DMEM F12 Research Articles

250 The Impact of Altering Fatty Acid Combinations in Vitro on Bovine Preadipocyte Hyperplasia

Abstract In the cattle industry, fat deposited on a carcass can have advantages and disadvantages depending on location and amount. Excess subcutaneous fat deposition reduces carcass red meat yield, while intramuscular fat (IMF) deposition, or marbling, increases carcass value and enhances tenderness and flavor. Two distinct adipocyte growth cycles can be attributed to total fat deposition: hypertrophy and hyperplasia. Hyperplasia refers to the adipogenic side of growth where there is an increase in preadipocyte cell number before cell differentiation. This study aimed to investigate the effects of altering fatty acid composition of cell culture media in vitro on preadipocyte hyperplasia. Subcutaneous fat was harvested from Angus-cross yearling steers (n = 4). Subcutaneous preadipocytes were isolated and propagated for a series of six passages. Preadipocytes were subsequently seeded, and eight different treatment mixtures with combinations of different fatty acids (C16:0, C18:0, C18:1cis-9, and C18:2cis-9,12) at varying ratios were added to the basal media (DMEM F12, 10% bovine fetal serum, 1% HEPES, 1% AB/AM) for a period of four days. Cell culture treatment media was changed daily. Each individual media mix was applied at the same total molar concentration (200 ug). At the end of the four-day treatment period, cells were harvested for RNA extraction and fatty acid analysis. The expression of critical adipogenic and lipogenic genes was evaluated. Treatments displayed differences (P < 0.05) in total fatty acid content (ug), which would suggest that the addition of fatty acids to media could push primary bovine preadipocytes into differentiation. Mix 8 (75% 18:1cis-9 - 25% 18:2cis-9,12) exhibited the greatest (P < 0.05) fatty acid content (ug), while mix 5 (50% C16:0- 50% C18:0) had the least (P < 0.05) fatty acid amount (ug). The expression of fatty acid binding protein-4 (FABP4) and stearoyl Co-A desaturase-1 (SCD-1) differed (P < 0.05) across treatments. The expression of peroxisome proliferating-activated receptor gamma (PPARy), sterol-regulatory element-binding protein (SREB-1c), and SREBP cleavage-activating protein (SCAP) were all downregulated (P < 0.05) in the fully saturated fatty acid mix, mix 5 (50% C16:0- 50% C18:0), compared with all other treatments. Based on the data, it appears that the addition of fatty acids at different ratios to media can affect molecular mechanisms, hyperplastic growth, and potentially the differentiation of primary bovine adipocytes. Additional examination of fatty acid supplementation in vitro could provide valuable insight into their effects on adipocyte growth and how it could be applied to alter fat deposition in vivo.

The effect of metformin on the metabolism of human vascular smooth muscle cells in high glucose conditions.

Metformin is widely used in type 2 diabetic patients as an antihyperglycemic drug. The aim of this study was to investigate the effect of metformin on the metabolism of vascular smooth muscle cells in high glucose conditions. The vascular smooth muscle cells were cultured in DMEM F12 containing glucose as high as 25mM. The preconditioned cells were then treated with metformin in doses of 1, 5, and 7mM for 24h. MTT method was used to determine cell viability. Biochemical parameters including lactate, glucose, total protein, creatinine, and triglyceride were measured in the cell culture after the treatment with metformin. Oil Red O staining method was used to stain the lipids in the cells. Metformin reduced significantly (p<0.001) VSMC proliferation in a concentration-dependent manner. With the increase of glucose uptake by VSMCs, the cell lipid deposition was not changed. Other biochemical parameters such as lactate, triglyceride, total protein, and creatinine were significantly changed in the cell culture (p<0.05). Metformin increased the glucose uptake impacting metabolic pathways in VSMCs. It also increased the lactate efflux and protein metabolism without the change in cellular lipid deposition in high glucose conditions.

