The multidirectional assessment of moderate caloric restriction and metformin treatment in obese patients.

Obesity treatment is often burdensome for patients. We used the combination of moderate caloric restriction (CR) with hypoglycemic metformin to assess their multidirectional effect in obese patients. One group was treated only with moderate CR (n=21) the second was treated with moderate CR and 800 mg metformin twice daily (n=23). Serum was drawn before and after treatment. The following parameters were monitored: anthropometric, cardiovascular, inflammatory, metabolic, and markers characteristic for thyroid, liver, pancreas, and kidney functions. Both tested groups did not significantly differ in most tested parameters after the treatment. Two groups reduced anthropometric parameters (body mass, body mass index (BMI), waist circumference) and fat mass but also muscle and fat-free mass, improving systolic blood pressure, insulin and leptin concentration, insulin sensitivity, leptin to adiponectin ratio, and inflammatory markers. Unfortunately, there was little impact on improving dyslipidemia and the thyroid and liver parameters. Free triiodothyronine (fT3) and gamma glutamyl transferase (GGT) activity were decreased in both groups, but triglycerides were reduced only in patients treated with moderate CR. Metformin with CR treatment decreases uric acid and aspartate aminotransferase (AspAT) activity. Metformin treatment with moderate CR in obese patients mainly improved insulin sensitivity, resulting in a reduction of patients with glucose intolerance, improved anthropometric, cardiovascular, and inflammatory mediators, and only slightly enhanced liver and thyroid function. No changes in kidney and pancreas function were observed during the treatment. In conclusion, eight weeks of CR alone and CR with metformin in obese adults improved anthropometric and metabolic markers, reduced muscle mass, fT3, GGT, proinflammatory, and CV parameters, and displayed no changes in kidney and pancreas function. The group treated with metformin after the treatment was still more obese and had higher C-reactive protein (CRP) and homeostasis model assessment-an index of insulin resistance (HOMA-IR), but despite this, considerably reduced the number of patients with glucose intolerance.

Soluble receptor for advanced glycation end products (sRAGE) correlates with obesity-related parameters, and it is not easy to be modified by moderate caloric restriction in obese humans.

Deficiency of a soluble form of the advanced glycation end products receptor (sRAGE) is implicated in obesity-induced complications. Serum sRAGE is inclined to be modified by changes in body weight. We analysed serum sRAGE concentrations in patients with obesity undergoing moderate calorie restriction, which mimics the real-life situation and is not harmful to obese humans. Serum sRAGE was measured by immunoassay in 50 patients with obesity who underwent calorie restriction by 300-500 kcal/day for 8 weeks. In effect calorie restriction resulted in an expected decrease in body weight (by 2.1 kg for an 8-week intervention, p<0.0001), as well as reduced systolic blood pressure, modified dyslipidemia (cholesterol, triglycerides), reduced obesity-related inflammation (tumor necrosis factor-alfa, interleukin-6, C-reactive protein), improved insulin sensitivity. However, it was not accompanied by any significant change in sRAGE concentration. There was a strong negative correlation between BMI and the sRAGE level. Accordingly, the levels of sRAGE were the highest in lean control. In conclusion: a modest weight reduction is unlikely to improve decreased sRAGE levels.

Sulodexide reduces glucose induced senescence in human retinal endothelial cells.

Chronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) ß-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (- 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.

N-acetylcysteine modulates effect of the iron isomaltoside on peritoneal mesothelial cells.

