Pawel Wolkow

Fibrin clot properties in acute ischemic stroke: relation to neurological deficit

INTRODUCTIONnHypercoagulable state occurs in patients with acute vascular events. We wondered whether clot structure/function is altered in acute ischemic stroke (AIS), like in acute myocardial infarction.nnnPATIENTS AND METHODSnIn 45 consecutive patients with AIS (24M, 21F), aged 67.4+/-10.9 years, and 45 healthy controls matched for age and sex, we investigated plasma fibrin clot structure/function by permeation, turbidity, and efficiency of fibrinolysis.nnnRESULTSnCompared to controls, AIS patients produced clots that had 30.5% less porous network (p<0.0001), were less susceptible to fibrinolysis (10.8% longer lysis time, p=0.001), were 20.5% more compact (p<0.0001), had 17.1% higher clot mass (p<0.0001), and showed increased (by 10.2%) overall fiber thickness (p<0.0001) with 8% shorter lag phase of fibrin formation (p=0.0002). Maximum rate of D-dimer release from clots was similar. Multiple regression analyses for all subjects (n=90) showed that being a stroke patient (p<0.0001), fibrinogen (p<0.0001) and lipoprotein(a) (p=0.0075) were independent predictors of clot permeability (model R2 0.79). Only fibrinogen (p<0.0001) and lipoprotein(a) (p=0.0026) predicted lysis time. All other fibrin parameters were predicted only by being a stroke patient. Clot compaction was associated with neurological deficit on admission (r=-0.81; p<0.0001) and at discharge (r=-0.69; p<0.0001). Patients with 0 or 1 point in the modified Rankin scale (n=19) had 13.3% higher clot permeability compared to the remainder (p=0.02).nnnCONCLUSIONSnThis study is the first to show that AIS is associated with unfavorably altered fibrin clot properties that might correlate with neurological deficit.

Variants of the adiponectin gene and type 2 diabetes in a Polish population

Several association studies of type 2 diabetes mellitus (T2DM) and adiponectin gene polymorphisms have been reported with conflicting results. Our aim was to search for the association of three polymorphisms (−11.391G>A, +45T>G, and +276G>T) in the adiponectin gene with T2DM and prediabetic quantitative traits in Polish Caucasians. The study groups comprised 495 unrelated T2DM cases and 435 controls. We compared the distribution of genotypes between study groups. In addition, genotype-quantitative trait analyses were also done in the controls. The study subjects were genotyped using the restriction fragment length polymorphism technique. The frequencies of the minor alleles were as follows: 10.6 versus 8.2% for −11.391G>A (pxa0=xa00.0722), 7.0 versus 8.0% for +45T>G (pxa0=xa00.48), and 15.5% in T2DM versus 19.8% in controls (pxa0=xa00.0145) for +276G>T, respectively. The difference for genotype distribution between the groups was statistically significant (pxa0=xa00.0247) for the +276G>T variant: 71.31 versus 62.99%, 26.46 versus 34.48% and 2.22 versus 2.53%, respectively, for GG, GT and TT. In quantitative traits analysis, the T allele of +276G>T was associated (pxa0<xa00.05) with lower insulin resistance (HOMA-IR, fasting insulin) among controls. Additionally, the A allele at position −11.391 was associated (pxa0<xa00.05) with higher insulin resistance (HOMA-IR, fasting insulin). In multiple regression analysis, all identified association remained significant after the inclusion in the model of gender, BMI and age. In addition, in this model, −11.391G>A and +276G>T were independently associated with T2DM. Finally, we conclude that the adiponectin gene polymorphisms are associated with T2DM and prediabetic quantitative traits in Polish Caucasians.

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.

Enhanced oxidative stress in hypertrophic cardiomyopathy

Elevated plasma levels of inflammation and endothelial dysfunction markers have been reported in patients with hypertrophic cardiomyopathy (HCM). The aim of the current study was to determine whether HCM is associated with enhanced oxidative stress. We enrolled 54 HCM patients with sinus rhythm, including 21 subjects with a left ventricular outflow tract (LVOT) obstruction (gradient >/= 30 mmHg), and 54 age- and sex-matched controls without cardiovascular diseases. Serum levels of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)), a stable marker of oxidative stress, were determined. Serum 8-iso-PGF(2alpha) levels were elevated in HCM patients compared with controls (35.4 +/- 10.2 vs. 29.9 +/- 9.9 pg/ml, p < 0.001). Patients with obstructive HCM displayed higher 8-iso-PGF(2alpha) levels compared with the non-obstructive HCM subgroup (41.6 +/- 12.7 vs. 31.4 +/- 5.4 pg/ml, p < 0.0001). Both anatomic (mitral-septal distance) and hemodynamic (subaortic gradient) indexes of LVOT obstruction, but not other echocardiographic variables, correlated with 8-iso-PGF(2alpha) levels (r = -0.43; p < 0.05 and r = 0.39; p < 0.05, respectively). This study is the first to show that HCM is characterized by enhanced oxidative stress as evidenced by higher 8-iso-PGF(2alpha), which achieves its highest values in the presence of LVOT obstruction in HCM patients.