OFFSPRING GENDER RATIO AFTER SPERM CO-INCUBATION WITH ENDOMETRIAL CELLS PRIOR IVF

To observe and compare the offspring gender ratio in couples who had co-incubation of spermatozoa with human endometrial cells prior IVF and patients with conventionally performed IVF. Observational. Data on the offspring gender of 106 couples who had sperm co-incubation with the partners’ endometrial cells prior IVF (study group) and 104 couples who reached labour after conventional IVF (control group) was recorded. Cells obtained from endometrial biopsies were cultivated in DMEM F12 10% FBS for 48 hours at 37ºC and 5%CO2. On the day of the follicular punction washed semen was incubated for 2 hours with the confluent endometrial cells followed by conventional IVF protocol. Statistical analysis was performed by Chi-square test using SPSS v.21. P<0.05 was considered significant. The conventional IVF group and the sperm co-incubation group were comparable in age, BMI, oocyte quality and number of unsuccessful ART procedures. The female to male ratio in the conventional IVF group was 1:1.08 (female:male) with 48.1% females and 51.9% males. Live births after sperm co-incubation with endometrial cells prior IVF resulted in significantly more female than male babies (63.2% vs. 36.8%, respectively, p<0.05) with gender ratio 1.71:1 (female:male). When the gender ratios were compared between the two groups, female proportion was significantly higher in the co-incubation group in comparison to the control group (p<0.05). Our data show that spermatozoa co-incubated with endometrial cells prior IVF skews the sex ratio of the offspring to favor females. This observation suggests that co-incubation of spermatozoa with endometrial cell culture may lead to preferential hyperactivation of human spermatozoa bearing X chromosome. More studies are needed to analyze the effect of the endometrial cell culture on spermatozoa.

Anticancer mechanisms determination of L‐aminoacids compounds serie on MDA‐MB‐231 cell line TNBC

Breast cancer represents the second most frequently diagnosed neoplasm worldwide of which approximately 15% is subclassified as triple negative breast cancer (TNBC) [1], characterized by the absence of estrogen receptors, progesterone and growth factor Her2/neu. TNBC is considered as one of the most aggressive cancers, due to its null response to current endocrine therapies and trastuzumab [2]. Breast cancer is a pathology associated with aging and unhealthy lifestyles, it has been proven through in vitro studies that MDA‐MB‐231 TNBC cells overexpress HDAC8 as a consequence of epigenetic deregulation mechanisms, linked to the high proliferative capacity and therefore, a high risk of recurrence and low survival [3]. In this context, our work group have evaluated a serie of compounds on the MDA‐MB‐231 cell line, these compounds have been designed, synthesized and evaluated as iHDAC8 by Docking. Therefore, this work presents the cytotoxicity and evaluation of the antitumor effect on the MDA‐MB‐231 cell line of three compounds derived from L‐amino acids, BisLys, GluEt and I1, which contain the isoindolinyl group and I1 which has a carboxybenzoyl group. To evaluate citotoxicity of BisLys, GlutEt and I1 compounds, MDA‐MB‐231 cells were seeded and incubated at 37°C in a 5% CO2 with DMEM F12 medium supplemented with FBS. Once the cells reached 95% confluence they were treated for 24 hours adding 8 different concentrations of each of the compounds; DMEM F12 medium was used as a negative control and SAHA as a positive control. The proliferation and viability of TNBC cells was quantified by the colorimetric for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Type of cell death whether it was necrosis or apoptosis was determined using Annexine V and propidium iodide by flow cytometry. Finally, the blocked cell cycle progression by those compounds was determined using Cayman 10009349 Kit by flow cytometry. The BisLys, GluEt and I1 compounds showed IC50 values of 0.83, 0.66 and I1 3.42 mM, respectively, and inhibited cell proliferation and viability in a dose‐dependent manner on the MDA‐MB‐231 cell line of TNBC. The GluEt compound induced death mainly by necrosis and the BisLys and I1 compounds by apoptosis. The BisLys, GluEt and I1 compounds induced cell cycle arrest in a dose‐dependent manner at the expense of the subG0/G1 phase. The compounds that induced the greatest apoptosis in MDA‐MB‐231 cells were BisLys and I1 which makes them leading compounds in suppressing the growth of MDA‐MB‐231 TNBC cells.

Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells

BackgroundThis study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) in vitro tracking of labeled L. major in the fibroblast cells. MethodsDil crystal and AO were used to stain L. major in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 106 parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The stained promastigote was incubated with fibroblasts for 6–8 h. The presence of stained parasites with AO in the fibroblast was assessed using a fluorescence microscope. 1 × 106/mL promastigote of L. major was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 μg/mL and it was kept for 20 min at the room temperature. Subsequently, after washing it in PBS for seven times, it was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the precipitate containing stained promastigote was suspended in fresh DMEM F12 with fibroblasts at 37 °C for 6 h. The presence of stained parasites with Dil in fibroblast was assessed using a fluorescence microscope. Fibroblast characterization was undertaken by a real-time polymerase chain reaction (PCR). ResultsAcridine orange staining assisted in detection of the live parasite in the fibroblast cells. Free promastigote looked green before entering into the fibroblasts after 12 h culture. The parasite entered the cytoplasm of fibroblasts at the beginning of the exposure and gradually entered the nucleus of the fibroblast. The fibroblast nucleus was entirely stained green by AO. The L. major appeared green under the fluorescent microscope. Dil staining revealed that the internalized parasites with red/orange color were localized within the cytoplasm after 6-hours and the nucleus of the fibroblasts after 72-hours following culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative for CD106 and integrin alpha 11. ConclusionThe fluorescent dye Dil staining is a safe, easy to use, inexpensive and fast method for labeling of the Leishmania parasite in the fibroblast cells. Acridine orange staining could be useful for tracing the parasites in the fibroblasts too. In this study, both Dil and AO were compared and considered as suitable vital dyes for identifying labeled Leishmania in the fibroblast in vitro, but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.

