Anna Wójtowicz

Nitric Oxide Induces the Synthesis of Vascular Endothelial Growth Factor by Rat Vascular Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1beta (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with L-arginine (10 mmol/L) or tetrahydrobiopterin (BH(4); 100 micromol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 micromol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1beta induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1beta-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-arginine or BH(4) increased NO generation by L-NAME- or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH(4). The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 micromol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.

Valproate-induced alterations in testosterone, estradiol and progesterone secretion from porcine follicular cells isolated from small- and medium-sized ovarian follicles

The aim of the present study was to investigate whether long-term exposure to valproate (VPA) alters follicular steroidogenesis and whether or not this effect is dependent on the degree of follicular development. Small- and medium-sized follicles were obtained from pig ovaries collected, respectively, at days 8-10 and 14-16 of oestrus cycle. Theca interna and granulosa cells were isolated from follicles and placed in the same well in the ratio 1 : 3 with or without the VPA in doses of 100, 300 and 500 micro g ml(-1). The culture medium was changed after 2, 4, 6 and 8 days. In both types of follicles, VPA caused a significant and dose-dependent reduction in both testosterone and estradiol secretion from follicular cells. In small-sized follicles, the testosterone to oestrogen ratio increased at all doses used and after all lengths of time in culture. In medium-sized follicles, a significant increase in the testosterone to oestrogen ratio was only observed at the highest dose level. All doses of VPA caused a marked inhibition of progesterone secretion after 48 hours while during long-term VPA exposure progesterone gradually increased demonstrating luteinization of cells. In conclusion, the present study demonstrates a direct effect of VPA on steroidogenesis. The effect seems to differ to some extent depending on the follicular stage of development. The elevated ratio of testosterone to estradiol suggests that VPA inhibits the conversion of testosterone to estradiol.

Comparison of Reproductive Effects of Levetiracetam and Valproate Studied in Prepubertal Porcine Ovarian Follicular Cells

Summary:  Purpose: Long‐term valproate (VPA) treatment has been associated with reproductive endocrine disorders characterized by hyperandrogenism and polycystic changes in the ovaries in women with epilepsy. Levetiracetam (LEV) is a promising, new antiepileptic drug that may represent an alternative to VPA for many patients. Here the effect of LEV and VPA on basal and gonadotropin‐stimulated steroid secretion from prepubertal porcine ovarian follicular cells was compared and the conversion of testosterone to estradiol is measured.

Effect of PCB 126 and PCB 153 on incidence of apoptosis in cultured theca and granulosa cells collected from small, medium and large preovulatory follicles

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.

Valproate irreversibly alters steroid secretion patterns from porcine follicular cells in vitro

The aim of the present study was to investigate whether exposure of porcine ovarian follicular cells to clinically relevant concentrations of valproate affected steroid production in vitro and to which extent these effects were reversed by removal of the drug from the culture medium. Small and medium follicles were obtained from ovaries collected, respectively, at days 8-10 and 14-16 of the estrous cycle. Theca and granulosa cells were collected from follicles and co-cultured in one well. To show whether the effect of valproate was reversible, cells were cultured for 24, 48, or 72 h with valproate 100 or 250 microg/ml. The medium was then changed to fresh medium without drugs for an additional 24, 48, or 72 h. Valproate added to the culture medium caused a significant reduction of estradiol secretion with concomitant increase in both testosterone and progesterone secretion by small follicles. In medium-sized follicles, 100 microg/ml valproate was without effect on estradiol secretion while 250 microg/ml caused a small, but statistically significant decrease. The effects of valproate on steroid secretion patterns were irreversibly independent of concentration, exposure time, and time of restoration after drug exposure up to 72 h in both small and medium follicles. In conclusion, the present study demonstrated that even short-term valproate treatment disrupted follicular steroidogenesis in isolated ovarian follicular cells resulting in increased testosterone and progesterone and decreased estradiol secretion. It was not possible to reverse the steroidogenic effects of valproate by removing the drug from the cell cultures.

