A systematic review of the natural history and biomarkers of primary lecithin:cholesterol acyltransferase deficiency

Syndromes associated with LCAT deficiency, a rare autosomal recessive condition, include fish-eye disease (FED) and familial LCAT deficiency (FLD). FLD is more severe and characterized by early and progressive chronic kidney disease (CKD). No treatment is currently available for FLD, but novel therapeutics are under development. Furthermore, although biomarkers of LCAT deficiency have been identified, their suitability to monitor disease progression and therapeutic efficacy is unclear, as little data exist on the rate of progression of renal disease. Here, we systematically review observational studies of FLD, FED, and heterozygous subjects, which summarize available evidence on the natural history and biomarkers of LCAT deficiency, in order to guide the development of novel therapeutics. We identified 146 FLD and 53 FED patients from 219 publications, showing that both syndromes are characterized by early corneal opacity and markedly reduced HDL-C levels. Proteinuria/hematuria were the first signs of renal impairment in FLD, followed by rapid decline of renal function. Furthermore, LCAT activity toward endogenous substrates and the percentage of circulating esterified cholesterol (EC%) were the best discriminators between these two syndromes. In FLD, higher levels of total, non-HDL, and unesterified cholesterol were associated with severe CKD. We reveal a nonlinear association between LCAT activity and EC% levels, in which subnormal levels of LCAT activity were associated with normal EC%. This review provides the first step toward the identification of disease biomarkers to be used in clinical trials and suggests that restoring LCAT activity to subnormal levels may be sufficient to prevent renal disease progression.     

Concentration dependent cholesteryl-ester and wax-ester structural relationships and meibomian gland dysfunction

BackgroundWith dry eye, the ratio of cholesteryl ester (CE) to wax ester (WE) decreases substantially in meibum, but the functional and structural consequences of this change are speculative. The aim of this study is to confirm this finding and to bridge this gap in knowledge by investigating the effect of varying CE/WE ratios on lipid structure and thermodynamics.MethodsInfrared spectroscopy was use to quantify CE and WE in human meibum and to measure hydrocarbon chain conformation and thermodynamics in a cholesteryl behenate, stearyl stearate model system.ResultsThe CE/WE molar ratio was 36% lower for meibum from donors with dry eye due to meibomian gland dysfunction compared with meibum from donors without dry eye. CE (5 mol %) dramatically increased the phase transition temperature of pure WE from -0.12 °C to 63 °C in the mixture. Above 5 mol % CB, the phase transition temperature increased linearly, from 68.5 °C to 85 °C. In the ordered state, CE caused an increase in lipid order from about 72% trans rotamers to about 86% trans rotamers. Above 10% CE, the hydrocarbon chains were arranged in a monoclinic geometry.ConclusionsThe CE/WE is lower in meibum from donors with dry eye due to meibomian-gland dysfunction. Major conformational changes in the hydrocarbon chains of wax and cholesteryl ester mixtures begin to occur with just 5% CB and above.General significanceCE-WE interactions may be important for in understanding lipid layer structure and functional relationships on the surface of tears, skin and plants.     

Associations of ApoE4 status and DHA supplementation on plasma and CSF lipid profiles and entorhinal cortex thickness

Apolipoprotein ε allele 4 (APOE4) influences the metabolism of polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The entorhinal cortex (EC) in the brain is affected early in Alzheimer's disease and is rich in DHA. The purpose of this study is to identify the effect of APOE4 and DHA lipid species on the EC. Plasma and cerebrospinal fluid (CSF) lipidomic measurements were obtained from the DHA Brain Delivery Pilot, a randomized clinical trial of DHA supplementation (n = 10) versus placebo (n = 12) for six months in nondemented older adults stratified by APOE4 status. Wild-type C57B6/J mice were fed a high or low DHA diet for 6 months followed by plasma and brain lipidomic analysis. Levels of phosphatidylcholine DHA (PC 38:6) and cholesterol ester DHA (CE 22:6) had the largest increases in CSF following supplementation (P < 0.001). DHA within triglyceride (TG) lipids in CSF strongly correlated with corresponding plasma TG lipids, and differed by APOE4, with carriers having a lower increase than noncarriers. Changes in plasma PC DHA had the strongest association with changes in EC thickness in millimeters, independent of APOE4 status (P = 0.007). In mice, a high DHA diet increased PUFAs within brain lipids. Our findings demonstrate an exchange of DHA at the CSF-blood barrier and into the brain within all lipid species with APOE having the strongest effect on DHA-containing TGs. The correlation of PC DHA with EC suggests a functional consequence of DHA accretion in high density lipoprotein for the brain.     

Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis

High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.     

Lipoprotein size is a main determinant for the rate of hydrolysis by exogenous LPL in human plasma

LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietin-like proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.     

Associations between insulin-like growth factor binding protein-2 and lipoprotein kinetics in men

Low circulating concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) have been associated with dyslipidemia, notably with high triglyceride (TG) levels. However, the determinants by which IGFBP-2 influences lipoprotein metabolism, especially that of TG-rich lipoproteins (TRLs), are poorly understood. Here, we aimed to assess the relationships between IGFBP-2 levels and lipoprotein production and catabolism in human subjects. Fasting IGFBP-2 concentrations were measured in the plasma of 219 men pooled from previous lipoprotein kinetics studies. We analyzed production rate and fractional catabolic rates of TRLapoB-48, and LDL-, IDL-, and VLDLapoB-100 by multicompartmental modeling of l-[5,5,5-D3] leucine enrichment data after a 12 h primed constant infusion in individuals kept in a constant nutritional steady state. Subjects had an average BMI of 30 kg/m², plasma IGFBP-2 levels of 157 ng/ml, and TG of 2.2 mmol/l. After adjustments for age and BMI, IGFBP-2 levels were negatively associated with plasma TG (r = ?0.29; P < 0.0001) and positively associated with HDL-cholesterol (r = 0.26; P < 0.0001). In addition, IGFBP-2 levels were positively associated with the fractional catabolic rate of VLDLapoB-100 (r = 0.20; P < 0.01) and IDLapoB-100 (r = 0.19; P < 0.05) and inversely with the production rate of TRLapoB-48 (r = ?0.28; P < 0.001). These correlations remained statistically significant after adjustments for age, BMI, and the amount of fat given during the tracer infusion. These findings show that the association between low plasma IGFBP-2 and high TG concentrations could be due to both an impaired clearance of apoB-100-containing VLDL and IDL particles and an increased production of apoB-48-containing chylomicrons. Additional studies are necessary to investigate whether and how IGFBP-2 directly impacts the kinetics of TRL.     

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.     

Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.     

Development of simple and effective PCR based assay to detect PCCA mutation (c.425G > A) among Saudi carriers and functional study of the homozygous PCCA mutations

The aim of this study is to develop a rapid and effective method to screen for Saudi carriers of one of the most common propionic acidemia mutations (c.425G > A) and to study the functional impact of this mutation. Using allele-specific primers, we have developed a qPCR assay that clearly distinguishes heterozygotes from mutated and wild type homozygotes that overcome the dependence on labor-intensive gene sequencing. We show here that (i) qPCR rapid test has strong accuracy in detecting (c.425G > A) mutation in heterozygotes and homozygotes individuals and that the Ct-value cut-offs were estimated to be and 23.37 ± 0.04 (CV-6 %, 95 %CI-7.25) for homozygote, 25.06 ± 0.02 (CV-3.5 %, 95 %CI-7.85) for heterozygote PCCA c.425G > A mutation and 29.55 ± 0.002 (CV-11 %, 95 %CI-1.41) for PCCA wild type; (ii) the incidence of PA heterozygotes/carriers in Saudi population is about 550/100,000; (iii) skin fibroblast assays show that homozygote c.425G > A mutation induced propionyl-CoA carboxylase activity abrogation, (iv) PA patients showed an increased level of propionyl carnitine C3 in blood and 3-hydroxy propionic acid and methyl citrate in urine. Conclusion: qPCR represent an effective strategy to assess for PCCA mutation carriers in the Saudi population and we believe that will help in preventing homozygosity in the population after been implemented in pre-marriage screening program.     

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.     

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ?stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.     

Apolipoprotein F concentration,activity, and the properties of LDL controlling ApoF activation in hyperlipidemic plasma

