Matrix metalloproteinase-9 mediated shedding of syndecan-4 in glomerular endothelial cells

Background - Diabetic nephropathy is the most common cause of end‐stage renal failure in the western world and Asia. The mechanisms are not fully elucidated, but disruption of glomerular endothelial glycocalyx and shedding of its components including syndecans has been implicated. Aims - We hypothesize that reduced glomerular filtration in diabetes is caused by disruption of endothelial glycocalyx in glomeruli, including increased shedding of syndecan‐4. The aim of this study was to determine the effects of experimental diabetic conditions by means of hyperglycemia and IL‐1β exposure on syndecan‐4 shedding in GEnC, and to investigate regulation of shedding by sheddases. Results - We found that in GEnC the expression of syndecan‐4 is higher than that of the other syndecans. In polarized GEnC, apical shedding of syndecan‐4 and syndecan‐4 gene expression was increased by 60% after IL‐1β‐stimulation, but not affected by hyperglycemic conditions. This was accompanied by a 50% increase in MMP9 gene expression in IL‐1β‐stimulated cells but not hyperglycemia. MMP9 knockdown reduced syndecan‐4 shedding by 50%. Conclusion - IL‐1β but not hyperglycemia increases the shedding of syndecan‐4 from GEnC in an MMP9‐dependent manner. This provides a potential mechanism of GEnC damage in diabetes and other inflammatory conditions

Matrix metalloproteinase-9 mediated shedding of syndecan-4 in glomerular endothelial cells

Background - Diabetic nephropathy is the most common cause of end‐stage renal failure in the western world and Asia. The mechanisms are not fully elucidated, but disruption of glomerular endothelial glycocalyx and shedding of its components including syndecans has been implicated. Aims - We hypothesize that reduced glomerular filtration in diabetes is caused by disruption of endothelial glycocalyx in glomeruli, including increased shedding of syndecan‐4. The aim of this study was to determine the effects of experimental diabetic conditions by means of hyperglycemia and IL‐1β exposure on syndecan‐4 shedding in GEnC, and to investigate regulation of shedding by sheddases. Results - We found that in GEnC the expression of syndecan‐4 is higher than that of the other syndecans. In polarized GEnC, apical shedding of syndecan‐4 and syndecan‐4 gene expression was increased by 60% after IL‐1β‐stimulation, but not affected by hyperglycemic conditions. This was accompanied by a 50% increase in MMP9 gene expression in IL‐1β‐stimulated cells but not hyperglycemia. MMP9 knockdown reduced syndecan‐4 shedding by 50%. Conclusion - IL‐1β but not hyperglycemia increases the shedding of syndecan‐4 from GEnC in an MMP9‐dependent manner. This provides a potential mechanism of GEnC damage in diabetes and other inflammatory conditions

Glucosamine exposure reduces proteoglycan synthesis in primary human endothelial cells in vitro

Purpose: Glucosamine (GlcN) supplements are promoted for medical reasons, for example, for patients with arthritis and other joint-related diseases. Oral intake of GlcN is followed by uptake in the intestine, transport in the circulation and thereafter delivery to chondrocytes. Here, it is postulated to have an effect on synthesis and turnover of extracellular matrix constituents expressed by these cells. Following uptake in the intestine, serum levels are transiently increased, and the endothelium is exposed to increased levels of GlcN. We investigated the possible effects of GlcN on synthesis of proteoglycans (PGs), an important matrix component, in primary human endothelial cells. Methods: Primary human endothelial cells were cultured in vitro in medium with 5 mM glucose and 0–10 mM GlcN. PGs were recovered and analysed by western blotting, or by SDS-PAGE, gel chromatography or ion-exchange chromatography of 35S-PGs after 35S-sulphate labelling of the cells. Results: The synthesis and secretion of 35S-PGs from cultured endothelial cells were reduced in a dose- and time-dependent manner after exposure to GlcN. PGs are substituted with sulphated glycosaminoglycan (GAG) chains, vital for PG function. The reduction in 35S-PGs was not related to an effect on GAG chain length, number or sulphation, but rather to the total expression of PGs. Conclusion: Exposure of endothelial cells to GlcN leads to a general decrease in 35S-PG synthesis. These results suggest that exposure to high levels of GlcN can lead to decreased matrix synthesis, contrary to what has been claimed by supporters of such supplements

