Bioactive Sphingolipids, Complement Cascade, and Free Hemoglobin Levels in Stable Coronary Artery Disease and Acute Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) and coronary artery bypass graft (CABG) surgery are associated with a pathogen-free inflammatory response (sterile inflammation). Complement cascade (CC) and bioactive sphingolipids (BS) are postulated to be involved in this process. AIM: The aim of this study was to evaluate plasma levels of CC cleavage fragments (C3a, C5a, and C5b9), sphingosine (SP), sphingosine-1-phosphate (S1P), and free hemoglobin (fHb) in AMI patients treated with primary percutaneous coronary intervention (pPCI) and stable coronary artery disease (SCAD) undergoing CABG. PATIENTS AND METHODS: The study enrolled 37 subjects (27 male) including 22 AMI patients, 7 CABG patients, and 8 healthy individuals as the control group (CTRL). In the AMI group, blood samples were collected at 5 time points (admission to hospital, 6, 12, 24, and 48 hours post pPCI) and 4 time points in the CABG group (6, 12, 24, and 48 hours post operation). SP and S1P concentrations were measured by high-performance liquid chromatography (HPLC). Analysis of C3a, C5a, and C5b9 levels was carried out using high-sensitivity ELISA and free hemoglobin by spectrophotometry. RESULTS: The plasma levels of CC cleavage fragments (C3a and C5b9) were significantly higher, while those of SP and S1P were lower in patients undergoing CABG surgery in comparison to the AMI group. In both groups, levels of CC factors showed no significant changes within 48 hours of follow-up. Conversely, SP and S1P levels gradually decreased throughout 48 hours in the AMI group but remained stable after CABG. Moreover, the fHb concentration was significantly higher after 24 and 48 hours post pPCI compared to the corresponding postoperative time points. Additionally, the fHb concentrations increased between 12 and 48 hours after PCI in patients with AMI. CONCLUSIONS: Inflammatory response after AMI and CABG differed regarding the release of sphingolipids, free hemoglobin, and complement cascade cleavage fragments.

Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs).

The role of blood proteinases in the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not well understood. As previously reported, activation of the complement cascade (ComC) and cleavage of C5 by C5 convertase are enabling events in the release of C5a that plays a crucial role in the egress of HSPCs from bone marrow (BM) into peripheral blood (PB) and explains why C5-deficient mice are poor mobilizers. Here we provide evidence that during granulocyte colony-stimulating factor- and AMD3100-induced mobilization, not only the ComC but also two other evolutionarily ancient proteolytic enzyme cascades, the coagulation cascade (CoaC) and the fibrynolytic cascade (FibC), become activated. Activation of all three cascades was measured by generation of C5a, decrease in prothrombin time and activated partial thromboplastin time as well as an increase in the concentrations of plasmin/antiplasmin and thrombin/antithrombin. More importantly, the CoaC and FibC, by generating thrombin and plasmin, respectively, provide C5 convertase activity, explaining why mobilization of HSPCs in C3-deficient mice, which do not generate ComC-generated C5a convertase, is not impaired. Our observations shed more light on how the CoaC and FibC modulate stem cell mobilization and may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, as it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (for example, infections, tissue/organ damage or strenuous exercise) and show a circadian rhythm of activation, they must be involved in both stress-induced and circadian changes in HSPC trafficking in PB.

Clinical analysis of systemic and adipose tissue levels of selected hormones/adipokines and stromal-derived factor-1.

Recent studies demonstrated that selected hormones/adipokines may be involved into the regulation of bone metabolism and bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) mobilization in humans. Interestingly, in obese individuals significantly higher numbers of spontaneously circulating stem cells are also observed. Therefore in this study we comprehensively examined plasma and AT (subcutaneous and visceral/omental) levels of hormones/adipokines involved in HSPCs mobilization in lean, overweight and obese individuals as well as verified their potential associations with concentrations of HSPCs chemoattractant, stromal-derived factor-1 (SDF-1). Blood and AT samples (35 subcutaneous and 35 omental) were obtained from individuals undergoing elective surgery. Plasma and AT-derived interstitial fluid levels of resistin, visfatin, osteocalcin and SDF-1 were measured using ELISA. In our study obese patients had almost significantly (P<0.06) higher plasma visfatin and resistin levels as well as lower osteocalcin concentrations (P<0.04) than lean individuals. Osteocalcin and resistin concentrations were strongly associated with levels of SDF-1 and metalloproteinases (MMP 2 and 9). AT levels of all examined substances were significantly lower than the corresponding levels in the plasma (in all cases at least P<0.05), and depot-specific differences in the concentrations of these factors were found only in terms of osteocalcin and SDF-1. In addition, subcutaneous and visceral/omental concentrations of osteocalcin and visfatin, but not of resistin, were associated with values of such parameters as age, body mass or adiposity indexes (BMI and BAI, respectively) and/or waist-to-hip ratio (WHR). In summary, our study showed that in obese individuals the biochemical constellation of adipokines/hormones involved in the process of HSPCs mobilization resembles this observed during pharmacological HSPCs mobilization. Moreover, our study offers further indirect translational evidence for existence of a biochemical cross-talk between bone and AT metabolism (so called - bone-fat- axis) in humans.

