Decidual mesenchymal stem/stromal cells from preeclamptic patients secrete endoglin, which at high levels inhibits endothelial cell attachment invitro.

INTRODUCTION: In preeclampsia (PE), inadequate remodelling of spiral arterioles in the decidua basalis causes oxidative stress and subsequent increased release of antiangiogenic soluble endoglin (sENG) into the maternal circulation. Decidual mesenchymal stem/stromal cells (DMSCs) reside adjacent to endothelial cells in this vascular niche. Surprisingly, DMSCs express membrane-bound ENG (CD105). PE-affected DMSCs (PE-DMSCs) are abnormal and due to reduced extravillous invasion, more of them are present, but the significance of this is not known. METHODS: DMSCs were isolated and characterised from normotensive control and severe-PE placentae. Extracellular vesicle (EV) types, shed microvesicles (sMV) and exosomes, were isolated from DMSC conditioned media (DMSCCM), respectively. Secretion of ENG by DMSCs was assessed by ELISA of DMSCCM, with and without EV depletion. The effects of reducing ENG concentration, by blocking antibody, on human umbilical vein endothelial cell (HUVEC) attachment were assessed by xCELLigence real-time functional assays. RESULTS: ENG was detected in DMSCCM and these levels significantly decreased when depleted of exosomes and sMV. There was no significant difference in the amount of ENG secreted by control DMSCs and PE-DMSCs. Blocking ENG in concentrated DMSCCM, used to treat HUVECs, improved endothelial cell attachment. DISCUSSION: In normotensive pregnancies, DMSC secretion of ENG likely has a beneficial effect on endothelial cells. However, in PE pregnancies, shallow invasion of the spiral arterioles exposes more PE-DMSC derived sources of ENG (soluble and EV). The presence of these PE-DMSCs in the vascular niche contributes to endothelial cell dysfunction.

Severe abdominal aortic calcification in older men is negatively associated with DKK1 serum levels: the STRAMBO study.

CONTEXT: Experimental data show that dickkopf-1 (DKK1) may be involved in the regulation of arterial calcification. However, clinical data on the association between serum DKK1 levels and severity of abdominal aortic calcification (AAC) are scarce. OBJECTIVE: Our aim was to determine the association between serum DKK1 concentration and AAC severity in men. DESIGN: This is a cross-sectional analysis in the STRAMBO cohort. SETTING: The cohort was recruited from the general population. PARTICIPANTS: We examined 1139 male volunteers aged 20 to 87 years. No specific exclusion criteria were used. INTERVENTIONS: We collected blood samples and assessed AAC severity on the lateral spine scans obtained by a Discovery A Hologic device using the semiquantitative Kauppila score. MAIN OUTCOME MEASURES: We tested the hypothesis that low DKK1 levels are associated with AAC severity in men. RESULTS: In men aged 20 to 60 years, serum DKK1 levels were not associated with other variables. In men aged 60 years and older, lower DKK1 levels were associated with higher odds of severe AAC (AAC score >5). After adjustment for confounders, odds of severe AAC increased with decreasing DKK1 levels (odds ratio = 1.42, 95% confidence interval = 1.13-1.79, P < .005) and was higher below vs above the median DKK1 level (odds ratio = 2.19, 95% confidence interval = 1.37-3.49, P < .005). Heavy smoking, hypertension, ischemic heart disease, and elevated levels of fibroblast growth factor 23 were associated with severe AAC significantly, independently of DKK1 and additively with low DKK1 levels. CONCLUSION: In older men, lower serum DKK1 levels are associated with severe AAC regardless of age and other potential confounders.

Serum myostatin levels are negatively associated with abdominal aortic calcification in older men: the STRAMBO study.

