Microvesicles released from ectopic endometrial foci as a potential biomarker of endometriosis.

OBJECTIVES: Angiogenesis is engaged in endometriosis. It is regulated by regulatory factors and cytokines, transported in microvesicles. The purpose was to investigate the presence of MVs with vascular endothelial growth factor (VEGF) and metalloproteinase-9 (MMP-9) in peripheral blood and peritoneal fluid of women operated on for endometrioma or teratoma Material and methods:Microvesicles (MVs) were determined in blood samples and peritoneal fluid samples collected from women aged 20-60 years operated on for endometriosis (test group) and teratoma (control group). The final investigations were performed on 47 patients, who qualified for the study based on the meticulous inclusion criteria. MVs were analyzed by flow cytometry (FACS) using annexin V, antibodies for molecules characteristic of cells from endometriosis foci (keratin 18 (K18), CD105, CD146), and antibodies for intraepithelial vascular growth factor VEGF and metalloproteinase-9 (MMP-9). The sample was double "reading" using flow cytometry (FACSCantoII). RESULTS: Cytometry analysis confirmed MVs' presence in plasma and peritoneal fluid collected from patients with both endometriosis and teratomas. A statistically significant higher level of AnnexinV (+) MVs were observed in plasma samples of endometriosis patients. In the control group, there was a higher percentage of double-positive VEGF (+)/MMP-9 (+) and single MMP-9 (+) positive MVs in the serum. In the peritoneal fluid higher frequency of double-positive VEGF (+)/MMP-9 (+) MVs were found in the control group. However, the amount of VEGF (+) / MMP-9 (+) MVs object did not enable to differentiate between the test and control groups. The study was the first, in which MVs were confirmed in plasma and peritoneal fluid in benign adnexa tumors. CONCLUSIONS: Microvesicles are present in peripheral blood and peritoneal fluid samples collected from patients with endometriosis and teratomas. Microvesicles with proangiogenic factors (VEGF and MMP-9) are more abundant in blood and peritoneal fluid samples from patients with teratomas.

Interleukin-4 during post-exercise recovery negatively correlates with the production of phagocyte-generated oxidants.

Exhaustive run induced a biphasic oxidative response of circulating phagocytes in 16 amateur sportsmen. The first phase involved an increment just after exercise of enhanced whole blood chemiluminescence normalized per phagocyte count, whereas in the second phase a decrement from 1 h post-exercise and ongoing till 24 h. We tested whether plasma Interleukin IL-4, IL-8, IL-10 and Tumor Necrosis Factor α concentrations change in response to exhaustive run and whether there are associations between their levels and delta resting. Moreover, IL-8 and IL-10 significantly increased immediately post-exercise and after 1 h, but later normalized. Tumor necrosis factor α rose by 1.1-times only just after exercise. However, none of these cytokines showed any correlation with the investigated chemiluminescence. Exercise did not alter plasma concentrations of IL-4. However, pre-exercise IL-4 negatively correlated with measured luminescence just after exercise (ρ = -0.54, p < 0.05), and also tended to be negatively associated with decrements of the second phase at 1 h post-exercise ρ = -0.45, p = 0.08. It is suggested that plasma IL-4, by a negative association with blood phagocytes oxidants production, could be involved in the maintenance of proper balance between oxidants and anti-oxidants during strenuous exercise and post-exercise recovery.

Longitudinal and Comparative Measures of Serum Chitotriosidase and YKL-40 in Patients With Idiopathic Pulmonary Fibrosis.

Background: Although chitin is absent in humans, chitinases are present in healthy subjects and show dysregulated expression in a variety of diseases resulting from abnormal tissue injury and repair responses. It was shown that chitotriosidase (chitinase 1/CHIT1) and structurally-related chitinase 3-like 1 protein (CHI3L1/YKL-40) play important roles in the pathobiology of idiopathic pulmonary fibrosis (IPF), however little is known about their longitudinal serum levels and relationship to clinical measures in IPF. Methods: The present study is the first to evaluate serial measurements of serum CHIT1 activity and YKL-40 concentrations in patients with IPF starting antifibrotic treatment and followed up for 24 months. In addition, baseline serum CHIT1 and YKL-40 were compared between patients with IPF and control subjects, and possible CHIT1 and YKL-40 relationships to longitudinal clinical assessments in IPF were explored. Results: Baseline serum CHIT1 activity and YKL-40 concentrations were significantly elevated in patients with IPF compared to control subjects and showed similar discriminatory ability in distinguishing IPF from controls. No significant differences between the median serum CHIT1 activity and YKL-40 concentration measured over a study follow-up were noted. We found significantly elevated baseline serum CHIT1 activity in the progressors compared with the stables in the first year, while significantly increased baseline serum CHIT1 activity was noted in the stables compared to the progressors in the second year. Additionally, we observed a significant negative correlation between a change in serum YKL-40 concentration and a change in forced vital capacity (FVC) % predicted (% pred.) in the stables subgroup, whereas, a change in serum CHIT1 activity correlated negatively with a change in FVC% pred. in the progressors subgroup. Conclusions: This explorative study findings add further evidence that CHIT1 and YKL-40 are upregulated in patients with IPF, and suggest that longitudinally stable serum CHIT1 activity and YKL-40 concentration levels may potentially be associated with the antifibrotic treatment response. In addition, our findings are supporting the possible role of CHIT1 and YKL-40 as candidate diagnostic and prognostic biomarkers in IPF. Further research is needed to validate present study findings.

