scholarly journals Identification of androgen receptor phosphorylation in the primate ovary in vivo

Reproduction ◽  
2010 ◽  
Vol 140 (1) ◽  
pp. 93-104 ◽  
Author(s):  
Iain J McEwan ◽  
Dagmara McGuinness ◽  
Colin W Hay ◽  
Robert P Millar ◽  
Philippa T K Saunders ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Androgen Receptor ◽  
Granulosa Cells ◽  
Gnrh Antagonist ◽  
Follicular Phase ◽  
Surface Epithelium ◽  
Control Group ◽  
Target Tissue ◽  
Receptor Phosphorylation

The androgen receptor (AR) is a member of the nuclear receptor superfamily, and is important for both male and female reproductive health. The receptor is a target for a number of post-translational modifications including phosphorylation, which has been intensively studied in vitro. However, little is known about the phosphorylation status of the receptor in target tissues in vivo. The common marmoset is a useful model for studying human reproductive functions, and comparison of the AR primary sequence from this primate shows high conservation of serines known to be phosphorylated in the human receptor and corresponding flanking amino acids. We have used a panel of phosphospecific antibodies to study AR phosphorylation in the marmoset ovary throughout the follicular phase and after treatment with GNRH antagonist or testosterone propionate. In normal follicular phase ovaries, total AR (both phosphorylated and non-phosphorylated forms) immunopositive staining was observed in several cell types including granulosa cells of developing follicles, theca cells and endothelial cells lining blood vessels. Receptor phosphorylation at serines 81, 308, and 650 was detected primarily in the granulosa cells of developing follicles, surface epithelium, and vessel endothelial cells. Testosterone treatment lead to a modest increase in AR staining in all stages of follicle studied, while GNRH antagonist had no effect. Neither treatment significantly altered the pattern of phosphorylation compared to the control group. These results demonstrate that phosphorylation of the AR occurs, at a subset of serine residues, in a reproductive target tissue in vivo, which appears refractory to hormonal manipulations.

scholarly journals Thrombospondin-1 Expression Is Increased during Follicular Atresia in the Primate Ovary

Endocrinology ◽  
2007 ◽  
Vol 149 (1) ◽  
pp. 185-192 ◽  
Author(s):  
Fiona H. Thomas ◽  
Helen Wilson ◽  
Audrey Silvestri ◽  
Hamish M. Fraser
Keyword(s):  
Endothelial Cells ◽  
Granulosa Cells ◽  
Follicular Phase ◽  
Follicular Atresia ◽  
Gonadotropin Secretion ◽  
Antral Follicles ◽  
Cd36 Expression ◽  
Mrna And Protein Expression ◽  
Vegf Trap

Thrombospondin (TSP)-1 is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression as well as expression of its receptor CD36 in the marmoset ovary and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on d 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (d 10). TSP-1 mRNA and protein were present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly but may influence TSP-1 activity via down-regulation of the CD36 receptor.

Exosomes from Adipose-Derived Mesenchymal Stem Cells Overexpressing Stanniocalcin-1 Promote Reendothelialization after Carotid Endarterium Mechanical Injury

2020 ◽  
Author(s):  
Kun Liu ◽  
Huihua Shi ◽  
Zhiyou Peng ◽  
Xiaoyu Wu ◽  
Weimin Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Mechanical Injury ◽  
Molecular Probe ◽  
Blue Staining ◽  
Control Group ◽  
Lateral Migration ◽  
Post Injury

