Lecithin:cholesterol Acyltransferase Research Articles

Systematic evaluation of lecithin:Cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: Regulatory role of charged residues at positions 4 and 7.

Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and β-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT β-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.

Lutein and Zeaxanthin Enhance, Whereas Oxidation, Fructosylation, and Low pH Damage High-Density Lipoprotein Biological Functionality.

High-density lipoproteins (HDLs) are key regulators of cellular cholesterol homeostasis but are functionally altered in many chronic diseases. The factors that cause HDL functional loss in chronic disease are not fully understood. It is also unknown what roles antioxidant carotenoids play in protecting HDL against functional loss. The aim of this study was to measure how various disease-associated chemical factors including exposure to (1) Cu2+ ions, (2) hypochlorous acid (HOCL), (3) hydrogen peroxide (H2O2), (4) sialidase, (5) glycosidase, (6) high glucose, (7) high fructose, and (8) acidic pH, and the carotenoid antioxidants (9) lutein and (10) zeaxanthin affect HDL functionality. We hypothesized that some of the modifications would have stronger impacts on HDL particle structure and function than others and that lutein and zeaxanthin would improve HDL function. HDL samples were isolated from generally healthy human plasma and incubated with the corresponding treatments listed above. Cholesterol efflux capacity (CEC), lecithin-cholesterol acyl transferase (LCAT) activity, and paraoxonase-1 (PON1) activity were measured in order to determine changes in HDL functionality. Median HDL particle diameter was increased by acidic pH treatment and reduced by HOCl, high glucose, high fructose, N-glycosidase, and lutein treatments. Acidic pH, oxidation, and fructosylation all reduced HDL CEC, whereas lutein, zeaxanthin, and sialidase treatment improved HDL CEC. LCAT activity was reduced by acidic pH, oxidation, high fructose treatments, and lutein. PON1 activity was reduced by sialidase, glycosidase, H2O2, and fructose and improved by zeaxanthin and lutein treatment. These results show that exposure to oxidizing agents, high fructose, and low pH directly impairs HDL functionality related to cholesterol efflux and particle maturation, whereas deglycosylation impairs HDL antioxidant capacity. On the other hand, the antioxidants lutein and zeaxanthin improve or preserve both HDL cholesterol efflux and antioxidant activity but have no effect on particle maturation.

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.

Impaired HDL function and endothelial barrier stability in severe anaphylaxis

Growing evidence demonstrates the importance of high- and low-density lipoprotein cholesterol in certain immune and allergy-mediated diseases. This study aimed to evaluate levels of high- and low-density lipoprotein cholesterol and apolipoproteins (Apo) A1 and B in serum from a cohort of patients presenting with hypersensitivity reactions. We further assessed the function of high-density lipoprotein particles as well as their involvement in the molecular mechanisms of anaphylaxis. Lipid profile determination was performed in paired (acute and baseline) serum samples from 153 patients. Thirty-eight experienced a non-anaphylactic reaction and 115 had an anaphylactic reaction (88 moderate and 27 severe). Lecithin cholesterol acyl transferase activity was assessed in patient serum, and we also evaluated macrophage cholesterol efflux in response to the serum samples. Lastly, the effect of anaphylactic-derived HDL particles on the endothelial barrier was studied. For detailed Methods, please see the Methods section in this article's Online Repository at www.jacionline.org. Severe anaphylactic reactions show statistically significant low levels of HDL-C, LDL-C, ApoA1, and ApoB, which points to a possible role as biomarkers. Specifically, HDL particles play a protective role in cardiovascular diseases. Using functional human sera-cell assays, we observed an impaired capacity of ApoB-depleted serum to induce macrophage cholesterol efflux in severe anaphylactic reactions. In addition, purified HDL particles from human anaphylactic sera failed to stabilize and maintain the endothelial barrier. These results encourage further research on HDL functions in severe anaphylaxis, which may lead to new diagnostic and therapeutic strategies.