OBJECTIVE: To assess the association between abdominal aortic calcification (AAC) and serum levels of myostatin, a negative regulator of skeletal muscle mass, which has been implicated in the development of atherosclerotic lesions in mice. DESIGN AND PATIENTS: We assessed AAC semiquantitatively from the lateral spine scans obtained using dual energy X-ray absorptiometry in 1071 men aged 20-87 years. Serum myostatin levels were measured by an immunoassay that detects all myostatin forms. RESULTS: Total myostatin serum levels did not differ between men with or without self-reported ischemic heart disease, hypertension, or diabetes mellitus. Total serum myostatin levels were higher in men with higher serum calcium levels and lower in men with higher serum concentrations of highly sensitive C-reactive protein. Men with AAC had lower myostatin levels compared with men without AAC. Prevalence of AAC (AAC score > 0) was lower in the highest myostatin quartile compared with the three lower quartiles (P < 0.05). After adjustment for confounders, odds of AAC (AAC score > 0) were lower (OR=0.62; 95% confidence interval (95% CI), 0.45-0.85; P< 0.005) for the fourth myostatin quartile vs the three lower quartiles combined. In the sub-analysis of 745 men aged 60 years, the results were similar: AAC prevalence was lower in the highest myostatin quartile compared with the three lower quartiles combined (OR=0.54; 95% CI, 0.38-0.78; P<0.001). CONCLUSIONS: In older men, total myostatin serum levels are inversely correlated with AAC. Further studies are needed to investigate mechanisms underlying this association and to assess utility of myostatin as a cardiovascular marker.

Serum level of the phosphaturic factor FGF23 is associated with abdominal aortic calcification in men: the STRAMBO study.

CONTEXT: Calcification inhibitor deficiencies, mineral imbalance, and phenotypic transformation of vascular cells to osteogenic cells initiate and sustain vascular calcification. Fibroblast growth factor-23 (FGF23) is a key molecule regulating mineral homeostasis. OBJECTIVE: Our objective was to assess the association of serum FGF23 levels with mineral metabolism parameters and abdominal aortic calcification (AAC) in men. DESIGN: This was a cross-sectional analysis in the STRAMBO cohort. SETTING: Men holding a private health insurance cover with Mutuelle de Travailleurs de la Région Lyonnaise were included in the study. PARTICIPANTS: Participants included male volunteers aged 20-87 (n = 1130). INTERVENTIONS: Nonfasting blood collection was done. AAC was semiquantitatively assessed from vertebral fracture assessment scans obtained using dual-energy x-ray absorptiometry. MAIN OUTCOME MEASURES: We evaluated the association between FGF23 concentration and AAC severity in men. RESULTS: In 350 men aged 60 yr or younger, FGF23 levels decreased with age (r = -0.21; P < 0.001) but were not associated with any other parameter. In 780 men aged over 60 yr, serum FGF23 correlated with age (r = 0.37; P < 0.001) and, after adjustment for confounders, with glomerular filtration rate (r = -0.31; P < 0.001) and PTH levels (r = 0.25; P < 0.001). After adjustment for confounders, self-reported ischemic heart disease, diabetes mellitus as well as higher concentrations of C-reactive protein and osteoprotegerin were all associated with higher FGF23 levels. After adjustment for confounders, subjects in the highest FGF23 quartile had higher prevalence of severe AAC compared with the three lower quartiles combined (odds ratio = 1.88; 95% confidence interval = 1.22-2.85; P < 0.005). CONCLUSIONS: In healthy older men, circulating FGF23 is associated with parameters of mineral metabolism, including bone metabolism-regulating cytokines, and with severe AAC independent of traditional risk factors.

Cortical bone status is associated with serum osteoprotegerin concentration in men: the STRAMBO study.