Intravenous (i.v.) iron supplementation is used in patients on chronic peritoneal dialysis (pd). Iron induced intraperitoneal inflammation observed in our previous studies with iron sucrose may deteriorate the function of the peritoneum as the dialysis membrane. We evaluated effect iron compound, iron-isomaltoside-100 (IIS) on the peritoneal mesothelial cells (MC). We studied the effect of iv treatment with IIS ± N-acetylcysteine (NAC) on the dialysate parameters and function of MC. In 7 uremic pd patients IIS 200 mg was infused i.v. ± NAC 600 mg. Afterward, a 4 hours exchange was performed with Dianeal 1.5%. As a control dialysate exchange preceding IIS treatment was used. Inflammatory parameters of the drained dialysates as well as the dialysates and IIS effects on MC were evaluated in ex vivo experiments. Intravenous infusion of IIS resulted in an increase of the dialysate Fe (+147%, P < 0.01). Concentrations of the dialysates inflammatory mediators were increased: interleukin-6 (IL-6) +39%, P < 0.02, monocyte chemoattractant protein-1(MCP1) +50%, P < 0.02, and hyaluronan (HA) +64%, P < 0.02. Simultaneous i.v. infusion of NAC prevented increase of the dialysate inflammatory mediators. Dialysates collected after IIS treatment induced oxidative stress in MC (+29%, P < 0.05) and stimulated IL-6 synthesis (+64%, P < 0.05) in MC; no such effect was seen in dialysates obtained after simultaneous IIS and NAC i.v. treatment. IIS used as the additive to culture medium stimulated synthesis in MC of IL6 (+76%, P < 0.001) and plasminogen activator inhibitor-1 (PAI-1) (28%, P < 0.001) whereas synthesis of tissue plasminogen activator (t-PA) was reduced (-16%, P < 0.001). These changes were prevented in the presence of NAC 1 mmol/L. Intravenous administration of IIS results in the mild stimulation of intraperitoneal inflammation. IIS changes MC phenotype to the inflammatory one with reduced fibrinolytic activity. These effects are prevented by NAC.

Evaluation of Copeptin during Pulmonary Exacerbation in Cystic Fibrosis.

Copeptin was found to be a stable biomarker of inflammation and stress response in cardiac, renal, metabolic, and respiratory conditions such as pneumonia. The aim of this study was to investigate the copeptin levels in biological fluids (serum and sputum supernatant) of cystic fibrosis pediatric patients during pulmonary exacerbation and remission and to investigate the possible influence of copeptin levels on disease severity and quality of life. Copeptin serum concentrations were measured in 28 pediatric cystic fibrosis (CF) patients: 13 in stable condition and 15 during pulmonary exacerbation. In 10 CF patients, copeptin was also measured in the sputum. In all the patients, we assessed complete blood count, BMI, sputum culture, lung function, and chest imaging (with Brasfield score). The severity of symptoms was assessed using the Shwachman-Kulczycki (SK) score, and the quality of life was assessed with the Cystic Fibrosis Quality of Life Questionnaire-Revised (CFQ-R). Copeptin concentrations in serum and sputum supernatant was measured using an ELISA kit. Statistical analysis was done in Statistica v.12. Serum and sputum copeptin levels were higher in CF patients during pulmonary exacerbation than in a stable period, but the differences were not significant (p = 0.58 and p = 0.13, respectively). Copeptin did not correlate significantly with any clinical, laboratory, or spirometry markers of exacerbation. There was, however, a significant inverse correlation between the serum copeptin level and symptoms severity (r = -0.77, p = 0.008) and radiological changes (r = -0.5626, p = 0.036) during pulmonary exacerbation in pediatric CF patients. Copeptin also inversely correlated with the quality of life domains in CF patients: vitality and eating habits, mostly loss of appetite (p = 0.031 and p = 0.016, respectively). Copeptin may be useful to identify patients with a higher risk of deterioration to improve their outcomes.

Genes involved in hormone metabolism and cellular response in human ovarian granulosa cells.

Endocrinal interactions are one of the most crucial regulatory mechanisms that maintain the state of homeostasis in humans. Processes such as oogenesis, folliculogenesis, menstruation and pregnancy remain under hormonal control. A key role in folliculogenesis is played by granulosa cells. Moreover, granulosa cells take part in corpus luteum formation after ovulation. Because of that, it is important to understand the ways in which the granulosa cells, associated with those processes, respond to hormonal stimulus. In the present study, a transcriptomic analysis of human granulosa cells (GCs) was carried out with the use of expression microarrays. The results were validated by RT-qPCR. The total RNA was isolated after 1st, 7th, 15th and 30th days of long-term primary cultures. The main focus of this work was placed on the genes belonging to "Response to estradiol", "Response to follicle-stimulating-hormone", "Cellular response to hormone stimulus", "Cellular hormone metabolic process" and "Hormone biosynthetic process" gene ontology groups. These groups of genes have been associated with GC hormone metabolism and cellular response to hormones. Eighty genes belonging to these groups were identified. Those that were members of more than one of the analyzed gene ontology groups, or exhibited unique expression patterns, were selected for further analysis. All of the selected genes were described, with their expression patterns detailed. In this manuscript, two gene expression patterns have been described. The first one showed large downregulation of genes in the later stages of culture, with the second one presenting upregulation of expression after day 1 of IVC. The present research was focused on six genes found to be the most important for steroidogenesis: STAR, POR, CYP11A1, ADM, GCLC, IL1B, as well as three genes of higher expression at the later stages of long-term in vitro culture: NR2F2, BMP4, COL1A1. The main goal of the presented study was to select genes involved in response to hormonal stimulus and hormone metabolism in GC long-term in vitro culture.

