute coronary syndrome, to investigate if these changes are related to the compromised capacity of HDL (high-density lipoprotein) to promote endothelial nitric oxide (NO) production, and to assess if rhLCAT (recombinant human LCAT) can rescue the defective vasoprotective HDL function. Approach and Results- Thirty ST-segment-elevation myocardial infarction (STEMI) patients were enrolled, and plasma was collected at hospital admission, 48 and 72 hours thereafter, at hospital discharge, and at 30-day follow-up. Plasma LCAT concentration and activity were measured and related to the capacity of HDL to promote NO production in cultured endothelial cells. In vitro studies were performed in which STEMI patients' plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in endothelial cells. The plasma concentration of the LCAT enzyme significantly decreases during STEMI with a parallel significant reduction in LCAT activity. HDL isolated from STEMI patients progressively lose the capacity to promote NO production by endothelial cells, and the reduction is related to decreased LCAT concentration. In vitro incubation of STEMI patients' plasma with rhLCAT restores HDL ability to promote endothelial NO production, possibly related to significant modification in HDL phospholipid classes. Conclusions- Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during acute coronary syndrome. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in acute coronary syndrome.

LCAT Enzyme Replacement Therapy Reduces LpX and Improves Kidney Function in a Mouse Model of Familial LCAT Deficiency.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.

Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid

Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

Familial lecithin:cholesterol acyltransferase deficiency: First-in-human treatment with enzyme replacement

Humans with familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) have extremely low or undetectable high-density lipoprotein cholesterol (HDL-C) levels and by early adulthood develop many manifestations of the disorder, including corneal opacities, anemia, and renal disease. To determine if infusions of recombinant human LCAT (rhLCAT) could reverse the anemia, halt progression of renal disease, and normalize HDL in FLD. rhLCAT (ACP-501) was infused intravenously over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg/kg), then 3.0 or 9.0 mg/kg every 1 to 2 weeks for 7 months in a maintenance phase. Plasma lipoproteins, lipids, LCAT levels, and several measures of renal function and other clinical labs were monitored. LCAT concentration peaked at the end of each infusion and decreased to near baseline over 7 days. Renal function generally stabilized or improved and the anemia improved. After infusion, HDL-C rapidly increased, peaking near normal in 8 to 12 hours; analysis of HDL particles by various methods all revealed rapid sequential disappearance of preβ-HDL and small α-4 HDL and appearance of normal α-HDL. Low-density lipoprotein cholesterol increased more slowly than HDL-C. Of note, triglyceride routinely decreased after meals after infusion, in contrast to the usual postprandial increase in the absence of rhLCAT infusion. rhLCAT infusions were well tolerated in this first-in-human study in FLD; the anemia improved, as did most parameters related to renal function in spite of advanced disease. Plasma lipids transiently normalized, and there was rapid sequential conversion of small preβ-HDL particles to mature spherical α-HDL particles.

Familial LCAT deficiency: from pathology to enzyme replacement therapy

Lecithin:cholesterol acyltransferase (LCAT) synthesizes most of the plasma cholesteryl esters, and plays a major role in HDL metabolism. Mutations in the LCAT gene cause two syndromes, familial LCAT deficiency and fish-eye disease, both characterized by severe alterations in plasma lipoprotein profile. Renal disease is the major cause of morbidity and mortality in familial LCAT deficiency cases, but an established therapy is not currently available. The present therapy of LCAT deficiency is mainly aimed at correcting the dyslipidemia associated with the disease and at delaying evolution of chronic nephropathy. LCAT deficiency represents a candidate disease for enzyme replacement therapy. In vitro and in vivo studies proved the efficacy of recombinant human LCAT in correcting dyslipidemia, and recombinant human LCAT is presently under development.

9. AAV-Mediated Gene Transfer as a Therapeutic Approach for Familial LCAT Deficiency

Lecithin Cholesterol Acyltransferase (LCAT) is a plasma enzyme secreted by the liver and responsible for esterification of free cholesterol to cholesteryl ester in the blood compartment. Familial LCAT deficiency (FLD) is an autosomal recessive disorder caused by loss-of-function mutations of the LCAT gene and is characterized by extremely low levels of high density lipoprotein cholesterol (HDL-C), anemia, corneal opacities, and chronic progressive renal disease leading to end-stage kidney disease as early as the third decade of life. Currently, there is no specific treatment for FLD. The fact that patients with even very low levels of plasma LCAT activity do not develop kidney disease make FLD an attractive candidate for liver-directed gene therapy with adeno-associated viral (AAV) vectors. LCAT is activated by apolipoprotein A-I (apoA-I) and this interaction is highly species specific. Therefore, LCAT KO mice were crossed with human apoA-I transgenic (hApoAITg) mice in order to facilitate experiments involving expression of human LCAT. LCAT KO/hApoAI transgenic mice received a single IP injection of AAV8 encoding human LCAT in a dose range of 5e10 GC/kg to 5e11 GC/kg. HDL-C increased in a dose-dependent fashion, from 32 mg/dL in the control group to 103 mg/dL at the dose of 1.5e11 GC/kg and 473 mg/dL at the dose of 5e11 GC/kg. Plasma% cholesterol ester, a sensitive indicator of LCAT activity, was normalized at the low dose of 1.5e11 GC/kg. Thus, we demonstrate that low doses of AAV8-hLCAT provide stable expression of the LCAT enzyme and correct dyslipidemia in a ‘humanized’ mouse model of LCAT deficiency. Studies designed to demonstrate prevention of kidney disease in this mouse model are ongoing. These proof-of-concept studies provide a foundation for further development of AAV-based gene therapy for familial LCAT deficiency.

