👤 Leo Joosten

The presence of low-grade inflammation has been reported in people with type 2 diabetes and related to the development of (macro)vascular complications. Whether systemic inflammation is present in type 1 diabetes and linked to long-term complications remains unknown. We used a targeted proteomics approach to compare inflammation in people with type 1 diabetes and type 2 diabetes with control subjects and linked these proteins to diabetes related characteristics and complications. We included 233 participants with type 1 diabetes, 387 participants with type 2 diabetes and 150 healthy controls. Plasma was collected and used to determine high sensitive C-reactive proteins (hs-CRP) and an additional 92 inflammatory proteins using the Olink proteomics platform. Compared to healthy controls, 41 circulating inflammatory proteins were higher in type 1 diabetes (FDR < 0.05) and 64 inflammatory proteins in type 2 diabetes (FDR < 0.05) (including CXCL5, IL-15RA, MCP-4 and AXIN1 for both groups). HbA Both type 1 diabetes and type 2 diabetes are associated with increased circulating inflammatory protein concentrations, but the increase is more pronounced in type 2 diabetes. These results suggest both differences in drivers of inflammation between type 1 diabetes and type 2 diabetes as well as potential similarities in pathways involved in the development of diabetes-associated complications. Show less

Former critically ill children show an epigenetic age deceleration 2 years after paediatric intensive care unit (PICU) admission as compared with normally developing healthy children, with stunted growth in height 2 years further in time as physical correlate. This was particularly pronounced in children who were 6 years or older at the time of critical illness. As this age roughly corresponds to the onset of adrenarche and further pubertal development, a relation with altered activation of endocrine pathways is plausible. We hypothesised that children who have been admitted to the PICU, sex- and age-dependently show long-term abnormal DNA methylation within genes involved in steroid hormone synthesis or steroid sulphation/desulphation, possibly aggravated by in-PICU glucocorticoid treatment, which may contribute to stunted growth in height further in time after critical illness. In this preplanned secondary analysis of the multicentre PEPaNIC-RCT and its follow-up, we compared the methylation status of genes involved in the biosynthesis of steroid hormones (aldosterone, cortisol and sex hormones) and steroid sulphation/desulphation in buccal mucosa DNA (Infinium HumanMethylation EPIC BeadChip) from former PICU patients at 2-year follow-up (n = 818) and healthy children with comparable sex and age (n = 392). Adjusting for technical variation and baseline risk factors and corrected for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 23 CpG sites (within CYP11A1, POR, CYB5A, HSD17B1, HSD17B2, HSD17B3, HSD17B6, HSD17B10, HSD17B12, CYP19A1, CYP21A2, and CYP11B2) and 4 DNA regions (within HSD17B2, HSD17B8, and HSD17B10) that were mostly hypomethylated. These abnormalities were partially sex- (1 CpG site) or age-dependent (7 CpG sites) and affected by glucocorticoid treatment (3 CpG sites). Finally, multivariable linear models identified robust associations of abnormal methylation of steroidogenic genes with shorter height further in time, at 4-year follow-up. Children who have been critically ill show abnormal methylation within steroidogenic genes 2 years after PICU admission, which explained part of the stunted growth in height at 4-year follow-up. The abnormalities in DNA methylation may point to a long-term disturbance in the balance between active sex steroids and mineralocorticoids/glucocorticoids after paediatric critical illness, which requires further investigation. Show less

Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project. Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 × 10 Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis. Show less

Somatostatin receptor targeting is considered the standard nuclear medicine technique for visualization of neuroendocrine tumors (NET). Since not all NETs over-express somatostatin receptors, the search for novel targets, visualizing these NETs, is ongoing. Many NETs, expressing low somatostatin receptor levels, express glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPR). Here, we evaluated the performance of [Lys Show less