Spectrophotometric methods as a novel screening approach for analysis of dihydropyrimidine dehydrogenase activity before treatment with 5-fluorouracil chemotherapy.

5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutics in the treatment of malignancies originating from breast, prostate, ovarian, skin and gastrointestinal tissues. Around 80% of administered dose of 5-FU is catabolized by dihydropirymidine dehydrogenase (DPD). Patients, in whom a deficiency or insufficient activity of this enzyme is observed, are at great risk of development of severe, even lethal, 5-FU toxicity. According to recent studies, so far over 30 mutations of DPYD gene, which are associated with DPD deficiency/insufficiency, have already been discovered. Currently, there are several analytical methods used for measurements of DPD activity. However, in this paper we report a novel, simple, economical and more accessible spectrophotometric method for measurements of DPD activity in the peripheral blood mononuclear cells (PBMCs) that was developed and validated on analysis of 200 generally healthy volunteers aged 22-63. We present two spectrophotometric protocols in this study, and as a reference method we used already described reverse phase high-performance liquid chromatography (RP HPLC) analysis. Basing on our findings, we conclude that spectrophotometric methods may be used as a screening protocol preceding 5-FU-based chemotherapy. Nevertheless, before introduction into clinical reality, our results should be confirmed in further larger studies.

Growth factor content in PRP and their applicability in medicine.

This paper reviews available reports on the advantages and possibilities of clinical use of platelet-rich plasma preparations, with particular emphasis on platelet growth factors. Platelets, an important reservoir of growth factors in the body, play an important role in many processes such as coagulation, immune response, angiogenesis and the healing of damaged tissues. Numerous proteins are contained in the alpha-granules of platelets: platelet-derived growth factor (PDGF), transforming growth factor (TGF), platelet factor interleukin (IL), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor IGF and fibronectin. The development of methods and systems for blood and cell sorting (e.g. CAPSS - compact advanced platelet sequestration system Elektromedics 500, PCCS - platelet concentrate collection system Curasan) have made it possible to obtain significant concentrations of platelets (even by 338 percent) and high concentrations of growth factors, in a form of sterile mass that can be used immediately for clinical purposes. Platelet-rich plasma (PRP; autologous platelet-rich plasma - APRP) are platelet concentrates made of autogenous blood with a high number of platelets in a small volume of plasma. The clinical efficacy of platelet concentrates depends mainly on the number of platelets and the concentration of their growth factors, which act as transmitters in most processes in tissues, particularly in healing where they are responsible for proliferation, differentiation, chemotaxis and tissue morphogenesis. They operate as part of autocrine, paracrine and endocrine mechanisms. Growth factors derived from centrifuged blood were first used in patients with chronic skin ulcers. The clinical use of PRP for a wide variety of applications has been reported mostly in oral and maxillo-facial surgery, orthopedic surgery, treatment of soft tissue diseases and injuries, treatment of burns, hard-to-heal wounds, tissue engineering and implantology.

Adenine, guanine and pyridine nucleotides in blood during physical exercise and restitution in healthy subjects.