Cholesterol esterification is hampered in alzheimer’s disease and cholesteryl esters composition is consequently altered

Introduction and Aim: Several epidemiological studies indicate a strong inverse association between the risk of developing Alzheimer’s disease (AD) and plasma HDL-C levels. The mechanism by which plasma HDL influence the pathogenesis and progression of AD is still unsolved and since cholesterol esterification is a crucial step in HDL metabolism it could be involved. The purpose of this study was to evaluate cholesterol esterification and HDL subclasses in plasma and cerebrospinal fluid (CSF) of Alzheimer’s Disease (AD) patients. Methods: The study enrolled 70 AD patients and 74 cognitively-normal controls comparable for age and sex. Lipids and lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF using assays set for measurement in plasma, which were appropriately modified for CSF. Results: AD patients have normal plasma lipids, but significantly reduced unesterified cholesterol and unesterified/total cholesterol ratio. Lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate (CER), two measures of the efficiency of the esterification process, were reduced by 29% and 16%, respectively, in plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls, but the content of small discoidal preβ-HDL particles was significantly reduced. In agreement with the reduced preβ-HDL particles, cholesterol efflux capacity mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients’ plasma. The CSF unesterified to total cholesterol ratio was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma unesterified cholesterol and unesterified/total cholesterol ratio with Aβ1-42 CSF content. Conclusions: Taken together data indicate that cholesterol esterification is hampered in plasma and CSF of AD patients, and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF Aβ1-42).

Essential phospholipids impact cytokine secretion and alter lipid-metabolizing enzymes in human hepatocyte cell lines

BackgroundEssential phospholipids (EPL) are hepatoprotective.MethodsThe effects on interleukin (IL)-6 and -8 secretion and on certain lipid-metabolizing enzymes of non-cytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), polyenylphosphatidylcholine (PPC), and phosphatidylinositol (PtdIns) (both at 0.1 and 1 mg/ml), compared with untreated controls, were assessed in human hepatocyte cell lines (HepG2, HepaRG, and steatotic HepaRG).ResultsLipopolysaccharide (LPS)-induced IL-6 secretion was significantly decreased in HepaRG cells by most phospholipids, and significantly increased in steatotic HepaRG cells with at least one concentration of EPL and PtdIns. LPS-induced IL-8 secretion was significantly increased in HepaRG and steatotic HepaRG cells with all phospholipids. All phospholipids significantly decreased amounts of fatty acid synthase in steatotic HepaRG cells and the amounts of acyl-CoA oxidase in HepaRG cells. Amounts of lecithin cholesterol acyltransferase were significantly decreased in HepG2 and HepaRG cells by most phospholipids, and significantly increased with 0.1 mg/ml PPC (HepaRG cells) and 1 mg/ml PtdIns (steatotic HepaRG cells). Glucose-6-phosphate dehydrogenase activity was unaffected by any phospholipid in any cell line.ConclusionsEPL, PPC, and PtdIns impacted the secretion of pro-inflammatory cytokines and affected amounts of several key lipid-metabolizing enzymes in human hepatocyte cell lines. Such changes may help liver function improvement, and provide further insights into the EPL’s mechanism of action.

A Study of the Relationship between Serum Magnesium Levels and Lipid Profile in Hypertensive patients on Diuretics