Abstract Background and objective: Endothelial cell inflammation caused by mechanical injury of endovascular treatment remains the major obstacle to reendothelialization, which leads to arterial restenosis. We investigated the reendothelialization effect of exosomes from adipose-derived mesenchymal stem cells (ADSC) overexpressing Stanniocalcin-1 (STC-1).Methods: Primary ADSCs were extracted from the adipose tissue of the inguinal area of C57/BL mice. ADSCs were transfected with lentivirus vectors containing STC-1. Exosomes were purified from culture medium using the Exo-Quick kit and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot. PHK-26 as molecular probe was used to track the exosomes engulfed by mice arterial endothelial cells (MAEC). The role of STC-1-ADSC-Exosome (S-ADSC-Exo) in MAECs was verified through scratch test and tube forming experiment. Carotid endarterium mechanical injury was induced by insertion with a guidewire into the common carotid artery lumen. Exosomes were administered by tail vein injection. Content of Reactive oxygen species (ROS) was measured using commercial kits. Carotid arteries were harvested for histological examination, immunofluorescence staining, and Evan’s blue staining.Results: Transfection of STC-1 significantly enhanced STC-1 levels in ADSCs, their exosomes, and MAECs. Compared with the control group and the ADSC-Exo group, STC-1 enriched exosomes markedly enhanced STC-1 level, inhibited the expression of NLRP3, Caspase-1, and IL-1β in MAECs, exhibited good lateral migration capacity, and promoted angiogenesis. Exosome-pretreating groups exhibited lower levels of ROS than those of controls. In vivo administration of S-ADSC-Exo had reendothelialization effect on post-injury carotid endarterium as evidenced by thinner arterial wall, low-expressed NLRP3, and more living endothelial cells.Conclusions: The reendothelialization effect of exosomes from ADSCs on post-injury carotid endarterium can be enhanced by genetic modification to contain elevated STC-1.

scholarly journals Utilizing a Kidney-Targeting Peptide to Improve Renal Deposition of a Pro-Angiogenic Protein Biopolymer

Pharmaceutics ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 542 ◽  
Author(s):  
Fakhri Mahdi ◽  
Alejandro R. Chade ◽  
Gene L. Bidwell
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Endothelial Cell Proliferation ◽  
Microvascular Endothelial Cell ◽  
Similar Distribution ◽  
Target Tissue ◽  
Cell Binding ◽  
Targeting Peptide

Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP–VEGF. The ELP–VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP–ELP, ELP–VEGF, and KTP–ELP–VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP–ELP–VEGF was equipotent to ELP–VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP–ELP–VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP–ELP–VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP–VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.

63 EQUINE FOLLICLES MODULATE CORTISOL LEVELS AND CAPABILITY OF OOCYTES TO ADAPT TO STRESS SITUATIONS

2016 ◽  
Vol 28 (2) ◽  
pp. 161
Author(s):  
D. Scarlet ◽  
N. Ille ◽  
G. D. A. Gastal ◽  
B. G. Alves ◽  
S. O. Paiva ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
Plasma Concentrations ◽  
Reproductive Function ◽  
Control Group ◽  
Target Tissue ◽  
Preovulatory Follicles ◽  
Before And After ◽  
Cortisol Levels ◽  
Systemic Stress

Glucocorticoids are mediators of the systemic stress response. Acute or chronic stress characterised by high cortisol concentrations in the periphery impairs reproductive function in a variety of species and therefore may affect fertility. The ovary has been shown to be a target tissue for glucocorticoids in many species, including the mare. This study hypothesised that the equine ovary possesses internal mechanisms to modulate cortisol activity and that supraphysiologic levels of glucocorticoids do not affect oocyte IVM rates. Light horse mares (n = 9) were used in this study. Growing follicles from an induced follicular wave were divided into the following groups: G1: 5–9 mm, G2: 10–14 mm, G3: 15–19 mm, G4: 20–24 mm, and G5: ≥25 mm. Follicular fluid (FF) and compact cumulus‐oocyte complexes (COCs) were obtained by ultrasound-guided transvaginal aspiration. Blood samples were collected at the beginning and the end of every aspiration session. Cortisol (DE1887, Demeditec, Kiel-Wellsee, Germany), progesterone (ADI-901–011, Enzo Life Sciences, Farmingdale, NY, USA), and corticosteroid binding globulin (CBG, MBS047353, MyBioSource, San Diego, CA, USA) concentrations were determined by ELISA. COCs (n = 80) were randomly distributed to either the control group (DMEM-F12+ medium) or the following hydrocortisone treatment groups: 0.1 µg mL–1, 1 µg mL–1, 5 µg mL–1, 10 µg mL–1. Maturation rate was assessed 30 h after incubation. Statistical analysis was performed with the SPSS Statistics 22 software. Data were analysed using one-way ANOVA, Pearson correlation, and chi-squared test. Cortisol (115.4 ± 13.3 ng mL–1) and progesterone (22.1 ± 3.1 ng mL–1) FF concentrations were higher (P < 0.05) in G5 follicles than in all other groups, and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol (118.6 ± 7.8 v. 120.3 ± 12.2 ng mL–1), progesterone (2.4 ± 0.5 v. 2.5 ± 0.4 ng mL–1), and CBG (11.1 ± 5.1 v. 9.9 ± 3.2 µg mL–1) did not differ before and after follicle aspiration. However, plasma CBG and progesterone were negatively correlated (r = –0.56; P < 0.01). Maturation rates did not differ among groups, regardless of the hydrocortisone concentration added to the culture medium. Our results demonstrated higher cortisol concentrations in preovulatory follicles in vivo, suggesting its importance for oocyte maturation. The greater unbound cortisol available in the FF of preovulatory follicles can be indicative of the displacement of cortisol from CBG in favour of progesterone. Furthermore, equine oocytes were capable of surviving cortisol concentrations 100 times higher than those physiologically present in preovulatory follicles. This finding suggests the ability of equine oocytes to modulate cortisol levels and adapt to stress situations.