Abstract 128: Recombinant Lecithin Cholesterol Acyltransferase Generates Mature High Density Lipoprotein with Increased Capacity to Efflux Cholesterol via ABCG1 in Cynomolgus Monkeys

Introduction: Lecithin cholesterol acyltransferase (LCAT) esterifies free cholesterol to cholesteryl esters on the HDL particle and is considered a key component of reverse cholesterol transport. We have developed a recombinant human LCAT fusion protein with high LCAT activity and characterized the effects of this molecule on lipoprotein metabolism and cholesterol efflux in cynomolgus monkeys. Methods: Cynomolgus monkeys received a single injection of rhLCAT at 0, 0.5, 1, or 5 mpk. Blood samples were drawn for analysis at pre-dose and up to 5 days after administration. HDL-C and apoAI were measured using standard clinical analyzers, apoE was measured by ELISA, and rhLCAT was measured by MSD immunoassay. ApoAI containing HDL subfractions were determined by two-dimensional non-denaturing gradient gel electrophoresis as described in the literature. Cholesterol efflux was measured ex vivo from ABCA1, ABCG1, or SRB1 transfected BHK cells, or J774 mouse macrophages using apoB depleted serum. Results: After administration, serum rhLCAT concentrations increased dose proportionately. HDL-C increased rapidly, beginning within the first 6 hours after dosing, and was dose dependent with maximum increases of 87% observed in the 1 and 5 mpk dose groups. ApoAI concentrations increased by as much as 15% and 21% in the 0.5 and 1 mpk groups, respectively, but decreased in the 5 mpk group by as much as 23%. The ratio of HDL-C to apoAI increased dose dependently indicating the formation of HDL particles containing increasingly more cholesterol. 2D gels confirmed the formation of large, primarily α-1, HDL particles. ApoE on HDL particles increased dose dependently with large increases observed following the 1 mpk (169%) and 5 mpk (910%) doses. Changes in the HDL particle were accompanied by changes in the ability of HDL to efflux cholesterol from cells. ABCG1 mediated efflux increased by as much as 21% and 24% in the 1 and 5 mpk dose groups, respectively. Conversely, efflux via ABCA1, SRB1, and from J774 cells was reduced dose dependently. Conclusions: The administration of rhLCAT in cynos resulted in the formation of large HDL particles that were enriched in cholesterol and ApoE. These larger particles showed increased capacity to efflux cholesterol via the ABCG1 transporter.

Characteristic, polymorphism and expression distribution of LCAT gene in a Mongolian gerbil model for hyperlipidemia

This study aims to evaluate the genetic basis and activity of lecithin cholesterol acyltransferase (LCAT) in a novel Mongolian gerbil model for hyperlipidemia. Gerbils may be susceptible to high fat and cholesterol (HF/HC) diets, which can rapidly lead to the development of hyperlipidemia. Approximately 10–30% of gerbils that are over 8months old and fed controlled diets spontaneously develop hyperlipidemia. Using the HF/HC diet model, we detected triglycerides (TG), total cholesterol (TC), HDL (high density lipoprotein)-C, LDL (low density lipoprotein)-C and LCAT in both old (>8months) and young gerbils. The TC and HDL-C levels were two times higher in old gerbils compared with young gerbils (P<0.01). However, in the old group the LCAT activity fell slightly compared with the normal lipidemia group. It is reasonable to hypothesize that this may be associated with single nucleotide polymorphisms of the LCAT gene. We cloned this gene to investigate the sensitivity of the gerbil to the HF/HC diet and spontaneous hyperlipidemia. The entire LCAT gene was cloned by splicing sequences of RACE (rapid amplification of cDNA ends) and nest-PCR products (AN: KC533867.1). The results showed that the 3683base pair gene consists of six exons and five introns. The LCAT protein consists of 444 amino acid (AA) residues, which are analogous to the human LCAT gene, and includes 24 signal peptide AA and 420 mature protein AA. Expression of LCAT was detected in the kidney, spleen and adrenal tissue, apart from the liver, by immunohistochemistry. The abundance of the protein was greater in the older group compared with the control group. Polymorphisms were analyzed by PCR-SSCP (PCR-single-strand conformation polymorphism) but none were found in 444 animals of the ZCLA closed population (a Chinese cultured laboratory gerbil population).

A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy

A kindred affected with fish eye disease (FED) from Oklahoma is reported. Two probands with corneal opacification had mean levels of high-density lipoprotein (HDL) cholesterol (C), apolipoprotein (apo) A-I, and apoA-I in very large alpha-1 HDL particles that were 9%, 17%, and 5% of normal, whereas their parents and 1 sibling had values that were 61%, 77%, and 72% of normal. The probands had no detectable lipoprotein-X, and had mean low-density lipoprotein cholesterol (LDL-C) and triglyceride levels that were elevated. Their mean lecithin cholesterol acyltransferase (LCAT) activities, cholesterol esterification rates, and free cholesterol levels were 8%, 42%, and 258% of normal, whereas their parents and 1 sibling had values that were 55%, 49%, and 114% of normal. The defect was due to 1 common variant in the LCAT gene in exon 1: c101t causing a proline34leucine substitution and a novel mutation c1177t causing a threonine37methionine substitution, with the former variant being found in the father and 1 sibling, and the latter mutation being found in the mother, and both mutations being present in the 2 probands. FED is distinguished from familial LCAT deficiency (FLD) by the lack of anemia, splenomegaly, and renal insufficiency as well as normal or increased LDL-C. Both FLD and FED cases have marked HDL deficiency and corneal opacification, and FED cases may have premature coronary heart disease in contrast to FLD cases. Therapy, using presently available agents, in FED should be to optimize LDL-C levels, and 1 proband responded well to statin therapy. The investigational use of human recombinant LCAT as an enzyme source is ongoing.