Maximal physical exertion is accompanied by increased degradation of purine nucleotides in muscles with the products of purine catabolism accumulating in the plasma. Thanks to membrane transporters, these products remain in an equilibrium between the plasma and red blood cells where they may serve as substrates in salvage reactions, contributing to an increase in the concentrations of purine nucleotides. In this study, we measured the concentrations of adenine nucleotides (ATP, ADP, AMP), inosine nucleotides (IMP), guanine nucleotides (GTP, GDP, GMP), and also pyridine nucleotides (NAD, NADP) in red blood cells immediately after standardized physical effort with increasing intensity, and at the 30th min of rest. We also examined the effect of muscular exercise on adenylate (guanylate) energy charge--AEC (GEC), and on the concentration of nucleosides (guanosine, inosine, adenosine) and hypoxanthine. We have shown in this study that a standardized physical exercise with increasing intensity leads to an increase in IMP concentration in red blood cells immediately after the exercise, which with a significant increase in Hyp concentration in the blood suggests that Hyp was included in the IMP pool. Restitution is accompanied by an increase in the ATP/ADP and ADP/AMP ratios, which indicates an increase in the phosphorylation of AMP and ADP to ATP. Physical effort applied in this study did not lead to changes in the concentrations of guanine and pyridine nucleotides in red blood cells.

Prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) synthesis is regulated by conjugated linoleic acids (CLA) in human macrophages.

Conjugated linoleic acid (CLAs) are positional and geometric isomers of linoleic acid with have a potential anti-atherosclerotic and anti-inflammation properties. Metabolites of arachidonic acid--prostaglandins and thromboxans--are endogenous mediators of inflammation. Prostaglandin E(2) and thromboxan A(2) which are a products of two izoformes of cyclooxygenases (COX-1 and COX-2) in macrophages, play an important role in this process. COX-1 is a constitutive enzyme, whereas the COX-2 is inducible and its amount in the cell rapidly increases during inflammation (e.g. via NF kappaB pathway). The aim of the study was to test the effect of CLAs on cyclooxygenases (COX-1 and COX-2) activity, their mRNA expression and protein content in macrophages. Additionally the active form of the kappaB (NF kappaB) transcription factor was measured. For the experiments monocytes from monocytic cell line (THP-1) and from human venous blood were used. Monocytes were differentiated to macrophages and cultured with 30 muM CLAs or linoleic acid for 48 h. The COX-1 and COX-2 products - PGE(2) and TXB(2), were measured by ELISA method. The enzymes (COX-s) activity were estimated by spectroscopic method. mRNA expression and protein analysis were analysed by real-time PCR and Western blot technique. In macrophages cultured with CLAs reduction of TXB(2) and PGE(2) concentration was observed. Significant change in COX-2 expression in cells cultured with trans-10, cis-12 CLA (in macrophages obtained from peripheral blood) was observed. COX-1 inhibition was resulting from competition of CLA and linoleic acid with arachidonic acid.

Exchange of unsaturated fatty acids between adipose tissue and atherosclerotic plaque studied with artificial neural networks.

The linoleic C18:2 (n-6) and linolenic C18:3 (n-3) are recognized as essential components of the diet. Free radical peroxidation of essential fatty acids (EFAs) present in lipoproteins produces oxidized low-density lipoproteins which play a critical role in the development of atherosclerosis. The accumulation of EFAs in the vascular wall and correlations between their content in the adipose tissue and atherosclerotic plaque have been confirmed. The present study was undertaken to determine the usefulness of a neural network for studying the exchange between tissues of linoleic, alpha-linolenic, and arachidonic acids-three fatty acids with a well-understood metabolism. Atheromatous plaques, adipose tissue, and serum were obtained from 31 patients who underwent surgery due to atherosclerotic stenosis of the abdominal aorta, iliac or femoral arteries. Fatty acids were extracted and separated as methyl esters using gas chromatography. Statistical analysis was done with STATISTICA neural networks package. Several correlations reported previously were corroborated and factors modifying the content of individual EFAs in adipose tissue and atherosclerotic plaque were revealed. Artificial neural networks (ANNs) were used to determine factors modifying the content of linoleic, alpha-linolenic, and arachidonic acids in human atheromatous plaques. The mechanism of exchange of some fatty acids between the adipose tissue, atheromatous plaque, and plasma is discussed. The results provide evidence for an effective mechanism of tissue uptake and turnover of linoleic acid. Reduced plasma levels of this acid are compensated by release from adipose tissue and atheromatous plaque. While alpha-linolenic acid is continuously taken up by the plaque, adipose tissue absorbs this acid to a certain level only. The dynamics of exchange of arachidonic acid between adipose tissue and atheromatous plaque reflects a minor role for adipose tissue in determining plaque content of this acid, suggesting that "de novo" synthesis is the chief source of arachidonic acid in plaques.