Increased postoperative myeloperoxidase concentration associated with low baseline antioxidant capacity as the risk factor of delirium after cardiac surgery.

BACKGROUND: Though risk factors of postoperative delirium are well described, its pathophysiology is still undiscovered. The primary objective of the current study is to assess whether increased pre- and postoperative myeloperoxidase (MPO) levels are associated with postoperative delirium in the population of cardiac surgery patients. The secondary objective is to evaluate the correlation between MPO levels and serum antioxidant capacity (AC). METHODS: The patients' cognitive status was assessed one day preoperatively with the use of the Mini-Mental State Examination Test and the Clock Drawing Test. A diagnosis of major depressive disorder and anxiety disorders was established based on DSM-5 criteria. Blood samples for MPO and AC levels were collected both pre- and postoperatively. The Confusion Assessment Method for the Intensive Care Unit was used to screen for a diagnosis of delirium. RESULTS: Delirium occurred in 34% (61 of 177) of patients. Multivariable logistic regression analysis revealed that increased postoperative MPO concentration was independently associated with postoperative delirium development, and negatively correlated with lower baseline serum AC. CONCLUSIONS: Cardiac surgery patients with less efficient antioxidative mechanisms experience a higher postoperative peak of serum MPO, which in turn may predispose to postoperative delirium development.KEY MESSAGESMPO is a lysosomal enzyme with strong pro-oxidative and pro-inflammatory properties.Cardiac surgery patients who have increased concentration of postoperative MPO are at significantly higher risk of postoperative delirium development.This higher level of postoperative MPO is negatively correlated with baseline antioxidant capacity (AC).It can be hypothesized that individuals with decreased baseline AC experience a higher peak of MPO post-surgery due to less efficient antioxidative mechanisms, which in turn contributes to postoperative delirium development.

Oxidative stress and soluble receptor for advanced glycation end-products play a role in the pathophysiology of delirium after cardiac surgery.

Coronary-artery bypass graft (CABG) surgery is known to improve cardiac function and decrease mortality, albeit, this method of treatment is also associated with a neuropsychiatric complications including postoperative delirium. The pathophysiology of delirium after cardiac surgery remains poorly understood. Thus, the purpose of this study was to investigate whether oxidative stress reflected by decreased preoperative and postoperative plasma antioxidant activity is independently associated with delirium after cardiac surgery. The second aim was to assess whether decreased antioxidant activity is stress-related or mediated by other pathologies such as major depressive disorder (MDD), anxiety disorders, and cognitive impairment. Furthermore, the putative relationship between pre- and postoperative soluble receptor for advanced glycation end-products (sRAGE) overexpression and plasma antioxidant capacity was evaluated. The patients cognitive status was assessed 1 day preoperatively with the use of the Mini-Mental State Examination Test and the Clock Drawing Test. A diagnosis of MDD and anxiety disorders was established on the basis of DSM-5 criteria. Blood samples for antioxidant capacity and sRAGE levels were collected both preoperatively and postoperatively. The Confusion Assessment Method for the Intensive Care Unit was used within the first 5 days postoperatively to screen for a diagnosis of delirium. Postoperative delirium was diagnosed in 34% (61 of 177) of individuals. Multivariate logistic regression analysis revealed that low baseline antioxidant capacity was independently associated with postoperative delirium development. Moreover, increased risk of delirium was observed among patients with a preoperative diagnosis of MDD associated with antioxidant capacity decreased postoperatively. According to receiver operating characteristic analysis, the most optimal cutoff values of the preoperative and postoperative antioxidant capacity that predict the development of delirium were 1.72 mM and 1.89 mM, respectively. Pre- and postoperative antioxidant capacity levels were negatively correlated with postoperative sRAGE concentration (Spearman's Rank Correlation - 0.198 and - 0.158, p < 0.05, respectively). Patients with decreased preoperative antioxidant activity and those with depressive episodes complicated with lower postoperative antioxidant activity are at significantly higher risk of delirium after cardiac surgery development. sRAGE overexpression may be considered as protective mechanism against increased oxidative stress and subsequent cell damage.

