An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 µmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

Plasma AGE and Oxidation Products, Renal Function, and Preeclampsia in Pregnant Women with Type 1 Diabetes: A Prospective Observational Study.

Aims: We aimed to determine whether plasma advanced glycation end products or oxidation products (AGE/oxidation-P) predict altered renal function and/or preeclampsia (PE) in pregnant women with type 1 diabetes. Methods: Prospectively, using a nested case-control design, we studied 47 pregnant women with type 1 diabetes, of whom 23 developed PE and 24 did not. Nineteen nondiabetic, normotensive pregnant women provided reference values. In plasma obtained at ~12, 22, and 32 weeks' gestation (visits 1, 2, and 3; V1-V3), we measured five AGE products (carboxymethyllysine (CML), carboxyethyl-lysine (CEL), methylglyoxal-hydroimidazolone (MGH1), 3-deoxyglucosone hydroimidazolone (3DGH), and glyoxal-hydroimidazolone (GH1)) and four oxidation products (methionine sulfoxide (MetSO), 2-aminoadipic acid (2-AAA), 3-nitrotyrosine (3NT), and dityrosine (DT)), by liquid chromatography/mass spectroscopy. Clinical outcomes were "estimated glomerular filtration rate" (eGFR) at each visit and onset of PE. Results: In diabetic women, associations between AGE/oxidation-P and eGFR were found only in those who developed PE. In this group, CEL, MGH1, and GH1 at V2 and CML, CEL, MGH1, and GH1 at V3 were inversely associated with contemporaneous eGFR, while CEL, MGH1, 3DGH, and GH1 at V2 were inversely associated with eGFR at V3 (all p < 0.05). There were no associations of plasma AGE or oxidation-P with pregnancy-related development of proteinuria or PE. Conclusions: Inverse associations of second and early third trimester plasma AGE with eGFR among type 1 diabetic women who developed PE suggest that these patients constitute a subset susceptible to AGE-mediated injury and thus to cardiorenal complications later in life. However, AGE/oxidation-P did not predict PE in type 1 diabetic women.

Comparisons of α2-Adrenergic Agents, Medetomidine and Xylazine, with Pentobarbital for Anesthesia: Important Pitfalls in Diabetic and Nondiabetic Rats.

Purpose: Anesthesia is necessary to conduct rodent electroretinograms (ERGs). We evaluated utility of the α2-agonist medetomidine versus xylazine for ERG studies in nondiabetic and diabetic rats. Pentobarbital was included as a comparator. Methods: Male Sprague-Dawley rats, with and without streptozotocin (STZ)-induced diabetes, were anesthetized with medetomidine (1 mg/kg), xylazine (10 mg/kg) (both with ketamine 75 mg/kg), or pentobarbital (70 mg/kg). The depth of anesthesia was assessed, and if adequate, scotopic ERGs were recorded. Blood glucose was monitored. Results: In nondiabetic rats, all three agents induced satisfactory anesthesia, but with differing durations: medetomidine > pentobarbital > xylazine. ERG responses were similar under medetomidine and xylazine, but relatively reduced under pentobarbital. Both α2-agonists (but not pentobarbital) elicited marked hyperglycemia (peak values 316.1 ± 42.6 and 300.3 ± 29.5 mg/dL, respectively), persisting for 12 h. In diabetic rats, elevated blood glucose concentrations were not affected by any of the agents, but the depth of anesthesia under medetomidine and xylazine was inadequate for ERG recording. Conclusions: In nondiabetic rats, medetomidine and xylazine elicited comparable effects on ERGs that differ from pentobarbital, but both perturbed glucose metabolism, potentially confounding experimental outcomes. In STZ-diabetic rats, neither α2-agent provided adequate anesthesia, while pentobarbital did so. Problems with α2-anesthetic agents, including medetomidine, must be recognized to ensure meaningful interpretation of experimental results.

Modelling preeclampsia: a comparative analysis of the common human trophoblast cell lines.

