Urocortin 2 - a protective effect in hypertension?

Urocortin 2, an endogenous selective ligand for the corticotropin-releasing hormone receptor type 2, has been suggested to exert cardioprotective effects. We analyzed the possible relationship between the level of Ucn2 and specific indicators of cardiovascular risk factors in patients with untreated hypertension and in healthy subjects. Sixty seven subjects were recruited: 38 with newly diagnosed treatment-naive hypertension (with no pharmacological treatment - HT group) and 29 healthy subjects without hypertension (nHT group). We evaluated ambulatory blood pressure monitoring, Ucn2 levels and metabolic indices. Multivariable regression analyses were performed to assess the effects of gender, age, and Ucn2 levels on metabolic indices or blood pressure (BP) level. Log of Ucn2 levels were higher in healthy subjects than in hypertensive patients (2.44±0.7 versus 2.09±0.66, p<.05) and correlated inversely with 24-hour diastolic blood pressure, and both night-time systolic and diastolic blood pressure regardless of age and gender (R2=0.06; R2=0.06; R2=0.052; respectively). Furthermore, Ucn2 levels inversely correlated with cholesterol and low-density cholesterol (LDL) concentrations in healthy subjects only. Ucn2 was independently related to total cholesterol (but not to LDL) regardless of age, gender and the presence of hypertension (R2=0.18). However, we did not find any relationship between urocortin 2, body mass index or waist-hip ratio as well as parameters of glucose metabolism. Our data indicates that higher levels of urocortin 2 are related to more favorable lipid profiles and lower blood pressure.

DNA methylation analysis of negative pressure therapy effect in diabetic foot ulcers.

OBJECTIVE: Negative pressure wound therapy (NPWT) has been used to treat diabetic foot ulcerations (DFUs). Its action on the molecular level, however, is only partially understood. Some earlier data suggested NPWT may be mediated through modification of local gene expression. As methylation is a key epigenetic regulatory mechanism of gene expression, we assessed the effect of NPWT on its profile in patients with type 2 diabetes (T2DM) and neuropathic non-infected DFUs. METHODS: Of 36 included patients, 23 were assigned to NPWT and 13 to standard therapy. Due to ethical concerns, the assignment was non-randomized and based on wound characteristics. Tissue samples were obtained before and 8 ± 1 days after therapy initiation. DNA methylation patterns were checked by Illumina Methylation EPIC kit. RESULTS: In terms of clinical characteristics, the groups presented typical features of T2DM; however, the NPWT group had significantly greater wound area: 16.8 cm2 vs 1.4 cm2 (P = 0.0003). Initially only one region at chromosome 5 was differentially methylated. After treatment, 57 differentially methylated genes were found, mainly located on chromosomes 6 (chr6p21) and 20 (chr20p13); they were associated with DNA repair and autocrine signaling via retinoic acid receptor. We performed differential analyses pre treatment and post treatment. The analysis revealed 426 differentially methylated regions in the NPWT group, but none in the control group. The enrichment analysis showed 11 processes significantly associated with NPWT, of which 4 were linked with complement system activation. All but one were hypermethylated after NPWT. CONCLUSION: The NPWT effect on DFUs may be mediated through epigenetic changes resulting in the inhibition of complement system activation.

Urocortin 2 in patients with hypertension treated with angiotensin converting enzyme inhibitors or angiotensin receptor blockers.

Urocortin 2 (Ucn2) - corticotropin-releasing hormone receptor 2 signalling has favourable effects in the cardiovascular system, including vasodilation, lowering of blood pressure and systemic peripheral resistance, increase in cardiac output and cardiac contractility, as well as cardioprotection against ischemia-reperfusion injury. Vasodilation and lowering of blood pressure seem to be very interesting and important effects, but their mechanism and interaction with the antihypertensive drugs have not been evaluated. The aim of the present study was to assess the relationship between Ucn2 concentration and antihypertensive therapy in patients with primary hypertension. We examined a group of 65 patients with primary hypertension receiving at least 3 antihypertensive drugs. In all of them plasma level of Ucn2, anthropometric measurements, biochemical tests, ambulatory blood pressure monitoring (ABPM), and echocardiography were performed. There were no differences in Ucn2 level related to beta-blockers, calcium channel blockers or diuretics, but we observed that in patients treated with angiotensin converting enzyme inhibitors (ACEI) (n = 52) serum Ucn2 levels were significantly higher than in patients treated with angiotensin-receptor blockers (ARBs) (n = 13) (10.93 versus 5.56 ng/mL; P < 0.05). Moreover, we did not observe any differences in terms of blood pressure on ABPM, biochemical measurements, left ventricular mass index, or presence of diabetes. In addition, in a small subgroup receiving alpha-blockers we also found a lower level of Ucn2, with coexisting higher systolic blood pressure at night, higher left ventricle mass index (LVMI) and more frequent occurrences of diabetes compared to non-alpha-blockers. Our findings suggest that the hypotensive action of renin-angiotensin-aldosterone system blockade may be related to the urocortin system. Ucn2 may be an important element in the mosaic of blood pressure-lowering factors in patients treated for essential hypertension.

Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia.

Blood is considered to be a sterile microenvironment, in which bacteria appear only periodically. Previously used methods allowed only for the detection of either viable bacteria with low sensitivity or selected species of bacteria. The Next-Generation Sequencing method (NGS) enables the identification of all bacteria in the sample with their taxonomic classification. We used NGS for the analysis of blood samples from healthy volunteers (n = 23) and patients with sepsis (n = 62) to check whether any bacterial DNA exists in the blood of healthy people and to identify bacterial taxonomic profile in the blood of septic patients. The presence of bacterial DNA was found both in septic and healthy subjects; however, bacterial diversity was significantly different (P = 0.002) between the studied groups. Among healthy volunteers, a significant predominance of anaerobic bacteria (76.2 %), of which most were bacteria of the order Bifidobacteriales (73.0 %), was observed. In sepsis, the majority of detected taxa belonged to aerobic or microaerophilic microorganisms (75.1 %). The most striking difference was seen in the case of Actinobacteria phyla, the abundance of which was decreased in sepsis (P < 0.001) and Proteobacteria phyla which was decreased in the healthy volunteers (P < 0.001). Our research shows that bacterial DNA can be detected in the blood of healthy people and that its taxonomic composition is different from the one seen in septic patients. Detection of bacterial DNA in the blood of healthy people may suggest that bacteria continuously translocate into the blood, but not always cause sepsis; this observation can be called DNAemia.

Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 µM), thiorphan (3 µM), or vehicle and incubated for 15 minutes with ANG I (1 µM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1-9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

Polymorphism in the chemokine receptor 7 gene (CCR7) is associated with previous myocardial infarction in patients undergoing elective coronary angiography.

Coronary artery disease (CAD) remains a major cause of death in developed countries. Both environmental and, less known, genetic factors contribute to progression of CAD to myocardial infarction (MI). Immune system is activated in patients with CAD through dendritic cells (DCs), which present plaque antigens to T lymphocytes. Production of proinflammatory cytokines by activated T cells contributes to plaque rupture in MI. Chemokine receptor 7 (CCR7) on DCs is required for their chemotaxis from plaque to lymph nodes. This makes possible an interaction of DCs with T lymphocytes and initiation of specific immune response. We hypothesized that single nucleotide polymorphisms (SNPs) in CCR7 gene locus are associated with previous MI in patients with CAD. To test this hypothesis, we genotyped six SNPs from the CCR7 gene locus in 300 consecutive patients, admitted for elective coronary angiography. We performed univariate-, multivariate- (including potential confounders) and haplotype-based tests of association of SNPs with previous MI and results of angiography. Allele A of rs17708087 SNP was associated with previous MI. This association remained significant after adjustment for age, sex, smoking, hypercholesterolaemia and drugs used by patients (odds ratio 2.13, 95% confidence interval: 1.13-3.86). Therefore, we conclude that CCR7 gene locus harbours a polymorphism that modifies risk of MI in patients with CAD. Replication of this association could be sought in a prospective cohort of initially healthy individuals.

Perindoprilat changes ANG (1-9) production in renal arteries isolated from young spontaneously hypertensive rats after ANG I incubation.

We used mass spectrometry to quantitate production of angiotensinogen metabolites in renal artery of 3- and 7-month-old Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Tissue fragments were incubated for 15 min in oxygenated buffer, with added angiotensin I. Concentrations of angiotensins I (ANG I), II (ANG II), III (ANG III), IV (ANG IV), angiotensin (1-9) [ANG (1-9)], angiotensin (1-7) [ANG (1-7)], and angiotensin (1-5) [ANG (1-5)], excreted into the buffer during experiment, were measured using liquid chromatography-mass spectrometry (LC/MS) and expressed per mg of dry tissue. Effects of pretreatment with 10 microM perindoprilat on the production of ANG I metabolites were quantitated. Background production of any of ANG I metabolites differed neither between WKY and SHR rats nor between 3- and 7-month-old rats. Perindoprilat pretreatment of renal arteries resulted, as expected, in decrease of ANG II production. However, renal arteries of 7-month-old SHR rats were resistant to ACE inhibitor and did not change ANG II production in response to perindoprilat. In renal arteries, taken from 3-month-old rats, pretreated with perindoprilat, incubation with ANG I, resulted in the level of ANG (1-9) significantly higher in SHR than WKY rats. Our conclusion is that in SHR rats, sensitivity of renal artery ACE to perindoprilat inhibition changes with age.