Effects of levetiracetam and valproate on reproductive endocrine function studied in human ovarian follicular cells

Purpose:  Recent animal studies have indicated possible reproductive endocrine effects of levetiracetam (LEV). The aim of the present study was to investigate possible reproductive endocrine effects of LEV compared to valproate (VPA) using human ovarian follicular cells.

Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles.

The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)β protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were ‘Mjosa’ extracted from burbot liver, which contains a high level of PBDEs, and ‘Marine mix’, extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to ‘Marine mix’ and ‘Mjosa mix’ at doses of 3.6 and 1.4 μg ml−1, respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by ‘Marine mix’ and down-regulation of ERβ by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both ‘Marine mix’ and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) ‘Marine mix’ is a mixed-type CYP inducer; (2) ‘Mjosa mix’ is an inducer of ERβ and CYP2B; and (3) both ‘Marine mix’ and ‘Mjosa mix’ stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19

Time-dependent action of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and its metabolite DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) on human chorionic gonadotropin and progesterone secretion

Explants of human placenta were used to study the effects of two isomers of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) [p,p′-DDT and o,p′-DDT] and their DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) metabolites [p,p′-DDE and o,p′-DDE] on the secretion of progesterone and human chorionic gonadotropin (hCG). Explants were treated with 1, 10, 100 or 1000 ng/ml of each compound for 24 h or 72 h. We found opposite effects (stimulatory after short-term and inhibitory after long-term exposure) of all compounds on progesterone secretion. However, both short- and long-term exposure to all investigated compounds caused decreased hCG secretion. In conclusion, we suggest the existence of a local axis between steroid hormones and hCG in placenta. DDT (which has estrogenic properties) increases progesterone secretion and consequently decreases hCG secretion. After long-term exposure, the low level of hCG is insufficient to stimulate progesterone.

Comparison of FTIR-ATR and Raman spectroscopy in determination of VLDL triglycerides in blood serum with PLS regression

Hypertriglyceridemia, related with triglyceride (TG) in plasma above 1.7mmol/L is one of the cardiovascular risk factors. Very low density lipoproteins (VLDL) are the main TG carriers. Despite being time consuming, demanding well-qualified staff and expensive instrumentation, ultracentrifugation technique still remains the gold standard for the VLDL isolation. Therefore faster and simpler method of VLDL-TG determination is needed. Vibrational spectroscopy, including FT-IR and Raman, is widely used technique in lipid and protein research. The aim of this study was assessment of Raman and FT-IR spectroscopy in determination of VLDL-TG directly in serum with the isolation step omitted. TG concentration in serum and in ultracentrifugated VLDL fractions from 32 patients were measured with reference colorimetric method. FT-IR and Raman spectra of VLDL and serum samples were acquired. Partial least square (PLS) regression was used for calibration and leave-one-out cross validation. Our results confirmed possibility of reagent-free determination of VLDL-TG directly in serum with both Raman and FT-IR spectroscopy. Quantitative VLDL testing by FT-IR and/or Raman spectroscopy applied directly to maternal serum seems to be promising screening test to identify women with increased risk of adverse pregnancy outcomes and patient friendly method of choice based on ease of performance, accuracy and efficiency.

What Distinguishes the Host Galaxies of Radio-loud and Radio-quiet AGNs?

We compare the optical properties of the host galaxies of radio-quiet (RQ) and radio-loud (RL) Type 2 active galactic nuclei (AGNs) to infer whether the jet production efficiency depends on the host properties or is determined just by intrinsic properties of the accretion flows. We carefully select galaxies from SDSS, FIRST, and NVSS catalogs. We confirm previous findings that the fraction of RL AGNs depends on the black-hole (BH) masses, and on the Eddington ratio. The comparison of the nature of the hosts of RL and RQ AGNs, therefore, requires pair-matching techniques. Matching in BH mass and Eddington ratio allows us to study the differences between galaxies hosting RL and RQ AGNs that have the same basic accretion parameters. We show that these two samples differ predominantly in the host-galaxy concentration index, morphological type (in the RL sample the frequency of elliptical galaxies becoming larger with increasing radio loudness), and nebular extinction (galaxies with highest radio loudness showing only low nebular extinction). Contrary to some previous studies, we find no significant difference between our radio-loud and radio-quiet samples regarding merger/interaction features.