ABSTRACT
Theca cells (TCs) play an important role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells aromatization to estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo TCs remain unclear. In the present study, the impacts of hypoxic conditions (5% oxygen) on androgen synthesis in buffalo TCs were examined. The results showed that hypoxia improved both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in buffalo TCs. Hypoxic conditions promoted the sensitivity of buffalo TCs to LH. Furthermore, inhibition of PI3K/AKT signaling pathway reduced both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in hypoxia-cultured buffalo TCs. Besides, inhibition of PI3K/AKT signaling pathway lowered the sensitivity of buffalo TCs to LH under hypoxic conditions. This study indicated that hypoxia enhanced the steroidogenic competence of buffalo TCs main through activating PI3K/AKT signaling pathway and subsequently facilitating the responsiveness of TCs to LH. This study provides a basis for further exploration of ovarian endocrine mechanism for steroidogenesis.
Subject(s)
Buffaloes , Theca Cells , Animals , Cells, Cultured , Female , Granulosa Cells , Hypoxia/veterinary , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
Bone marrow-derived mesenchymal stem cells (BMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that BMSCs were prone to malignant transformation of mesenchymal-to-epithelial transition in vitro, which can cause many barriers to further application of BMSCs. The transforming growth factor ß (TGF-ß) signaling pathway has been widely studied as the most important signaling pathway involved in regulating mesenchymal features of BMSCs. However, the effects of the TGF-ß signaling pathway on mesenchymal characteristics of buffalo BMSCs (bBMSCs) remain unclear. In the present study, the impacts of the growth factor, TGF-ß1, on cell proliferation, apoptosis, migration, and karyotype of bBMSCs were tested. Besides, the effects of TGF-ß1 on pluripotency, mesenchymal markers, and epithelial-to-mesenchymal transition (EMT)-related gene expression of bBMSCs were also examined. Results showed that the suitable concentration and time of TGF-ß1 treatment (2 ng/mL and 24 hours) promoted cell proliferation and significantly reduced cell apoptosis (p < 0.05) in bBMSCs. The cell migration capacity and normal karyotype rate of bBMSCs were significantly (p < 0.05) improved under TGF-ß1 treatment. The expression levels of pluripotency-related genes (Sox2 and Nanog) and mesenchymal markers (N-cadherin and Fn1) were significantly (p < 0.05) up-regulated under TGF-ß1 treatment. Furthermore, TGF-ß1 activated the EMT process, thereby contributing to significantly enhancing the expression levels of EMT-related genes (Snail and Slug) (p < 0.05), which in turn improved maintenance of the mesenchymal nature in bBMSCs. Finally, bBMSCs underwent self-transformation more easily and efficiently and exhibited more characteristics of mesenchymal stem cells under TGF-ß1 treatment. This study provides theoretical guidance for elucidating the detailed mechanism of the TGF-ß signaling pathway in mesenchymal feature maintenance of bBMSCs and is of significance to establish a stable culture system of bBMSCs.
Subject(s)
Cell Differentiation , Epithelial-Mesenchymal Transition , Mesenchymal Stem Cells/cytology , Transforming Growth Factor beta1/pharmacology , Animals , Apoptosis , Buffaloes , Cell Movement , Cell Proliferation , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Signal TransductionABSTRACT
Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.
Subject(s)
Animals , Rats , Cell Cycle , Cell Line , Cell Movement , Graft Occlusion, Vascular , Drug Therapy , Metabolism , Pathology , Muscle, Smooth, Vascular , Metabolism , Pathology , Myocytes, Smooth Muscle , Metabolism , Pathology , Protein Kinase Inhibitors , Pharmacology , Purines , PharmacologyABSTRACT
Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.
ABSTRACT
Abnormal enhanced transmural dispersion of repolarization (TDR) plays an important role in the maintaining of the severe ventricular arrhythmias such as torsades de pointes (TDP) which can be induced in long-QT (LQT) syndrome. Taking advantage of an in vitro rabbit model of LQT2, we detected the effects of KN-93, a CaM-dependent kinase (CaMK) II inhibitor on repolarization heterogeneity of ventricular myocardium. Using the monophasic action potential recording technique, the action potentials of epicardium and endocardium were recorded in rabbit cardiac wedge infused with hypokalemic, hypomagnesaemic Tyrode's solution. At a basic length (BCL) of 2000 ms, LQT2 model was successfully mimicked with the perfusion of 0.5 μmol/L E-4031, QT intervals and the interval from the peak of T wave to the end of T wave (Tp-e) were prolonged, and Tp-e/QT increased. Besides, TDR was increased and the occurrence rate of arrhythmias like EAD, R-on-T extrasystole, and TDP increased under the above condition. Pretreatment with KN-93 (0.5 μmol/L) could inhibit EAD, R-on-T extrasystole, and TDP induced by E-4031 without affecting QT interval, Tp-e, and Tp-e/QT. This study demonstrated KN-93, a CaMKII inhibitor, can inhibit EADs which are the triggers of TDP, resulting in the suppression of TDP induced by LQT2 without affecting TDR.
Subject(s)
Animals , Rabbits , Action Potentials , Anti-Arrhythmia Agents , Pharmacology , Arrhythmias, Cardiac , Benzylamines , Pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Electrocardiography , Electrophysiologic Techniques, Cardiac , Endocardium , Heart , In Vitro Techniques , Long QT Syndrome , Pericardium , Piperidines , Pharmacology , Protein Kinase Inhibitors , Pharmacology , Pyridines , Pharmacology , Sulfonamides , Pharmacology , Torsades de PointesABSTRACT
Abnormal enhanced transmural dispersion of repolarization (TDR) plays an important role in the maintaining of the severe ventricular arrhythmias such as torsades de pointes (TDP) which can be induced in long-QT (LQT) syndrome. Taking advantage of an in vitro rabbit model of LQT2, we detected the effects of KN-93, a CaM-dependent kinase (CaMK) II inhibitor on repolarization heterogeneity of ventricular myocardium. Using the monophasic action potential recording technique, the action potentials of epicardium and endocardium were recorded in rabbit cardiac wedge infused with hypokalemic, hypomagnesaemic Tyrode's solution. At a basic length (BCL) of 2000 ms, LQT2 model was successfully mimicked with the perfusion of 0.5 μmol/L E-4031, QT intervals and the interval from the peak of T wave to the end of T wave (Tp-e) were prolonged, and Tp-e/QT increased. Besides, TDR was increased and the occurrence rate of arrhythmias like EAD, R-on-T extrasystole, and TDP increased under the above condition. Pretreatment with KN-93 (0.5 μmol/L) could inhibit EAD, R-on-T extrasystole, and TDP induced by E-4031 without affecting QT interval, Tp-e, and Tp-e/QT. This study demonstrated KN-93, a CaMKII inhibitor, can inhibit EADs which are the triggers of TDP, resulting in the suppression of TDP induced by LQT2 without affecting TDR.