Transcriptomic profile of cell cycle progression genes in human ovarian granulosa cells.

The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

Sulodexide modulates the dialysate effect on the peritoneal mesothelium.

Peritoneal membrane damage during chronic peritoneal dialysis is the main cause of that treatment failure. Preservation of the mesothelial cells (MC) is important for the survival of the peritoneum. Evaluation of dialysates effect on the function of MC and potential modification of that effect by sulodexide (heparin 80% and dermatan sulfate 20%). Dialysate effluents, after the overnight exchange with dianeal 1.5% dextrose, were collected from 7 continuous ambulatory peritoneal dialysis (CAPD) patients, and their effect ± sulodexide 0.5 LRU/mL on genes expression, secretory activity and protein synthesis in MC was studied. Exposure of MC to the studied dialysates caused intracellular oxidative stress and significantly increased expression of the genes regulating the synthesis of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-ß), vascular cell adhesion molecule 1 (VCAM-1) and vascular endothelial growth factor (VEGF). Secretion of the studied molecules from MC treated with dialysates was increased: by 96% for IL-6 (P < 0.01), 34% for MCP-1(P < 0.01), 24% for TGF-ß (P < 0.01), 27% for VCAM-1 (P < 0.01), and by 15% for VEGF (P < 0.01). Sulodexide reduced the stimulatory effect of the dialysates on the intracellular generation of free radicals, genes expression and secretory activity of MC. These cells exposed to the dialysates showed increased synthesis of total protein (by 216%, P < 0.005) and collagen (by 264%, P < 0.005), as compared to standard culture medium. Supplementation of the dialysates with sulodexide resulted in weaker stimulation of collagen synthesis (-21% versus dialysate). We concluded that peritoneal dialysate changes the genes expression and phenotype of MC to a proinflammatory, profibrotic and proangiogenic one. Sulodexide reduces these negative effects of the dialysate.

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.

Long-term sequelae after lung abscess in children - Two tertiary centers' experience.

AIM: The aim of the study was to describe the epidemiology and clinical characteristic of children hospitalized with pneumonia complicated by lung abscess, as well as to evaluate the long-term sequelae of the disease. METHODS: A retrospective review of medical records of all patients treated for pulmonary abscess in two tertiary centers was undertaken. Pulmonary function tests and lung ultrasound were performed at a follow-up. RESULTS: During the study period, 5151 children with pneumonia were admitted, and 49 (0.95%) cases were complicated with lung abscess. In 38 (77.5%) patients, lung abscess was treated solely with antibiotics, and in nine cases (16.3%) surgically. In 21 (51.21%) children complete radiological regression was documented. The mean time for radiological abnormalities regression was 84.14 ± 51.57 days, regardless of the treatment mode. Fifteen patients were followed up at 61.6 ± 28.3 months after discharge. Lung ultrasound revealed minor residual abnormalities: pleural thickening, subpleural consolidations and line B artefacts in 11 (73.3%) children. Pulmonary function tests results were abnormal in eight (53.3%) patients, the most frequent abnormality being hyperinflation. We did not find a restrictive disorder in any of the children. There were no deaths in our study. CONCLUSIONS: Lung abscess is a rare but severe complication of pneumonia in children. Most children recover uneventfully with no significant long-term pulmonary sequelae.

L-2-oxothiazolidine-4-carboxylic acids slow down dialysate-induced senescence in human peritoneal mesothelial cells.