Heparins Inhibit Tumor Angiogenesis by Sequestering Fibroblast Growth Factor (FGF) from Tumor Vascular Endothelial Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2662-2662
Author(s):  
Shannon L. Smiley ◽  
Dale O. Henry ◽  
Shang-Chiung Chen ◽  
Michael K.K. Wong
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Tumor Angiogenesis ◽  
Experimental Models ◽  
Control Group ◽  
Molecular Evidence ◽  
Dependent Manner ◽  
Fibroblast Growth

Abstract The association between cancer and thromboembolic disease is a well-known phenomenon and contributes to the morbidity and mortality of cancer patients. Clinical studies of thrombosis in these patients show that heparins may have beneficial effects on survival. Antithrombotic agents have been shown to exert an anti-tumor effect in various experimental models however the underlying mechanism remains unknown. We show that heparins inhibit in vivo tumor angiogenesis and offer molecular evidence that heparins exert an anti-angiogenic effect by directly sequestering fibroblast growth factor (FGF) from its receptor on tumor derived endothelial cells (TDECs). NIH-3T3 fibroblasts were stably transfected with an expression construct that results in the constitutive excretion of FGF-1 (Clone C). Clone C gives rise to aggressive and highly angiogenic xenograft tumors. Clone C was inoculated into nude mice and therapeutic doses of Low Molecular Weight (LMW) heparins were injected daily beginning on Day 2. Tumors in the control group were grossly angiogenic and highly vascularized. In contrast, the heparin treated tumors were pale and possessed only scant peri-tumoral vessels. In order to assess the biologic mechanism of this, murine TDECs were isolated and cultured as previously published. Unfractionated and LMW heparins inhibit FGF-induced TDEC mitogenesis in a concentration- and time-dependent manner. FGF overcame and rescued heparin-induced inhibition suggesting that an FGF-heparin interaction is responsible. In order to test the hypothesis that heparin strips and sequesters FGF off its receptor on TDECs, we used a FGF protein fused to a hemagglutinin peptide tag at the carboxyl-terminus end (FGF-HA). FGF-HA is biologically identical to wild type FGF, but its detection limit is 10X more sensitive. FGF-HA was allowed to bind to FGFR on TDECs. These cells were subsequently incubated with Heparin covalently linked to Sepharose beads (Heparin-Sepharose) or to Sepharose alone. These beads were removed, and TDEC growth analyzed prospectively. Heparin-Sepharose treatment results in significant TDEC growth inhibition as compared to incubation with Sepharose alone. Western blot analysis shows that FGF was sequestered only on the Heparin-Sepharose beads. Conclusion: The anti-angiogenic mechanism of heparins resides, in part, in its ability to sequester angiogenic cytokines such as FGF from its receptor on tumor endothelium.

scholarly journals Expression and Epigenetic Change of the AR and FSHR Genes in the Granulosa Cells of Endometriosis Patients

2012 ◽  
Vol 4 ◽  
pp. GEG.S9877 ◽  
Author(s):  
Mika Hayashi ◽  
Yoshiki Yamashita ◽  
Atsushi Hayashi ◽  
Yoko Yoshida ◽  
Sachiko Kawabe ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Pcr Analysis ◽  
Control Group ◽  
Epigenetic Change ◽  
Human Granulosa Cells ◽  
Significant Difference ◽  
Gynecological Diseases