Kaempferol, but not resveratrol inhibits angiotensin converting enzyme.

Inhibition of angiotensin converting enzyme (ACE) has proved to be beneficial in the treatment of various cardiovascular disorders. The aim of this study was to evaluate ACE inhibitory potential of two polyphenolic compounds with different structures: resveratrol (present in high quantities in French wine) and kaempferol (abundant in greens), using method of liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) for ex vivo measurement of angiotensin I to angiotensin II conversion by ACE in aortic tissue of Wistar-Kyoto rats. In this setting, kaempferol (10-30-100 microM), but not resveratrol (10-30-100 microM) appeared to inhibit dose-dependently conversion of Ang I to Ang II. Although the mechanism of ACE inhibition by kaempferol remains to be elucidated, this observation may help in search or designing of new classes of ACE inhibitors.

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.

Paraoxonase gene polymorphisms and sporadic ALS.

BACKGROUND: The human paraoxonase (PON) gene family consists of three members, PON1, PON2, and PON3, located adjacent to each other on chromosome 7. PON catalytic activity may be influenced by frequent amino acid variants. Chronic exposure to certain chemicals or to environmental factors causing enhanced lipid peroxidation metabolized by paraoxonases may be a risk factor for sporadic ALS (sALS). OBJECTIVE: The aim of this study was to examine the association between PON1 Q192R, PON1 L55M, and PON2 C311S functional polymorphisms and the risk of sALS in a Polish population. METHODS: The authors included 185 patients with a definite or probable diagnosis of sALS (El Escorial Criteria) and 437 healthy controls of similar age and gender. The paraoxonase polymorphisms were studied by PCR and restriction enzyme digestion. RESULTS: Using logistic regression analyses, the C allele of the C311S polymorphism was associated with sALS in dominant and additive models, whereas the R allele of the Q192R polymorphism was associated with sALS in recessive, additive, and dominant models. The authors compared the distribution of haplotypes between cases and controls. The R-C haplotype was overrepresented among cases (odds ratio 3.44, 95% CI: 1.55 to 7.62, p = 0.002). CONCLUSIONS: Frequent amino acid variants in the paraoxonase 1 and paraoxonase 2 genes are associated with sporadic ALS in a Polish population.

Pharmacology at the Jagiellonian University in Kracow, short review of contribution to global science and cardiovascular research through 400 years of history.

Pharmacology at the Jagiellonian University was taught since the foundation of the University in 1364 under then used names of medical botany and pharmacognosy. The first in Poland Division of Pharmacology and Pharmacognosy was created in 1857. Modern era in the history of pharmacology in Krakow starts in 1929 when Prof. Janusz Supniewski became the Head of the Department. He was the relentless researcher whose scientific interests were as diverse as ranging from anticancer chemotherapeutics, antibiotics, and oral hypoglycemic drugs to lipid-lowering agents. Skills of organic chemistry synthesis were of paramount importance for the scientific achievements. Prof. Supniewski died in 1964, leaving the Department well equipped in instruments. He raised in his laboratory many eminent scientists who later became heads of pharmacology departments throughout Poland. In 1964, the Head of the Department of Pharmacology became Prof. Ryszard Gryglewski. Under his leadership the Department focused scientific efforts on various aspects of cardiovascular pharmacology. Prof. Gryglewski established collaborations with many of the best pharmacology researchers in the world, including Sir John Vane, Nobel Prize laureate. Prof. Gryglewski's assistants had ample opportunity to train in these laboratories and to bring these skills back to Krakow. Prof. Gryglewski and his team published many articles in the most prestigious scientific journals. The most important themes included discovery of prostacyclin, role of nitric oxide and of free radicals for vascular biology. Since 2003, when Prof. Gryglewski retired, the Department of Pharmacology has been led by Prof. Ryszard Korbut.

Polymorphisms in the gene encoding hepatocyte nuclear factor-4alpha and susceptibility to type 2 diabetes in a Polish population.