P1628Angiopoietin-like protein (Angptl) 2 secreted from epicardial adipose tissue induces atrial myocardial fibrosis

Abstract Background Using excised human left atrial appendage samples, we previously demonstrated that epicardial adipose tissue (EAT) are highly associated with atrial myocardial fibrosis as a substrate of atrial fibrillation (AF). We also reported the relationship between Angptl2 in EAT and atrial fibrosis. However, the mechanism is not clear. The purpose is to clarify the mechanisms underlying the effect of EAT on the atrial myocardium. Methods Human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) samples were obtained from 6 cases (2 females, 70.2±13.2 years). 50 mg of EAT and SAT were quickly washed with PBS and centrifuged 1min at 1200rpm. After 3 times this procedures, adipose tissues were cultured in DMEM F12 medium with Fetal bovine Serum (FBS) overnight. After pre-incubation, EAT and SAT tissues were washed and centrifuge d three times and cultured in medium without FBS for 24hours. Finally, we collected oozed medium (conditioned medium) and used for experiments. Concentrations of Angptl2 in conditioned medium were measured by ELISA. To study the effects of conditioned medium, we used “organo-culture” system. Isolated atrium from 8week old male Sprague-Dawley rats were placed on the porous membrane with the endothelial face toward the membrane. After that, loading medium (conditioned medium:culture medium = 1:4), culture medium (control), or recombinant Angptl2 were dropped onto the epicardial face of the atrium once a day and incubated for 7 days (37°C, 5% CO2). Then, histological and immunohistochemical analysis were performed. We also performed quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis. Next, we isolated and cultured neonatal rat fibroblast and loaded Angptl2 for 24 hours.After collected these cells, we performed western blotting analysis. Results Atria organo-culture incubated for 7 days with conditioned medium showed global fibrosis. At epicardial side, fibrotic area of EAT group was significantly greater compared to that of SAT and control group (P&lt;0.05). mRNA of Col1a1, col3a1 and TGFβ1 were significantly increased in EAT group compared with the SAT and control group. And, the concentration of conditioned medium created from EAT was significant higher than that from SAT (P&lt;0.05). Then, we dropped 500 ng/ml of recombinant Angptl2 onto the rat atria. Fibrotic area of Angptl22 group significantly greater than that of control with increasing number of α-SMA positive cells, and mRNA of col3a1 and TGFβ1 were significantly increased in Angptl2 group compared with control group. In cultured fibroblasts, α-SMA and p-ERK expression were increased in Angptl2 group measured by western blotting analysis. Conclusions Our results demonstrated that EAT rather than SAT induces atrial myocardial fibrosis. There is a possibility that Angptl2 effused from EAT plays a part in atrial fibrosis thought EAT paracrine effect. Acknowledgement/Funding ONO PHARMACEUTICAL CO

Zinc oxide nanoparticle induced neurotoxic potential upon interaction with primary astrocytes

As obvious from the basic prerequisite of any particle in nanoscale, Zinc oxide nanoparticles (ZnO NPs) possess numerous tunable properties distinct from their bulk formulations. Emerging innovations in various sectors of nanotechnology are exploiting ZnO NPs largely. This inturn picks up the occasions of human exposure irrespective of the application fields. Although the platform of nanotoxicology has been garnished with nano-bio interaction studies using different cell lines, a few are existing so far comprising primary cells which symbolize realistic in vivo environment. The present study addresses the neurotoxic potential of ZnO NPs using primary astrocytes isolated from post-natal 0–2 day old rat pups. Cells were cultured and maintained in DMEM F12 followed by purification. ZnO NPs generated by wet chemical method was then characterized both physico chemically and biologically. All of the techniques confirmed homogenous distribution of NPs and ensured enough colloidal stability. Bio-nano interaction studies commence on cell viability assays (MTT and NRU) and both of which confirmed dose and time dependent cytotoxicity. Alterations within cellular morphology, cytoskeletal arrangement, lysosomal stability, mitochondrial membrane potential (MMP) and caspase activation were evaluated by standardized techniques. All of the assays substantiated significant toxic consequences in astrocytes with characteristic hall marks. Apoptotic cell death was noted without any deformations of nuclear material. A comparative toxicity study using ZnO NPs, ZnCl2 and ZnO bulk form was performed which confirmed nanospecific toxicity of ZnO NPs. Overall study evidently provide cautious information that ZnO NPs is capable of eliciting serious neuronal tissue damages which can turn out to be fatal during prolonged exposure.