Hypertension (HTN) is a chronic universal health problem. It is considered an important changeable risk factor for morbidity and mortality. Diuretics are among the first-line medications for HTN treatment. Both thiazide-type diuretics and loop diuretics promote urinary loss of electrolytes such as magnesium. Magnesium has been studied for its important involvement in many physiological processes and biochemical pathways. It plays a significant role in regulating the activity of enzymes required in lipids and lipoproteins metabolism like HMG-CoA Reductase, lipoprotein lipase (LPL), and lecithin cholesterol acyl transferase (LCAT). It is well documented that dyslipidemia is a general threat to health and should be determined early to manage cardiovascular disease (CVD) and prevent its complications. The goal of the current study was to investigate the association between serum magnesium levels and lipid profile components in hypertensive patients on long-term diuretics therapy. The study was conducted on 50 adult hypertensive patients and 25 matched healthy subjects at Tishreen University Hospital (TUH) of Lattakia City in Syria. The study showed a statistically significant decrease in serum magnesium concentrations in the patients group (1.87±0.16mg/dl) versus the controls (2.40±0.41mg/dl) (P<0.05). A significant negative correlation between magnesium and triglycerides, total cholesterol, LDL cholesterol was identified, while a significant positive correlation with HDL cholesterol was found. In conclusion, this study has showed that hypomagnesemia is related to increased levels of TC, LDL-c, TG and decreased levels of HDL-c, therefore, an increased risk of dyslipidemia in hypertensive patients on diuretics.

Contemporary hypolipemic therapy: possibilities of novel hypolipemic drugs in correction of lipid metabolism disorders. Review

Dyslipidemia plays a role of significant risk factor for development and progression of cardiovascular diseases. Today several classes of hypolipemic drugs with evidenced effectiveness are used. But some patients don’t tolerate medicines with high efficacy or can’t achieve the targeted blood lipid profile levels even in case of the use of maximal doses or combined therapy. This necessitates the creation of new hypolipidemic agents.
 The review is devoted to discussion of new directions of influence on impaired lipid metabolism through creation of new classes of hypolipemic drugs with some perspectives of effective correction of lipid factors of cardiovascular risk (CVR) in cases of impossibility to achieve the targeted levels of low‑density lipoprotein cholesterol (LDL‑C) or there is a residual CVR caused by elevated serum triglyceride (TG) levels and/or low levels of high‑density lipoprotein cholesterol (HDL‑C).
 Among the novel drugs for hypercholesterolemia (HCE) correction, considerations are given for some mechanisms of action and efficacy of application of proprotein convertase subtilisin‑kexine type 9 (PCSK9) inhibitors as well as bempedoic acid (adenosine triphosphate citrate lyase inhibitor) and inclisiran, a small synthesized interfering ribonucleic acid (RNA) with its ability to blockade genes‑regulators of hepatic PCSK9 activity. The novel agents for hypertriglyceridemia correction, discussed in the review, include icosapent ethyl (a high purified and firm ester of ethyl‑eicosapentoenic acid), pemafibrate (the first selective modulator of α‑receptors activated by peroxisome proliferator), apolipoprotein C‑III (ApoC‑III) inhibitors (volasenorsen, olesarsen), mipomersen (antisense nucleotide for matrix RNA of apolipoprotein B‑100), lomitapide (inhibitor of microsomal TG transfer protein) and inhibitors of angiopoietin‑like protein 3 (ANGPTL3) (evinacumab, vupanorsen). Among the new approaches to HDL‑C elevation an attention is paid to the experimental and clinical investigations of recombinant HDL, agents with recombinant lecithin‑cholesterol‑acyl‑transferase (LCAT), and apolipoprotein A‑I (ApoA‑I) mimetic peptides. For the lowering of blood lipoprotein(a) (LP(a)) level the possibilities of pelacarsen, an antisense oligonucleotide for matrix RNA of apolipoprotein(a) (apo(a)) contained in LP(a) molecule and olpasiran, a small interfering RNA for LP(a) molecule inhibited apo(a) synthesis are uncovered. A high probability of clinical application in the nearest future will have novel drugs approved by US Food and Drug Administration (FDA) and European Medicines Agency (EMA) such as bempedoic acid and inclisiran for HCE correction as well as lomitapide for the treatment of familial HCE.

