Soluble syndecan-1 and glycosaminoglycans in preeclamptic and normotensive pregnancies.

Preeclampsia, an important cause of maternal and fetal morbidity and mortality, is associated with increased sFLT1 levels and with structural and functional damage to the glycocalyx contributing to endothelial dysfunction. We investigated glycocalyx components in relation to preeclampsia in human samples. While soluble syndecan-1 and heparan sulphate were similar in plasma of preeclamptic and normotensive pregnant women, dermatan sulphate was increased and keratan sulphate decreased in preeclamptic women. Dermatan sulphate was correlated with soluble syndecan-1, and inversely correlated with blood pressure and activated partial thromboplastin time. To determine if syndecan-1 was a prerequisite for the sFlt1 induced increase in blood pressure in mice we studied the effect of sFlt1 on blood pressure and vascular contractile responses in syndecan-1 deficient and wild type male mice. The classical sFlt1 induced rise in blood pressure was absent in syndecan-1 deficient mice indicating that syndecan-1 is a prerequisite for sFlt1 induced increase in blood pressure central to preeclampsia. The results show that an interplay between syndecan-1 and dermatan sulphate contributes to sFlt1 induced blood pressure elevation in pre-eclampsia.

High-Throughput Screen Fails to Identify Compounds That Enhance Residual Enzyme Activity of Mutant N-Acetyl-α-Glucosaminidase in Mucopolysaccharidosis Type IIIB.

BACKGROUND: In the severe neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB or Sanfilippo disease type B), deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in accumulation of heparan sulfate. Patients present with a severe, rapidly progressing phenotype (RP) or a more attenuated, slowly progressing phenotype (SP). In a previous study, residual NAGLU activity in fibroblasts of SP patients could be increased by culturing at 30°C, probably as a result of improved protein folding and lysosomal targeting under these conditions. Chaperones are molecules which influence protein folding and could therefore have therapeutic potential in SP MPSIIIB patients. Here we studied the effects of 1,302 different compounds on residual NAGLU activity in SP MPSIIIB patient fibroblasts including 1,280 approved compounds from the Prestwick Chemical Library. METHODS: Skin fibroblasts of healthy controls, an SP MPSIIIB patient (homozygous for the temperature sensitive mutation p.S612G) and an RP MPSIIIB patient (homozygous for the p.R297* mutation and non-temperature sensitive), were used. A high-throughput assay for measurement of NAGLU activity was developed and validated, after which 1,302 different molecules were tested for their potential to increase NAGLU activity. RESULTS: None of the compounds tested were able to enhance NAGLU activity. CONCLUSIONS: This high-throughput screen failed to identify compounds that could enhance residual activity of mutant NAGLU in fibroblasts of SP MPSIIIB patients with temperature sensitive mutations. To therapeutically simulate the positive effect of lower temperatures on residual NAGLU activity, first more insight is needed into the mechanisms underlying this temperature dependent increase.

Processing of mutant N-acetyl-α-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature.

BACKGROUND: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype. Previous studies showed significantly higher NAGLU activity in skin fibroblasts of SP patients when cultured at 30°C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. METHODS: Fibroblasts from controls, one RP patient (homozygous for the p.R297* mutation) and three SP MPSIIIB patients (homozygous for the mutation p.S612G or p.R643C, or compound heterozygous for the mutations p.A72_G79dup8 and p.R565Q) were cultured at temperatures ranging from 37°C to 27°C and harvested at different time points to assess NAGLU activity, mRNA and protein levels, and NAGLU glycosylation. Intracellular localization of wild-type and mutant mCherry-tagged NAGLU was analyzed by immunofluorescence. RESULTS: In control fibroblasts NAGLU was present as a 85kDa precursor and a 82kDa mature form. In SP patients' fibroblasts cultured at 37°C, only the 85kDa form was detected. Culturing at lower temperatures resulted in higher NAGLU mRNA levels, increased levels of both precursor and mature NAGLU protein and improved processing. The formation of mature NAGLU corresponded with higher NAGLU activity levels. CONCLUSION: We show that the NAGLU protein consists of a precursor and a mature form and that in SP MPSIIIB patients' fibroblasts only the precursor protein is present at 37°C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing.

Residual N-acetyl-α-glucosaminidase activity in fibroblasts correlates with disease severity in patients with mucopolysaccharidosis type IIIB.

BACKGROUND: Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare genetic disorder in which the deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU) results in the accumulation of heparan sulfate (HS), leading to progressive neurocognitive deterioration. In MPS IIIB a wide spectrum of disease severity is seen. Due to a large allelic heterogeneity, establishing genotype-phenotype correlations is difficult. However, reliable prediction of the natural course of the disease is needed, in particular for the assessment of the efficacy of potential therapies. METHODS: To identify markers that correlate with disease severity, all Dutch patients diagnosed with MPS IIIB were characterised as either rapid (RP; classical, severe phenotype) or slow progressors (SP; non-classical, less severe phenotype), based on clinical data. NAGLU activity and HS levels were measured in patients' fibroblasts after culturing at different temperatures. RESULTS: A small, though significant difference in NAGLU activity was measured between RP and SP patients after culturing at 37 °C (p < 0.01). Culturing at 30 °C resulted in more pronounced and significantly higher NAGLU activity levels in SP patients (p < 0.001) with a NAGLU activity of 0.58 nmol.mg-1.hr-1 calculated to be the optimal cut-off value to distinguish between the groups (sensitivity and specificity 100 %). A lower capacity of patients' fibroblasts to increase NAGLU activity at 30 °C could significantly predict for the loss of several disease specific functions. CONCLUSION: NAGLU activity in fibroblasts cultured at 30 °C can be used to discriminate between RP and SP MPS IIIB patients and the capacity of cells to increase NAGLU activity at lower temperatures correlates with disease symptoms.