Anticancer mechanisms determination of N‐(o‐carboxybenzoyl)‐derived compounds of L‐amino acids serie on MDA‐MB‐231 cell line of TNBC

Breast cancer is ranked as the second most frequent neoplasm worldwide [1], of which approximately 15% is subclassified as triple negative breast cancer (TNBC), characterized by absence of estrogen, progesterone and HER2/neu receptors. TNBC has been identified as one of the most aggressive cancers, due to its null response to endocrine therapies and trastuzumab [2]. Breast cancer is a pathology associated with aging and unhealthy lifestyles, which has been proven through studies that demonstrate MDA‐MB‐231 TNBC cells overexpress HDAC8 as a consequence of epigenetic dysregulation mechanisms linked to the high level proliferative capacity, resulting into high risk of recurrence and low survival [3]. Because all above our work group has focused in the design by Docking technique and synthesis of new HDAC inhibitor (HDACi) compounds as a therapeutic target for the TNBC. In this investigation, N‐(o‐carboxybenzoyl) derived compounds of L‐amino acids were used to determine their cytotoxicity by inhibiting the proliferation and viability of the cell line MDA‐MB‐231, as well as elucidate the possible mechanisms of cell death induced by the compounds BisLys, GlutEt and I1.MethodsTo evaluate overall cellular toxicity of BisLys, GlutEt and I1 compounds, MDA‐MB‐231 cells were seeded and incubated at 37°C in a 5% CO2, DMEM F12 medium supplemented with FBS. Compounds were added in 8 different concentrations, DMEM F12 medium was used as a negative control and SAHA as a positive control. The proliferation and viability of TNBC cells was quantified by the colorimetric for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Type of cell death whether it was necrosis or apoptosis was determined using Annexine V and propidium iodide by flow cytometry. Finally, the blocked cell cycle progression by those compounds was determined using Cayman 10009349 Kit by flow cytometry.ResultsThe compounds BisLys and Glut Et decreased cell proliferation via apoptosis after 24h of treatment and blocked cell cycle progression at G1 phase with a concurrent decrease in S and G2/M phase. On the other hand, compound I1 did not significantly decrease cell proliferation after 24 hours of treatment. However, it shared the blocked cell cycle progression at G1 phase in the same way as the BisLys and Glut Et compounds.ConclusionCompounds BisLys and Glut Et were able to inhibit the proliferation of the MDA‐MB‐231 cell line and induced blocked cell cycle and cell death mainly by apoptosis, so they could be considered as potentially therapeutic compounds.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishment of pantropic spotted dolphin (Stenella attenuata) fibroblast cell line and potential influence of polybrominated diphenyl ethers (PBDEs) on cytokines response

The presence of polybrominated diphenyl ethers (PBDEs) in the aquatic environment is an issue of major concern which may be a cause of increasing prevalence and severity of diseases in marine mammals. Although, cell culture model development and in vitro investigation approach is a prime need of time to progress immunotoxic research on aquatic mammals. In this study, we stablished fibroblast cell line (pantropic spotted dolphin) to assess the potential effects of PBDEs on cytokines response. Cells were grown in 6 well cell culture plate and complete media (DMEM and Ham's F12 nutrient mixture, fetal bovine serum, antibiotic and essential amino acids) was provided. The primary culture of (PSP-LWH) cells identification was achieved by vimentin (gene and protein) expressions. Karyotyping revealed pantropic spotted dolphin chromosomes 20 pairs with XX. Transfection was achieved by SV40 LT antigen and transfected cells were expended for passages. Stability of cell line was confirmed at various passages intervals using RT-PCR, western blotting and immunofluorescence methods. After confirmation, cell line was exposed to BDE-47 (250 ng/ml), BDE-100 (250 ng/ml) and BDE-209 (1000 ng/ml), with control group (PBS), positive control DMSO (0.1%) and negative control LPS (500 ng/ml) for 24 h. The ELISA results showed significant increase in IL-6 in BDE- 100 and BDE-209 while IL-1β and IL-8 were found higher in BDE-47 and BDE-100. TNFα and IL-10 secretion was noted higher in control and positive control groups. Altogether, these results emphasize importance of transfected (PSP-LWHT) cell line in aquatic research and potential effects of PBDEs on fibroblast provides evident to understand immune modulating effects of PBDEs in marine mammals. The impact of PBDEs on dolphin’s fibroblast cells immune response and altered cytokine response have been presented for the first time.