Preeclampsia remains a challenge without an effective therapy. Evidence supports targetability of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), which are released excessively from the placenta under ischemic and hypoxic stresses. We compared four trophoblast cell lines, BeWo, Jar, Jeg-3, and HTR-8/SVneo, in order to identify a suitable model for drug screening. Cultured trophoblasts were exposed to 1% oxygen vs. normoxia for 24-48 hr; human umbilical vein and aortic endothelial cells were included for comparison. Supernatant sFlt-1 and sEng concentrations were measured by ELISA, and sFlt-1 mRNA expression determined by RT-PCR. Cellular responses to experimental therapeutics were explored. All four trophoblast lines secreted sEng, which did not increase by hypoxia. BeWo, Jar, and Jeg-3 exhibited significantly enhanced expression of sFlt-1 i13 and e15a mRNA in response to hypoxia; however, only BeWo released a detectable level of sFlt-1 protein, which was doubled by hypoxia. In contrast, hypoxia decreased sFlt-1 mRNA expression and protein release in HTR-8/SVneo, similarly to endothelial cells. The cellular mechanism involved HIFα. BeWo responded to representative agents similarly to human primary placental tissues in the literature. These data support that the BeWo-hypoxia model mimics a key pathogenic mechanism of preeclampsia and has potential value for translational drug discovery.

Effects of modified lipoproteins on human trophoblast cells: a role in pre-eclampsia in pregnancies complicated by diabetes.

INTRODUCTION: Pre-eclampsia (PE) is increased ~4-fold by maternal diabetes. Elevated plasma antiangiogenic factors, soluble fms-like tyrosine kinase (sFLT-1) and soluble endoglin (sENG), precede PE onset. We investigated whether diabetes-related stresses, modified lipoproteins and elevated glucose enhance trophoblast sFLT-1 and sENG release and/or alter placental barrier function and whether oxidized low-density lipoprotein (Ox-LDL) is in placental tissue. RESEARCH DESIGN AND METHODS: HTR8/SVneo cells were exposed to 'heavily-oxidized, glycated' LDL (HOG-LDL) versus native LDL (N-LDL) (10-200 mg protein/L) for 24 hours ±pretreatment with glucose (30 mmol/L, 72 hours). Concentrations of sFLT-1 and sENG in supernatants (by ELISA) and expressions of sFLT-1-I13 and sFLT-1-E15A isoforms, endoglin (ENG) and matrix metalloproteinase-14 (MMP-14; by RT-PCR) were quantified. For barrier studies, JAR cells were cultured in Transwell plates (12-14 days), then exposed to LDL. Transepithelial electrical resistance (TEER) was measured after 6, 12 and 24 hours. In placental sections from women with and without type 1 diabetes, immunostaining of apolipoprotein B100 (ApoB, a marker of LDL), Ox-LDL and lipoxidation product 4-hydroxynonenal was performed. RESULTS: HOG-LDL (50 mg/L) increased sFLT-1 (2.7-fold, p<0.01) and sENG (6.4-fold, p<0.001) in supernatants versus N-LDL. HOG-LDL increased expression of sFLT-1-I13 (twofold, p<0.05), sFLT-1-E15A (1.9-fold, p<0.05), ENG (1.6-fold, p<0.01) and MMP-14 (1.8-fold, p<0.05) versus N-LDL. High glucose did not by itself alter sFLT-1 or sENG concentrations, but potentiated effects of HOG-LDL on sFLT-1 by 1.5-fold (p<0.05) and on sENG by 1.8-fold (p<0.01). HOG-LDL (200 mg/L) induced trophoblast barrier impairment, decreasing TEER at 6 hours (p<0.01), 12 hours (p<0.01) and 24 hours (p<0.05) versus N-LDL. Immunostaining of term placental samples from women both with and without diabetes revealed presence of intravillous modified lipoproteins. CONCLUSION: These findings may explain, in part, the high risk for PE in women with diabetes. The trophoblast culture model has potential for evaluating novel therapies targeting barrier dysfunction.

