HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression

2016 ◽  
Vol 16 (3) ◽  
pp. 203-211 ◽  
Author(s):  
Christophe Louis Depoix ◽  
Olivier Flabat ◽  
Frédéric Debiève ◽  
Corinne Hubinont
Keyword(s):  
Protein Expression ◽  
In Vitro Culture ◽  
Oxygen Tension ◽  
Mrna And Protein Expression ◽  
Effect Of Oxygen

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-β1

2017 ◽  
Vol 42 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
Xu Lin ◽  
Xintng Zhen ◽  
Haiting Huang ◽  
Haohao Wu ◽  
Yanwu You ◽  
...  
Keyword(s):  
Protein Expression ◽  
Antisense Oligonucleotides ◽  
Negative Control ◽  
Signal Pathway ◽  
Caspase 9 ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Background/Aims: Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-β1 and to determine further molecular mediators involved in the effects of miR-155. Methods: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-β1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-β1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. Results: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-β1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. Conclusions: TGF-β1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs

2013 ◽  
Vol 61 (1) ◽  
pp. 85-98 ◽  
Author(s):  
Anna Nynca ◽  
Dominika Słonina ◽  
Olga Jablońska ◽  
Barbara Kamińska ◽  
Renata Ciereszko
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
Oestrogen Receptor ◽  
Receptor Expression ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
Mrna And Protein Expression ◽  
Induced Changes ◽  
Reproductive Processes

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

scholarly journals Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.

1998 ◽  
Vol 9 (12) ◽  
pp. 2283-2290
Author(s):  
B Beck-Schimmer ◽  
B Oertli ◽  
T Pasch ◽  
R P Wüthrich
Keyword(s):  
Protein Expression ◽  
Renal Injury ◽  
De Novo ◽  
Kidney Diseases ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Mrna And Protein Expression ◽  
Renal Tubular

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that accumulates in the renal interstitium in immune-mediated kidney diseases. The functional significance of such HA deposition in the kidney has not been elucidated. Several studies have suggested that HA may exhibit proinflammatory effects. Since chemokines such as monocyte chemoattractant protein-1 (MCP-1) play an important role in the recruitment of leukocytes in renal injury, this study tested whether HA and its fragments could promote MCP-1 production by renal parenchymal cells. Mouse cortical tubular cells were stimulated with fragmented HA or with high molecular weight HA (Healon) in vitro and were examined for MCP-1 expression. Fragmented HA, but not Healon, increased MCP-1 mRNA within 30 min with a peak after 2 h. In addition, a 10-fold increase of MCP-1 protein in the supernatant was found after a 6-h stimulation with fragmented HA. The enhanced MCP-1 mRNA and protein expression in response to HA was dose-dependent between 1 and 100 microg/ml. Upregulation of MCP-1 protein production could be blocked by preincubation with actinomycin D or cycloheximide, suggesting that MCP-1 mRNA and protein expression in response to HA are based on de novo synthesis. The HA-stimulated MCP-1 production was also inhibited with anti-CD44 antibodies, suggesting that MCP-1 is upregulated at least in part by signaling through CD44. In summary, fragmented HA markedly stimulates renal tubular MCP-1 production by mechanisms that involve binding to the HA receptor CD44. It is hypothesized that the accumulation of HA in immune renal injury could participate in the recruitment and activation of inflammatory cells in vivo through production of MCP-1.

scholarly journals HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation

2021 ◽  
Author(s):  
Yong-Xiang Chen ◽  
Chunhua Shi ◽  
Jingti Deng ◽  
Catherine Diao ◽  
Nadia Maarouf ◽  
...  
Keyword(s):  
Protein Expression ◽  
Immune Complex ◽  
Cholesterol Metabolism ◽  
Plaque Burden ◽  
Blood Levels ◽  
Igg Antibodies ◽  
Amyloid A ◽  
Cholesterol Levels ◽  
Mrna And Protein Expression

Objective: Elevated HSP27 (heat shock protein 27) levels predict relative freedom from cardiovascular events. Over-expression or twice daily subcutaneous injections of human HSP27 in ApoE −/− mice reduces blood and plaque cholesterol levels, as well as inflammation and atherosclerotic plaque burden. Antibodies to HSP27 are present in human blood, and the purpose of the current studies is to explore their role. Approach and Results: Blood levels of both HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared with patients with cardiovascular disease. ApoE −/− mice fed a high-fat diet and vaccinated with recombinant HSP25 (rHSP25, murine ortholog) show increased levels of anti-HSP25 IgG antibodies and reductions in plasma cholesterol and atherogenesis. Moreover, rHSP25 vaccination markedly lowered serum amyloid A levels as well as hepatic macrophage abundance and inflammatory cytokine expression. The effects of the HPS25 vaccination on cholesterol metabolism are divergent: increased hepatic LDLR (low-density lipoprotein receptor) mRNA and protein expression and reduced plasma PCSK9 (proprotein convertase subtilisin/kexin type 9) levels—despite no effect on PCSK9 expression. In vitro, the HSP27 immune complex upregulates hepatocyte LDLR mRNA and protein expression independent of intracellular cholesterol levels and increases LDLR promoter activity. The increase in LDLR expression by the HSP27 immune complex is dependent upon activation of the NF-κB (nuclear factor κ light chain enhancer of activated B cells) pathway. Hepatocyte PCSK9 protein levels are reduced after HSP27 immune complex treatment in vitro despite only minor transient effects on gene expression. Conclusions: HSP27 immunotherapy represents a novel means of lowering cholesterol and PCSK9 levels, primarily due to augmentation of LDLR expression and is associated with marked reductions in inflammation.

scholarly journals Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts

Rheumatology ◽  
2019 ◽  
Vol 59 (9) ◽  
pp. 2258-2263 ◽  
Author(s):  
Tiago Carvalheiro ◽  
Beatriz Malvar Fernández ◽  
Andrea Ottria ◽  
Barbara Giovannone ◽  
Wioleta Marut ◽  
...  
Keyword(s):  
Protein Expression ◽  
Therapeutic Target ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Healthy Controls ◽  
Multiple Organs ◽  
Extracellular Matrix Components ◽  
Mrna And Protein Expression

Abstract Objectives SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. Methods Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. Results Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling. Conclusion These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

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