Peritoneal dialysis induces an intraperitoneal inflammatory reaction, which in the long term may cause deterioration of the peritoneal structure and function as the dialysis membrane. We studied the effect of the overnight effluent dialysate from patients on chronic peritoneal dialysis on aging of the human peritoneal mesothelial cells in an in vitro model of replicative cellular senescence. In the control group cells were cultured in the standard medium and in the studied groups in culture medium mixed 1:1 v/v with the dialysate ± L-2-oxothiazolodine-4-carboxylic acid 1 mmol/L (OTZ). OTZ was used as the precursor for the synthesis of glutathione in these cells. Dialysate accelerated senescence of the mesothelial cells as reflected by elongation of their population doubling time, reduced expression of KI-67 gene, and increased ß-galactosidase activity. Also, expression of the genes regulating the production of the inflammatory mediators (interleukin-6, monocyte chemoattractant protein-1, metalloproteinase-2, hyaluronan), proangiogenic (VEGF) and profibrotic (fibronectin) factors was increased in that group. At the same time, these cells secreted more inflammatory mediators. Simultaneous treatment of the cells with the dialysate and OTZ slowed down their senescence, whose intensity was similar to that in the control group. The results presented in this manuscript prove that the intraperitoneal inflammatory reaction induced by repeated infusions of the dialysis fluid accelerates the senescence of the mesothelial cells, which may result in fibrosis and neoangiogenesis within the peritoneum. Simultaneous supplementation of the cells with a glutathione precursor (OTZ) may prevent the development of these pathological changes.

Weight loss-dependent and -independent effects of moderate calorie restriction on endothelial cell markers in obesity.

Endothelial cell dysfunction in obesity can be reduced by calorie restriction (CR), however it is unclear whether this benefit requires a concomitant weight loss or is it simply related to the reduced calorie intake per se. In our study serum was drawn from 41 obese women who were undergoing an 8-week dietary intervention with 15 - 30% energy deficit, and from 48 age- and sex-matched controls of normal weight. Serum was analysed for biomarkers of endothelial cell function, oxidative stress and inflammation. Compared with non-obese individuals, the obese patients had lower serum levels of nitric oxide (NO), adiponectin, and decreased serum antioxidant status. They also had significantly higher levels of adhesive molecules, thrombomodulin (TM), von Wilebrand factor (vWF), asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and leptin. To further characterize the effect of moderate CR, the patients were ranked into two comparable groups according to the extent of weight loss - below and above the median (-5.8 kg). A moderate dietary intervention did not correct adiponectin, antioxidant status, vWF, TM, and plasminogen activator inhibitor-1 (PAI-1) but ameliorated changes in other parameters. Only changes in NO and - to a lesser degree - in sE-selectin showed a clear relationship with the magnitude of weight reduction. By contrast, a beneficial reduction in TNF-α occurred equally in patients who lost more or less weight after caloric restriction. We concluded that moderate calorie restriction could still improve several parameters of endothelial cell function irrespective of whether it was accompanied by changes in body mass. However, a significant improvement in nitric oxide, a key mediator of endothelial well-being, requires a substantial reduction in body weight.

The role of mTOR inhibitors and HMG-CoA reductase inhibitors on young and old endothelial cell functions, critical for re-endothelialisation after percutaneous coronary intervention: an in vitro study.

Percutaneous coronary intervention (PCI) has become a standard treatment in patients with acute coronary syndrome. However, it is associated with endothelial cell denudation, which may predispose to in-stent thrombosis and restenosis. Pharmacological methods which prevent restenosis can delay post-PCI re-endothelialisation. We have therefore examined how atorvastatin (HMG-CoA reductase inhibitor), sirolimus and everolimus (mTOR inhibitors) affect young and old endothelial cell functions which are responsible for wound healing after PCI. Replicative senescence was induced by serial passages of human umbilical vein endothelial cells (HUVECs). The cells which were examined at their first passages and last passages were designated as 'young' and 'old' respectively. Young and old endothelium were grown to confluence and were wounded by scraping. Scratch healing in the presence or absence of atorvastatin (AT), rapamycin (SR) and everolimus (EV) was monitored by time-lapse microscopy. In addition cells were assessed for viability (MTT assay), migration (chemotaxis chamber), proliferation (3H-thymidine), and cytokine production (immunoassays). Senescent endothelial cells produce more proinflammatory cytokines, angiogenic VEGF and extracellular matrix proteins. They stop proliferating and have diminished migration. When compared to young endothelium, they have similar viability and can regenerate wounds in comparable time. The drugs that have been tested have anti-inflammatory properties but even after pretreatment old cells still produced significantly higher concentration of tested mediators in comparison with young ones. In the concentration obtained in serum after stent implantation, mTOR inhibitors in dose-dependent manner reduced cell proliferation, migration and wound healing. Reduced healing is more pronounced in young endothelium. Atorvastatin, at clinically relevant concentration, is safe for young and old cells. Atorvastatin, sirolimus and everolimus inhibited the secretion of pro-inflammatory mediators in young and old endothelium. In concentrations seen in serum during standard therapy, rapalogs impair endothelial cell regeneration after injuries mimicking those occurring during PCI, while atorvastatin does not affect the healing.