Effects of Modified Low-Density Lipoproteins and Fenofibrate on an Outer Blood-Retina Barrier Model: Implications for Diabetic Retinopathy.

Purpose: There is a lack of treatment for early diabetic retinopathy (DR), including blood-retina barrier (BRB) breakdown. The robust clinical benefit of fenofibrate in DR provides an opportunity to explore disease mechanisms and therapeutic targets. We have previously found that modified lipoproteins contribute to DR and that fenofibrate protects the inner BRB. We now investigate (1) whether modified lipoproteins elicit outer BRB injury and (2) whether fenofibrate may alleviate such damage. Methods: Human retinal pigment epithelium ARPE-19 cells were cultured in semipermeable transwells to establish a monolayer barrier and then exposed to heavily oxidized, glycated low-density lipoprotein (HOG-LDL, 25-300 mg/L, up to 24 h) versus native (N)-LDL. Transepithelial electric resistance (TEER) and FITC-dextran permeability were measured. The effects of fenofibrate, its active metabolite fenofibric acid, and other peroxisome proliferator-activated receptor (PPARα) agonists (gemfibrozil, bezafibrate, and WY14643) were evaluated, with and without the PPARα antagonist GW6471 or the adenosine monophosphate-activated protein kinase (AMPK) inhibitor Compound C. Results: HOG-LDL induced concentration- and time-dependent barrier impairment, decreasing TEER and increasing dextran leakage, effects that were amplified by high glucose. Fenofibric acid, but not fenofibrate, gemfibrozil, bezafibrate, or WY14643, attenuated barrier impairment. This effect was reversed significantly by Compound C, but not by GW6471. Conclusions: Modified lipoproteins elicited outer BRB injury in an experimental model, which was reduced by fenofibric acid through a PPARα-independent, AMPK-mediated mechanism. These findings suggest a protective role of fenofibric acid on the outer BRB in diabetic retina.

Differential regulation of a placental SAM68 and sFLT1 gene pathway and the relevance to maternal vitamin D sufficiency.

OBJECTIVE: The goal of this study was to determine if an axis of placental gene expression associated with early onset and severe preeclampsia (EOSPE) was operative in term pregnancy and correlated with vitamin D sufficiency. METHODS: qPCR analysis of NKX2-5, SAM68, sFLT1 and membrane bound VEGFR1/FLT1 mRNA expression was conducted in placentas from 43 subjects enrolled in a vitamin D3 pregnancy supplementation trial. Pair-wise rank order correlations between patient-specific gene expression levels were calculated, and their relationship to maternal 25(OH)D status was assessed by a two-sample Wilcoxon test. Additionally, we probed the mechanistic link between SAM68 and sFLT1 using siRNA depletion in a human trophoblast cell line model. RESULTS: Positive and highly significant correlations were found between SAM68 vs. sFLT1 and SAM68 vs. FLT1 expression levels, as were significant and differential correlations between the expression of these genes and perinatal 25(OH)D status. The variability when stratified by race/ethnicity was qualitatively distinct from those previously observed in EOSPE. Mechanistic studies confirmed a functional role for SAM68 protein in the regulation of sFLT1 expression. NKX2-5 expression was not significantly correlated with sFLT1 or SAM68 expression in these samples, suggesting that its expression may be significant at earlier stages of pregnancy or be restricted to pathological settings. CONCLUSIONS: These data further support our overarching hypothesis that SAM68 expression is a key determinant of VEGFR1 isoform expression in the placenta, and provide additional insights into how this gene pathway may be differentially deployed or modified in normal and pathological pregnancies.

Vitamin D Metabolites and Binding Protein Predict Preeclampsia in Women with Type 1 Diabetes.