Longitudinal analysis of clinical and laboratory biomarkers in a patient with familial lecithin: Cholesterol acyltransferase deficiency (FLD) and accelerated eGFR decline: A case study

Familial lecithincholesterol acyltransferase (LCAT) deficiency (FLD) is an ultra-rare autosomal recessive disease characterized by very low HDL-C levels, corneal opacity, anemia, and progressive renal disease. The rate and severity of renal disease are variable across FLD patients and the biomarkers and risk factors for disease progression are poorly understood. Here we report a 30 year-long comparative analysis of the clinical and laboratory biomarkers in an FLD patient with accelerated renal decline, who underwent 2 kidney and one liver transplantations. Results show that elevated TG and non-HDL-C levels may promote the formation of LpX and accelerate renal function decline, whereas markers of anemia may be early predictors. Conversely, corneal opacity progresses at a steady rate and does not correlate with lipid, hematologic, or renal biomarkers. Our study suggests that monitoring of markers of anemia may aid the early detection and timely management of kidney disease with conservative therapies. Furthermore, it suggests that controlling hypercholesterolemia and hypertriglyceridemia may help improve renal disease prognosis.

Targeting host-specific metabolic pathways-opportunities and challenges for anti-infective therapy.

Microorganisms can takeover critical metabolic pathways in host cells to fuel their replication. This interaction provides an opportunity to target host metabolic pathways, in addition to the pathogen-specific ones, in the development of antimicrobials. Host-directed therapy (HDT) is an emerging strategy of anti-infective therapy, which targets host cell metabolism utilized by facultative and obligate intracellular pathogens for entry, replication, egress or persistence of infected host cells. This review provides an overview of the host lipid metabolism and links it to the challenges in the development of HDTs for viral and bacterial infections, where pathogens are using important for the host lipid enzymes, or producing their own analogous of lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) thus interfering with the human host's lipid metabolism.

A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Effects of Thyroid Powder on Tadpole (Lithobates catesbeiana) Metamorphosis and Growth: The Role of Lipid Metabolism and Gut Microbiota.

A low metamorphosis rate of amphibian larvae, commonly known as tadpoles, limits the farming production of bullfrogs (Lithobates catesbeiana). This study aimed to examine the effects of processed thyroid powder as a feed additive on tadpole metamorphosis, lipid metabolism, and gut microbiota. Five groups of tadpoles were fed with diets containing 0 g/kg (TH0), 1.5 g/kg (TH1.5), 3 g/kg (TH3), 4.5 g/kg (TH4.5), and 6 g/kg (TH6) thyroid powder for 70 days. The results showed that TH increased the average weight of tadpoles during metamorphosis, with the TH6 group having the highest values. The TH4.5 group had the highest metamorphosis rate (p < 0.05). Biochemical tests and Oil Red O staining showed that the lipid (triglyceride) content in the liver decreased after TH supplementation, especially at doses higher than 1.5 g/kg. RT-qPCR revealed that TH at doses higher than 4.5 g/kg significantly up-regulated the transcriptional expression of the pparα, accb, fas, fadd6, acadl, and lcat genes, which are related to lipid metabolism (p < 0.05). These results showed that TH seems to simultaneously promote the synthesis and decomposition of lipid and fatty acids, but ultimately show a decrease in lipids. As for the gut microbiota, it is noteworthy that Verrucomicrobia increased significantly in the TH4.5 and TH6 groups, and the Akkermansia (classified as Verrucomicrobia) was the corresponding genus, which is related to lipid metabolism. Specifically, the metabolic pathways of the gut microbiota were mainly enriched in metabolic-related functions (such as lipid metabolism), and there were significant differences in metabolic and immune pathways between the TH4.5 and TH0 groups (p < 0.05). In summary, TH may enhance lipid metabolism by modulating the gut microbiota (especially Akkermansia), thereby promoting the growth of tadpoles. Consequently, a supplementation of 4.5 g/kg or 6 g/kg of TH is recommended for promoting the metamorphosis and growth of tadpoles.

