Maureen Sampson

Detection and Quantitation of Serum Mesothelin, a Tumor Marker for Patients with Mesothelioma and Ovarian Cancer

Purpose: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. Experimental Design: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. Results: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. Conclusions: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.

High Pre-β1 HDL Concentrations and Low Lecithin: Cholesterol Acyltransferase Activities Are Strong Positive Risk Markers for Ischemic Heart Disease and Independent of HDL-Cholesterol

BACKGROUND We hypothesized that patients with high HDL-cholesterol (HDL-C) and ischemic heart disease (IHD) may have dysfunctional HDL or unrecognized nonconventional risk factors. METHODS Individuals with IHD (Copenhagen University Hospital) and either high HDL-C (n = 53; women >or=735 mg/L; men >or=619 mg/L) or low HDL-C (n = 42; women <or=387 mg/L; men <or=341 mg/L) were compared with individuals without IHD (Copenhagen City Heart Study) matched by age, sex, and HDL-C concentrations (n = 110). All participants had concentrations within reference intervals for LDL-C (<1600 mg/L) and triglyceride (<1500 mg/L), and none were treated with lipid-lowering medications. Pre-beta(1) HDL and phospholipid transfer protein concentrations were measured by using commercial kits and lecithin:cholesterol acyltransferase (LCAT) activity by using a proteoliposome cholesterol esterification assay. RESULTS Pre-beta(1) HDL concentrations were 2-fold higher in individuals with IHD vs no IHD in both the high [63 (5.7) vs 35 (2.3) mg/L; P < 0.0001] and low HDL-C [49 (5.0) vs 27 (1.5) mg/L; P = 0.001] groups. Low LCAT activity was also associated with IHD in the high [95.2 (6.7) vs 123.0 (5.3) micromol x L(-1) x h(-1); P = 0.002] and low [93.4 (8.3) vs 113.5 (4.9) micromol x L(-1) . h(-1); P = 0.03] HDL-C groups. ROC curves for pre-beta(1) HDL in the high-HDL-C groups yielded an area under the curve of 0.71 (95% CI: 0.61-0.81) for predicting IHD, which increased to 0.92 (0.87-0.97) when LCAT was included. Similar results were obtained for low HDL-C groups. An inverse correlation between LCAT activity and pre-beta(1) HDL was observed (r(2) = 0.30; P < 0.0001) in IHD participants, which was stronger in the low HDL-C group (r(2) = 0.56; P < 0.0001). CONCLUSIONS IHD was associated with high pre-beta(1) HDL concentrations and low LCAT levels, yielding correct classification in more than 90% of the IHD cases for which both were measured, thus making pre-beta(1) HDL concentration and LCAT activity level potentially useful diagnostic markers for cardiovascular disease.

Effect of sofosbuvir and ribavirin treatment on peripheral and hepatic lipid metabolism in chronic hepatitis C virus, genotype 1-infected patients.

Hepatitis C virus (HCV) modulates intrahepatic cholesterol biosynthetic pathways to promote viral replication. Chronic HCV infection is associated with altered metabolism, including dyslipidemia and insulin resistance (IR), which contributes to disease progression and influences response to therapy. To further understand the impact of HCV infection on host metabolism, we examined changes in serum lipid profiles and intrahepatic expression of lipid‐related genes during interferon (IFN)‐free treatment of chronic HCV, genotype 1 infection with sofosbuvir and ribavirin (RBV), and explored associations with treatment outcome. Serum lipids (total cholesterol, low‐density lipoprotein [LDL], high‐density lipoprotein [HDL], and triglycerides [TGs]) and hemoglobin A1C (HbA1C) were measured during treatment, while gene expression of lipid‐related genes was assessed using paired pre‐ and end‐of‐treatment (EOT) liver biopsies from 8 patients (n = 7 sustained virologic response [SVR]; n = 1 relapse) and unpaired EOT liver biopsies from 25 patients (n = 17 SVR; n = 8 relapse). Serum LDL concentration and particle size increased early in therapy, whereas TG concentration and very‐low‐density lipoprotein particle size decreased concomitantly, irrespective of treatment outcome. Whereas LDL increased in patients regardless of treatment outcome, average LDL concentration was lower at baseline and post‐treatment in patients who relapsed. Analysis of paired liver biopsies revealed altered expression of genes associated with lipid transport, assembly, and signaling. In unpaired EOT liver biopsies, intrahepatic expression of fatty acid metabolism and lipid transport genes was lower in patients who experienced treatment relapse. Conclusion: Clearance of HCV using an IFN‐free antiviral regimen results in rapid changes in peripheral and intrahepatic metabolic pathways, implicating a direct effect of HCV replication on lipid homeostasis. (Hepatology 2015;61:790–801)

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.