Elevated Monocyte Chemoattractant Protein-1 as the Independent Risk Factor of Delirium after Cardiac Surgery. A Prospective Cohort Study.

BACKGROUND: The pathogenesis of postoperative delirium is largely unknown. The primary objective of this study is to assess whether increased levels of monocyte chemoattractant protein-1 (MCP-1) and high-sensitivity C-reactive protein (hsCRP) are associated with postoperative delirium in patients who have undergone cardiac surgery. The secondary objective is to investigate whether any association between raised inflammatory biomarkers levels and delirium is related to surgical and anesthetic procedures or mediated by pre-existing psychiatric conditions associated with raised pro-inflammatory markers levels. METHODS: The patients were screened for cognitive impairment one day preoperatively with the use of the Mini-Mental State Examination Test and the Clock Drawing Test. A diagnosis of major depressive disorder (MDD) and anxiety disorders was established on the basis of Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria. Blood samples were collected pre- and postoperatively for hsCRP and chemokine levels. RESULTS: Postoperative delirium developed in 34% (61 of 177) of patients. Both pre- and postoperative hsCRP, and preoperative MCP-1 levels were associated with postoperative delirium in univariate comparisons; p = 0.001; p < 0.001; p < 0.001, respectively. However, according to a multivariable logistic regression analysis, only a raised MCP-1 concentration before surgery was independently associated with postoperative delirium, and related to advancing age, preoperative anxiety disorders and prolonged intubation. CONCLUSIONS: The present study suggests that an elevated preoperative MCP-1 concentration is associated with delirium after cardiac surgery. Monitoring of this inflammatory marker may reveal the cardiovascular disease (CVD) patients who are at risk of neuropsychiatric syndromes development.

Overexpression of chitotriosidase and YKL-40 in peripheral blood and sputum of healthy smokers and patients with chronic obstructive pulmonary disease.

Background: Despite the absence of endogenous chitin in humans, chitinases are present in the serum of healthy subjects and their levels are increased in a variety of chronic inflammatory conditions. It has been shown that chitotriosidase and structurally related chitinase-like protein-YKL-40 contribute to the pathogenesis of COPD. However, details regarding the relation of their systemic and local airways levels remain unknown. Objectives: To examine peripheral blood and sputum chitotriosidase and YKL-40 expression in smokers and patients with COPD. Methods: Forty patients with COPD, 20 healthy smokers and 10 healthy never-smokers were studied. Serum and induced sputum chitotriosidase protein and activity levels, YKL-40 concentrations, and their gene expression in sputum cells and peripheral blood mononuclear cells (PBMC) were evaluated. Results: Both chitotriosidase protein levels and activity were higher in sputum obtained from COPD subjects compared to healthy never-smokers (P<0.05 and P<0.01, respectively). A similar pattern was observed for PBMC chitotriosidase mRNA expression (P<0.001). YKL-40 serum concentrations were elevated in healthy smokers and COPD subjects compared to healthy never-smokers (P<0.001 and P<0.01, respectively). In sputum, YKL-40 levels were increased in COPD compared to healthy never-smokers (P<0.01). PBMC YKL-40 mRNA expression was increased in COPD and healthy smokers compared to healthy never-smokers (P<0.0001). No associations were found between chitotriosidase or YKL-40 peripheral blood levels and sputum levels. Conclusions: Our results demonstrate that chitotriosidase and YKL-40 are overexpressed in peripheral blood and airways in both healthy smokers and COPD subjects which may indicate smoking-related activation of macrophages, neutrophils, and epithelial cells.

Effect of thalidomide affecting VEGF secretion, cell migration, adhesion and capillary tube formation of human endothelial EA.hy 926 cells.