Betaine supplementation alleviates corticosterone-induced hepatic cholesterol accumulation through epigenetic modulation of HMGCR and CYP7A1 genes in laying hens

Excessive corticosterone (CORT) exposure could cause hepatic cholesterol accumulation in chickens and maternal betaine supplementation could decrease hepatic cholesterol deposition through epigenetic modifications in offspring chickens. Nevertheless, it remains uncertain whether providing betaine to laying hens could protect CORT-induced hepatic cholesterol accumulation via epigenetic mechanisms. This study aimed to examine the effects of dietary betaine on plasma and hepatic cholesterol contents, expression of cholesterol metabolic genes, as well as DNA methylation on their promoters in the liver of laying hens exposed to CORT. A total of 72 laying hens at 130 d of age were randomly divided into 3 groups: control (CON), CORT, and CORT+betaine (CORT+BET) groups. The experiment lasted for 35 d. Chickens in CON and CORT groups were fed a basal diet, whereas the CORT+BET group chickens were fed the basal diet supplemented with 0.1% betaine for 35 d. On d 28 of the experiment, chickens in CORT and CORT+BET groups received daily subcutaneous injections of CORT (4.0 mg/kg body weight), whereas the CON group chickens were injected with an equal volume of solvent for 7 d. The results showed that CORT administration led to a significant increase (P < 0.05) in the contents of cholesterol in plasma and liver, associated with activation (P < 0.05) of sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), lecithin-cholesterol acyltransferase (LCAT) and low-density lipoprotein receptor (LDLR) genes expression, and inhibition of cholesterol-7-alpha hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) genes expression in the liver compared to the CON. In contrast, CORT-induced up-regulation of HMGCR mRNA and protein abundances and downregulation of CYP7A1 mRNA and protein abundances were completely normalized (P < 0.05) by betaine supplementation. Besides, CORT injection led to significant hypomethylation (P < 0.05) on HMGCR promoter and hypermethylation (P < 0.05) on CYP7A1 promoter. Moreover, dietary betaine rescued (P < 0.05) CORT-induced changes in methylation status of HMGCR and CYP7A1 genes promoters. These results indicate that dietary betaine addition protects laying hens from CORT-induced hepatic cholesterol accumulation via epigenetic modulation of HMGCR and CYP7A1 genes.

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.

Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment. First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin-cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients. Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells. Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA. While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours. Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Seasonal Factors Are Associated with Activities of Enzymes Involved in High-Density Lipoprotein Metabolism among Pregnant Females in Ghana

BackgroundSmall-quantity lipid-based nutrient supplements (SQ-LNS) during pregnancy and postnatally were previously shown to improve high-density lipoprotein (HDL) cholesterol efflux capacity (CEC) and length in the children of supplemented mothers at 18 mo of age in the International Lipid-Based Nutrient Supplements (iLiNS) DYAD trial in Ghana. However, the effects of SQ-LNS on maternal HDL functionality during pregnancy are unknown. ObjectiveThe goal of this cross-sectional, secondary outcome analysis was to compare HDL function in mothers supplemented with SQ-LNS vs. iron and folic acid (IFA) during gestation. MethodsHDL CEC and the activities of 3 HDL-associated enzymes were analyzed in archived plasma samples (N = 197) from a subsample of females at 36 weeks of gestation enrolled in the iLiNS-DYAD trial in Ghana. Correlations between HDL function and birth outcomes, inflammatory markers C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), and the effects of season were explored to determine the influence of these factors on HDL function in this cohort of pregnant females. ResultsThere were no statistically significant differences in HDL CEC, plasma lecithin-cholesterol acyltransferase (LCAT) activity, cholesteryl ester transfer protein (CETP) activity, or phospholipid transfer protein (PLTP) activity between mothers supplemented with SQ-LNS compared with IFA control, and no statistically significant relationships between maternal HDL function and childbirth outcomes. LCAT activity was negatively correlated with plasma AGP (R = -0.19, P = 0.007) and CRP (R = -0.28, P < 0.001), CETP and LCAT activity were higher during the dry season compared to the wet season, and PLTP activity was higher in the wet season compared to the dry season. ConclusionsMothers in Ghana supplemented with SQ-LNS compared with IFA during gestation did not have measurable differences in HDL functionality, and maternal HDL function was not associated with childbirth outcomes. However, seasonal factors and markers of inflammation were associated with HDL function, indicating that these factors had a stronger influence on HDL functionality than SQ-LNS supplementation during pregnancy. Clinical Trial Registry numberThe study was registered as NCT00970866. https://clinicaltrials.gov/study/NCT00970866

