Katarzyna Oszajca

The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients

Lung cancer is a particular challenge in oncology. More than 1 million new cases occur worldwide every year and despite many clinical trials and modern diagnostic techniques, long-term survival rate has only marginally improved. The aim of the current research is to explore new molecular prognostic factors and identify new targets for anticancer therapy. Current evidence shows that angiogenesis is controlled by several angiogenic factors including VEGF and BMP-2. It has been also demonstrated that VEGF plays a key role in this process that is essential in carcinogenesis. Our study has shown that the expressions of the VEGF, BMP-2 and BMP-4 mRNAs were significantly higher (7.1-fold, 25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues (real-time RT-PCR). Analysis based on the Pearsons correlation coefficient indicated the positive correlation between VEGF and BMP-2 gene expression, whereas no significant correlation between VEGF and BMP-4 gene expression was found. The mean+/-standard deviation serum level of VEGF was 423+/-136 pg/ml. Significant differences in the serum levels of VEGF between patients with T1 tumors and patients with T2, T3 or T4 tumors were observed. Patients with T2, T3 and T4 tumors, respectively, had 1.6-fold, 1.8-fold and 2.3-fold greater serum levels of VEGF than their peers with T1 tumors. In current study patients homozygous for the 936T allele of the +936C/T VEGF gene polymorphism had 12-fold lower VEGF gene expression and 1.3-fold lower VEGF serum level than patients homozygous for the 936C allele. In conclusion, our findings underline the importance of the two angiogenic factors namely VEGF and BMP-2 as well as +936C/T VEGF gene polymorphism in the evaluation of lung cancer patients.

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant - An in-vivo study

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Effect of oxidative stress on the expression of t-PA, u-PA, u-PAR, and PAI-1 in endothelial cells.

In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-kappaB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-kappaB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-kappaB and AP-1 are involved in this regulation.

Serum MicroRNAs as Potential Biomarkers of AMD

Background Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Circulating microRNAs (miRNAs) in serum have emerged as novel candidate biomarkers for many diseases. The aim of the present study was to identify a serum microRNA (miRNA) expression profile specific for dry and wet forms of AMD. Material/Methods Serum miRNA expression was first screened using TaqMan® Human MicroRNA Array A (Applied Biosystems). An extensive, self-validated, individual, quantitative RT-PCR (qRT-PCR) study was then performed on a cohort of 300 AMD patients (150 wet form and 150 dry form) and 200 controls. The Mann-Whitney U test and nonparametric Spearman’s rank correlation coefficient were used for statistical analysis. Results miRNA expression analysis revealed increased expression of miR661 and miR3121 in serum of patients with dry AMD and miR4258, miR889, and Let7 in patients with wet form. Expression of analyzed miRNA was not observed or remained at low level in controls. Conclusions Differences in miRNA serum profile exist between patients with wet and dry form of AMD, which indicates miRNAs as potential biomarkers of AMD. Further studies should be performed to confirm its significance in clinical practice.

The study of t-PA, u-PA and PAI-1 genes polymorphisms in patients with abdominal aortic aneurysm

The most important feature of abdominal aortic aneurysm (AAA) pathogenesis is an enzymatic degradation of elastic lamellae and extracellular matrix proteins particularly with participation of matrix metalloproteinases. Plasmin, which is responsible for the dissolution of fibrin in blood vessels, plays also a key role in the cascade for activation of the metalloproteinases. The purpose of this study was to evaluate the influence of selected polymorphisms in genes coding for tissue plasminogen activator (−7351 C/T polymorphism), urokinase-type plasminogen activator (1788 C/T polymorphism) and plasminogen activator inhibitor 1 (−675 4G/5G and −844 G/A polymorphism) on the susceptibility to AAA. We performed a case–control study of 153 polish patients hospitalized due to AAA and compared them with matched healthy control subjects. The polymorphisms were ascertained through genotyping by polymerase chain reaction and restriction digestion of amplified fragments or through high-resolution melting analysis. In this study we have found lower frequency of wild-type GG genotype of the −844G/A PAI-1 polymorphism in cases than in controls, what may suggest the protective effect of this genotype for the risk of AAA development. None of the remaining polymorphisms tested were associated with AAA occurrence.

Association analysis of genetic polymorphisms of factor V, factor VII and fibrinogen β chain genes with human abdominal aortic aneurysm

Increased activity of the coagulation system is associated with the increased risk of many arterial thrombotic diseases and atherosclerosis. The purpose of this study was to evaluate the influence of selected polymorphisms in genes coding for coagulation factor V (1691 G/A, the so-called Leiden mutation), factor VII (−323 0/10 bp insertion/deletion) and fibrinogen β chain (−455 G/A) on the risk of abdominal aortic aneurysm, a particular form of atherothrombosis. We conducted a case-control study of 153 Polish patients hospitalized due to abdominal aortic aneurysm (AAA) and compared the results to those obtained from matched healthy control subjects. The polymorphisms were ascertained through genotyping by polymerase chain reaction and restriction digestion of amplified fragments. The study revealed that individuals carrying heterozygous genotype GA for the fibrinogen β chain −455 G/A mutation had at least a 2-fold greater likelihood of AAA development compared to control subjects (OR=3.01; 95% CI 1.83–4.96). The cases possessing homozygous mutant genotype (AA) had no significant risk of developing AAA compared to the control subjects (OR=1.12; 95% CI 0.33–2.44; p=0.83). Concerning factor V 1691 G/A and factor VII −323 0/10 bp mutations, we did not find any statistically significant correlation between them and AAA occurrence. In conclusion, we suggest that the −455G/A polymorphism of the fibrinogen β chain gene is a potential genetic marker to identify the risk of AAA.