Lipoprotein profile, plasma Ischemia Modified Albumin and LDL density change in the course of postprandial lipemia. Insights from the LIPGENE study

Abstract Postprandial lipemia is associated with elevated risk of cardiovascular disease. Very little data exists regarding postprandial response in subjects with metabolic syndrome (MetS). The current study was conducted within the LIPGENE EU Integrated Project. Patients were randomized to one of the four isocaloric fatty meals (Oral Fat Tolerance Tests, OFTT): (A) high-fat, saturated fatty acid (SFA)-rich (HFSA), (B) high-fat, monounsaturated fatty acid (MUFA)-rich (HFMUFA), (C) low-fat, high-complex carbohydrate with 1.24 g high oleic sunflower oil supplement (LFHCC) and (D) low-fat high-complex carbohydrate with 1.24 g long chain n-3 poly-unsaturated fatty acid (LC n-3 PUFA) supplement (LFHCCn-3). The total and incremental areas under the curve (tAUC and iAUC) of plasma lipid and lipoprotein, Ischemia Modified Albumin (IMA) and LDL density were examined in patients with MetS to define effect of OFTT. All types of OFTT transiently increased plasma triglyceride and LDL density (LDLdens). It was paralleled by temporal decrease in total cholesterol (TC), LDL cholesterol (LDL-C), and HDL cholesterol (HDL-C). This last effect was partly alleviated in LFHCCn-3 test. A reversible increase of IMA was statistically significant only in the course of HSFA and HMUFA tests. EPA and DHA supplement in combined high complex-carbohydrate meal may attenuate adverse effect of tested meal on LDL particle profile and plasma ischemia modified albumin. No expected associations between measures of central adiposity (waist, WHR), adipose tissue insulin resistance (Adipo-IR), and postprandial responses of TG, TC, LDL-C, HDL-C, LDLdens and IMA/Alb ratio were found in subgroup analysis. Trial registration: ClinicalTrials.gov identifier: NCT00429195.

The effect of the plasma n-3/n-6 polyunsaturated fatty acid ratio on the dietary LDL phenotype transformation – Insights from the LIPGENE study

BACKGROUND & AIMSnLDL phenotype B is associated with obesity, insulin resistance, hypertriglyceridemia and oxidative stress. The effect of plasma n-3/n-6 PUFA ratio on LDL phenotype transformation was investigated.nnnMETHODSnPatients with metabolic syndrome (n=99) received one of four isocaloric diets: (A) High-fat (38% energy) SFA-rich diet (HSFA); (B) High-fat (38% energy), MUFA-rich diet (HMUFA); (C), low-fat (LF) (28% energy), high-complex carbohydrate diet with 1.24g/d oleic sunflower oil (LFHCC) and (D): low-fat (28% energy), high-complex carbohydrate diet, with 1.24g/d LC n-3 PUFA (LFHCC n-3) for 12 weeks. Analysis of plasma lipid profile and LDL phenotype was done pre- and post-interventions.nnnRESULTSnPost-dietary change of LDL density was a main effect observed in all groups. LFHCC n-3 and HFMUFA diets resulted in favorable alteration of LDL phenotype from B to A and decreased LDL density. In contrast, increased LDL density was observed in HSFA and LFHCC groups. The plasma pre-n3/n6 PUFA, Apo E change and pre-Apo CIII/CII ratios explained in 65% the post-dietary change of LDL density in diet LFHCC n-3 consumers.nnnCONCLUSIONSnStudy demonstrates efficacy of dietary n-3 PUFA to modify pro-atherogenic to less atherogenic LDL phenotype in patients with metabolic syndrome. Study identifier at ClinicalTrials.gov was NCT00429195.

The rs2200733 variant on chromosome 4q25 is a risk factor for cardioembolic stroke related to atrial fibrillation in Polish patients.

BACKGROUND AND PURPOSEnA few single nucleotide polymorphisms (SNPs) on chromosome 4q25, associated with atrial fibrillation (AF), are risk factors for ischaemic stroke. We studied the significance of the SNP rs2200733 on chromosome 4q25 in different types of cardioembolic (CE) stroke.nnnMATERIAL AND METHODSnWe genotyped 428 controls and 301 CE stroke patients, among whom 197 (65.4%) presented with high risk sources of embolism (CE stroke related to AF) and 104 with medium risk sources (CE stroke unrelated to AF). The SNP rs2200733 was analysed using real-time polymorphism chain reaction.nnnRESULTSnBoth univariate and multivariate regression analyses showed that the studied variant affected risk of all CE strokes or CE strokes related to AF in recessive and additive mo-dels. The two types of CE stroke differed significantly in demographics and distribution of vascular risk factors.nnnCONCLUSIONSnThe SNP rs2200733 on chromosome 4q25 is a risk factor for CE stroke related to AF only.

