Explore the vast range of titles available.

Triglyceride (TG) metabolism is highly regulated by angiopoietin-like protein (ANGPTL) family members [Y. Q. Chen et al., J. Lipid Res. 61, 1203–1220 (2020)]. During feeding, ANGPTL8 forms complexes with the fibrinogen-like domain-containing protein ANGPTL4 in adipose tissue to decrease ANGPTL3/8- and ANGPTL4-mediated lipoprotein lipase (LPL)-inhibitory activity and promote TG hydrolysis and fatty acid (FA) uptake. The ANGPTL4/8 complex, however, tightly binds LPL and partially inhibits it in vitro. To try to reconcile the in vivo and in vitro data on ANGPTL4/8, we aimed to find novel binding partners of ANGPTL4/8. To that end, we performed pulldown experiments and found that ANGPTL4/8 bound both tissue plasminogen activator (tPA) and plasminogen, the precursor of the fibrinolytic enzyme plasmin. Remarkably, ANGPTL4/8 enhanced tPA activation of plasminogen to generate plasmin in a manner like that observed with fibrin, while minimal plasmin generation was observed with ANGPTL4 alone. The addition of tPA and plasminogen to LPL-bound ANGPTL4/8 caused rapid, complete ANGPTL4/8 cleavage and increased LPL activity. Restoration of LPL activity in the presence of ANGPTL4/8 was also achieved with plasmin but was blocked when catalytically inactive plasminogen (S760A) was added to tPA or when plasminogen activator inhibitor-1 was added to tPA + plasminogen, indicating that conversion of plasminogen to plasmin was essential. Together, these results suggest that LPL-bound ANGPTL4/8 mimics fibrin to recruit tPA and plasminogen to generate plasmin, which then cleaves ANGPTL4/8, enabling LPL activity to be increased. Our observations thus reveal a unique link between the ANGPTL4/8 complex and plasmin generation.

Skeletal muscle atrophy and impaired muscle function are associated with lower health-related quality of life, and greater disability and mortality risk in those with chronic kidney disease (CKD). However, the pathogenesis of skeletal dysfunction in CKD is unknown. We used a slow progressing, naturally occurring, CKD rat model (Cy/+ rat) with hormonal abnormalities consistent with clinical presentations of CKD to study skeletal muscle signaling. The CKD rats demonstrated augmented skeletal muscle regeneration with higher activation and differentiation signals in muscle cells (i.e. lower Pax-7; higher MyoD and myogenin RNA expression). However, there was also higher expression of proteolytic markers (Atrogin-1 and MuRF-1) in CKD muscle relative to normal. CKD animals had higher indices of oxidative stress compared to normal, evident by elevated plasma levels of an oxidative stress marker, 8-hydroxy-2' -deoxyguanosine (8-OHdG), increased muscle expression of succinate dehydrogenase (SDH) and Nox4 and altered mitochondria morphology. Furthermore, we show significantly higher serum levels of myostatin and expression of myostatin in skeletal muscle of CKD animals compared to normal. Taken together, these data show aberrant regeneration and proteolytic signaling that is associated with oxidative stress and high levels of myostatin in the setting of CKD. These changes likely play a role in the compromised skeletal muscle function that exists in CKD.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.

In three phase 3 trials, ixekizumab, an anti–IL-17A monoclonal antibody, was effective in the treatment of patients with moderate-to-severe plaque psoriasis. Adverse events included neutropenia, candida infections, and inflammatory bowel disease. Psoriasis is a chronic inflammatory disease that is mediated by aberrant immune responses and driven by self-perpetuating cytokine networks. 1 , 2 Advances in understanding the pathogenic cytokine network of psoriasis have led to the development of new treatments 3 – 5 that provide greater efficacy in terms of complete skin clearance. 6 – 9 The motivation to completely clear psoriasis plaques from the skin of patients has grown in response to accumulating evidence that residual skin disease can affect a patient’s health-related quality of life 10 – 12 similar to that associated with chronic conditions such as type 2 diabetes. 13 Ixekizumab, a recombinant, high-affinity, humanized, IgG4-κ monoclonal . . .

