Diet in Prevention and Treatment of Endometriosis: Current State of Knowledge.

PURPOSE OF REVIEW: Endometriosis (EM) is a chronic gynecological disease that affects about 10% of women worldwide. It is characterized by the implantation of endometrial cells at ectopic sites. The most common symptom of EM is painful menstruation, which can often lead to chronic pelvic pain that significantly worsens the quality of life. Because some disease-related processes, such as inflammation, hormonal activity, menstrual cycle, or prostaglandin metabolism, can be modified by diet, nutrition may have a significant impact on development and treatment of EM. The purpose of this article was to overview the current knowledge regarding the dietary management of endometriosis. RECENT FINDINGS: The attention of researchers has so far concentrated mainly on the role of nutrition in the risk of developing EM, while less attention has been paid to examining the use of diet in the treatment of the disease. Current studies focus primarily on various dietary components that have antiproliferative, anti-inflammatory, antioxidant, analgesic, and estrogen-lowering properties. Exploring different ways of coping with endometriosis can make a significant contribution to improving the quality of life of women at risk or diagnosed with EM.

Errate: Dysregulation of Inflammation, Oxidative Stress, and Glucose Metabolism-Related Genes and miRNAs in Visceral Adipose Tissue of Women with Type 2 Diabetes Mellitus.

The authors informed the journal that errors occurred in their manuscript, and were not noticed by the authors during the proofreading. Corrections: 1. Figure 1, top entry: "Predipocytes" should read "Preadipocytes". 2. Figure 3, chart "TIGAR": "-9" value on y axis should read "-8". 3. Figure 4, chart "let-7g-5p": the upper "-4" value on y axis should read "0". 4. Figure 5: the title of the bottom right chart should read "TIGAR". 5. Figure 6, chart "miR-26a-5p": the values on y axis should read from the top: 2, 1, 0, -1, -2. 6. Figure 6, chart "miR-374a-5p": the values on y axis should read from the top: 0, -1, -2, -3, -4. 7. Table 4., in the 6 rows from the bottom: in column "miRNAs", "hsa-miR-21-5" should read "hsa-miR-21-5p". 8. Supplementary Table1, 1st column on the left: "TG-HDL" should read "TG/HDL" Reference: Adam Wróblewski, Justyna Strycharz, Katarzyna Oszajca, Piotr Czarny, Ewa Swiderska, Tomasz Matyjas, Andrzej Zieleniak, Monika Rucinska, Lech Pomorski, Józef Drzewoski, Agnieszka Sliwinska, Janusz Szemraj: Dysregulation of Inflammation, Oxidative Stress, and Glucose Metabolism-Related Genes and miRNAs in Visceral Adipose Tissue of Women with Type 2 Diabetes Mellitus. Med Sci Monit, 2023; 29: e939299. DOI: 10.12659/MSM.939299.

Dysregulation of Inflammation, Oxidative Stress, and Glucose Metabolism-Related Genes and miRNAs in Visceral Adipose Tissue of Women with Type 2 Diabetes Mellitus.

BACKGROUND Human visceral adipose tissue (VAT), now identified as an endocrine organ, plays a significant role in impaired fasting glucose and diabetes through the deregulated metabolism and adipogenesis of visceral adipocytes in obesity. Our study focuses on exploring the link between inflammation, oxidative stress, and glucose metabolism-associated genes with corresponding miRNAs in human visceral adipocytes and VAT from individuals with glucose metabolism disorders. MATERIAL AND METHODS We examined the expression of ATM, NFKB1, SOD2, INSR, and TIGAR, along with their related miRNAs using PCR, in two contexts:1 - During the three-stage visceral adipogenesis under normal glucose levels (5.5 millimoles), intermittent, and chronic hyperglycemia (30 millimoles).2 - In visceral adipose tissue from subjects (34 F, 18 M) with normal glucose metabolism, impaired fasting glucose, and type 2 diabetes mellitus. RESULTS Both chronic and intermittent hyperglycemia similarly influenced ATM, NFKB1, TIGAR, SOD2, INSR gene expression in visceral adipocytes, with corresponding changes in a few tested miRNAs (eg, let-7g-5p, miR-145-5p, miR-21-5p). Anthropometric and biochemical parameters led us to focus on female subjects. Our results showed transactivation of NFKB1, TIGAR, miR-10b-5p, miR-132-3p, miR-20a-5p, miR-21-5p, and miR-26a-5p exclusively in type 2 diabetes mellitus. Upregulated molecules (excluding miR-10b-5p and miR-20a-5p) positively correlated with glucose metabolism markers. CONCLUSIONS The genes studied may undergo miRNA interferences and hyperglycemic memory in visceral adipocytes under hyperglycemic conditions. VAT from women with type 2 diabetes mellitus, but not with impaired fasting glucose, showed transactivated miRNAs and a molecular dysregulation of TIGAR and NFKB1, possibly enhancing inflammation, oxidative stress, and disrupted glucose metabolism. These findings highlight the epigenetic and molecular disturbances in VAT related to glucose metabolism abnormalities. However, additional research is necessary to further understand their biological significance.