A Cholesterol-Lowering VLP Vaccine that Targets PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that controls cholesterol homeostasis by enhancing endosomal and lysosomal degradation of the low-density lipoprotein receptor (LDL-R). Mutations that cause increased activity of PCSK9 are associated with hypercholesterolemia, atherosclerosis and early cardiovascular disease (CVD), whereas individuals with loss-of-function mutations in PCSK9 are apparently healthy but are hypocholesterolemic and have a dramatically decreased risk of CVD. In this study, we generated virus-like particle (VLP)-based vaccines targeting PCSK9. Mice and macaques vaccinated with bacteriophage VLPs displaying PCSK9-derived peptides developed high titer IgG antibodies that bound to circulating PCSK9. Vaccination was associated with significant reductions in total cholesterol, free cholesterol, phospholipids, and triglycerides. A vaccine targeting PCSK9 may, therefore, be an attractive alternative to monoclonal antibody-based therapies.

Olive Oil Polyphenols Decrease LDL Concentrations and LDL Atherogenicity in Men in a Randomized Controlled Trial

BACKGROUND Olive oil polyphenols have shown protective effects on cardiovascular risk factors. Their consumption decreased oxidative stress biomarkers and improved some features of the lipid profile. However, their effects on LDL concentrations in plasma and LDL atherogenicity have not yet been elucidated. OBJECTIVE Our objective was to assess whether the consumption of olive oil polyphenols could decrease LDL concentrations [measured as apolipoprotein B-100 (apo B-100) concentrations and the total number of LDL particles] and atherogenicity (the number of small LDL particles and LDL oxidizability) in humans. METHODS The study was a randomized, cross-over controlled trial in 25 healthy European men, aged 20-59 y, in the context of the EUROLIVE (Effect of Olive Oil Consumption on Oxidative Damage in European Populations) study. Volunteers ingested 25 mL/d raw low-polyphenol-content olive oil (LPCOO; 366 mg/kg) or high-polyphenol-content olive oil (HPCOO; 2.7 mg/kg) for 3 wk. Interventions were preceded by 2-wk washout periods. Effects of olive oil polyphenols on plasma LDL concentrations and atherogenicity were determined in the sample of 25 men. Effects on lipoprotein lipase (LPL) gene expression were assessed in another sample of 18 men from the EUROLIVE study. RESULTS Plasma apo B-100 concentrations and the number of total and small LDL particles decreased (mean ± SD: by 5.94% ± 16.6%, 11.9% ± 12.0%, and 15.3% ± 35.1%, respectively) from baseline after the HPCOO intervention. These changes differed significantly from those after the LPCOO intervention, which resulted in significant increases of 6.39% ± 16.6%, 4.73% ± 22.0%, and 13.6% ± 36.4% from baseline (P < 0.03). LDL oxidation lag time increased by 5.0% ± 10.3% from baseline after the HPCOO intervention, which was significantly different only relative to preintervention values (P = 0.038). LPL gene expression tended to increase by 26% from baseline after the HPCOO intervention (P = 0.08) and did not change after the LPCOO intervention. CONCLUSION The consumption of olive oil polyphenols decreased plasma LDL concentrations and LDL atherogenicity in healthy young men. This trial was registered at www.controlled-trials.com as ISRCTN09220811.

The differentiating agent phenylacetate increases prostate-specific antigen production by prostate cancer cells

