Use of mucolytics in COPD: A Delphi consensus study.

BACKGROUND: International guidelines recommend mucolytic agents as add-on therapy in selected patients with COPD because they may reduce exacerbations and improve health status. As the evidence varies among mucolytic agents, we used the Delphi method to assess consensus amongst an international panel of COPD experts on mucolytics use in COPD. METHODS: 53 COPD experts from 12 countries were asked to complete an online questionnaire and rate their agreement with 15 statements using a 5-point scale. The mucolytic agents evaluated were carbocysteine, erdosteine and N-acetylcysteine (NAC). Data were collected anonymously and consensus presented using descriptive statistics. RESULTS: The 47 respondents reached consensus on the statements. They agreed that regular treatment with mucolytic agents effectively reduces the frequency of exacerbations, reduces the duration of mild-to-moderate exacerbations, and can increase the time to first exacerbation and symptom-free time in COPD patients. Consensus was consistently highest for erdosteine. The experts agreed that all three mucolytics display antioxidant and anti-inflammatory activity. Erdosteine and NAC were thought to improve the efficacy of some classes of antibacterial drugs. All three mucolytics were considered effective for the short-term treatment of symptoms of acute exacerbations when added to other drugs. The panel agreed that approved doses of mucolytic agents have favorable side-effect profiles and can be recommended for regular use in patients with a bronchitic phenotype. CONCLUSIONS: Consensus findings support the wider use of mucolytic agents as add-on therapy for COPD. However, the differences in pharmacological actions and clinical effectiveness must be considered when deciding which mucolytic to use.

Comorbidities of COPD in Bulgarian Patients - Prevalence and Association with Severity and Inflammation.

BACKGROUND: wis a disease with constantly rising economic and social burden; it is associated with multiple comorbidities which contribute to the disease severity. AIM: To investigate the prevalence of co-morbidities in COPD patients and their association with the disease severity and CRP levels. PATIENTS AND METHODS: We conducted a retrospective study among 338 COPD patients (mean age 65.2±7.6 years) with assessment of comorbidities, spirometry measurements and serum levels of CRP. In 183 patients we found metabolic syndrome (MS) according to IDF criteria. RESULTS: We found prevalence of cardiovascular diseases (CVD) of 73.5% (hyper-tension 70.4%, CHF 47.4%, ishemic heart disease 37.5%, and cardiac arrhythmias 12.6%), with higher prevalence in patients with more severe disease. We found prevalence of type 2 diabetes of 21.1%, and 12.4% prevalence of bronchiectasis. In a subpopulation of the patients we found 48.1% prevalence of MS and the serum levels of CRP were significantly higher in patients with COPD and MS compared to those without the syndrome: 7.4 (3.14 - 11. 54) mg/ml vs 4.06 (2.64 - 6.93) mg/ml, p=0.006. CONCLUSION: The present study suggests high prevalence of CVD comorbidities in COPD patients and association with the disease severity. Metabolic syndrome is a common comorbidity and is associated with increased inflammatory response.

Role of Aminophospholipids in the Formation of Lipid Rafts in Model Membranes.

The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of Lo domains.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes.

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process.

Testosterone replacement therapy improves erythrocyte membrane lipid composition in hypogonadal men.

AIM: The aim of this study was to investigate the effects of testosterone replacement therapy (TRT) on erythrocyte membrane (EM) lipid composition and physico-chemical properties in hypogonadal men. METHODS: EM isolated from three patients before and after TRT with injectable testosterone undecanoate or testosterone gel were used for analysis of the phospholipid and fatty acid composition, cholesterol/phospholipid ratio, membrane fluidity, ceramide level and enzyme activities responsible for sphingomyelin metabolism. RESULTS: TRT induced increase of phosphatidylethanolamine (PE) in the EMs and sphingomyelin. Reduction of the relative content of the saturated palmitic and stearic fatty acids and a slight increase of different unsaturated fatty acids was observed in phosphatidylcholine (PC). TRT also induced decrease of the cholesterol/total phospholipids ratio and fluidization of the EM. DISCUSSION: The TRT induced increase of PE content and the reduction of saturation in the PC acyl chains induced alterations in the structure of EM could result in higher flexibility of the erythrocytes. The increase of the SM-metabolizing enzyme neutral sphingomyelinase, which regulates the content of ceramide in membranes has a possible impact on the SM signaling pathway. CONCLUSION: We presume that the observed effect of TRT on the composition and fluidity of the EM contributes for improvement of blood rheology and may diminish the thrombosis risk. Larger studies are needed to confirm the findings of this pilot study.

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions.

Complementary biophysical approaches were used to study the structural organization of plasma membrane lipids obtained from fibroblasts cultured as two-dimensional (2D) monolayer and in tissue-like three-dimensional (3D) conditions. Fluorescence microscopy experiments demonstrated different domain patterns for 2D and 3D plasma membrane lipid extracts. ESR demonstrated that 3D lipid extract is characterized with lower order parameter than 2D in the deep hydrophobic core of the lipid bilayer. Higher cholesterol and sphingomyelin content in 3D extract, known to increase the order in the glycerophospholipid matrix, was not able to compensate higher fatty acid polyunsaturation of the phospholipids. The interfacial region of the bilayer was probed by the fluorescent probe Laurdan. A higher general polarization value for 3D extract was measured. It is assigned to the increased content of sphingomyelin, cholesterol, phosphatidylethanolamine and phosphatidylserine in the 3D membranes. These results demonstrate that cells cultured under different conditions exhibit compositional heterogeneity of the constituent lipids which determine different structural organization of the membranes.

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix.

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.