The Impact of Short-Term Shark Liver Oil Supplementation on the Fatty Acid Composition of Erythrocyte Membranes.

Fatty acid (FA) balance is strictly related to human health. The composition of fatty acids in lipid membranes seems to be influenced by diet. Shark liver oil (SLO) supplementation has been widely used recently in the prevention and treatment of human diseases. We analyzed the impact of short-term SLO supplementation on certain biochemical parameters and erythrocyte FA composition in a group of young healthy women. Our results showed that 6 weeks of SLO supplementation led to a significant decrease in C-reactive protein levels in sera and intracellular cholesterol levels in peripheral blood mononuclear cells. SLO supplementation caused a significant increase in the content of the polyunsaturated omega-3 FAs: docosahexaenoic acid, docosapentaenoic acid and α-linolenic acid. In the group of omega-6 FAs, we observed a significant elevation of arachidonic and dihomo-gamma-linoleic acid content. Due to these alterations, the omega-3 index increased significantly from 3.6% (before) to 4.2% (after supplementation). We also observed the impact of SLO supplementation on the membrane fluidity index. The ratio between saturated and unsaturated FAs decreased significantly from 13.1 to 9.9. In conclusion, our results show that even short-term SLO supplementation can improve human erythrocyte fatty acid composition and other parameters that may have health-promoting consequences.

Assessment of the correlation between oxidative stress and expression of MMP-2, TIMP-1 and COX-2 in human aortic smooth muscle cells.

INTRODUCTION: Smooth muscle cells (SMCs) are considered to be the main producer of matrix metalloproteinase-2 (MMP-2) participating primarily in extracellular matrix (ECM) remodeling. Any disturbances in ECM structure may underlie the pathogenesis of many cardiovascular diseases and contribute to angiogenesis, cancer development, invasion or metastasis. The purpose of the study was to examine the effect of oxidative stress on the expression of MMP-2, its tissue inhibitor type 1 (TIMP-1) and cyclooxygenase-2 (COX-2) in human aortic smooth muscle cells (HASMCs). MATERIAL AND METHODS: HASMCs were treated with exogenously applied H2O2 or TNF-α. N-acetylcysteine (NAC) was used as an antioxidant. Gene expression levels were measured by real-time PCR and the protein levels were determined using ELISA assay. RESULTS: The studies revealed no association between oxidative stress and either mRNA quantity or protein secretion of MMP-2 and TIMP-1. However, we found markedly reduced (p < 0.001) MMP-2 secretion in cells incubated with NAC. HASMCs stimulated with TNF-α demonstrated a significantly increased COX-2 mRNA level as well as enzyme activity. H2O2-induced cells showed lowered COX-2 activity in comparison to untreated cells. MMP-2 and TIMP-1 expression did not change after COX-2 inhibition with DuP-697. CONCLUSIONS: We did not find any effect of oxidative stress on expression of MMP-2 and TIMP-1 in HASMCs. However, COX-2 mRNA and protein level were elevated in these conditions. There was no correlation between COX-2 activity and MMP-2 and TIMP-1 mRNA expression or protein secretion.

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.

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.

BACKGROUND: 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. METHODS: We performed a case-control study of 99 Polish children hospitalized due to IH and compared them with matched healthy control subjects. The polymorphisms were ascertained through genotyping by PCR-RFLP assay, PCR-HRM, or the allelic discrimination method. RESULTS: The study revealed a lower odds of infantile hemangioma in individuals with GG genotype or G allele for +405 G/C VEGF-A polymorphism (ORdis = 0.52, P = 0.023 and ORdis  = 0.63, P = 0.025, respectively). No association was observed for the remaining VEGF and VEGFR-2 polymorphisms and IH risk. CONCLUSIONS: In our study, none of the investigated VEGF-A and VEGFR-2 genes polymorphisms was found to be an independent prognostic marker for infantile hemangioma. However, there is evidence that individuals carrying at least one G allele of +405 G/C VEGF-A polymorphism have significantly lower risk of IH.

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 AND 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.

Treatment with platelet-derived growth factor (PDGF) and rock inhibitors is related to declined nerve growth factor (NGF) signaling in an experimental model of rat pulmonary hypertension.

BACKGROUND: Recent studies reveal that nerve growth factor (NGF) plays a critical role in the pathobiology of pulmonary hypertension (PH). The aim of the present study is to clarify the relationship between NGF signaling and treatment with PDGF or ROCK inhibitors in an animal model of PH. METHODS: Lung tissues were obtained from animals with monocrotaline (MCT)-challenged PH which had been administered long term imatinib, fasudil or statin. Reversal of disease was indicated by decreases in right ventricle pressure (RVP) and hypertrophy. NGF expression was examined at the mRNA and protein levels using quantitative real-time PCR reaction and ELISA. RESULTS: MCT significantly increased NGF mRNA and protein content in lung tissue. ROCK inhibitor (fasudil) and PDGF inhibitor (imatinib) caused significant decreases in NGF mRNA and protein content when administered alone, with no further effects noted when used in combination. CONCLUSION: The beneficial reversal of MCT-mediated effects in PH caused by PDGF or ROCK inhibition may be also partially mediated by decreased NGF signaling.