The risk for preeclampsia (PE) is enhanced ~4-fold by the presence of maternal type 1 diabetes (T1DM). Vitamin D is essential for healthy pregnancy. We assessed the total, bioavailable, and free concentrations of plasma 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), and vitamin D binding protein (VDBP) at ~12, ~22, and ~32 weeks' gestation ("Visits" (V) 1, 2, and 3, respectively) in 23 T1DM women who developed PE, 24 who remained normotensive, and 19 non-diabetic, normotensive women (reference controls). 25(OH)D deficiency was more frequent in diabetic than non-diabetic women (69% vs. 22%, p < 0.05), but no measure of 25(OH)D predicted PE. By contrast, higher 1,25(OH)2D concentrations at V2 (total, bioavailable, and free: p < 0.01) and V3 (bioavailable: p < 0.05; free: p < 0.01), lower concentrations of VDBP at V3 (p < 0.05), and elevated ratios of 1,25(OH)2D/VDBP (V2, V3: p < 0.01) and 1,25(OH)2D/25(OH)D (V3, p < 0.05) were all associated with PE, and significance persisted in multivariate analyses. In summary, in women with T1DM, concentrations of 1,25(OH)2D were higher, and VDBP lower, in the second and third trimesters in women who later developed PE than in those who did not. 1,25(OH)2D may serve as a new marker for PE risk and could be implicated in pathogenesis.

Haptoglobin Phenotype Modulates Lipoprotein-Associated Risk for Preeclampsia in Women With Type 1 Diabetes.

CONTEXT: The incidence of preeclampsia (PE) is increased in women with diabetes (∼20% vs ∼5% in the general population), and first trimester lipoprotein profiles are predictive. Haptoglobin (Hp), a protein with functional genetic polymorphisms, has antioxidant, anti-inflammatory, and angiogenic effects. Among people with diabetes, the Hp 2-2 phenotype is associated with cardiorenal disease. OBJECTIVE: To investigate whether Hp phenotype is associated with PE in type 1 diabetes mellitus (T1DM) and/or modulates lipoprotein-associated risks. DESIGN AND SETTING: Multicenter prospective study of T1DM pregnancy. PATIENTS: Pregnant women with T1DM (normal albuminuria, normotensive at enrolment, n = 47) studied at three visits, all preceding PE onset: 12.3 ± 1.9, 21.8 ± 1.5, and 31.5 ± 1.6 weeks' gestation (mean ± SD). MAIN OUTCOME MEASURES: Hp phenotype and lipoprotein profiles in women with (n = 23) vs without (n = 24) subsequent PE. RESULTS: Hp phenotype did not predict PE, but lipoprotein associations with subsequent PE were confined to women with Hp 2-2, in whom the following associations with PE were observed: increased low-density lipoprotein (LDL) cholesterol, LDL particle concentration, apolipoprotein B (APOB), triacylglycerol/high-density lipoprotein (HDL) cholesterol ratio, and APOB/apolipoprotein AI (APOA1) ratio; decreased HDL cholesterol, APOA1, large HDL particle concentration, and peripheral lipoprotein lipolysis (all P < 0.05). In women with one or two Hp-1 alleles, no such associations were observed. CONCLUSIONS: In women with T1DM, although Hp phenotype did not predict PE risk, lipoprotein-related risks for PE were limited to those with the Hp 2-2 phenotype. Hp phenotype may modulate PE risk in diabetes.

Correction to: Discovery and Identification of an Endogenous Metabolite of Tramiprosate and Its Prodrug ALZ-801 that Inhibits Beta Amyloid Oligomer Formation in the Human Brain.

Page 856: Fig. 6 was an incorrect duplication of Fig. 5. The correct figure is given below.

Discovery and Identification of an Endogenous Metabolite of Tramiprosate and Its Prodrug ALZ-801 that Inhibits Beta Amyloid Oligomer Formation in the Human Brain.