Serglycin in Quiescent and Proliferating Primary Endothelial Cells

Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development

Acute exercise increases syndecan-1 and -4 serum concentrations

The effects of acute and long-term exercise on syndecans and the relationship to insulin sensitivity are not fully explored. We aimed to examine the effects of acute and 12 weeks of exercise on (1) serum levels of syndecan-1 and -4, (2) gene expression related to syndecan synthesis and modification in skeletal muscle and adipose tissue, and (3) the relationship to insulin sensitivity. Sedentary men with (n = 13) or without (n = 13) dysglycemia underwent two 45 min acute bicycle tests interspersed by 12 weeks of exercise intervention. Euglycemic hyperinsulinemic clamp and mRNA-sequencing of skeletal muscle and adipose tissue biopsies were performed before and after intervention. Serum syndecan-1 and -4 levels were quantified before, immediately after and 2 h after bicycling. Syndecan-1 and -4 serum concentrations increased in response to acute physical exercise. Baseline syndecan-4 but not syndecan-1 concentrations were higher in dysglycemic compared to normoglycemic men, and correlated to change in insulin sensitivity, but did not change during the 12 weeks exercise intervention. Only syndecan-4 was expressed in skeletal muscle and adipose tissue. Adipose tissue mRNA levels of transcripts affecting syndecan structure and shedding were upregulated in dysglycemia, and muscle mRNA responded to long-term physical activity. The increase in serum syndecan-1 and -4 due to acute exercise suggest increased syndecan shedding and disruption of glycocalyx in response to increased blood flow. The higher syndecan-4 baseline serum levels in dysglycemia, association to insulin sensitivity, and changes in mRNA transcripts may suggest syndecan-4 involvement in muscle and adipose tissue response to exercise

Serglycin is part of the secretory repertoire of LPS-activated monocytes

Monocytes play multiple roles in the immune system, and are active in both acute and chronic diseases. Patients exposed to bacterial infections depend on monocytes in defense reactions, but excessive immune reactions may also cause morbidity through systemic inflammatory responses. Few studies have addressed the importance of proteoglycans, and in particular, the hematopoietic serglycin, in such monocyte immune reactions. Adherent primary monocytes were cultured in absence and presence of LPS. Media were analyzed by ELISA for detection of serglycin. Lysed cell fractions were used to determine the mRNA level of serglycin. Monocytes were also cultured on chamber slides to investigate if serglycin could be detected intracellularly by immunocytochemistry. Monocytes secreted serglycin, and LPS-stimulation increased the secretion. Secretion of inflammatory cytokines increased to a larger extent than serglycin. mRNA levels of serglycin were also increased, suggesting both increased expression and secretion. Immunocytochemistry revealed the presence of serglycin in intracellular vesicles, many destined for secretion. Serglycin containing vesicles increased in number and size when the cells were exposed to LPS. Intracellular vesicle localization and secretion of the proteoglycan serglycin is shown for the first time in primary human monocytes. Monocyte activation by LPS increased the expression and secretion of serglycin, suggesting roles for serglycin in inflammatory processes

NBEAL1 controls SREBP2 processing and cholesterol metabolism and is a susceptibility locus for coronary artery disease

Dysregulated cholesterol homeostasis promotes the pathology of atherosclerosis, myocardial infarction and strokes. Cellular cholesterol is mainly regulated at the transcriptional level by SREBP2, but also through uptake of extracellular cholesterol from low density lipoproteins (LDL) via expression of LDL receptors (LDLR) at the cell surface. Identification of the mechanisms involved in regulation of these processes are thus key to understand the pathology of coronary artery disease. Here, we identify the large and poorly characterized BEACH domain protein Neurobeachin-like (NBEAL) 1 as a Golgi- associated protein required for regulation of cholesterol metabolism. NBEAL1 is most abundantly expressed in arteries. Genetic variants in NBEAL1 are associated with decreased expression of NBEAL1 in arteries and increased risk of coronary artery disease in humans. We show that NBEAL1 regulates cholesterol metabolism by modulating LDLR expression in a mechanism involving interaction with SCAP and PAQR3 and subsequent SREBP2-processing. Thus, low expression of NBEAL1 may lead to increased risk of coronary artery disease by downregulation of LDLR levels