PO-270 Signalling pathways WNT, hedgehog and NOTCH in breast cancer with presence and absence of cancer stem cells

IntroductionEndometrial carcinoma is the most common malignancy of the female genital tract in developed countries. These tumours, usually are early diagnosed and associated to a good prognosis, although 20% of the cases, present an aggressive and invasive behaviour. Cancer stem cells (CSC) are defined as an undifferentiated tumour cell subpopulation involved in tumour initiation, resistance to therapy and metastatic phenotype. Several mechanisms are associated to resistance to therapy, such as pathways of aldehyde dehydrogenase (ALDH), an enzyme related with tumour size, lymphatic nodal invasion, recurrent disease, and poor prognosis. In endometrial cancer, the ALDH1 expression was related to tumorigenesis and chemoresistance. In our previous work, ALDH expression was associated with endometrial CSC phenotype.Material and methodsRL95-2, a human endometrial cancer cell line, was submitted to ALDH inhibitors, diethylaminobenzaldehyde (DEAB) 50–100 µM, all-trans retinoic acid (ATRA) 5–10 µM and JQ1 100–250 nM for 48 hours. The cytotoxicity was evaluated through MTT assay. ALDH expression was evaluated through western blot. Sphere formation assay was performed, cells were cultured in coated plates with poly(2-hydroxyethyl-methacrylate), with serum-free DMEM F12 medium with or without 100 µM DEAB, for 5 days. Every two days, spheres were supplemented with EGF and bFGF at a concentration of 10 ng/mL. To evaluate the sphere projection area, spheres obtained were photographed at a magnification of 400x and analysed with Image J software.Results and discussionsThe metabolic activity in of RL95-2 cells was not influenced by the presence of ALDH inhibitors. Cells submitted to 50 and 100 µM DEAB presented a decrease in ALDH relative expression compared with the control cells, of about 36% and 27%, respectively. Regarding the evaluation of sphere projection area, preliminary results showed that spheres submitted to 100 µM DEAB appears to harbour decreased dimensions than control spheres.ConclusionDEAB was the most promising ALDH inhibitor in endometrial cancer cells. This inhibitor also seems to influence the sphere profile considering spheres dimensions. In further studies, we aim to evaluate its influence in the response to cytostatics.FundingFCT (Portugal) PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440), Bolsa Liga Portuguesa Contra o Cancro/CIMAGO, CIMAGO n° 02/2017, Sociedade Portuguesa de Ginecologia/Bayer.

PO-269 Targeting ALDH inhibition in endometrial cancer cells

IntroductionEndometrial carcinoma is the most common malignancy of the female genital tract in developed countries. These tumours, usually are early diagnosed and associated to a good prognosis, although 20% of the cases, present an aggressive and invasive behaviour. Cancer stem cells (CSC) are defined as an undifferentiated tumour cell subpopulation involved in tumour initiation, resistance to therapy and metastatic phenotype. Several mechanisms are associated to resistance to therapy, such as pathways of aldehyde dehydrogenase (ALDH), an enzyme related with tumour size, lymphatic nodal invasion, recurrent disease, and poor prognosis. In endometrial cancer, the ALDH1 expression was related to tumorigenesis and chemoresistance. In our previous work, ALDH expression was associated with endometrial CSC phenotype.Material and methodsRL95-2, a human endometrial cancer cell line, was submitted to ALDH inhibitors, diethylaminobenzaldehyde (DEAB) 50–100 µM, all-trans retinoic acid (ATRA) 5–10 µM and JQ1 100–250 nM for 48 hours. The cytotoxicity was evaluated through MTT assay. ALDH expression was evaluated through western blot. Sphere formation assay was performed, cells were cultured in coated plates with poly(2-hydroxyethyl-methacrylate), with serum-free DMEM F12 medium with or without 100 µM DEAB, for 5 days. Every two days, spheres were supplemented with EGF and bFGF at a concentration of 10 ng/mL. To evaluate the sphere projection area, spheres obtained were photographed at a magnification of 400x and analysed with Image J software.Results and discussionsThe metabolic activity in of RL95-2 cells was not influenced by the presence of ALDH inhibitors. Cells submitted to 50 and 100 µM DEAB presented a decrease in ALDH relative expression compared with the control cells, of about 36% and 27%, respectively. Regarding the evaluation of sphere projection area, preliminary results showed that spheres submitted to 100 µM DEAB appears to harbour decreased dimensions than control spheres.ConclusionDEAB was the most promising ALDH inhibitor in endometrial cancer cells. This inhibitor also seems to influence the sphere profile considering spheres dimensions. In further studies, we aim to evaluate its influence in the response to cytostatics.FundingFCT (Portugal) PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440), Bolsa Liga Portuguesa Contra o Cancro/CIMAGO, CIMAGO n° 02/2017, Sociedade Portuguesa de Ginecologia/Bayer.