CONTEXT: Osteoprotegerin (OPG) is an inhibitor of bone resorption, but its relationship to bone microarchitecture remains unclear. OBJECTIVE: Our objective was to study the relationship between OPG concentration and bone microarchitecture in men. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional study of a population-based cohort of 1149 men aged 20-87 yr. INTERVENTIONS: We assessed bone microarchitecture at the distal radius and tibia by high-resolution peripheral quantitative computed tomography (XtremeCT Scanco) and measured serum OPG concentration and bone turnover markers: osteocalcin, bone-specific alkaline phosphatase, N-terminal extension type I collagen propeptide, C-terminal type 1 collagen telopeptide, and urinary deoxypyridinoline. MAIN OUTCOME MEASURES: Differences were assessed in bone microarchitectural parameters across the OPG quartiles in the models adjusted for age, weight, height, physical activity, ischemic heart disease, diabetes mellitus, calcium intake, serum levels of free testosterone, bioavailable 17ß-estradiol, PTH, 25-hydroxycholecalciferol, and creatinine. RESULTS: After adjustment for the confounders, men in the highest (fourth) quartile of OPG levels (>4.55 pmol/liter) had higher total cross-sectional area and trabecular area at the distal radius and distal tibia (3.3-6.0%, P < 0.05). At both skeletal sites, the highest OPG quartile was associated with lower cortical thickness (8.2%, P < 0.001, and 3.7%, P < 0.05) and volumetric bone mineral density (vBMD, 2.7%, P < 0.001, and 1.6%, P < 0.005) compared with the three lower quartiles combined. Associations of OPG level with trabecular vBMD, number, thickness, and distribution were not significant. Men in the fourth OPG quartile had higher levels of bone resorption markers (11.8-13.1%, P < 0.01-0.001). CONCLUSIONS: Men with higher serum OPG concentration had lower cortical thickness and vBMD, probably due to accelerated endo- and intracortical bone turnover, but higher cross-sectional area possibly due to periosteal apposition.

Role for alkaline phosphatase as an inducer of vascular calcification in renal failure?

Vascular calcification is associated with increased cardiovascular morbidity and mortality. A number of calcification inhibitors have been defined recently, including inorganic pyrophosphate (PP(i)), an important physicochemical inhibitor of hydroxyapatite crystal growth. Increased hydrolysis of PP(i) by tissue-nonspecific alkaline phosphatase (TNAP) may occur in renal failure and act to enhance mineralization of vessels.

Exploring the biology of vascular calcification in chronic kidney disease: what's circulating?

Chronic kidney disease (CKD) is associated with fatal cardiovascular consequences in part due to ectopic calcification of soft tissues particularly arteries, capillaries, and cardiac valves. An increasing body of evidence from experimental studies and in vivo data suggest that (I) a mineral imbalance with hyperphosphatemia and high-circulating calcium x phosphate product, (II) a deficiency of systemic or local calcification inhibitors, (III) death or 'damage' of vascular smooth muscle cells (VSMCs), and/or (IV) phenotypic transformation of VSMCs to osteo/chondrocytic cells may all act in concert to initiate and sustain vascular calcification. In CKD patients inhibitory systems are overwhelmed by a multitude of agents that induce VSMC damage and cell death resulting in the release of vesicles capable of nucleating basic calcium phosphate. Studies with genetically altered mice have identified both local and systemic calcification inhibitors that act to maintain VSMC differentiation or regulate vesicle properties. However, for many of these proteins the mechanisms and sites of action are still under investigation. In particular, it is unclear whether factors present in the circulation have an inhibitory role there and whether circulating levels of these proteins influence or are indicative of underlying disease processes in individual patients. A greater understanding of the origins and roles of potential circulating inhibitors may result in novel strategies aimed at the prevention or reversal of the life-limiting calcifying vasculopathies seen in CKD patients.

Cytokine-induced osteoprotegerin expression protects pancreatic beta cells through p38 mitogen-activated protein kinase signalling against cell death.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines play a crucial role in immune-mediated beta cell destruction, an essential mechanism in the pathogenesis of type 1 diabetes mellitus. Microarray analysis recently identified osteoprotegerin (OPG; now known as tumour necrosis factor receptor superfamily, member 11b [TNFRSF11B]) as a cytokine-induced gene in beta cells. The aim of the present study was to characterise the functional role and signalling pathways of OPG that are involved in cytokine-induced beta cell death. MATERIALS AND METHODS: As cellular models, the rat beta cell line INS-1E and human primary pancreatic islets were employed. The effects of IL-1beta and TNF-alpha on OPG expression were characterised by northern blot and immunoassay. The effect of OPG on beta cell survival was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Signalling pathways were evaluated by western blot analysis using antibodies against p38 mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. RESULTS: The INS-1E cell line and primary pancreatic islets expressed OPG mRNA and secreted OPG protein, both of which were enhanced by IL-1beta and TNF-alpha. Exposure to IL-1beta resulted in sustained phosphorylation of p38 MAPK in INS-1E cells and subsequent cell death. Administration of exogenous OPG prevented both IL-1beta-induced beta cell death and sustained p38 MAPK phosphorylation. CONCLUSIONS/INTERPRETATION: Our data indicate that cytokine-induced production of OPG may protect beta cells from further damage. This protective effect is, at least in part, mediated through inhibition of p38 MAPK phosphorylation. Thus OPG is an autocrine or paracrine survival factor for beta cells.