BACKGROUND: ALZ-801 is an oral, small-molecule inhibitor of beta amyloid (Aß) oligomer formation in clinical development for Alzheimer's disease (AD). ALZ-801 is a prodrug of tramiprosate with improved pharmacokinetic properties and gastrointestinal tolerability. During clinical studies, we discovered that the primary metabolite of tramiprosate and its prodrug ALZ-801, 3-sulfopropanoic acid (3-SPA), is an endogenous molecule in the human brain and present in the cerebrospinal fluid (CSF) of patients with AD and other neurodegenerative brain diseases. OBJECTIVE: The objectives of this research were to (1) identify and confirm the presence of 3-SPA in CSF samples from elderly, drug-naïve patients with memory deficits; (2) quantify the levels of 3-SPA in the CSF of patients with AD from tramiprosate phase III North American (NA) trial; (3) evaluate the in vitro anti-Aß42 oligomer activity of 3-SPA; and (4) characterize the pharmacokinetics and brain-penetration properties of 3-SPA. METHODS: Lumbar CSF samples from 64 drug-naïve patients with cognitive deficits (Mini-Mental State Examination [MMSE] score range 15-30) and six patients with AD treated with tramiprosate 150 mg twice daily in the phase III trial, at week 78, were analyzed. We used liquid chromatography-tandem mass spectrometry to confirm the structural molecular identity of endogenous 3-SPA with a 3-SPA reference standard and ion-mobility spectrometry-mass spectrometry with molecular dynamics to characterize interactions of 3-SPA with Aß42 monomers, and the resultant conformational alterations. Rat studies using oral (30 mg/kg) and intravenous (10 mg/kg) doses were conducted to characterize the pharmacokinetic properties and brain penetration of 3-SPA. RESULTS: We confirmed the presence of 3-SPA in the CSF of drug-naïve patients with cognitive deficits (mean concentration 11.7 ± 4.3 nM). The mean concentration of 3-SPA in patients with AD treated with tramiprosate was 135 ± 51 nM. In vitro studies revealed a multi-ligand interaction of 3-SPA with monomeric Aß42 that inhibits the aggregation of Aß42 into small oligomers. Comparisons of the molecular interactions of tramiprosate and 3-SPA with Aß42 are also presented. Furthermore, in rat preclinical studies, 3-SPA displayed 100% oral bioavailability and 25% brain penetration, indicating that the metabolite is well absorbed and crosses the blood-brain barrier. CONCLUSIONS: We confirmed the endogenous presence of 3-SPA, the major metabolite of tramiprosate, in the CSF of drug-naïve elderly patients with memory deficits due to AD and a variety of other neurodegenerative disorders. The levels of 3-SPA were up to 12.6-fold greater in patients with AD receiving tramiprosate than in drug-naïve patients. In addition, we showed that 3-SPA has potent anti-Aß oligomer activity, inhibiting aggregation of Aß42 into small oligomers with efficacy comparable to that of tramiprosate. 3-SPA displays excellent oral availability and brain penetration in rats, suggesting that the higher CSF concentrations of 3-SPA in the human brain after oral administration of ALZ-801 or tramiprosate (and subsequent conversion to 3-SPA) result from the penetration of the metabolite into the central nervous system. These data suggest that 3-SPA is an endogenous agent with potential activity stabilizing the conformational flexibility of Aß monomers that, in turn, inhibit Aß misfolding and formation of soluble toxic Aß oligomers in humans, thereby preventing the initial pathogenic step in the progression of AD. Clinical improvements observed in patients with AD carrying the ε4 allele of the apolipoprotein E gene in tramiprosate phase III studies may in part be explained by the therapeutic effects of excess levels of the metabolite in the brains of these patients. The potential protective role of 3-SPA in AD pathogenesis, as well as its therapeutic role in AD and other neurodegenerative disorders, warrants further investigation.

Subclinical First Trimester Renal Abnormalities Are Associated With Preeclampsia in Normoalbuminuric Women With Type 1 Diabetes.