The role of interleukin (IL-)32 in inflammatory conditions is well-established, however, the mechanism behind its role in atherosclerosis remains unexplained. Our group reported a promoter single nucleotide polymorphism in IL-32 associated with higher high-density lipoprotein (HDL) concentrations. We hypothesize that endogenous IL-32 in liver cells, a human monocytic cell line and carotid plaque tissue, can affect atherosclerosis by regulating (HDL) cholesterol homeostasis via expression of cholesterol transporters/mediators. Human primary liver cells were stimulated with recombinant human (rh)TNFα and poly I:C to study the expression of IL-32 and mediators in cholesterol pathways. Additionally, IL-32 was overexpressed in HepG2 cells and overexpressed and silenced in THP-1 cells to study the direct effect of IL-32 on cholesterol transporters expression and function. Stimulation of human primary liver cells resulted in induction of IL-32α, IL-32β and IL-32γ mRNA expression (p < 0.01). A strong correlation between the expression of IL-32γ and ABCA1, ABCG1, LXRα and apoA1 was observed (p < 0.01), and intracellular lipid concentrations were reduced in the presence of endogenous IL-32 (p < 0.05). Finally, IL32γ and ABCA1 mRNA expression was upregulated in carotid plaque tissue and when IL-32 was silenced in THP-1 cells, mRNA expression of ABCA1 was strongly reduced. Regulation of IL-32 in human primary liver cells, HepG2 and THP-1 cells strongly influences the mRNA expression of ABCA1, ABCG1, LXRα and apoA1 and affects intracellular lipid concentrations in the presence of endogenous IL-32. These data, for the first time, show an important role for IL32 in cholesterol homeostasis. Show less

Gout is associated with dyslipidaemia. Association of the apolipoprotein A1-C3-A4 gene cluster with gout has previously been reported in a small study. To investigate a possible causal role for this locus in gout, we tested the association of genetic variants from APOA1 (rs670) and APOC3 (rs5128) with gout. We studied data for 2452 controls and 2690 clinically ascertained gout cases of European and New Zealand Polynesian (Māori and Pacific) ancestry. Data were also used from the publicly available Atherosclerosis Risk in Communities study (n = 5367) and the Framingham Heart Study (n = 2984). Multivariate adjusted logistic and linear regression was used to test the association of single-nucleotide polymorphisms with gout risk, serum urate, triglyceride and high-density lipoprotein cholesterol (HDL-C). In Polynesians, the T-allele of rs670 (APOA1) increased (odds ratio, OR = 1.53, P = 4.9 × 10(-6)) and the G-allele of rs5128 (APOC3) decreased the risk of gout (OR = 0.86, P = 0.026). In Europeans, there was a strong trend to a risk effect of the T-allele for rs670 (OR = 1.11, P = 0.055), with a significant protective effect of the G-allele for rs5128 being observed after adjustment for triglycerides and HDL-C (OR = 0.81, P = 0.039). The effect at rs5128 was specific to males in both Europeans and Polynesians. Association in Polynesians was independent of any effect of rs670 and rs5128 on triglyceride and HDL-C levels. There was no evidence for association of either single-nucleotide polymorphism with serum urate levels (P ⩾ 0.10). Our data, replicating a previous study, supports the hypothesis that the apolipoprotein A1-C3-A4 gene cluster plays a causal role in gout. Show less

Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ∼3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design. Show less

Fibrin is a temporary matrix that not only seals a wound, but also provides a temporary matrix structure for invading cells during wound healing. Two naturally occurring fibrinogen variants, high molecular weight (HMW) and low molecular weight (LMW) fibrinogen, display different properties in supporting angiogenesis in vivo and in vitro. This study was aimed at investigating the functional characteristics and molecular mechanisms of human microvascular endothelial cells (HMVECs) cultured on HMW and LMW fibrin matrices. HMVECs on HMW fibrin matrices showed increased proliferation and tube formation as compared with their counterparts on unfractionated and LMW fibrin. Degradation of HMW fibrin was markedly enhanced by the presence of HMVECs, that of LMW fibrin was enhanced only slightly. However, the expression levels of fibrinolysis-regulating proteins and integrins were similar. Subsequent microarray analysis revealed that the expression of 377 genes differed significantly between HMVECs cultured on HMW fibrin and those cultured on LMW fibrin. Among these genes, UNC5B, DLL4 and the DLL4-Notch downstream targets Hey1, Hey2 and Hes1 showed increased expression in HMVECs on LMW fibrin. However, pharmacologic and genetic (DLL4 small interfering RNA) inhibition of DLL4-Notch signaling blunted rather than enhanced proliferation and tube formation by HMVECs on both fibrin variants. Heterogeneity in naturally occurring fibrinogen strongly influences endothelial cell proliferation and tube formation, and causes alterations in gene expression, including that of DLL4-Notch. The higher fibrinolytic sensitivity of HMW fibrin in the presence of HMVECs contributes to increased tube formation. Although the expression of DLL4-Notch was altered, it did not explain the enhanced tube formation in HMW fibrin. This study provides new perspectives for biological and tissue engineering applications. Show less