Vascular calcification and osteoporosis--from clinical observation towards molecular understanding.

Patients with osteoporosis frequently suffer from vascular calcification, which was shown to predict both cardiovascular morbidity/mortality and osteoporotic fractures. Various common risk factors and mechanisms have been suggested to cause both bone loss and vascular calcification, including aging, estrogen deficiency, vitamin D and K abnormalities, chronic inflammation and oxidative stress. Major breakthroughs in molecular and cellular biology of bone metabolism and the characterization of knockout animals with deletion of bone-related genes have led to the concept that common signaling pathways, transcription factors and extracellular matrix interactions may account for both skeletal and vascular abnormalities. For example, mice that lack the cytokine decoy receptor osteoprotegerin or the hormone Klotho display a combined osteoporosis-arterial calcification phenotype. In this review, we summarize the current data and evaluate potential mechanisms of the osteoporosis-arterial calcification syndrome. We propose a unifying hypothesis of vascular calcification that combines both active and passive mechanisms of vascular mineralization with aspects of bone resorption and age-related changes.

Tumour necrosis factor-related apoptosis-inducing ligand and osteoprotegerin serum levels in psoriatic arthritis.

OBJECTIVES: The degree of bone loss in patients with psoriatic arthritis (PsA) has not been well-defined. We tested the hypothesis, whether serum levels of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine and osteoprotegerin (OPG), an anti-osteoclastic cytokine, are associated with changes in biochemical markers of bone turnover or bone mineral density (BMD) in patients with PsA. METHODS: In a cross-sectional study, we evaluated biochemical markers of bone turnover, BMD and serum levels of TRAIL and OPG in 116 patients with PsA (mean age: 52+/-13 yrs). RESULTS: In patients with PsA, osteopenia was present in one-third of women and men, while osteoporosis was more frequent in men (10.2%) than in women (1.75%). Serum levels of TRAIL were significantly higher in patients with PsA (66.1+/-45.3 pmol/l) compared with controls (50.0+/-20.1 pmol/l, P<0.01), whereas OPG serum levels were not different. There were no associations between TRAIL or OPG serum levels with BMD and biochemical markers of bone turnover. However, TRAIL serum levels were associated with C-reactive protein (CRP) levels (R = 0.201, P<0.05), whereas OPG serum levels were associated with the erythrocyte sedimentation rate (R=0.215, P<0.05). CONCLUSION: In summary, BMD is decreased in one-third of patients with PsA, and predominantly men with PsA suffer from osteoporosis. While TRAIL serum levels are increased in PsA and correlated with CRP levels, neither TRAIL nor OPG serum levels are correlated with BMD or markers of bone metabolism.

Novel aspects on RANK ligand and osteoprotegerin in osteoporosis and vascular disease.

The clinical coincidence of osteoporosis and vascular disease has long indicated that common mediators may adversely affect bone metabolism and vascular integrity alike. Receptor activator of NF-kappaB ligand (RANKL) is an important cytokine for bone resorption that acts through its osteoclastic receptor, receptor activator of NF-kappaB (RANK), while osteoprotegerin serves as a decoy receptor that binds RANKL and prevents activation of RANK. Skeletal and vascular cells are sources and targets of RANKL and OPG both in vitro and in vivo. Modulation of the RANKL/RANK/OPG system in animals results in a skeletal and vascular phenotype, and administration of OPG may prevent osteoporosis and vascular calcification. Recent studies on OPG serum levels and gene polymorphisms also suggest an important role of this cytokine system in skeletal and vascular diseases. In summary, there is increasing evidence that RANKL and OPG may link the skeletal with the vascular system.

Alcohol and the heart.