OBJECTIVE: This study was conducted to determine the utility of tubular (urinary/plasma neutrophil gelatinase-associated lipocalin [NGAL] and urinary kidney injury molecule 1 [KIM-1]) and glomerular (estimated glomerular filtration rate [eGFR]) biomarkers in predicting preeclampsia (PE) in pregnant women with type 1 diabetes mellitus (T1DM) who were free of microalbuminuria and hypertension at the first trimester. RESEARCH DESIGN AND METHODS: This was a prospective study of T1DM pregnancy. Maternal urinary and plasma NGAL, urinary KIM-1 (ELISA of frozen samples), and eGFR (Chronic Kidney Disease Epidemiology Collaboration equation) were determined at three study visits (V1: 12.4 ± 1.8; V2: 21.7 ± 1.4; V3: 31.4 ± 1.5 weeks' gestation [mean ± SD]) in 23 women with T1DM with subsequent PE (DM+PE+), 24 who remained normotensive (DM+PE-), and, for reference, in 19 normotensive pregnant women without diabetes (DM-). The groups with diabetes were matched for age, diabetes duration, and parity. All subjects were normotensive and free of microalbuminuria or albuminuria at V1. All study visits preceded the onset of PE. RESULTS: Urinary creatinine-corrected NGAL (uNGALcc, ng/mg) was significantly elevated at V1 in DM+PE+ vs. DM+PE- women (P = 0.01); this remained significant after exclusion of leukocyte-positive samples (5 DM+PE+ and 2 DM+PE-) (P = 0.02). Accounting for BMI, HbA1c, and total daily insulin dose, a doubling of uNGALcc at V1 conferred a sevenfold increase in risk for PE (P = 0.026). In contrast, neither plasma NGAL nor urinary KIM-1 predicted PE. Also at V1, eGFR was elevated in DM+PE+ vs. DM+PE- (P = 0.04). CONCLUSIONS: Early tubular and glomerular dysfunction may predict PE in first trimester women with T1DM, even if free of microalbuminuria. These data suggest that subclinical renal tubular and glomerular injury, if present early in pregnancy, may predispose women with T1DM to PE.

Clinical Pharmacokinetics and Safety of ALZ-801, a Novel Prodrug of Tramiprosate in Development for the Treatment of Alzheimer's Disease.

BACKGROUND: ALZ-801 is an orally available, valine-conjugated prodrug of tramiprosate. Tramiprosate, the active agent, is a small-molecule ß-amyloid (Aß) anti-oligomer and aggregation inhibitor that was evaluated extensively in preclinical and clinical investigations for the treatment of Alzheimer's disease (AD). Tramiprosate has been found to inhibit ß-amyloid oligomer formation by a multi-ligand enveloping mechanism of action that stabilizes Aß42 monomers, resulting in the inhibition of formation of oligomers and subsequent aggregation. Although promising as an AD treatment, tramiprosate exhibited two limiting deficiencies: high intersubject pharmacokinetic (PK) variability likely due to extensive gastrointestinal metabolism, and mild-to-moderate incidence of nausea and vomiting. To address these, we developed an optimized prodrug, ALZ-801, which retains the favorable efficacy attributes of tramiprosate while improving oral PK variability and gastrointestinal tolerability. In this study, we summarize the phase I bridging program to evaluate the safety, tolerability and PK for ALZ-801 after single and multiple rising dose administration in healthy volunteers. METHODS: Randomized, placebo-controlled, phase I studies in 127 healthy male and female adult and elderly volunteers included [1] a single ascending dose (SAD) study; [2] a 14-day multiple ascending dose (MAD) study; and [3] a single-dose tablet food-effect study. This program was conducted with both a loose-filled capsule and an immediate-release tablet formulation, under both fasted and fed conditions. Safety and tolerability were assessed, and plasma and urine were collected for liquid chromatography-mass spectrometry (LC-MS) determination and non-compartmental PK analysis. In addition, we defined the target dose of ALZ-801 that delivers a steady-state plasma area under the curve (AUC) exposure of tramiprosate equivalent to that studied in the tramiprosate phase III study. RESULTS: ALZ-801 was well tolerated and there were no severe or serious adverse events (AEs) or laboratory findings. The most common AEs were transient mild nausea and some instances of vomiting, which were not dose-related and showed development of tolerance after continued use. ALZ-801 produced dose-dependent maximum plasma concentration (C max) and AUC exposures of tramiprosate, which were equivalent to that after oral tramiprosate, but with a substantially reduced intersubject variability and a longer elimination half-life. Administration of ALZ-801 with food markedly reduced the incidence of gastrointestinal symptoms compared with the fasted state, without affecting plasma tramiprosate exposure. An immediate-release tablet formulation of ALZ-801 displayed plasma exposure and low variability similar to the loose-filled capsule. ALZ-801 also showed excellent dose-proportionality without accumulation or decrease in plasma exposure of tramiprosate over 14 days. Based on these data, 265 mg of ALZ-801 twice daily was found to achieve a steady-state AUC exposure of tramiprosate equivalent to 150 mg twice daily of oral tramiprosate in the previous phase III trials. CONCLUSIONS: ALZ-801, when administered in capsule and tablet forms, showed excellent oral safety and tolerability in healthy adults and elderly volunteers, with significantly improved PK characteristics over oral tramiprosate. A clinical dose of ALZ-801 (265 mg twice daily) was established that achieves the AUC exposure of 150 mg of tramiprosate twice daily, which showed positive cognitive and functional improvements in apolipoprotein E4/4 homozygous AD patients. These bridging data support the phase III development of ALZ-801in patients with AD.