Heparan sulfate derived disaccharides in plasma and total urinary excretion of glycosaminoglycans correlate with disease severity in Sanfilippo disease.

BACKGROUND: Sanfilippo disease (Mucopolysaccharidosis III) is a neurodegenerative lysosomal disorder characterized by accumulation of the glycosaminoglycan heparan sulfate (HS). MPS III has a large phenotypic variability and early assessment of disease severity is difficult. We investigated the correlation between disease severity and the plasma concentration of HS (pHS, defined by the sum of the heparan sulfate derived disaccharides obtained after enzymatic digestion) and urinary total GAGs level (uGAGs, measured by the dimethylene blue test) in a cross-sectional cohort of 44 MPS III patients. METHODS: Disease severity was established on the basis of the age of complete loss of independent walking and of full loss of speech in all patients. Hazard ratios (HR) were obtained with cox-regression analysis. In order to allow prediction of a severe phenotype based on a cut-off value for pHS, patients were divided in two groups (severely affected and less severely affected) based on predictive mutations or on the age of full loss of speech. Receiver operator characteristics (ROC) were obtained for pHS. RESULTS: pHS and uGAGs were independently and linearly associated with an increased risk of speech loss with a HR of 1.8 (95 % CI 1.3-2.7) per 500 ng/ml increase of HS in plasma (p = 0.002), and a HR of 2.7 (95 % CI 1.6-4.4) per 10 mg/mmol creatinine increase of uGAGs (p < 0.001). pHS and uGAGS were less strongly associated with loss of walking. The area under the ROC curve for pHS was 0.85, indicating good discrimination. CONCLUSION: pHS and uGAGs may be useful biomarkers for prediction of severity in MPS III.

Effects of short-chain galacto- and long-chain fructo-oligosaccharides on systemic and local immune status during pregnancy.

Nondigestible oligosaccharides can positively influence health via various mechanisms. During pregnancy, supplementation of nondigestible oligosaccharides has positive effects on hypertension and metabolism and may be used to ameliorate pregnancy-related metabolic disturbances. In the nonpregnant state, nondigestible oligosaccharides have been shown to induce a tolerogenic immune response mediated by T-regulatory cells. Since relatively little is known about the effects of nondigestible oligosaccharides on the immune system during pregnancy, pregnant mice were supplemented with a specific mixture of short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS; ratio 9:1). Systemic and local immune parameters were analyzed on day 18 of pregnancy. This study shows that, compared with virgin mice, scGOS/lcFOS supplementation appears to elicit a more tolerogenic immune reaction in pregnant mice and supplementation does not increase the Th1-dependent delayed type hypersensitivity response in pregnant mice as it does in virgin mice.

Perinatal programming of murine immune responses by polyunsaturated fatty acids.

Linoleic acid and α-linolenic acid are essential fatty acids (eFAs) and have to be acquired from the diet. eFAs are the precursors for long-chain polyunsaturated fatty acids (lcPUFAs), which are important immune-modulating compounds. lcPUFAs can be converted into eicosanoids and other mediators. They affect membrane structure and fluidity and can alter gene expression. There has been a marked change in dietary fatty acid intake over the last several decades. Since eFAs are acquired from the diet and immune development occurs mainly perinatally, the maternal diet may influence fetal and neonatal eFA levels, and thereby lcPUFA status, and thus immune development and function. To study whether early exposure to eFAs can program immune function, mice were fed diets varying in the ratio of ω-3 to ω-6-eFAs during pregnancy and/or lactation. After weaning, pups received a Western-style diet. At 11 weeks of age, the effects of maternal diet on the offspring's allergic and vaccination responses were examined using the T-helper 2 driven ovalbumin-induced allergy model and the T-helper 1 driven influenza-vaccination model, respectively. Offspring of dams fed a high α-linolenic acid diet during lactation showed an enhanced vaccination response. As diets with either low or high ω-3/ω-6-eFA ratio attenuated the T-helper 2 allergic response, the high α-linolenic acid diet fed during lactation had the most pronounced effect. These results indicate that there is a programming effect of maternal diet on the offspring's immune response and that in mice the window of greatest susceptibility to maternal dietary intervention is the lactation/suckling period.

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation.

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.

L-carnitine is synthesized in the human fetal-placental unit: potential roles in placental and fetal metabolism.

Carnitine plays an indispensable role in fatty acid oxidation. Previous studies revealed that fetal carnitine is derived from the mother via transplacental transfer. Recent studies demonstrated the presence and importance of an active fatty acid oxidation system in the human placenta and in the human fetus. In view of these findings we decided to study carnitine metabolism in the fetal-placental unit by measuring carnitine metabolites, intermediary metabolites of carnitine biosynthesis, as well as the activity of carnitine biosynthesis enzymes in human term placenta, cord blood and selected embryonic and fetal tissues (5-20 weeks of development). Placenta contained low but detectable activity of gamma-butyrobetaine dioxygenase. This enzyme, which was considered to be expressed only in kidney, liver and brain, catalyzes the last step in the carnitine biosynthesis pathway. In addition, our results show that human fetal kidney, liver and spinal cord already have the capacity to synthesize carnitine. The ability of the placenta and fetus to synthesize carnitine suggests that in circumstances when maternal carnitine supply is limited, carnitine biosynthesis by the fetal-placental unit may supply sufficient carnitine for placental and fetal metabolism.

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways.

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.