ALCOHOLISM IN GENERAL: Alcoholism is one of the major health problems in the world. Alcohol consumption has an impact on different body systems like the central nervous system, the gastrointestinal tract, the hematopoetic organs, and the cardiovascular system. Alcohol interferes with other medications, and drinking can exacerbate a variety of medical illnesses. IMPACT ON THE HEART: In the heart, alcohol and its metabolite acetaldehyde confer a toxic effect on mitochondria as well as on the sarcoplasmatic reticulum, which is dependent on both the mean daily consumption and the duration of alcohol intake. A wide range of toxic effects of alcohol in distinct individuals can be observed and modest doses of alcohol can exert beneficial effects on the cardiovascular system probably by an increase in high density lipoprotein cholesterol (HDL) or changes in blood clotting mechanisms. Detrimental effects of alcohol on the heart comprise a decrease in myocardial contractility, hypertension, atrial and ventricular arrhythmias, and secondary non-ischemic dilated cardiomyopathy. After consuming large quantities of alcohol over years, alcoholic cardiomyopathy may develop, which presents with dilation and impaired contractility of the left or both ventricles. Endomyocardial biopsies of patients with alcoholic cardiomyopathy reveal in up to 30% of all cases myocarditis with lymphocytic infiltrates. TREATMENT: Abstinence after development of milder heart failure can stop progression or even reverse symptoms in some cases, otherwise severe heart failure ensues leading to a poor prognosis. Except abstinence, treatment of alcoholic cardiomyopathy is based on the regimen of therapy for heart failure to reduce the size of the dilated heart and to mitigate the symptoms of heart failure.

Differential stimulation of helper and cytotoxic T cells by dendritic cells after infection by Yersinia enterocolitica in vitro.

Dendritic cells (DC) are antigen-presenting cells crucial for initiating immune responses like sensitization of T cells to foreign antigens. We have previously shown that infection of DC by enteropathogenic Yersinia enterocolitica in vitro leads to a transient suppression in the immunostimulatory capacity for autologous enriched total T cells. In this study, we found that killed Yersinia could replace live bacteria in this aspect, and that yersinial lipopolysaccharide (LPS)-antigen could be detected intracellularly over a time course of 8 days. A suppressive effect on T cell proliferation after stimulation with Yersinia-infected compared to uninfected DC was seen for CD4+ T cells isolated by immunomagnetic separation techniques over the whole time course of 8 days, whereas CD8+ T cells followed to exhibit a suppressed proliferation rate starting on day 5 post infection till the end of the time course. In contrast, enriched total T cells stimulated by Yersinia-infected DC showed weaker proliferation till day 6 post infection compared to stimulation by uninfected DC, but not thereafter. Mixing of purified CD4+ and CD8+ T cells at day 8 post infection could reconstitute the effect seen for enriched total T cells. Thus, helper in concert with cytotoxic T cells might contribute to the immune responses, that are necessary for control of Yersinia-infections.

Dendritic cell function is perturbed by Yersinia enterocolitica infection in vitro.

Infection with Yersinia enterocolitica is the cause of intestinal or extraintestinal diseases. We investigated the role of dendritic cells (DC), the most potent antigen-presenting cell (APC), in the course of infection with Y. enterocolitica in vitro. For these studies, DC were isolated from human peripheral blood and infected with green fluorescent protein (GFP)-labelled Y. enterocolitica. Bacteria were found within DC by FACS analysis and viable bacteria could be cultured from lysed cells. Within 24 h after infection, DC upregulated CD83 and CD86 followed at day 3, indicating maturation of DC. In contrast, for MHC class II, a marked but transient downregulation was observed at day 3 after infection, and downregulation to a lesser extent for CD80 at day 5. To assess the immunostimulatory capacity of DC, viable infected and uninfected DC were incubated with autologous T cells in the presence of phytohemagglutinin A (PHA). T cell proliferation was significantly reduced at days 4-6 after infection but not thereafter, whereas nonpathogenic Escherichia coli was not able to mimick this suppressive effect of Y. enterocolitica. The same suppression could be observed when infected DC were used in a mixed leucocyte reaction with allogeneic T cells. Thus Y. enterocolitica is able to invade DC, does not induce necrosis or apoptosis, but affects maturation of DC. However, MHC class II-molecules are downregulated initially, which coincides with a diminished immunostimulatory capacity of DC infected with Y. enterocolitica. The diminished immunostimulatory capacity of DC following infection with Y. enterocolitica in vitro might impair or delay elimination of bacteria thereby contributing to pathogenesis of bacterial enteritis or extraintestinal manifestations such as reactive arthritis.