Angiogenesis, new blood vessel formation, is a multistep process, precisely regulated by pro-angiogenic cytokines, which stimulate endothelial cells to migrate, proliferate and differentiate to form new capillary microvessels. Excessive vascular development and blood vessel remodeling appears in psoriasis, rheumatoid arthritis, diabetic retinopathy and solid tumors formation. Thalidomide [alpha-(N-phthalimido)-glutarimide] is known to be a potent inhibitor of angiogenesis, but the mechanism of its inhibitory action remains unclear. The aim of the study was to investigate the potential influence of thalidomide on the several steps of angiogenesis, using in vitro models. We have evaluated the effect of thalidomide on VEGF secretion, cell migration, adhesion as well as in capillary formation of human endothelial cell line EA.hy 926. Thalidomide at the concentrations of 0.01 microM and 10 microM inhibited VEGF secretion into supernatants, decreased the number of formed capillary tubes and increased cell adhesion to collagen. Administration of thalidomide at the concentration of 0.01 microM increased cell migration, while at 10 microM, it decreased cell migration. Thalidomide in concentrations from 0.1 microM to 10 microM did not change cell proliferation of 72-h cell cultures. We conclude that anti-angiogenic action of thalidomide is due to direct inhibitory action on VEGF secretion and capillary microvessel formation as well as immunomodulatory influence on EA.hy 926 cells migration and adhesion.

[Vascular endothelial growth factor--structure and functions]. / Czynnik wzrostu sródblonka naczyn--budowa i funkcje.

Vascular endothelial cell growth factor (VEGF), originally described as a vascular permeability factor, is currently known as one of the main factors which regulate angiogenesis. It plays an important role in the regulation of normal as well as pathological angiogenesis. In this paper we try to shortly review the actual knowledge on VEGF protein family, its expression, VEGF receptors and role of VEGF in signal transduction. The aim of this review is also to summarize recent achievements in research on biological functions of vascular endothelial growth factor and their clinical applications.

Heat shock proteins and other components of cellular machinery for protein synthesis are up-regulated in vascular endothelial cell growth factor-activated human endothelial cells.

Proteome analysis of human umbilical endothelial cells was performed to identify proteins that are modified during vascular endothelial cell growth factor (VEGF)-induced transition from the quiescent into the proliferating-migrative phenotype. Subtractive analysis of two-dimensional gel patterns of human endothelial cells, before and after stimulation with VEGF(165), revealed differences in 85 protein spots. All proteins were identified by peptide sequencing and peptide mass fingerprinting using an electrospray spectrometer. The proteins identified were members of specific families including Ca(2+)-binding proteins, fatty-acid binding proteins, structural proteins, and chaperones. Remarkably, there was a massive activation of cellular machinery for both protein synthesis and protein degradation. Thus, among up-regulated proteins there were members of all groups of heat shock proteins (HSPs; HSP 27, HSP 60, HSP 70p5, HSP 70p8, HSP 90, and HSP 96) and some other proteins showing either chaperone activity or which participate in assembly of multimolecular structures (TCP-1, desmoplakins, junction plakoglobin, GRP 94, thioredoxin related protein, and peptidylprolyl isomerase). The increased expression of HSPs was confirmed at the mRNA level at different stages of treatment with VEGF. Similarly, components of the proteolytic machinery for the degradation of misfolded proteins (ER-60, cathepsin D, proteasome subunits, and protease inhibitor 6) were also up-regulated. On the other hand, changes in the expression of structural proteins (T-plastin, vimentin, alpha tubulin, actin, and myosin) could account, at least in part, for the different morphologies displayed by migrating endothelial cells. In summary, our data show that VEGF levels similar to those during physiological stresses induce a number of genes and multiple endogenous pathways seem to be engaged in restoring cellular homeostasis. To ensure cell survival, the molecular chaperones (the heat shock family of stress proteins) are highly up-regulated providing protein-folding machinery to repair or degrade misfolded proteins.

Expression of Integrins and Adhesive Properties of Human Endothelial Cell Line EA.hy 926.

Immortalized endothelial cell lines are very often used as a model of endothelium for studies of various processes connected with its functions. Among the hybrid cells, the EA.hy 926 cell line, derived by the fusion of HUVECs with the continuous human lung carcinoma cell line A549, is presently the best characterized macro-vascular endothelial cell line. Although EA.hy 926 cells retain several endothelial characteristics, our data show some differences between this cell line and primary human umbilical vein endothelial cells (HUVEC). Analysis of their proteomic pattern reveals that there are many proteins expressed only in the immortalized cell line, but several proteins of EA.hy 926 are missed when compared to HUVECs. We observed a distinct profile of integrin expression on the surface of both types of endothelial cells, that may be responsible for diminished EA.hy 926 adhesion and migration to selected adhesive proteins. Studies on proliferation and migration in the presence of VEGF showed lower growth factor responsiveness of EA.hy 926 in comparison with HUVECs, but hybrid endothelial cells can also be converted into a pro-angiogenic phenotype. These studies showed significant similarity of endothelial cell lines with primary HUVECs, but also pointed out marked phenotype differences.

Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells.

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.