Glomerular lipidosis as a feature of renal-limited macrophage activation syndrome in a transplanted kidney: a case report

BackgroundGlomerular lipidosis is a rare histological feature presenting the extensive glomerular accumulation of lipids with or without histiocytic infiltration, which develops under various conditions. Among its various etiologies, macrophage activation syndrome (MAS) is a condition reported to be associated with histiocytic glomerular lipidosis. Here we describe the first case of glomerular lipidosis observed in a renal allograft that histologically mimicked histiocytic glomerulopathy owing to MAS.Case presentationA 42-year-old man underwent successful living-donor kidney transplantation. However, middle-grade proteinuria and increased serum triglyceride levels indicative of type V hyperlipidemia developed rapidly thereafter. An allograft biopsy performed 6 months after the transplantation showed extensive glomerular infiltration of CD68+ foam cells (histiocytes) intermingled with many CD3+ T-cells (predominantly CD8+ cells). Furthermore, frequent contact between glomerular T-cells and histiocytes, and the existence of activated CD8+ cells (CD8+, HLA-DR+ cells) were observed by double immunostaining. There was no clinicopathological data suggesting lipoprotein glomerulopathy or lecithin cholesterol acyltransferase deficiency, both of which are well-known causes of glomerular lipidosis. The histological findings were relatively similar to those of histiocytic glomerulopathy caused by MAS. As systemic manifestations of MAS, such as fever, pancytopenia, coagulation abnormalities, hyperferritinemia, increased liver enzyme levels, hepatosplenomegaly, and lymphadenopathy were minimal, this patient was clinicopathologically diagnosed as having renal-limited MAS. Although optimal treatment strategies for MAS in kidney transplant patients remains unclear, we strengthened lipid-lowering therapy using pemafibrate, without modifying the amount of immunosuppressants. Serum triglyceride levels were normalized with this treatment; however, the patient’s extensive proteinuria and renal dysfunction did not improve. Biopsy analysis at 1 year after the transplantation demonstrated the disappearance of glomerular foamy changes, but the number of glomerular infiltrating cells remained similar.ConclusionTo our knowledge, this is the first reported case of glomerular lipidosis in a transplanted kidney. Increased interaction-activation of histiocytes (macrophages) and CD8+ T-cells, the key pathogenic feature of MAS, was observed in the glomeruli of this patient, who did not demonstrate overt systemic manifestations, suggesting a pathological condition of renal-limited MAS. The clinical effects of triglyceride-lowering therapy were limited, suggesting that hypertriglyceridemia was not the cause of but rather may be a consequence of renal-limited MAS.

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 &gt;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.