Sulodexide suppresses inflammation in patients with chronic venous insufficiency.

AIM: According to previously performed studies, inflammation plays a crucial role in vein wall and leg tissue injury related to chronic venous insufficiency (CVI) development. Sulodexide (SUL) is a balanced mix of glycosaminoglycans with potential anticoagulant and profibrinolytic activity, also protecting endothelial cells and suppressing inflammatory reactions in various vascular disease-related conditions. The goal of the present study was to evaluate the anti-inflammatory action of SUL in patients with CVI. METHODS: The study was performed on a group of 11 patients with chronic venous disease (stage C5 according to CEAP classification). The mean age of the patients was 58.4±7.7 years, and none of them were diabetic. The patients were treated for 8 weeks with orally-administered SUL (2 x 500 LSU/day). Blood samples were collected at the start and at the end of the study for measurement of MMP-9, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Additionally, the effect of the obtained serum samples on the function of human venous endothelial cells (HVEC) in in-vitro culture was evaluated. RESULTS: After treatment with SUL, the serum concentration of MMP-9 (ng/mL) decreased from 6.50±3.48 to 5.41±1.36, P<0.05, and the concentration of IL-6 (pg/mL) decreased from 11.5±3.4 to 10.1±2.3, P<0.005. There was also a trend of decreased serum MCP-1 (pg/mL) from 31.3±23.0 before treatment to 27.1±10.7 at the end. Intracellular generation of oxygen-derived free radicals in HVEC maintained in in-vitro culture was lower in the serum samples collected after treatment with SUL: 3.09±0.35 abs/µg protein vs. 3.63±0.32 abs/µg protein, at the start, P<0.05. Synthesis of IL-6 was lower in HVEC exposed in vitro to serum collected at the end of SUL treatment: 1.02±0.31 ng/µg cell protein vs. 1.32±0.41 ng/µg cell protein before SUL treatment. The proliferation rate of HVEC was similar in serum collected at the beginning and at the end of SUL treatment. CONCLUSION: We conclude that treatment with SUL in patients with CVI reduces intravascular inflammation and is protective for the endothelial cells and for the extracellular matrix changes related to metalloproteinase expression.

N-Acetylglucosamine modulates function of the skin fibroblasts.

BACKGROUND: Fibroblasts are an important component of the skin determining its properties. N-Acetylglucosamine (NAG) is the substrate for hyaluronan synthesis, and it also has anti-inflammatory and anti-senescent activity in mesothelial cells. METHODS: We tested in in vitro-cultured human skin fibroblasts how supplementation of culture medium with NAG 10 mmol L(-1) changes properties of these cells. RESULTS: Fibroblasts cultured in presence of NAG produced more proteins and that was mainly due to increased synthesis of collagen (+33% vs. control, P < 0.05). Hyaluronan synthesis was increased (+107% vs. control, P < 0.001), but interleukin-6 synthesis was reduced (-22% vs. control, P < 0.05). Fibroblasts cultured in medium with NAG 10 mmol L(-1) demonstrated improved ability to heal the injured layer of cells (+34% vs. control, P < 0.05). Additionally senescence of fibroblasts undergoing replicative ageing in the presence of NAG was less pronounced, as reflected by smaller increase in the population doubling time (-70% vs. control, P < 0.05). CONCLUSION: We conclude that NAG induced changes in the skin fibroblasts' properties maybe important for prevention of the age-dependent changes in its structure and function.

Sulodexide modifies intravascular homeostasis what affects function of the endothelium.