Circulating adipokines are associated with pre-eclampsia in women with type 1 diabetes.

AIMS/HYPOTHESIS: The incidence of pre-eclampsia, a multisystem disorder of pregnancy, is fourfold higher in type 1 diabetic than non-diabetic women; it is also increased in women with features of the metabolic syndrome and insulin resistance. In a prospective study of pregnant women with type 1 diabetes, we measured plasma levels of adipokines known to be associated with insulin resistance: leptin, fatty acid binding protein 4 (FABP4), adiponectin (total and high molecular weight [HMW]; also known as high molecular mass), retinol binding protein 4 (RBP4) and resistin and evaluated associations with the subsequent development of pre-eclampsia. METHODS: From an established prospective cohort of pregnant type 1 diabetic women, we studied 23 who developed pre-eclampsia and 24 who remained normotensive; for reference values we included 19 healthy non-diabetic normotensive pregnant women. Plasma adipokines were measured (by ELISA) in stored samples from three study visits (Visit 1- Visit 3) at different gestational ages (mean ± SD): Visit 1, 12.4 ± 1.8 weeks; Visit 2, 21.7 ± 1.4 weeks; and Visit 3, 31.4 ± 1.5 weeks. All the women were free of microalbuminuria and hypertension at enrolment. All study visits preceded the clinical onset of pre-eclampsia. RESULTS: In all groups, leptin, the ratio of leptin to total or HMW adiponectin, FABP4 concentration, ratio of FABP4 to total or HMW adiponectin and resistin level increased, while total and HMW adiponectin decreased, with gestational age. At Visit 1: (1) in diabetic women with vs without subsequent pre-eclampsia, leptin, ratio of leptin to total or HMW adiponectin, and ratio of FABP4 to total or HMW adiponectin, were increased (p < 0.05), while total adiponectin was decreased (p < 0.05); and (2) in normotensive diabetic vs non-diabetic women, total adiponectin was elevated (p < 0.05). At Visits 2 and 3: (1) the primary findings in the two diabetic groups persisted, and FABP4 also increased in women with subsequent pre-eclampsia (p < 0.05); and (2) there were no differences between the two normotensive groups. By logistic regression analyses after covariate adjustment (HbA1c, insulin kg-1 day-1 and gestational age), the best predictive models for pre-eclampsia were as follows: Visit 1, doubling of leptin, OR 9.0 (p < 0.01); Visit 2, doubling of the leptin:total adiponectin ratio, OR 3.7 (p < 0.05); and Visit 3, doubling of FABP4 concentration, OR 25.1 (p < 0.01). The associations were independent of BMI. CONCLUSIONS/INTERPRETATION: As early as the first trimester in type 1 diabetic women, adipokine profiles that suggest insulin resistance are associated with subsequent pre-eclampsia, possibly reflecting maternal characteristics that precede pregnancy. These associations persist in the second and third trimesters, and are independent of BMI. Insulin resistance may predispose women with type 1 diabetes to pre-eclampsia.

Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy.

AIMS/HYPOTHESIS: Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects. METHODS: Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein-LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 µmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis. RESULTS: Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100-200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25-200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis. CONCLUSIONS: Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Beneficial Effects of Berberine on Oxidized LDL-Induced Cytotoxicity to Human Retinal Müller Cells.

PURPOSE: Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal Müller cells. We now explore pathogenic effects of modified LDL on Müller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS: Confluent human Müller cells were exposed to in vitro-modified 'highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/without pretreatment with berberine (5 µM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 µM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-α), and glial cell activation (glial fibrillary acidic protein). RESULTS: Native-LDL had no effect on cultured human Müller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS: Berberine inhibits modified LDL-induced Müller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.

Extravascular modified lipoproteins: a role in the propagation of diabetic retinopathy in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to determine whether plasma lipoproteins, after leakage into the retina and modification by glycation and oxidation, contribute to the development of diabetic retinopathy in a mouse model of type 1 diabetes. METHODS: To simulate permeation of plasma lipoproteins into retinal tissues, streptozotocin-induced mouse models of diabetes and non-diabetic mice were challenged with intravitreal injection of human 'highly-oxidised glycated' low-density lipoprotein (HOG-LDL), native- (N-) LDL, or the vehicle PBS. Retinal histology, electroretinography (ERG) and biochemical markers were assessed over the subsequent 14 days. RESULTS: Intravitreal administration of N-LDL and PBS had no effect on retinal structure or function in either diabetic or non-diabetic animals. In non-diabetic mice, HOG-LDL elicited a transient inflammatory response without altering retinal function, but in diabetic mice it caused severe, progressive retinal injury, with abnormal morphology, ERG changes, vascular leakage, vascular endothelial growth factor overexpression, gliosis, endoplasmic reticulum stress, and propensity to apoptosis. CONCLUSIONS/INTERPRETATION: Diabetes confers susceptibility to retinal injury imposed by intravitreal injection of modified LDL. The data add to the existing evidence that extravasated, modified plasma lipoproteins contribute to the propagation of diabetic retinopathy. Intravitreal delivery of HOG-LDL simulates a stress known to be present, in addition to hyperglycaemia, in human diabetic retinopathy once blood-retinal barriers are compromised.

Trace elements as predictors of preeclampsia in type 1 diabetic pregnancy.

Preeclampsia (PE) affects approximately 5% of all pregnancies, but is increased several-fold in women with pre-gestational type 1 diabetes mellitus (T1DM). Increased oxidative stress and altered maternal plasma trace elements that modulate the antioxidant system have been implicated in PE. In non-diabetic women, increased plasma copper and iron and decreased manganese, selenium, and zinc have been associated with PE in cross-sectional studies. In a longitudinal study, we hypothesized that plasma levels of trace elements differ between T1DM women with vs. without subsequent PE. Samples were collected during the first (gestation 12.2 ± 1.9 weeks, [mean ± SD]), second (21.6 ± 1.5 weeks), and third (31.5 ± 1.7 weeks) trimesters of pregnancy, all before the onset of PE. We compared 23 T1DM women who subsequently developed PE with 24 T1DM women who remained normotensive; and we included 19 non-diabetic (non-DM) normotensive pregnant women as reference controls. Trace elements were measured using inductively coupled plasma mass spectroscopy. In T1DM women with subsequent PE vs normotensive, only plasma zinc was significantly higher at the first trimester, while copper:zinc and copper:high-density lipoprotein cholesterol ratios were higher throughout gestation (all P < .05). These findings persisted after adjustment for covariates. Higher copper:zinc ratios may contribute to oxidative stress in T1DM women who develop PE. Ratios of pro- to anti-oxidant factors may predict risk for PE in diabetic pregnancies more effectively than individual trace element levels.

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes.

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0-200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.