Recently, several association studies of type 2 diabetes mellitus (T2DM) and the hepatocyte nuclear factor (HNF)-4alpha gene were reported with conflicting results. Our aim was to search for association between two polymorphisms of HNF-4alpha and T2DM in Polish Caucasians. The study groups comprised of 461 T2DM cases and 366 controls. Genotype-quantitative trait analyses were based on the oral glucose tolerance test (OGTT), glucose and insulin results, and comprised 310 glucose-tolerant subjects. All individuals were genotyped for two HNF-4alpha polymorphisms. The frequencies of the minor alleles were as follows: 19.2% in T2DM vs. 17.6% in controls for rs2144908; and 20.6% vs. 20.1% for rs4810424, respectively. The distributions of alleles, genotypes, and haplotypes of the HNF-4alpha polymorphisms did not differ between the study groups (lowest P = 0.41). None of the examined SNPs showed an association in control subjects with quantitative traits of fasting plasma glucose, fasting insulin, as well as plasma glucose and insulin 2 hours after glucose load in OGTT. We conclude that both examined polymorphisms in HNF-4alpha are not associated with T2DM and prediabetic phenotypes in Polish Caucasian study groups of this size.

Involvement and dual effects of nitric oxide in septic shock.

Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.

Protective role of pulmonary nitric oxide in the acute phase of endotoxemia in rats.

We present for the first time direct continuous assay of NO concentration (porphyrinic sensor) in the lung parenchyma of Sprague-Dawley rats in vivo during endotoxemia. Intravenous infusion of lipopolysaccharide (LPS, 2 mg x kg(-1) x min(-1) for 10 minutes) stimulated an acute burst of NO from constitutive NO synthase (NOS) that peaked 10 to 15 minutes after the start of LPS infusion, mirroring a coincident peak drop in arterial pressure. NO concentration declined over the next hour to twice above pre-LPS infusion NO levels, where it remained until the rats died, 5 to 6 hours after LPS infusion. The chronic drop in arterial pressure observed from 70 minutes to 6 hours after the start of LPS infusion was not convincingly mirrored by a chronic increase in NO concentration, even though indirect NO assay (Griess method, assaying NO decay products NO2-/NO3-) showed that NO production was increasing as a result of continuous NO release by inducible NOS. A NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA, 10 mg/kg i.v.) injected 45 minutes before LPS infusion, resulted in sudden death accompanied by macroscopically/microscopically diagnosed symptoms similar to acute respiratory distress syndrome <25 minutes after the start of LPS infusion. Pharmacological analysis of this L-NNA+LPS model by replacing L-NNA with 1-amino-2-hydroxy-guanidine (selective inhibitor of inducible NOS) or by pretreatment with S-nitroso-N-acetyl-penicillamine (NO donor), camonagrel (thromboxane synthase inhibitor), or WEB2170 (platelet-activating factor receptor antagonist) indicated that in the early acute phase of endotoxemia, LPS stimulated the production of cytoprotective NO, cytotoxic thromboxane A2, and platelet-activating factor.

Pneumotoxicity of lipopolysaccharide in nitric oxide-deficient rats is limited by a thromboxane synthase inhibitor.

Both nitric oxide and arachidonic acid metabolites have been implicated in pathogenesis of septic shock. We have recently described a model of endotoxin-induced acute lung injury in rats in which nitric oxide synthase is inhibited. The possible interplay between nitric oxide and eicosanoids (thromboxane A2, prostacyclin) in this model have been presently studied. Animals were randomly assigned to four experimental groups which received the following treatment. 1. Lipopolysaccharide (LPS) infusion only, 2 mg.kg-1min-1 during 10 min (LPS group). 2. N omega-Nitro-L-Arginine 10 mg.kg-1 (L-NNA, nitric oxide synthase inhibitor) pretreatment followed by LPS infusion (L-NNA + LPS group). 3. L-NNA and camonagrel 25 mg.kg-1 (CAM, thromboxane synthase inhibitor) pretreatment followed by LPS infusion (L-NNA + CAM + LPS group). 4. L-NNA and iloprost 0.3 microgram.kg-1.min-1(ILO, stable analog of prostacyclin) pretreatment followed by LPS infusion (L-NNA + ILO + LPS group). LPS infusion resulted in a biphasic response in mean arterial blood pressure. A transient but deep fall in arterial blood pressure was followed by a long-lasting hypotension that led to death after 278 +/- 49 min. L-NNA + LPS rats died within 22 +/- 5 min among the symptoms of systemic hypotension and acute lung injury. In L-NNA + CAM + LPS group a significant attenuation of early phase of hypotension occurred and survival time was comparable with that of the LPS group (298 +/- 68 min). In rats of the L-NNA + ILO + LPS group survival time increased insignificantly to 48 +/- 41 min. It is concluded that immediate deleterious effects of lipopolysaccharide in NO-deficient rats are at least partially mediated by thromboxane A2 while prostacyclin cannot replace NO in its pneumoprotective action.