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 AND 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.

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 AND 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, ß, 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.

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 ß 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 ß 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 ß 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 ß chain gene is a potential genetic marker to identify the risk of AAA.

The Bcl I single nucleotide polymorphism of the human glucocorticoid receptor gene h-GR/NR3C1 promoter in patients with bronchial asthma: pilot study.

Bcl I in the promoter polymorphism observed within h-GR/NR3C1 gene may play an important role in the development of bronchial asthma and resistance to GCs in the severe bronchial asthma. The aim of the investigation was to study the correlation between this h-GR/NR3C1 gene polymorphism and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 70 healthy volunteers and 59 asthma patients. Structuralized anamnesis, spirometry and allergy skin prick tests were performed in all participants. Genotyping was carried out with PCR-RFLP method. In healthy, non-atopic population variants of Bcl I: GG, GC, CC were found with frequency 0.129/0.471/0.400, respectively. In asthma patients Bcl I: GG, GC, CC occurred with respective frequencies of 0.410/0.462/0.128. Chi-square analysis revealed a significantly different (P<0.05) distribution between cases and controls for the Bcl I polymorphism. The Bcl I polymorphism of h-GR/NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of severe form and resistance to GCs in Polish population.

Frequencies of Bcl I, E22E, and N363S of h-GR/NR3C1 restriction fragment length polymorphisms of glucocorticoid receptor gene in Polish adult population.

BACKGROUND: Polymorphism of the gene encoding the glucocorticoid receptor - h-GR/NR3C1 demonstrates close genetic and biochemical correlation with etiopathogenesis of metabolic, cardiovascular, hematologic, and mental disorders. The natural variability of DNA sequence within the h-GR gene affects both the conformation and the activity of glucocorticoid receptors. Modifications of the amino acid receptor structure give rise to disturbances of the receptor-hormone complex interaction with many genes responsible for normal cellular function. Transactivation, or transrepression, of the genes encoding proteins synthesized within the framework of cellular response to glucocorticosteroids is 1 among many molecular pathways leading to the development of resistance to anti-inflammatory drugs. MATERIAL/METHODS: A group of 70 healthy participants, with no history of asthma or atopic conditions, qualified for the study. Genotyping was accomplished using PCR-RFLP method. RESULTS: In healthy, nonatopic population, within the h-GR gene promoter, a polymorphism of Bcl I: GG, GC, and CC occurring with 0.129/0.471/0.40 frequency, were identified. Two polymorphisms were identified in exon 2 at 1220 and 198 position in the h-GR gene: N363S (AA, AG occurring with 0.9/0.1 frequency) and ER22/23EK (GG, GC occurring with 0.843/0.157 frequency). CONCLUSIONS: The frequency of polymorphisms occurring within the h-GR gene has not been assessed to date in the Polish population. The present study is the first attempt of such estimation. The study is an introduction to more-detailed analysis of the correlation between the occurrence of h-GR gene polymorphisms, and the development of severe, steroid-resistant bronchial asthma.

Manganese superoxide dismutase (MnSOD) gene (Ala-9Val, Ile58Thr) polymorphism in patients with age-related macular degeneration (AMD).

BACKGROUND: Oxidative stress is involved in the pathogenesis of many chronic disorders including cancer, inflammation, and neurologic diseases. Reactive oxygen species (ROS) may play a major role in age-related macular degeneration (AMD). This study investigated the mRNA and protein profiles of manganese superoxide dismutase MnSOD in patients with AMD and healthy controls, while examining its genetic sequence polymorphism (Ala-9Val, Ile58Thr). Our intent was to find a correlation between the expression of MnSOD genes and nucleotide sequence polymorphisms encoded in the gene of the dry and wet form of AMD. MATERIAL/METHODS: We examined 300 unrelated AMD patients and 300 unrelated healthy controls who gave free consent to participate in the study. The MnSOD gene polymorphisms were determined by PCR/RFLP method. We also used real-time RT-PCR and ELISA methods to estimate expression of MnSOD mRNA and protein. RESULTS: There were statistically significant differences in the genotype distribution between patients with AMD and controls. Our results showed positive correlations between gene sequence polymorphism and the level of MnSOD mRNA and protein expression. The Ala-9Ala genotype and alanine allele (Ala-9Val sequence polymorphism) is much more frequent in AMD patients than in healthy subjects. Healthy controls who are homozygotes Val/Val and heterozygotes Ala/Val showed lower expression of the MnSOD gene as compared to homozygote Ala/Ala. The lowest expression of MnSOD (homozygotes Val/Val and heterozygotes Ala/Val for wet and dry form of AMD) was noted in patients with AMD. CONCLUSIONS: These data suggest a genetic role of MnSOD polymorphism in the development of age-related degeneration.

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 Pearson's 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.

Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant.

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.

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.

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.