Monika Gora

Altered Gene Expression Pattern in Peripheral Blood Mononuclear Cells in Patients with Acute Myocardial Infarction

Background Despite a substantial progress in diagnosis and therapy, acute myocardial infarction (MI) is a major cause of mortality in the general population. A novel insight into the pathophysiology of myocardial infarction obtained by studying gene expression should help to discover novel biomarkers of MI and to suggest novel strategies of therapy. The aim of our study was to establish gene expression patterns in leukocytes from acute myocardial infarction patients. Methods and Results Twenty-eight patients with ST-segment elevation myocardial infarction (STEMI) were included. The blood was collected on the 1st day of myocardial infarction, after 4–6 days, and after 6 months. Control group comprised 14 patients with stable coronary artery disease, without history of myocardial infarction. Gene expression analysis was performed with Affymetrix Human Gene 1.0 ST microarrays and GCS3000 TG system. Lists of genes showing altered expression levels (fold change >1.5, p<0.05) were submitted to Ingenuity Pathway Analysis. Gene lists from each group were examined for canonical pathways and molecular and cellular functions. Comparing acute phase of MI with the same patients after 6 months (stable phase) and with control group we found 24 genes with changed expression. In canonical analysis three pathways were highlighted: signaling of PPAR (peroxisome proliferator-activated receptor), IL-10 and IL-6 (interleukin 10 and 6). Conclusions In the acute phase of STEMI, dozens of genes from several pathways linked with lipid/glucose metabolism, platelet function and atherosclerotic plaque stability show altered expression. Up-regulation of SOCS3 and FAM20 genes in the first days of myocardial infarction is observed in the vast majority of patients.

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)‐like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO‐like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR‐B, and here report our findings on the location of IDR‐A and its relationship to IDR‐B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR‐B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR‐A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR‐A was found to lie immediately adjacent to IDR‐B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR‐B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.

Gene expression profiling reveals potential prognostic biomarkers associated with the progression of heart failure

BackgroundHeart failure (HF) is the most common cause of morbidity and mortality in developed countries. Here, we identify biologically relevant transcripts that are significantly altered in the early phase of myocardial infarction and are associated with the development of post-myocardial infarction HF.MethodsWe collected peripheral blood samples from patients with ST-segment elevation myocardial infarction (STEMI): n = 111 and n = 41 patients from the study and validation groups, respectively. Control groups comprised patients with a stable coronary artery disease and without a history of myocardial infarction. Based on plasma NT-proBNP level and left ventricular ejection fraction parameters the STEMI patients were divided into HF and non-HF groups. Microarrays were used to analyze mRNA levels in peripheral blood mononuclear cells (PBMCs) isolated from the study group at four time points and control group. Microarray results were validated by RT-qPCR using whole blood RNA from the validation group.ResultsSamples from the first three time points (admission, discharge, and 1 month after AMI) were compared with the samples from the same patients collected 6 months after AMI (stable phase) and with the control group. The greatest differences in transcriptional profiles were observed on admission and they gradually stabilized during the follow-up. We have also identified a set of genes the expression of which on the first day of STEMI differed significantly between patients who developed HF after 6 months of observation and those who did not. RNASE1, FMN1, and JDP2 were selected for further analysis and their early up-regulation was confirmed in HF patients from both the study and validation groups. Significant correlations were found between expression levels of these biomarkers and clinical parameters. The receiver operating characteristic (ROC) curves indicated a good prognostic value of the genes chosen.ConclusionsThis study demonstrates an altered gene expression profile in PBMCs during acute myocardial infarction and through the follow-up. The identified gene expression changes at the early phase of STEMI that differentiated the patients who developed HF from those who did not could serve as a convenient tool contributing to the prognosis of heart failure.

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

BackgroundThe yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism.ResultsStatins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis.ConclusionStatins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KMs for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.

miR-22-5p revealed as a potential biomarker involved in the acute phase of myocardial infarction via profiling of circulating microRNAs

Acute myocardial infarction (AMI) is a life-threatening episode of coronary artery disease. Recently, circulating myocardial-derived microRNAs (miRNAs) have been reported as potential biomarkers of infarction. The present study aimed to identify differentially expressed miRNAs in patients with ST-segment elevation myocardial infarction that could be potentially dysregulated in response to early myocardial damage. miRNA expression profile analysis was performed using the Serum/Plasma Focus miRNA Polymerase Chain Reaction (PCR) panel of Exiqon A/S (Vedbaek, Denmark) on plasma samples of patients on the first day of AMI (admission) and on samples from the identical patients collected six months following AMI. Selected miRNAs were validated by reverse transcription‑quantitative PCR (RT‑qPCR) using independent patients with AMI and a control group of patients with a stable coronary artery disease. Thirty‑two species of plasma miRNA were differentially expressed (P<0.05) on admission compared with six months following AMI. Subsequent validation in an independent patient group confirmed that miR‑133b and miR‑22‑5p were significantly up‑regulated in the serum of patients with AMI. The receiver operating characteristic (ROC) curve analysis demonstrated a diagnostic utility for miR-22-5p, which has not previously been reported to be associated with AMI. Among the selected miRNAs, miR‑22‑5p represents a novel promising biomarker for the diagnosis of AMI.

Interindividual variability of atorvastatin treatment influence on the MPO gene expression in patients after acute myocardial infarction

Myeloperoxidase (MPO) and C-reactive protein (CRP) may play critical roles in generation of oxidative stress and the development of the systemic inflammatory response. The aim of the study was to determine the effect of atorvastatin therapy on the MPO gene expression and its plasma level in relation to lipids level lowering and an anti-inflammatory response in patients after acute myocardial infarction. The research material was represented by 112 samples. Thirty-eight patients with first AMI receiving atorvastatin therapy (40 mg/day) and followed up for one month were involved in the study. The relative MPO gene expression in peripheral blood mononuclear cells (PBMCs) was examined using RT-qPCR in 38 patients before-, 38 patients after-therapy and in 36 patients as the control group. The plasma concentrations of MPO and serum concentrations of biochemical parameters were determined using commercially available diagnostic tests. After one month of atorvastatin therapy, in 60.5% patients a decrease of MPO gene expression, whereas in 39.5% patients an increase, was observed. The plasma MPO levels behaved in the same way as the MPO gene expression. However, the serum lipids and CRP concentrations were significantly lower after one month of atorvastatin therapy in both groups of patients - with decreased and increased MPO gene expression. Atorvastatin exhibited a different effect on MPO gene expression and its plasma level. Short-term atorvastatin therapy resulted in lipid lowering and anti-inflammatory activity in patients after AMI, independently of its effect on MPO gene expression. The molecular mechanisms of this phenomenon are not yet defined and require further research.

Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment

The main objective of the study is molecular and biological characterization of the human‐yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia.

Will global transcriptome analysis allow the detection of novel prognostic markers in coronary artery disease and heart failure

Coronary artery disease (CAD) is one of the leading causes of death in the developed countries. Myocardial infarction (MI) is an acute episode of CAD that results in myocardial injury and subsequent heart failure (HF). In the acute phase of MI several risk factors for future cardiovascular events have been found. The molecular mechanisms of these disorders are still unknown, but altered gene expression may play an important role in the development and progression of cardiovascular diseases. High-throughput techniques should greatly facilitate the elucidation of the mechanisms and provide novel insights into the pathophysiology of cardiovascular diseases. In this review we focus on the perspectives of gene-expression profiling conducted on cardiac tissues and blood for the determination of novel diagnostic and prognostic markers and therapeutic targets.