NADPH Oxidase 2 and Reactive Oxygen Species Mediate Pulmonary Arterial Smooth Muscle Cell Phenotypic Modulation Following Chronic Hypoxia

Chronic hypoxia (CH), which is associated with chronic obstructive pulmonary disease and high‐altitude exposure, increases pulmonary vascular resistance and promotes pulmonary hypertension through mechanisms that stimulate vasoconstriction and arterial remodeling. Vascular remodeling is characterized in part by abnormal migration of pulmonary arterial smooth muscle cells (PASMCs). Based upon previous studies implicating NADPH oxidase 2 (NOX‐2) in the development of CH‐induced pulmonary hypertension, we hypothesized that NOX‐2‐generated reactive oxygen species (ROS) enhance PASMC migration following CH. To test this hypothesis, we harvested primary PASMCs from control and chronically hypoxic (4 wk at 0.5 atm) rats, cultured them in 5% serum DMEM F12 growth media for 3 days and in 0.3% serum media for 24 hr, and then plated the cells onto 8 μm polycarbonate membrane Transwell inserts (Costar). After allowing the PASMCs to adhere for 1 hr, they were treated with the general NOX inhibitor apocynin (30 μM), the specific NOX‐2 inhibitor GSK2795039 (500 nM), the superoxide dismutase/catalase mimetic EUK‐134 (10 μM), or a vehicle control (0.1% dimethyl sulfoxide) for 24 hr. PASMCs were fixed for 20 min in 4% paraformaldehyde, washed with phosphate‐buffered saline (PBS), stained with 1% Coomassie, and washed again with PBS. Transwell inserts were imaged with an upright brightfield microscope (Nikon) before and after the top layer of PASMCs was removed with a cotton swab. Cell migration was expressed as the percent of total cells that migrated through the Transwell membrane. Our results indicate that CH significantly (p &lt; 0.05) enhances PASMC migration, while either inhibition of NOX‐2 or scavenging of ROS significantly blunts this response. We conclude that NOX‐2‐derived ROS contribute to enhanced PASMC migration following CH.Support or Funding InformationThis work was supported by NIH grants R01 HL132883 and T32 HL‐007736 (to T.C. Resta) and K12‐GM‐ 088021 (to A. Wandinger‐Ness).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Formulation of vitamin D encapsulated cinnamon oil nanoemulsion: Its potential anti-cancerous activity in human alveolar carcinoma cells.

Cinnamon oil is used for medicinal purpose since ancient time because of its antioxidant activity. Oil-in-water nanoemulsion (NE) of cinnamon oil was formulated using cinnamon oil, nonionic surfactant Tween 80 and water by ultrasonication technique. Phase diagram was constructed to investigate the influence of oil, water and surfactant concentration. Vitamin D encapsulated cinnamon oil NE was fabricated by wash out method followed by ultrasonication in similar fashion. The hydrodynamic size of cinnamon oil NE and vitamin D encapsulated cinnamon oil NE was observed as 40.52 and 48.96 nm in complete DMEM F12 media respectively. We focused on the cytotoxic and genotoxic responses of NEs in A549 cells in concentration dependent manner. We observed that both NEs induce DNA damage along with corresponding increase in micronucleus frequency that is evident from the comet and CBMN assay. Both the NEs arrested the cell cycle progression in G0/G1 phase, showed increased expression of Bax, capase-3 and caspase-9 and decrease expression of BcL2 proteins along with significant (p < 0.05) increase in apoptotic cell population and loss of mitochondrial membrane potential. NEs were also evaluated for bactericidal efficacy against E. coli. Thus, both NEs have cytotoxic, genotoxic and antibacterial potential and hence can also be used in food industry with cinnamon oil as carrier for lipophilic nutraceutical like vitamin D.