The prostatic‐specific antigen (PSA) is the tumor marker most widely relied upon for the monitoring of patients with prostate cancer. Recently, declines in the serum concentrations of PSA have been advocated as a surrogate marker of tumor response in clinical trials of investigational antitumor agents. We examined the hypothesis that this postulate may not apply to the evaluation of drugs such as phenylacetate, a differentiating agent endowed with mechanisms of action different from those of classic cytotoxic chemotherapy. Using human prostatic carcinoma LNCaP cells as a model, we show that phenylacetate induces PSA production despite inhibition of tumor cell proliferation. Incubation of LNCaP cultures with cytostatic doses of phenylacetate (3–10 mM) resulted in a three‐ to fourfoLd increase in PSA secretion per cell. This appears to result from upregulation of PSA gene expression, as indicated by elevated PSA mRNA steady‐state levels in treated cells. The increase in PSA production per cell was confirmed in rats bearing subcutaneous LNCaP tumor implants that were treated systemically with phenylacetate. Further comparative studies indicate that upregulation of PSA is common to various differentiation inducers, including all, trans‐retinoic acid, 1,25‐dihydroxyvitamin D3, and butyrate but is not induced by other antitumor agents of clinical interest such as suramin. We conclude that declines in PSA may be treatment specific and that the exclusive use of this criterion as a marker of disease response may mislead the proper evaluation of differentiating agents in prostate cancer patients.

Rosuvastatin Alters the Proteome of High Density Lipoproteins: Generation of alpha-1-antitrypsin Enriched Particles with Anti-inflammatory Properties

Statins lower plasma cholesterol by as much as 50%, thus reducing future cardiovascular events. However, the physiological effects of statins are diverse and not all are related to low density lipoprotein cholesterol (LDL-C) lowering. We performed a small clinical pilot study to assess the impact of statins on lipoprotein-associated proteins in healthy individuals (n = 10) with normal LDL-C (<130 mg/dL), who were treated with rosuvastatin (20 mg/day) for 28 days. Proteomic analysis of size-exclusion chromatography isolated LDL, large high density lipoprotein (HDL-L), and small HDL (HDL-S) fractions and spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool and included both increases and decreases in several proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the rosuvastatin treatment was an increase in α-1-antirypsin (A1AT) spectral counts associated with HDL-L particles. Quantitative measurement by ELISA confirmed an average 5.7-fold increase in HDL-L associated A1AT. Molecular modeling predictions indicated that the hydrophobic reactive center loop of A1AT, the functional domain responsible for its protease inhibitor activity, is likely involved in lipid binding and association with HDL was found to protect A1AT against oxidative inactivation. Cell culture experiments, using J774 macrophages, demonstrated that the association of A1AT with HDL enhances its antiprotease activity, preventing elastase induced production of tumor necrosis factor α. In conclusion, we show that statins can significantly alter the protein composition of both LDL and HDL and our studies reveal a novel functional relationship between A1AT and HDL. The up-regulation of A1AT on HDL enhances its anti-inflammatory functionality, which may contribute to the non-lipid lowering beneficial effects of statins.

Serum Apolipoprotein A-I and Large High-Density Lipoprotein Particles Are Positively Correlated with FEV1 in Atopic Asthma

RATIONALE Although lipids, apolipoproteins, and lipoprotein particles are important modulators of inflammation, varying relationships exist between these parameters and asthma. OBJECTIVES To determine whether serum lipids and apolipoproteins correlate with the severity of airflow obstruction in subjects with atopy and asthma. METHODS Serum samples were obtained from 154 atopic and nonatopic subjects without asthma, and 159 subjects with atopy and asthma. Serum lipid and lipoprotein levels were quantified using standard diagnostic assays and nuclear magnetic resonance (NMR) spectroscopy. Airflow obstruction was assessed by FEV1% predicted. MEASUREMENTS AND MAIN RESULTS Serum lipid levels correlated with FEV1 only in the subjects with atopy and asthma. Serum levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) were positively correlated with FEV1 in subjects with atopy and asthma, whereas a negative correlation existed between FEV1 and serum levels of triglycerides, low-density lipoprotein (LDL) cholesterol, apolipoprotein B (apoB), and the apoB/apoA-I ratio. NMR spectroscopy identified a positive correlation between FEV1 and HDLNMR particle size, as well as the concentrations of large HDLNMR particles and total IDLNMR (intermediate-density lipoprotein) particles in subjects with atopy and asthma. In contrast, LDLNMR particle size and concentrations of LDLNMR and VLDLNMR (very-low-density lipoprotein) particles were negatively correlated with FEV1 in subjects with atopy and asthma. CONCLUSIONS In subjects with atopy and asthma, serum levels of apoA-I and large HDLNMR particles are positively correlated with FEV1, whereas serum triglycerides, LDL cholesterol, and apoB are associated with more severe airflow obstruction. These results may facilitate future studies to assess whether apoA-I and large HDLNMR particles can reduce airflow obstruction and disease severity in asthma.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40–200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 μmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.