During the past decade, proprotein convertase subtilisin kexin type 9 (PCSK9) has been identified as a key regulator of serum LDL-cholesterol (LDL-C) levels. PCSK9 is secreted by the liver into the plasma and binds the hepatic LDL receptor, causing its subsequent degradation. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia that manifests with dramatically increased serum levels of LDL-C, while loss-of-function mutations in PCSK9 are associated with significantly decreased LDL-C and cardiovascular risk. Initial studies in animals and cultured cells demonstrated that statins increased PCSK9 mRNA expression, resulting in many research groups exploring the effect of statins on PCSK9 levels in humans. We first reported that statins increased human PCSK9 circulating protein levels. Additional researchers subsequently confirmed these observations, further prompting many laboratories including our own to examine the effect of other lipid lowering medications on PCSK9 levels. Our observation that fenofibrate (200 mg/day) significantly increased PCSK9 levels was confirmed by another laboratory, and an additional group demonstrated that ezetimibe also increased PCSK9 levels. It has become clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels. These observations almost certainly explain why these agents are not more effective in lowering LDL-C and suggest that efforts should be made toward the development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through decreasing or inhibiting PCSK9.

A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0-300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (p = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, p = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

Hepcidin reduces iron absorption by binding to the intestinal iron transporter ferroportin, thereby causing its degradation. Although short-term administration of testosterone or growth hormone (GH) has been reported to decrease circulating hepcidin levels, little is known about how hepcidin is influenced in human endocrine conditions associated with anemia. We used a sensitive and specific dual-monoclonal antibody sandwich immunoassay to measure hepcidin-25 in patients (a) during initiation of in vitro fertilization when endogenous estrogens were elevated vs. suppressed, (b) with GH deficiency before and after 12 months substitution treatment, (c) with hyperthyroidism before and after normalization, and (d) with hyperprolactinemia before and after six months of treatment with a dopamine agonist. In response to a marked stimulation of endogenous estrogen production, median hepcidin levels decreased from 4.85 to 1.43 ng/mL (p < 0.01). Hyperthyroidism, hyperprolactinemia, or GH substitution to GH-deficient patients did not influence serum hepcidin-25 levels. In humans, gonadotropin-stimulated endogenous estrogen markedly decreases circulating hepcidin-25 levels. No clear and stable correlation between iron biomarkers and hepcidin-25 was seen before or after treatment of hyperthyroidism, hyperprolactinemia or growth hormone deficiency.

Biotherapeutics have the potential to elicit unwanted immune responses that can lead to the production of anti-drug antibodies (ADA). It is critical that ADA responses are detected, characterized, and monitored to understand the safety and efficacy of a drug. ADA samples must remain stable in long- and short-term storage conditions to ensure reliable analysis. Whereas the stability of anti-vaccine antibodies has been well-studied, there are few reports examining the stability of anti-therapeutic antibodies using clinical samples. In this study, ADA samples from four clinical trials of antibody therapeutics were found to be stable after long-term storage (1–10 years) at -80°C and short-term storage (24 h to two weeks) at 4°C, 22°C, and 37°C. In addition, samples were stable after 16 freeze/thaw cycles. The results demonstrate the stability of ADA in clinical samples under various conditions. Consequently, the results observed herein suggest that the routine assessment of ADA sample stability may not be warranted. Graphical Abstract

