Gabriel Bodek

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

A Novel Approach of Targeted Ablation of Mammary Carcinoma Cells Through Luteinizing Hormone Receptors using Hecate-CGβ Conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CGβ conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CGβ-chain in vitro. To test the hypothesis that the Hecate-CGβ conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CGβ conjugate and Hecate alone. Cytotoxic effects of the Hecate-CGβ conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CGβ conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CGβ conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC50) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CGβ for in vivo trials.

Targeted Ablation of Prostate Carcinoma Cells Through LH Receptor Using Hecate-CGβ Conjugate: Functional Characteristic and Molecular Mechanism of Cell Death Pathway

A Hecate-CGβ conjugate (lytic peptide and β-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.

Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo-and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units ± SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 ± 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 ± 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 ± 2.5; ampulla 35.6 ± 4.2; infundibulum 24.7 ± 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.

Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites

BackgroundThe interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.MethodsThe primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated.ResultsWe received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release.ConclusionsTNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.

Identification of pluripotent cells in bovine uterus: in situ and in vitro studies.

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8-10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

Growth Repression in Diethylstilbestrol/Dimethylbenz[a]anthracene–Induced Rat Mammary Gland Tumor Using Hecate-CGβ Conjugate

Recently, we have shown that Hecate-CGβ conjugate, which is a fusion of the lytic peptide Hecate and a 15–amino acid fragment of the β-chain of chorionic gonadotropin (CGβ), selectively destroys mammary gland carcinoma cells that possess luteinizing hormone receptors (LHR) in vitro. We induced mammary gland tumors using combined prenatal exposure to synthetic diethylstilbestrol (DES) and additional postnatal exposure to dimethylbenz[a]anthracene (DMBA). Rats with tumors were equally randomized (10/group) and treated with either sham (control) or 12 mg/kg body wt of either Hecate or Hecate-CGβ once a week for 3 weeks by tail vein injections. One week after the last injection, rats were kilted. Reverse-transcription–nested polymerase chain reaction/Southern blotting revealed alternatively spliced mRNA for LHR in tumor tissues of 5 of 30 females, which was further confirmed by Western blot analysis. The percentage of tumor volume increase was lowest in the group treated with Hecate-CGβ (45.3 ± 27.6), compared with Hecate- and shamtreated, control group (324.8 ± 78.1 and 309.9 ± 51.2, respectively; P < 0.001). Hecate-CGβ induced a significant decrease in tumor burden compared with controls (9.5 ± 2.1 mg/g body wt vs. 21.6 ± 2.9; P < 0.01). A smaller reduction in tumor burden was also observed in Hecate-treated females (17.6 ± 1.6 mg/g body wt vs. 21.6 ± 2.9; P < 0.05). Our results prove the principle that Hecate-CGβ conjugate is able to repress mammary gland tumor growth, even in tumor tissues that lack or have very low levels of LHR. The mechanism of Hecate-CGβ conjugate action in repression of DES/DMBA-induced tumor growth needs to be further analyzed to clarify the molecular mechanisms of Hecate-CGβ conjugate action in vivo.

Evidence for the presence of stem/progenitor cells in porcine endometrium

A population of adult stem cells responsible for cyclic reconstructing and remodeling has been proposed to reside in the highly regenerative mammalian endometrium. Recently, stem/progenitor cells have been identified in the human and mouse endometrium, but less is known about these cells in livestock animals. Using Hoechst 33342 fluorescent dye staining and flow cytometry, we identified an emerging cell side population that may be responsible for the regeneration process of the porcine endometrium. The percentage of side‐population cells on Day 19 of the estrous cycle was significantly higher than that on Days 2–4. Moreover, single cells were able to seed clones that could differentiate into three independent mesenchymal‐cell lineages. We also demonstrated the expression of specific markers of self‐renewal cells on these side‐population cells and the presence of a population of cells among the stromal cells that possess markers for mesenchymal stem cells. These results indicate that the porcine endometrium contains a population of cells with the capacity for self‐renewal and a high rate of proliferation, which depend on the phase of the estrous cycle. These cells could potentially be involved in the cyclic reconstruction of the porcine endometrium. Mol. Reprod. Dev. 82: 182–190, 2015.

Immortalization of Swine Umbilical Vein Endothelial Cells (SUVECs) With the Simian Virus 40 Large-T Antigen

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G‐1410, using Simian virus 40 T‐antigen (SV40 T‐ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T‐ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G‐1410 cells did not differ morphologically from SUVECs. The G‐1410 cells exhibited positive staining for vascular endothelial (VE)‐cadherin and von Willebrand factor (vWF), and formed capillary‐like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T‐ag, these transformed G‐1410 cells have remained karyotypically normal and non‐tumorigenic. G‐1410 cells also responded to stimulation with VEGF, FGF‐2, and newborn calf serum. Moreover, G‐1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF‐A), and FGF‐2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G‐1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis. Mol. Reprod. Dev. 78:597–610, 2011.

Effects of seminal plasma and the presence of a conceptus on regulation of lymphocyte-cytokine network in porcine endometrium

Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. Seminal plasma induced long‐term modulatory effects and conceptus‐induced immune changes in leukocyte populations were measured by flow cytometry and mRNAs for various cytokines by quantitative reverse‐transcriptase PCR in porcine endometrium collected on Days 6 and 13 from cycling and pregnant animals or from animals given seminal plasma infusions. Seminal plasma infusion induced long‐term modulatory effects, resulting in significantly more endometrial FoxP3‐positive T‐regulatory and T‐helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T‐cytotoxic and T‐null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13‐days post‐infusion, although animals on Day 13 of pregnancy did show significantly more T‐cells (of any type investigated). Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony‐stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by implanting conceptuses. Mol. Reprod. Dev. 2014.