Background Endometriosis is one of the most common gynecological diseases associated with infertility. Endometriosis may affect the androgen receptor (AR) mRNA expression in human granulosa cells and the methylation of the promoter region of AR. We investigated 28 patients with endometriosis and 47 subjects without endometriosis undertaking IVF treatment. Methods Granulosa cells were obtained from 28 patients with endometriosis and 47 subjects without endometriosis as a control. Expressions of AR and FSHR mRNA were then evaluated by OneStep real-time PCR analysis, and the level of methylation of the promoter region was qualified by methylation-specified PCR (MSP). Results The expression of AR mRNA in the endometriosis group was statistically lower than that in the control group. As well, FSHR mRNA expression in the control group showed a positive correlation with AR mRNA expression; however, there was no such correlation in endometriosis patients. In the control group, AR mRNA expression was statistically higher in pregnant subjects compared with non-pregnant subjects; however, in the endometriosis group, no significant difference was identified. The promoter of AR was heavily methylated in all endometriosis cases; however, only 5 (45.4%) were methylated in the control group. Conclusion Lower AR mRNA expression and methylation of the AR promoter region might affect the expression of AR and FSHR the presence of endometriosis, thus leading to a disturbance in the regulation of AR and FSHR.

Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation In Vivo

2006 ◽  
Author(s):  
Samir Taneja ◽  
Susan Logan ◽  
Brian Dynlacht ◽  
Michael Garabedian ◽  
Derick Mitchell
Keyword(s):  
Androgen Receptor ◽  
Receptor Phosphorylation ◽  
Prostate Cell ◽  
Specific Regulation

scholarly journals Efficacy of antiangiogenic targeted toxins against glioblastoma multiforme

2006 ◽  
Vol 20 (4) ◽  
pp. E23 ◽  
Author(s):  
Walter A. Hall ◽  
Daniel A. Vallera
Keyword(s):  
Endothelial Cells ◽  
Glioblastoma Multiforme ◽  
Fusion Protein ◽  
Plasminogen Activator ◽  
Systemic Exposure ◽  
Treatment Modalities ◽  
Target Tissue ◽  
Targeted Toxins

Object Because the prognosis for patients with glioblastoma multiforme (GBM) remains poor, investigators have focused on developing new and more effective treatment modalities. Targeted toxins represent a new class of compounds composed of a potent protein toxin and a carrier ligand that will recognize cell surface antigens located on target tissue. A recombinant fusion protein was created that contains the translocation and catalytic portions of diphtheria toxin that are responsible for cell entry and killing, respectively, fused to the noninternalizing aminoterminal fragment portion of human plasminogen activator. This diptheria toxin–uPA fusion protein (DTAT) has the advantage over other fusion proteins of targeting malignant glioma cells and the endothelial cells of the neovasculature that express the urokinase-type plasminogen activator receptor (uPAR). Another protein, DTAT13, was synthesized to target uPAR on the neovasculature and the uPAR and interleukin-13 receptor-expressing GBM cells. The authors describe the in vitro and in vivo efficacy of DTAT and DTAT13 against GBM. Methods The in vitro cytotoxicity of DTAT and DTAT13 was measured using cell proliferation assays. In vivo studies were performed in which DTAT, DTAT13, or a control protein was injected directly into GBM flank tumors in athymic nude mice. Tumor volume was assessed over time and analyzed using the Student t-test. The systemic organ effects of DTAT and DTAT13 were examined functionally and histologically in tumor-free C57BL/6 mice. In vitro, DTAT and DTAT13 were found to be highly potent and selective against U118MG, U87MG, and U373MG GBM cell lines and human umbilical vein endothelial cells. In vivo, DTAT and DTAT13 both caused a statistically significant (p < 0.05) regression of U87MG GBM flank tumors when administered every other day at 10 μg/day for five doses. No tumor regression was seen in control animals. Both DTAT and DTAT13 had little effect on histological findings in the liver, kidney, spleen, and lungs. Serum analysis did not demonstrate an effect on blood urea nitrogen levels, but liver alanine aminotransferase levels rose to statistically significant (p = 0.046) but not life-threatening levels. Also, DTAT13 was less toxic than DTAT in studies of mortality rates. Conclusions Both DTAT and DTAT13 might have potential for clinical application against GBM because of their ability to target both the tumor cells and neovasculature simultaneously with an absence of serious systemic side effects. The discovery that DTAT13 was less toxic than DTAT indicated that the bispecific fusion protein might target a broader subset of antigenetically diverse patients with tumors while reducing the systemic exposure to toxin that would be necessary if two agents were administered separately.

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