Pavol Janega

Spice up the hypertension diet - curcumin and piperine prevent remodeling of aorta in experimental L-NAME induced hypertension

BackgroundIncrease of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension.MethodsWistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallorys phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated.ResultsThe increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself.ConclusionsAdministration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.

Matrix metalloproteinase 1 and circulating tumor cells in early breast cancer

BackgroundMatrix metalloproteinases (MMPs) are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. The aim of this study was to assess correlation between CTCs and tumor MMP1 in BC.MethodsStudy included 149 primary BC patients treated by surgery from March 2012 to March 2013. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSepTM selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of healthy donors (n = 60) were considered as CTC positive. Expression of MMP1 in surgical specimens was evaluated by immunohistochemistry.ResultsCTCs were detected in 24.2% patients. CTCs exhibiting only epithelial markers were present in 8.7% patients, whereas CTCs with epithelial-mesenchymal transition (EMT) markers (CTC_EMT) were observed in 13.4% of patients and CTCs co-expressing both markers were detected in 2.0% patients. Patients with CTC_EMT in peripheral blood had significantly increased expression of MMP1 in tumor cells (p = 0.02) and tumor associated stroma (p = 0.05) than those of patients without CTC_EMT. In multivariate analysis, CTC_EMT and tumor grade were independently associated with MMP1 expression in cancer cells, while CTC_EMT and Ki67 were independently associated with MMP1 expression in cancer associated stroma.ConclusionOur data suggest link between MMP1 and CTCs with EMT phenotype and support role of MMPs and EMT in tumor dissemination.

Regression of left ventricular hypertrophy and aortic remodelling in NO‐deficient hypertensive rats: effect of l‐arginine and spironolactone

Aim:  We investigated, whether the substrate for nitric oxide (NO) formation –l‐arginine – and the aldosterone receptor antagonist – spironolactone – are able to reverse alterations of the left ventricle (LV) and aorta in Nω‐nitro‐l‐arginine methyl ester (l‐NAME)‐induced hypertension.

Changes in sialic acid expression in the lung during intrauterine development of the human fetus

Sialic acid is a component of glycoproteins that influences enzymatic and receptor functions of cells. During proliferation and differentiation of tissues, sialic acid can serve as a recognition determinant in intercellular communication and interactions of cells with the extracellular matrix. In the present study, sialic acid expression in relation to developmental maturity of the lung has been studied. We analyzed 12 necroptic lung specimens from foetuses of different gestational ages from the 15th week to the neonate. Sections were stained histochemically using 3 lectins specific for sialic acid: Tritrichomonas mobilensis lectin (TML), specific for sialic acid without linkage preference, Sambucus nigra agglutinin (SNA), specific for alpha2,6-linked sialic acid, and Maackia amurensis leucoagglutinin (MAL), specific for alpha2,3-linked sialic acid. MAL positivity dominated over SNA positivity showing prevalence of alpha2,3-linked sialic acids to be homogeneously distributed in the lung at the canalicular stage of development. In more mature lungs, well-differentiated bronchial epithelium showed strong sialic acid expression of both linkages. Sialic acid with alpha2,6 linkage dominated in vascular endothelium. Our results showed a slight decrease in sialic acid expression in lungs with gestational age to a relative minimum before birth. Lectin staining of mature lung tissue showed intense sialic acid expression in alveolar epithelial type II cells. Changes in expression of specific sialic acids during differentiation of the lungs may be useful as marker of the degree of maturity of the foetus.

Alcohol based fixatives provide excellent tissue morphology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens.

Fixation techniques preserving morphological fidelity, protein antigenicity and integrity of nucleic acids can have a high impact on both basic and applied biomedical sciences and diagnostic pathology. Different types of mouse tissues were fixed with neutral buffered formalin, ethanol supplemented with acetic acid and modified methacarn (methanol-Carnoy) fixative. The alcohol-fixed samples were processed in an Autotechnicon tissue processor or in an incubator. The preservation of tissue morphology was assessed in all specimens and the immunoreactivity was evaluated with antibodies specific for proteins with nuclear, membrane or cytoplasmic localization. RNA was extracted from all groups of fixed hind limb skeletal muscle specimens and was assessed versus unfixed tissue for preservation of its quantity and quality by amplification of gene-specific fragments of different lengths. Both alcohol-based fixatives preserved the tissue architecture and the specificity of immunoreactivity in excellent quality; the trimming approach did not result in detectable differences. Oligonucleotide fragments of length between 108 and 577 base pairs were amplified from all groups of alcohol-fixed skeletal muscle specimens in amounts comparative to the unfixed muscle tissue. We conclude that both alcohol-based fixatives are an excellent tool for storage of tissue samples designed for immunohistochemical and mRNA expression studies when the access to fresh samples is limited.

Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine

In our work, we have evaluated efficiency of gene-directed enzyme/prodrug therapy (GDEPT) based on combination of fusion yeast cytosine deaminase (yCD) and 5-fluorocytosine (5FC) on model human medullary thyroid carcinoma (MTC) cell line TT. We determined the efficiency of this GDEPT approach in suicide and bystander cytotoxicity induction. We have shown significant bystander effect in vitro and 5FC administration resulted in potent antitumor effect in vivo. Furthermore, we have unraveled high efficiency of cell-mediated GDEPT, when human mesenchymal stromal cells (MSC) were used as delivery vehicles in direct cocultures in vitro. Nevertheless, effector MSC exhibited inhibitory effect on TT cell proliferation and abrogated TT xenotransplant growth in vivo. We suggest that yCD/5FC combination represents another experimental treatment modality to be tested in MTC and our data further support the exploration of MSC antitumor potential for future use in metastatic MTC therapy.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d‐dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT‐PCR assay for expression of epithelial (CK19) or epithelial‐mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme‐linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Sialic acid expression in autoimmune thyroiditis

Autoimmune diseases of the thyroid gland are among the most frequent endocrine disorders. The present study analyzes expression patterns of sialic acids in these diseases. Three lectins specific for sialic acids were used for the histochemical analysis of surgical specimens of the thyroid gland: Tritrichomonas mobilensis lectin that stains all types of sialic acids, Maackia amurensis leukoagglutinin that stains sialic acids with alpha2,3 linkage and Sambucus nigra agglutinin that stains sialic acids with alpha2,6 linkage. In autoimmune thyroiditis, there was a significant increase in sialic acid expression in epithelial cells, especially on luminal membranes of follicular cells. The alpha2,3 linkage dominated over the alpha2,6 linkage. Lymphocytes of patients with Hashimoto thyroiditis, especially in germinal centers, showed strong expression of alpha2,6-linked sialic acids on their cell membrane. Vascular endothelium was positive in all specimens. It can be concluded, that there is a significant increase in sialic acid expression in autoimmune diseases of the thyroid gland, predominantly of sialic acids with alpha2,3 linkage, whereas the sialylation pattern of lymphocytes in Hashimoto thyroiditis was also different.

Activation of Akt kinase accompanies increased cardiac resistance to ischemia/reperfusion in rats after short-term feeding with lard-based high-fat diet and increased sucrose intake

High-fat or high-carbohydrate food consumption contributes to changes in myocardial tolerance to ischemia. However, with respect to experimental models, most studies used diets with very high doses of cholesterol, saturated fatty acids, or fructose. In our study, we fed rats a high-fat diet based on lard in combination with administration of a sweet beverage (30% sucrose solution) (high-fat sucrose diet [HFS]). This diet was used to simulate the unhealthy dietary habit typical for developed countries. We hypothesized that the application of HFS diet for 48 days might initiate progression of pathologic changes in the heart associated with myocardial remodeling and activation of adaptive mechanisms. We investigated the influence of HFS diet on cardiac function and vulnerability to ischemia-reperfusion (I/R) injury in Langendorff-perfused rat hearts subjected to 30-minute global ischemia and 120-minute reperfusion as well as on Akt kinase and matrix metalloproteinases. We found lower food consumption in HFS group compared with controls, but a significant increase in visceral fat mass and concentrations of triacylglycerol, low-density lipoprotein, and very low-density lipoprotein cholesterol. Baseline heart functional parameters and their postischemic recovery were not affected by HFS diet. On the other hand, hearts of HFS group were more resistant to lethal I/R injury manifested by significantly smaller infarct size. In addition, there was lower content of collagen I and III in the left ventricle associated with Akt kinase activation and matrix metalloproteinase 9 up-regulation. In conclusion, feeding rats with HFS diet resulted in heart remodeling associated with activation of some adaptive mechanisms, which can contribute to modulation of myocardial resistance to I/R injury.

Effect of methotrexate on inflammatory cells redistribution in experimental adjuvant arthritis

The aim of this study was to evaluate the morphological changes in the spleen, the thymus and the knee joints of rats with experimental adjuvant arthritis induced by Mycobacterium butyricum in the incomplete Freund’s adjuvant and the effect of treatment with methotrexate (MTX). Particular attention was aimed on the redistribution of granulocytes in the tissues during the inflammatory process. Clinical parameters, e.g., joint edema, body weight and of gamma glutamyl transferase (GGT) activity as an inflammatory marker, have also been determined. Induction of adjuvant arthritis caused a significant decrease in granulocyte number in the spleen and vice versa a significant increase in the knee joints, but without significant changes in the thymus. Treatment with methotrexate reversed this phenomenon by increasing the granulocyte number in the spleen and decreasing it in knee joints. MTX decreased the joint edema as well as the activity of GGT in the spleen, modified the size of the white pulp of the spleen and increased the cortex/medulla ratio in the thymus. The observed changes support the anti-inflammatory and immunomodulatory properties of MTX supporting its use as the first-line medication in patients with rheumatoid arthritis.