PURPOSE: Sulodexide is a mixture of heparin and dermatan sulphate which has an antithrombotic action. It was shown that it has also direct effect on the endothelial cells. We tested the effect of sulodexide on the intravascular homeostasis in patients with peripheral vascular disease. METHODS: Sulodexide was infused iv. at a dose of 1200 Lipoprotein Lipase Releasing Units (LRU) in 10 patients with peripheral vascular disease. Blood samples were collected before the infusion and 1, 6 and 24 hours after the infusion. Inflammatory and fibrinolytic parameters were studied in the collected serum samples. Additionally, ex-vivo effect of the serum samples on in vitro function of the endothelial cells was studied. RESULTS: Infusion of sulodexide caused acute and transient peak of the Hepatocyte Growth Factor (HGF) concentration in blood and decrease of the Vascular Endothelial Growth Factor (VEGF) level, what, as we found in in vitro experiments, was due to adsorption of VEGF to endothelium. We found that HGF enhanced in vitro stimulating effect of VEGF on proliferation of the endothelial cells. Serum level of interleukin-6 was gradually decreased, whereas fibrinolytic activity of serum, reflected by t-PA/PAI-1 ratio, increased. Serum samples obtained from the studied patients suppressed oxidative stress and release of interleukin-6 in endothelial cells maintained in in vitro culture. CONCLUSION: Sulodexide reduces intravascular inflammation and suppresses inflammatory reaction in the endothelial cells; both effects are desirable in patients with peripheral vascular disease.

Atorvastatin does not impair endothelial cell wound healing in an in vitro model of vascular injury.

Percutaneous coronary intervention (PCI) became standard treatment modality for coronary revascularization. However, it is associated with endothelial cell denudation, which may predispose to in-stent thrombosis and restenosis. Since statins may inhibit vascular cell proliferation, one may fear that their early administration will delay post-PCI re-endothelialization. We have therefore employed an in vitro scratch assay to examine how atorvastatin affects endothelial cell wound healing. Human umbilical vein endothelial cells (HUVEC) were grown to confluence and wounded by scraping. Scratch healing in the presence or absence of atorvastatin was monitored by time-lapse photo-microscopy. In addition cells were assessed for viability (MTT assay), migration (chemotaxis chamber), proliferation ((3)H-thymidine and bromodeoxyuridine incorporation), and cytokine production (immunoassays). The exposure of HUVEC to atorvastatin resulted in a dose-dependent decrease in cell viability and proliferation. However, this effect was observed only at doses ≥1 µM, which is well above the concentrations seen in vivo. At clinically relevant doses (≤0.1 µM) atorvastatin did not impair wound closure, nor did it inhibit cell viability, proliferation, and migration. It did however reduce the constitutive and the stimulated release of cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1) and matrix proteins (fibronectin). We conclude that atorvastatin at doses corresponding to concentrations seen in serum during standard therapy does not impair endothelial cell regeneration after injuries mimicking those occurring during PCI. It does, however, inhibits the secretion of pro-inflammatory mediators.

Role of ADRB2 gene polymorphism in asthma and response to beta(2)-agonists in Polish children.

The aims of this study were: (1) to find associations of asthma with single-nucleotide polymorphisms (SNPs) within the ADRB2 gene: Arg16Gly, Gln27Glu, -1023 G/A, -367 T/C, -47 C/T ; (2) to define linkage disequilibrium in the gene region, basing on the analyzed SNPs; and (3) to analyze the importance of ADRB2 polymorphism for response to bronchodilator drugs in children diagnosed with bronchial asthma. We compared 113 asthmatic children and 123 healthy subjects from the Polish population. Genotyping was performed by PCR-RFLP. We found an association of the A allele of -1023A/G ADRB2 polymorphism with asthma (P = 0.024). No significant associations with other SNPs were detected. Moderate linkage was found between Gln27Glu and -47C/T polymorphisms in linkage disequilibrium analysis (D' = 0.85, r(2) = 0.429, LOD = 31.97). No significant differences were found in haplotype frequencies in comparison to the control group, implicating that they are not associated with susceptibility to asthma in the analyzed population. There was no significant correlation between the analyzed SNPs of the ADRB2 gene and the response to beta(2)-agonists. This is the first report providing suggestive evidence for association of -1023A/G ADRB2 polymorphism with an increased risk of asthma. The analyzed SNPs may not play a major role in response to beta(2)-agonists in asthmatic children.