New Type of BACE1 siRNA Delivery to Cells

Background Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. Material/Methods An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, β, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. Results Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP – INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. Conclusions Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity.

Association analysis of genetic polymorphisms and expression levels of selected genes involved in extracellular matrix turnover and angiogenesis with the risk of age-related macular degeneration

ABSTRACT Background: Age-related macular degeneration is a progressive eye disease affecting the macula and causing acute visual loss particularly in elder people. The aim of the study was an attempt to discern an influence of expression levels and functional genetic polymorphisms of selected genes related to the extracellular matrix turnover or neovascularization on age-related macular degeneration occurrence and progression. Methods: We conducted a case-control study of 200 polish patients with recognized age-related macular degeneration (dry and wet) and compared the results with those obtained from matched 100 healthy control subjects. TaqMan Genotyping Assays were employed to examine the following single nucleotide polymorphisms: matrix metalloproteinase (MMP)-2 −735C/T, MMP-7 −181A/G, MMP-9 −1702T/A, and −1562C/T; tissue inhibitors of metalloproteinase (TIMP)-2 −418G/C; vascular endothelial growth factor (VEGF) +405 G/C and +936 C/T, VEGFR-2 +1719 T/A and −271 G/A. Real-time polymerase chain reaction was assessed to determine the mRNA quantity. Serum levels of proteins were measured using enzyme-linked immunosorbent assay. Results: The single nucleotide polymorphism genotyping showed that TT genotype for MMP-9 −1702T/A and CC genotype for VEGF +936C/T increase markedly the risk of age-related macular degeneration but do not influence on its progression. Additionally, the possible protective effect of CC genetic variant in MMP-9 −1562C/T polymorphism against progression of age-related macular degeneration was observed. We also found significant differences in systemic expression levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, VEGFR-2, and pigment epithelium-derived factor between studied group. The research demonstrated evident differences in serum levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, and pigment epithelium-derived factor between wet and dry age-related macular degeneration patients. Conclusions: We can conclude that disturbances in angiogenic homeostasis and processes of extracellular matrix turnover occurring in age-related macular degeneration-affected ocular tissues may be reflected in changes in systemic expression levels of the investigated genes.

Single-nucleotide polymorphisms of VEGF-A and VEGFR-2 genes and risk of infantile hemangioma

Infantile hemangioma (IH) is the most common vascular tumor of childhood and infancy. It is distinguished by rapid proliferation of endothelial cells during the first year of life followed by spontaneous regression thereafter. One of the possible factors responsible for the IH development is vascular endothelial grow factor (VEGF). The purpose of this study was to evaluate the influence of selected polymorphisms in the genes coding for VEGF‐A (+405 G/C, rs2010963; +936 C/T, rs3025039) and its receptor VEGFR‐2 (+1416 T/A, rs1870377; –271 G/A, rs7667298) on the susceptibility to infantile hemangioma.

MicroRNA Expression Analysis in Serum of Patients with Congenital Hemochromatosis and Age-Related Macular Degeneration (AMD)

Background Congenital hemochromatosis is a disorder caused by mutations of genes involved in iron metabolism, leading to increased levels of iron concentration in tissues and serum. High concentrations of iron can lead to the development of AMD. The aim of this study was to analyze circulating miRNAs in the serum of congenital hemochromatosis patients with AMD and their correlation with the expression of genes involved in iron metabolism. Material/Methods Peripheral blood monolayer cells and serum were obtained from patients with congenital hemochromatosis, congenital hemochromatosis and AMD, AMD patients without congenital hemochromatosis, and healthy controls. Serum miRNAs expressions were analyzed by RT-PCR (qRT-PCR) using TaqMan MicroRNA probes, and proteins levels were measured by ELSA kits. Gene polymorphisms in TF and TFRC genes were determined using the TaqMan discrimination assay. Results Statistical analysis of the miRNAs expressions selected for further study the miR-31, miR-133a, miR-141, miR-145, miR-149, and miR-182, which are involved in the posttranscriptional expression of iron-related genes: TF, TFRI, DMT1, FTL, and FPN1. It was discovered that the observed changes in the expressions of the miRNAs was correlated with the level of protein in the serum of the analyzed genes. There were no statistically significant differences in the distribution of genotype and allele frequencies in TF and TFRC genes between analyzed groups of patients. Conclusions The differences studied in the miRNA serum profile, in conjunction with the changes in the analyzed protein levels, may be useful in the early detection of congenital hemochromatosis in patients who may develop AMD disease.