👤 Audrey H H Merry

Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2- but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2(-/-) mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2(-/-) tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length. Show less

Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20:5n-3) and DHA (C22:6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20:4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20:4n-6 to C20:3n-6 and C18:3n-6 to C18:2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence. We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors. The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20:5n-3 and C22:6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15-0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk. In this prospective cohort study, we observed a reduced CHD risk with an increased C20:4n-6 to C20:3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies. Show less

Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate. Show less