Apolipoprotein F (ApoF) modulates lipoprotein metabolism by selectively inhibiting cholesteryl ester transfer protein activity on LDL. This ApoF activity requires that it is bound to LDL. How hyperlipidemia alters total plasma ApoF and its binding to LDL are poorly understood. In this study, total plasma ApoF and LDL-bound ApoF were quantified by ELISA (n = 200). Plasma ApoF was increased 31% in hypercholesterolemic plasma but decreased 20% in hypertriglyceridemia. However, in donors with combined hypercholesterolemia and hypertriglyceridemia, the elevated triglyceride ameliorated the rise in ApoF caused by hypercholesterolemia alone. Compared with normolipidemic LDL, hypercholesterolemic LDL contained ～2-fold more ApoF per LDL particle, whereas ApoF bound to LDL in hypertriglyceridemia plasma was <20% of control. To understand the basis for altered association of ApoF with hyperlipidemic LDL, the physiochemical properties of LDL were modified in vitro by cholesteryl ester transfer protein ± LCAT activities. The time-dependent change in LDL lipid composition, proteome, core and surface lipid packing, LDL surface charge, and LDL size caused by these factors were compared with the ApoF binding capacity of these LDLs. Only LDL particle size correlated with ApoF binding capacity. This positive association between LDL size and ApoF content was confirmed in hyperlipidemic plasmas. Similarly, when in vitro produced and enlarged LDLs with elevated ApoF binding capacity were incubated with LPL to reduce their size, ApoF binding was reduced by 90%. Thus, plasma ApoF levels and the activation status of this ApoF are differentially altered by hypercholesterolemia and hypertriglyceridemia. LDL size is a key determinate of ApoF binding and activation.     

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.     

Bioactive lipid lysophosphatidic acid species are associated with disease progression in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with significant mortality. Prognostic biomarkers to identify rapid progressors are urgently needed to improve patient management. Since the lysophosphatidic acid (LPA) pathway has been implicated in lung fibrosis in preclinical models and identified as a potential therapeutic target, we aimed to investigate if bioactive lipid LPA species could be prognostic biomarkers that predict IPF disease progression. LPAs and lipidomics were measured in baseline placebo plasma of a randomized IPF-controlled trial. The association of lipids with disease progression indices were assessed using statistical models. Compared to healthy, IPF patients had significantly higher levels of five LPAs (LPA16:0, 16:1, 18:1, 18:2, 20:4) and reduced levels of two triglycerides species (TAG48:4-FA12:0, -FA18:2) (false discovery rate < 0.05, fold change > 2). Patients with higher levels of LPAs had greater declines in diffusion capacity of carbon monoxide over 52 weeks (P < 0.01); additionally, LPA20:4-high (≥median) patients had earlier time to exacerbation compared to LPA20:4-low (<median) patients (hazard ratio (95% CI)): 5.71 (1.17–27.72) (P = 0.031). Higher baseline LPAs were associated with greater increases in fibrosis in lower lungs as quantified by high-resolution computed tomography at week 72 (P < 0.05). Some of these LPAs were positively associated with biomarkers of profibrotic macrophages (CCL17, CCL18, OPN, and YKL40) and lung epithelial damage (SPD and sRAGE) (P < 0.05). In summary, our study established the association of LPAs with IPF disease progression, further supporting the role of the LPA pathway in IPF pathobiology.     

Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma

Ovarian cancer is one of the most lethal female cancers. For accurate prognosis prediction, this study aimed to investigate novel, blood-based prognostic biomarkers for high-grade serous ovarian carcinoma (HGSOC) using mass spectrometry–based proteomics methods. We conducted label-free liquid chromatography–tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed HGSOC (n = 20). Based on progression-free survival (PFS), the samples were divided into two groups: good (PFS ≥18 months) and poor prognosis groups (PFS <18 months). Proteomic profiles were compared between the two groups. Referring to proteomics data that we previously obtained using frozen cancer tissues from chemotherapy-naïve patients with HGSOC, overlapping protein biomarkers were selected as candidate biomarkers. Biomarkers were validated using an independent set of HGSOC plasma samples (n = 202) via enzyme-linked immunosorbent assay (ELISA). To construct models predicting the 18-month PFS rate, we performed stepwise selection based on the area under the receiver operating characteristic curve (AUC) with 5-fold cross-validation. Analysis of differentially expressed proteins in plasma samples revealed that 35 and 61 proteins were upregulated in the good and poor prognosis groups, respectively. Through hierarchical clustering and bioinformatic analyses, GSN, VCAN, SND1, SIGLEC14, CD163, and PRMT1 were selected as candidate biomarkers and were subjected to ELISA. In multivariate analysis, plasma GSN was identified as an independent poor prognostic biomarker for PFS (adjusted hazard ratio, 1.556; 95% confidence interval, 1.073–2.256; p = 0.020). By combining clinical factors and ELISA results, we constructed several models to predict the 18-month PFS rate. A model consisting of four predictors (FIGO stage, residual tumor after surgery, and plasma levels of GSN and VCAN) showed the best predictive performance (mean validated AUC, 0.779). The newly developed model was converted to a nomogram for clinical use. Our study results provided insights into protein biomarkers, which might offer clues for developing therapeutic targets.