FRI100 Lipoprotein X In Graft Vs. Host Disease Affecting Liver

Abstract Disclosure: D. Vedamurthy: None. C. Vitali: None. M. Cuchel: None. D. Soffer: None. D. Rader: None. Introduction: Lipoprotein-X (LpX) is an abnormal lipoprotein rich in phospholipid and unesterified cholesterol (UC) found in patients with cholestatic liver disease, lecithin-cholesterol acyl transferase (LCAT) deficiency, and after lipid emulsion infusion. LpX has been detected in patients with graft-versus-host disease (GVHD) of the liver. Cholestasis leads to the accumulation of bile salts and UC in the circulation, promoting the formation of LpX. This condition is often not well-recognized, partly because the detection of LpX is technically challenging and requires specialized laboratory assays. Case Description: A 66-year lady with acute myeloblastic leukemia (AML) underwent an allogeneic matched unrelated donor stem cell transplant (SCT) which was complicated by graft-versus-host disease (GVHD) affecting the liver, eyes, lungs, and skin. Past medical history includes hypertension. Family history was significant for coronary artery disease in the mother. She is a reformed smoker. Her post-transplant course was highly complicated with infections, GVHD affecting the liver, eyes, lungs, and skin, and vascular events including myocardial infarction and stroke. Laboratory testing revealed abnormal liver enzymes (ALT=268, AST=173, ALKP =402 U/L), and a markedly abnormal lipid profile (TC=500, Non-HDL-C=358, TG= 345 mg/dL). Her thyroid panel and viscosity were normal, but apolipoprotein B levels were discordantly low (77mg/dL). The ratio of ApoB: Non-HDL cholesterol was abnormally low (0.215). She was started on ezetimibe, evolocumab, and omega-3 fatty acids by her neurovascular team for cardiovascular risk reduction. A physical exam revealed a thin, frail woman with mild oral and nail GVHD changes. The rest of the exam was notable for the abdomen showing chronic patchy hyperpigmentation with sclerodermatous changes anteriorly and posteriorly. She had no significant focal neurologic deficits. Her lipid levels were improved with therapy, but TC, non-HDL-C, UC, and triglycerides levels were still elevated (TC=353, non-HDL=258, UC=163, TG=414 mg/dL), UC: TC ratio was high (0.46). FPLC separation of TRL-depleted plasma revealed the presence of LpX. LpX is not very atherogenic, and we recommended continuing current medical therapy as the serum viscosity was within normal limits. Conclusion: The presence of LpX should be suspected in the setting of GVHD accompanied by markedly elevated TC. LpX may cause hyperviscosity, which can induce end-organ ischemia. While the direct assessment of LpX is challenging and requires specialized techniques, ApoB and non-HDL-C levels are available in most clinical laboratories, and a very low ApoB: Non-HDL-C ratio in GVHD patients may indicate the presence of LpX. The treatment goal is to resolve the underlying causes of cholestasis. If hyperviscosity is present, Lp-X can be acutely treated by plasma exchange or lipoprotein apheresis. Presentation: Friday, June 16, 2023

Genomic evidence of sex chromosome aneuploidy and infection-associated genotypes in the tsetse fly Glossina fuscipes, the major vector of African trypanosomiasis in Uganda.

The primary vector of the trypanosome parasite causing human and animal African trypanosomiasis in Uganda is the riverine tsetse fly Glossina fuscipes fuscipes (Gff). Our study improved the Gff genome assembly with whole genome 10× Chromium sequencing of a lab reared pupae, identified autosomal versus sex-chromosomal regions of the genome with ddRAD-seq data from 627 field caught Gff, and identified SNPs associated with trypanosome infection with genome-wide association (GWA) analysis in a subset of 351 flies. Results from 10× Chromium sequencing greatly improved Gff genome assembly metrics and assigned a full third of the genome to the sex chromosome. Results from ddRAD-seq suggested possible sex-chromosome aneuploidy in Gff and identified a single autosomal SNP to be highly associated with trypanosome infection. The top associated SNP was ∼1100bp upstream of the gene lecithin cholesterol acyltransferase (LCAT), an important component of the molecular pathway that initiates trypanosome lysis and protection in mammals. Results suggest that there may be naturally occurring genetic variation in Gff in genomic regions in linkage disequilibrium with LCAT that can protect against trypanosome infection, thereby paving the way for targeted research into novel vector control strategies that can promote parasite resistance in natural populations.