Serglycin Is a Major Proteoglycan in Polarized Human Endothelial Cells and Is Implicated in the Secretion of the Chemokine GRO alpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [35S]sulfate labeling of polarized HUVEC. Interestingly, a major part of 35S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of &#12630 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene &#945 (GRO&#945/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-&#946-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GRO&#945 was reduced. The level of GRO&#945 in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion

Mediators linking maternal weight to birthweight and neonatal fat mass in healthy pregnancies

Abstract Context Lifestyle interventions have not efficaciously reduced complications caused by maternal weight on fetal growth, requiring insight into explanatory mediators. Objective We hypothesized that maternal mediators, including adiponectin, leptin, insulin, and glucose, mediate effects of pregestational BMI (pBMI) and gestational weight gain (GWG) on birthweight and neonatal fat mass percentage (FM%) through placental weight and fetal mediators, including insulin levels (Ifv) and venous-arterial glucose difference (ΔGfva). Hypothesized confounders were maternal age, gestational age, and parity. Methods A cross-sectional study of healthy mother-offspring-pairs (n = 165) applying the 4-vessel in vivo sampling method at Oslo University Hospital, Norway. We obtained pBMI, GWG, birthweight, and placental weight. FM% was available and calculated for a subcohort (n = 84). We measured circulating levels of adiponectin, leptin, glucose, and insulin and performed path analysis and traditional mediation analyses based on linear regression models. Results The total effect of pBMI and GWG on newborn size was estimated to be 30 g (range, 16-45 g) birthweight and 0.17 FM% (range, 0.04-0.29 FM%) per kg∙m–2 pBMI and 31 g (range, 18-44 g) and 0.24 FM% (range, 0.10-0.37 FM%) per kg GWG. The placental weight was the main mediator, mediating 25-g birthweight and 0.11 FM% per kg∙m–2 pBMI and 25-g birthweight and 0.13 FM% per kg GWG. The maternal mediators mediated a smaller part of the effect of pBMI (3.8-g birthweight and 0.023 FM% per kg∙m–2 pBMI) but not GWG. Conclusion Placental weight was the main mediator linking pBMI and GWG to birthweight and FM%. The effect of pBMI, but not GWG, on birthweight and FM%, was also mediated via the maternal and fetal mediators

Gender differences in the association of syndecan-4 with myocardial infarction: The population-based Tromso Study

Background and aims: Cardiovascular disease is a common cause of morbidity and mortality, with gender differences in pathophysiology. The endothelial glycocalyx maintains vascular integrity, and glycocalyx shedding reflects endothelial dysfunction and early atherosclerosis. Syndecan-1 and -4 are components of the glycocalyx, and increased serum levels indicate glycocalyx damage. We hypothesised that increased serum syndecan-1 and -4 were independently associated with myocardial infarction (MI), ischaemic stroke and all-cause mortality in men and women from a general population. Methods: Using a case-cohort design, we included 1495 participants from the Tromsø Study 2001-02. Syndecan-1 and -4 were measured in serum. Baseline variables also included age, gender, cardiovascular risk factors and urinary albumin-creatinine ratio (ACR). Hazard ratios were assessed using multivariable Cox regression models. Results: Between baseline in 2001-02 and December 2007 fatal or non-fatal MI was experienced by 328 and ischaemic stroke by 191 subjects, and 423 participants died. Syndecan-4 was independently associated with MI (hazard ratio per 10 ng/mL increase 1.32; 95% confidence interval 1.06–1.63), but not ischaemic stroke and mortality, and the associations were unchanged by adjustment for urinary ACR. Interaction between syndecan-4 and sex was borderline significant, and in gender-specific analysis, syndecan-4 was associated with MI in women only. Syndecan-1 was not associated with any endpoint. Conclusions: Syndecan-4 was associated with incident MI, and the association was stronger in women than in men. This suggests a link between endothelial glycocalyx shedding and coronary heart disease in women. Use of syndecan-4 as a risk marker in clinical setting needs further investigation