Infection of dendritic cells by enterobacteriaceae.

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in initiation and modulation of specific immune responses. Various pathogens like viruses or bacteria are able to persist inside DC. In this study we investigated the ability of the Gram-negative bacteria Salmonella typhimurium and Escherichia coli to infect DC. DC isolated from peripheral blood of healthy donors were infected with wild-type S. typhimurium and a nonpathogenic E. coli stool isolate. Association of bacteria with DC was assessed by labeling of the bacteria with green fluorescent protein. Both Gram-negative bacteria were associated with DC as evidenced by microscopy and flow cytometry. The intracellular location could be confirmed by lysis of DC and subsequent determination of colony-forming units on agar plates, which showed a rapid decline in viable Gram-negative bacteria 6 h after infection, being by far more pronounced for E. coli than for S. typhimurium. Testing the stimulation of T cells by infected versus uninfected but otherwise identically treated human immature DC in a mitogen-dependent T cell proliferation assay, we found that S. typhimurium. but not E. coli exhibited a suppressive effect on T cell stimulation, being most significant on days 3-5 after infection. Thus, suppression of dendritic cell function was associated with an enteropathogenic bacterium, S. typhimurium, which can cause severe forms of enteritis. The bacteria with normally mild or no gastric symptoms, E. coli, had no influence on stimulation of T cells by DC.

Human decidua contains potent immunostimulatory CD83(+) dendritic cells.

Dendritic cells (DCs) are sentinel cells of the immune system important in initiating antigen-specific T-cell responses to microbial and transplantation antigens. DCs are particularly found in surface tissues such as skin and mucosa, where the organism is threatened by infectious agents. The human decidua, despite its proposed immunosuppressive function, hosts a variety of immunocompetent CD45 cells such as natural killer cells, macrophages, and T cells. Here we describe the detection, isolation, and characterization of CD45(+), CD40(+), HLA-DR(++), and CD83(+) cells from human early pregnancy decidua with typical DC morphology. CD83(+) as well as CD1a(+) cells were found in close vicinity to endometrial glands, with preference to the basal layer of the decidua. In vitro, decidual CD83(+) cells could be enriched to approximately 30%, with the remainder of cells encompassing DC-bound CD3(+) T cells. Stimulation of allogeneic T cells in a mixed leukocyte reaction by the decidual cell fraction enriched for CD83(+) cells, was similar to that obtained with blood monocyte-derived DCs, demonstrating the potent immunostimulatory capacity of these cells. Decidual DCs with morphological, phenotypic, and functional characteristics of immunostimulatory DCs might be important mediators in the regulation of immunological balance between maternal and fetal tissue, leading to successful pregnancy.

[New possibilities of donor selection for bone marrow and organ transplantation by HLA typing and genomic DNA]. / Neue Möglichkeiten der Spenderauswahl für die Knochenmark- und Organtransplantation durch HLA-Typisierung aus genomischer DNA.

For one year we determined HLA class II antigens from all patients with renal and haematological disorders and their healthy family members both by using serological methods and DNA typing (SSO, SSP). The rate of discrepancies between the results of serological DR typing and DRB1 typing was 10.8% (13/120). We were able to demonstrate that DNA typing for class II antigens leads to definite results even for patients with a poor cell quality or with lymphocytopenia where serological typing is often impossible. Furthermore, DNA typing from patients with haematological disorders is very important, especially if a bone marrow transplantation is considered. In addition to MLC testing, DRB1, DQB1 and DPB1 typing should be carried out for the patient and the potential donor. DNA typing is also recommended for patients waiting for a kidney transplantation particularly if they were serologically typed as 'homozygous', as the chance to receive an HLA-compatible organ is much higher for a heterozygous individual.