A Single Nucleotide Polymorphism in MGEA5 Encoding O-GlcNAc–selective N -Acetyl-β- d Glucosaminidase Is Associated With Type 2 Diabetes in Mexican Americans Donna M. Lehman 1 , Dong-Jing Fu 2 , Angela B. Freeman 2 , Kelly J. Hunt 1 , Robin J. Leach 3 4 , Teresa Johnson-Pais 4 , Jeanette Hamlington 1 , Thomas D. Dyer 5 , Rector Arya 1 , Hanna Abboud 1 , Harald H.H. Göring 6 , Ravindranath Duggirala 6 , John Blangero 6 , Robert J. Konrad 5 and Michael P. Stern 1 1 Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 2 Lilly Research Laboratories, Indianapolis, Indiana 3 Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 4 Department of Pediatrics, University of Texas Health Science Center, San Antonio, Texas 5 Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas 6 Department of Nephrology, University of Texas Health Science Center, San Antonio, Texas Address correspondence and reprint requests to Donna M. Lehman, Department of Medicine/Clinical Epidemiology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, Texas 78229. E-mail: lehman{at}uthscsa.edu Abstract Excess O-glycosylation of proteins by O-linked β- N -acetylglucosamine (O-GlcNAc) may be involved in the pathogenesis of type 2 diabetes. The enzyme O-GlcNAc–selective N -acetyl-β- d glucosaminidase (O-GlcNAcase) encoded by MGEA5 on 10q24.1-q24.3 reverses this modification by catalyzing the removal of O-GlcNAc. We have previously reported the linkage of type 2 diabetes and age at diabetes onset to an overlapping region on chromosome 10q in the San Antonio Family Diabetes Study (SAFADS). In this study, we investigated menangioma-expressed antigen-5 (MGEA5) as a positional candidate gene. Twenty-four single nucleotide polymorphisms (SNPs), identified by sequencing 44 SAFADS subjects, were genotyped in 436 individuals from 27 families whose data were used in the original linkage report. Association tests indicated significant association of a novel SNP with the traits diabetes ( P = 0.0128, relative risk = 2.77) and age at diabetes onset ( P = 0.0017). The associated SNP is located in intron 10, which contains an alternate stop codon and may lead to decreased expression of the 130-kDa isoform, the isoform predicted to contain the O-GlcNAcase activity. We investigated whether this variant was responsible for the original linkage signal. The variance attributed to this SNP accounted for ∼25% of the logarithm of odds. These results suggest that this variant within the MGEA5 gene may increase diabetes risk in Mexican Americans. LD, linkage disequilibrium LOD, logarithm of odds MGEA5, menangioma-expressed antigen-5 O-GlcNAc, O-linked β-N-acetylglucosamine O-GlcNAcase, O-GlcNAc–selective N-acetyl-β-d glucosaminidase SAFADS, San Antonio Family Diabetes Study SNP, single nucleotide polymorphism Footnotes Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org . Accepted December 9, 2004. Received September 30, 2004. DIABETES

Background: We measured plasma PCSK9 concentrations in healthy men with a PCSK9 (proprotein convertase subtilisin/kexin type 9) loss-of-function variant (p.R46L), in statin-treated patients with a clinical diagnosis of familial hypercholesterolemia (FH) and carrying a PCSK9 gain-of-function mutation (p.D374Y), and in statin-treated patients with FH due to different genetic causes. Methods: PCSK9 was measured with a previously described ELISA. Results: In 81 healthy middle-aged Caucasian men, the PCSK9 concentration was significantly associated with the concentrations of total cholesterol (r = 0.42; P < 0.0001), LDL cholesterol (r = 0.34; P = 0.01), and triglycerides (r = 0.25; P = 0.02). In p.R46L carriers, mean (SD) concentrations of PCSK9 were 15% lower than in RR individuals [65.5 μg/L (21.6 μg/L) vs 77.5 μg/L (18.2 μg/L); P = 0.03]. In patients with the p.D374Y variant (n = 7), the mean PCSK9 concentration was significantly lower than in the combined group of patients with an LDLR (low density lipoprotein receptor) mutation (n = 25), an APOB [apolipoprotein B (including Ag(x) antigen)] variant encoding p.R3527Q (n = 6), or no detectable mutation (n = 14) [96.4 μg/L (42.5 μg/L) vs 151.6 μg/L (69.6 μg/L); P = 0.02]. Two of the 14 patients with no mutation had PCSK9 concentrations below the mean for p.D374Y carriers; sequencing of the PCSK9 gene and promoter revealed no mutations. Among 409 FH patients, we identified 6 carriers of the promoter variant −287G>A (1.5%), a frequency similar to that (1.0%) previously reported for 2772 healthy men in the UK. In neither group was the −287G>A variant associated with differences in lipid traits. Conclusions: The loss-of-function p.R46L variant is associated with the expected lower concentrations of circulating PCSK9; the gain-of-function p.D374Y mutation is also associated with lower concentrations, presumably because of the higher affinity of this variant for the LDL receptor and its more rapid clearance. In treated FH patients, a low plasma PCSK9 concentration does not appear to be a useful screening tool for identifying novel PCSK9 mutations.