MAST CELLS CONTENT IN THE STRUCTURES OF THE ISCHEMIC MODEL WOUND ON THE 12TH DAY OF REGENERATIVE POTENTIAL STIMULATION BY AUTO- AND HETEROFIBROBLASTS AND DERMAL EQUIVALENT

Mast cell is a traditional cell for wound healing, but their content in the wound after transplantation of autoand heterofibroblasts and dermal equivalent on the basis of these cells remains poorly studied. Aim. To study the morphological structure, collagen formation, angiogenesis and content of mast cells in biopsy materials of the newly formed epidermis and dermis on the 12th day of their recovery in a model ischemic wound after administration of auto- and heterofibroblasts and after transplantation of dermal equivalent with heterofibroblasts. Materials and methods . The study was performed on 28 white mature mice of the C57 / B1 line aged up to 1 year. 0.4 ml of suspension with 1.33 million fibroblasts in growth medium DMEM F12 (Lonza) and in dermal equivalent were administered around and into the bottom of skin surgical model wound in the scapular region. The scar was embedded in paraffin and stained with hematoxylin and eosin, according to Weigert-Van Gieson, and kit for mast cells staining (Biovitrum). Results . On the 12th day epidermis is thicker after introduction of autofibroblasts, but it is more differentiated after transplantation of dermal equivalent with heterofibroblasts. Angiogenesis is also most active after the administration of autofibroblasts. Fibroblasts production of collagen fibers in granulation tissue, angiogenesis and the content of functionally active mast cells indicates the most favorable influence of autofibroblasts transplantation on the wound healing. Conclusion. The effect of dermal equivalent with heterofibroblasts differs from exposure autofibroblasts by several percent: the area of collagen fibers – by 2%, the area of blood vessels − by 5%, the index of degranulation of mast cells – by 4%.

Проліферативна активність мезенхімальних стовбурових клітин щура за впливу культурального середовища

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. With published data we are known about the effectiveness of using stem cells in degenerative processes in the organs. Therefore, we can argue about expediency of use cell technologies for the treatment degenerative pathology’s. MSCs seem to represent a future powerful tool in regenerative medicine, therefore their research are particularly important in clinical research. Goal of the work . Investigate the proliferative activity of MSCs depending on the type used by culture media and to establish the ability of mesenchymal stem cells to differentiate into kardiomyogenic direction under the influence of exogenous factors. Materials and methods . In studies using MSCs, obtained from 12-day not linear rats. We compared the impact of three different culture media on proliferative activity derived cells. We investigated the ability of MSCs to differentiate into kardiomyogenic direction under the influence of 5 - azacytidine. Results of research and discussion. Was investigated influence of three different composition culture medium on proliferative activity of MSCs. Was defined the better culture medium. Proved that mesenchymal stem cells are able of aimed differentiation at kardiomyogenic under the direction of transcription factor 5 azacytidine. The data confirmed the morphological assessment of culture and research at the molecular level. Conclusions. Cultivation the rat mesenchymal stem cells in DMEM F12 - D8437 and ECT - 20% with the addition of antibiotic-antimycotics 10 ml / cm3 provide the highest proliferative activity. Mesenchymal stem cells are capable of aimed cardiomyogenic differentiation under the direction of transcription factor 5 azacytidine.

Corneal lenticules as an ex‐vivo model to study keratocyte biology

PurposeKeratocytes show differential gene expression in culture media; and are extensively used to study wound healing, corneal disease biology and response to topical drugs. However, mono‐layer culture cannot replicate the 3‐dimensional biological environment of corneal stroma. Hence, we propose to establish corneal lenticule as an ex‐vivo model to study keratocyte biology for corneal diseases, drug response studies and in wound healing experiments.MethodsSMILE surgery was performed using the VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany). After the refractive lenticule of intra‐stromal corneal tissue was created using the femtosecond cutting procedure, it was dissected and separated through the side‐cut opening and removed manually. SMILE lenticules from patients were obtained in MK media and were transferred to DMEM F12 with 10% FBS and 1% PSA. The media was replenished every 24 h and lenticules were harvested at 0 h, 24 h, 48 h, 78 h and 96 h. Gene Expression analysis was performed for pro‐fibrotic genes (fibronectin, ɑ‐sma, vimentin, TGF‐β and TGF‐βR2), pro‐inflammatory (IL‐6 and TNF‐ɑ) and structural genes (Col1‐A1, Col4‐A1and Col5‐A1).ResultsOur results demonstrate that lenticules remain metabolically active in culture media for long periods of time as evident from the varying expression of different pro‐fibrotic, pro‐inflammatory and structural genes after 0 h, 24 h, 48 h, 78 h and 96 h of culture. Furthermore, linear regression analyses show that clinical parameters like lenticule thickness do not affect the expression profile of the various genes by the keratocytes contained in the lenticule.ConclusionsIn conclusion, lenticule can be used as an ex‐vivo model to study keratocyte biology in various corneal diseases and for drug testing.