The –A162G polymorphism of the PON1 gene and the risk of sporadic amyotrophic lateral sclerosis

BACKGROUND AND PURPOSEnSporadic amyotrophic lateral sclerosis (sALS) is a devastating neurodegenerative disease, which results from complex genetic and environmental interactions. Recent studies have reported an association between several polymorphisms of the PON1 and PON2 genes and risk of sALS. The aim of the present study was to identify an association between the -A162G polymorphism of the promoter region of the human PON1 gene and the risk of sALS in a Polish population.nnnMATERIAL AND METHODSnWe included 259 patients with a diagnosis of definite or probable sALS (76 bulbar onset, 183 limb onset) and 694 healthy controls matched for age and sex. The diagnosis of ALS was established according to El Escorial criteria. The polymorphism was studied by Single Nucleotide Polymorphism Real-Time Polymerase Chain Reaction analysis.nnnRESULTSnNo overall difference in the PON1 -A162G geno-type and allele distribution was seen between cases and controls (all p > 0.05). There was, however, a difference in the A allele frequency when the bulbar onset group was compared to the controls (p = 0.03), but this significance disappeared after the Bonferroni correction.nnnCONCLUSIONSnThe results did not show that the -A162G polymorphism of the PON1 gene is a risk factor of sALS in a Polish population; it may affect, however, bulbar onset of the disease.

Paraoxonase 1 gene polymorphisms do not influence the response to treatment in Alzheimer's disease.

Background: Acetylcholinesterase inhibitors (AChEIs) are the treatment of choice for patients with Alzheimer’s disease (AD). However, their efficacy is moderate and differs from patient to patient. Recent studies suggest that the Q192R variant of the paraoxonase 1 gene (PON1) might affect individual susceptibility to these drugs. Methods: We investigated the influence of 3 single nucleotide polymorphisms (SNPs) in PON1 (rs 662, rs 854560, rs 705381) and the APOE common polymorphism in 101 Polish patients with late-onset AD in response to treatment with AChEIs. Results: No significant differences were observed between carriers and non-carriers of the PON1 SNPs or the APOE common polymorphism in terms of treatment response. These results did not change after stratification of APOE status. Conclusion: Our results suggest that both the investigated PON1 and APOE common SNPs do not influence treatment response to AChEIs in patients with AD.

\"Mitochondrial GWAS and Association of Nuclear - Mitochondrial Epistasis with BMI in T1DM Patients\"

Mitochondria are organelles whose main role is energy production and might influence obesity. They are the only organelles with their own genome. Here we have genotyped 435 patients with type 1 diabetes using Illumina Infinium Omni Express Exome-8 v1.4 arrays and performed mitoGWAS on BMI. We have analyzed additive interactions between mitochondrial and nuclear variants in genes known to be associated with mitochondrial functioning (MitoCarta2.0) and confirmed and refined the results on external cohorts - Framingham Heart Study (FHS) and GTEx data. The linear mixed model analysis was performed using the GENESIS package in R/Bioconductor We have found a nominal association between rs28357980 localized to MT-ND2 and BMI (β=−0.69, p=0.056). This was confirmed on 1889 patients from FHS cohort (β =−0.312, p=0.047). Next, we have searched for additive interactions between mitochondrial and nuclear variants. MT-ND2 variants interacted with variants in SIRT3, ATP5B, CYCS, TFB2M and POLRMT genes. TFB2M is a mitochondrial transcription factor and together with TFAM creates transcription promoter complex for mitochondrial polymerase POLRMT. We have found that the interaction between rs3021088 of MT-ND2 gene and rs6701836 in TFB2M has led to BMI decrease (inter_pval=0.0241), while interaction of rs3021088in MT-ND2 and rs41542013 in POLRMT gene led to BMI increase (inter_pval=0.0004). The influence of these interactions on BMI was confirmed on external cohorts. Here, we have shown that variants in mitochondrial genome as well as additive interactions between mitochondrial and nuclear SNPs influence BMI in T1DM and general cohorts. Author summary Obesity is an epidemic of our times. It is known that it results from an imbalance between energy intake and its expenditure, while mitochondria are organelles whose main role is energy production. They are the only organelles that contain their own genome. Thus, we have genotyped 435 patients with type 1 diabetes and looked on single mitochondrial variant influence as well as on additive interactions between mitochondrial and nuclear variants which might affect BMI. Our analysis has shown, that rs28357980 localized to MT-ND2 is associated with BMI. Next, we looked whether variants in this gene, which builds complex I of the electron transport chain, might interact with nuclear variants and together they modify obesity risk. We focused mainly on mitochondrial biogenesis and found that interactions between variants in TFB2M (rs6701836) or POLRMT (rs41542013) and MT-ND2 (rs3021088) affect patients BMI. TFB2M is a mitochondrial transcription factor which, together with TFAM, creates transcription promoter complex and enables transcription by mitochondrial polymerase POLRMT. The obtained results were also confirmed and refined on external cohorts - Framingham Heart Study (FHS) and GTEx data. Thus, we have shown that variations in mitochondrial genome and its interactions with nuclear variants might have an influence on BMI.