Corneal lenticules as an ex‐vivo model to study keratocyte biology

PurposeKeratocytes show differential gene expression in culture media; and are extensively used to study wound healing, corneal disease biology and response to topical drugs. However, mono‐layer culture cannot replicate the 3‐dimensional biological environment of corneal stroma. Hence, we propose to establish corneal lenticule as an ex‐vivo model to study keratocyte biology for corneal diseases, drug response studies and in wound healing experiments.MethodsSMILE surgery was performed using the VisuMax femtosecond laser system (Carl Zeiss Meditec AG, Jena, Germany). After the refractive lenticule of intra‐stromal corneal tissue was created using the femtosecond cutting procedure, it was dissected and separated through the side‐cut opening and removed manually. SMILE lenticules from patients were obtained in MK media and were transferred to DMEM F12 with 10% FBS and 1% PSA. The media was replenished every 24 h and lenticules were harvested at 0, 24, 48, 78 and 96 h. Gene Expression analysis was performed for pro‐fibrotic genes (fibronectin, α‐sma, vimentin, TGF‐β and TGF‐βR2), pro‐inflammatory (IL‐6 and TNF‐α) and structural genes (Col1‐A1, Col4‐A1and Col5‐A1).ResultsOur results demonstrate that lenticules remain metabolically active in culture media for long periods of time as evident from the varying expression of different pro‐fibrotic, pro‐inflammatory and structural genes after 0, 24, 48, 78 and 96 h of culture. Furthermore, linear regression analyses show that clinical parameters like lenticule thickness do not affect the expression profile of the various genes by the keratocytes contained in the lenticule.ConclusionsIn conclusion, lenticule can be used as an ex‐vivo model to study keratocyte biology in various corneal diseases and for drug testing.

Differentiation of Human Mesenchymal Stem Cells to Otic Progenitor‐like Cells

Objectives: Differentiate mesenchymal stem cells to otic progenitor-like cells. Methods: We isolated and cultured the stromal vascular fraction (SVF) of 3 samples of human adipose tissue. We differentiated human adipose stem cells (hASCs) to otic lineage, using a differentiation medium containing DMEM HG, F12, N2, B27 and different growth factors like FGF3, FGF10, EGF, and IGF-1. The differentiated cells were fixed and RNA extracted at day 10 and 38 to analyze the expression of otic progenitor, neuronal or inner ear hair cell markers. Results: After 10 days of differentiation we detected otic placode markers expression: Pax2 (90.9 ± 0.16%), Pax8 (44.2 ± 0.03%), and BMP7 (86.4 ± 0.24%). Brn3.c (24.2 ± 0.05%), MyoVIIa (10.8 ± 0.05%), and BIII tubulin (28.8 ± 0.37%) were also detected, consistent with the presence of progenitors of sensorineural and inner ear hair cells. At day 38, an increase of inner ear hair cell markers (33.2 ± 0.04% MyoVIIa, 33.9 ± 0.3% Brn3.c and 14.3% Math1), a decreased of otic progenitor markers (46.2% Pax2 and 15.9% Pax8) and absence of neural markers were seen. The same pattern was seen at mRNA level. Conclusions: Even though these results are preliminary and a protocol improvement is necessary, this work demonstrates the ability of hASCs to differentiate into otic lineage, opening a door to a future therapeutic option for sensorineural hearing loss.

AB0134 Are salivary mucins able to trigger a pro-inflammatory response?

BackgroundSjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease that affects lacrimal and salivary glands, whereby SS-patients complain of eye and mouth dryness. Salivary-acinar-cells of SS-patients display alterations in cell...

Curcumin Effect on MMPs and TIMPs Genes in a Breast Cancer Cell Line

Curcumin (CM) possesses anti-cancer activity against a variety of tumors. Matrix metalloproteinases (MMPs) play an important role in remodeling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. Control of MMP and TIMP activity are now of great significance. In this study, the effect of CM is investigated on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells cultured in a mixture of DMEM and Ham's F12 medium and treated with different concentrations of CM (10, 20 and 40 μM for various lengths of time. Reverse transcription followed by quantitative real time PCR was used to detect the gene expression levels of MMPs and TIMPs in CM-treated versus untreated cases and the data were analyzed by one-way ANOVA. At high concentrations of curcumin, TIMP-1, -2, -3 and -4 genes were up-regulated after 48 hours of treatment, their over-expression being accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner. These results suggest that curcumin plays a role in regulating cell metastasis by inhibiting MMP-2 and MMP-9 and up-regulating TIMP1 and TIMP4 gene expression in breast cancer cells.