Actin Gene Expression Research Articles

Evaluation of actin as a reference for quantitative gene expression studies in Emiliania huxleyi (Prymnesiophyceae) under ocean acidification conditions

ABSTRACT Gene expression studies of marine phytoplankton under ocean acidification conditions are frequently based on relative measurements, with actin commonly used as a reference gene. Evidence from other organisms suggests that actin gene expression may be regulated by environmental conditions, compromising the role of actin as a reference gene. In this work the reliability of actin as a reference gene for ocean acidification experimental conditions (high CO2 vs low CO2) in two different metabolic states (acclimated metabolism vs perturbed metabolism) for the coccolithophore Emiliania huxleyi was tested. The transcriptional response of the actin (act) is compared with the expression of specific target genes associated with inorganic carbon uptake (α-carbonic anhydrase: αca1) and assimilation (RuBisCO: rbcL), which was regulated under the experimental conditions. Our results showed act expression instability in experimental conditions, evidencing that act is not a reliable reference gene for studies assessing the effect of ocean acidification on Emiliania huxleyi. Furthermore, when the act-based normalization was quantitatively tested, rbcL and αca1 expression were compromised, leading us to conclude that absolute gene expression quantification should be considered as a potentially reliable alternative for studying gene expression under ocean acidification conditions.

Cytoskeletal Protein Zyxin Inhibits the Activity of Genes Responsible for Embryonic Stem Cell Status

Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.

Human umbilical cord mesenchymal stem cells ameliorate skin fibrosis development in a mouse model of bleomycin-induced systemic sclerosis.

Mesenchymal stem cell (MSC) infusion has become a novel therapeutic strategy for complex autoimmune diseases; however, few detailed studies have been performed to investigate the benefit and mechanism of MSC treatment on systemic sclerosis (SSc). The present study aimed to evaluate the therapeutic effect of human umbilical cord derived-MSCs (UC-MSCs) on bleomycin-induced SSc in mice and explore the potential underlying mechanism. The murine SSc model was established by daily subcutaneous injection of bleomycin for 4 weeks, followed with two UC-MSC infusions every 7 days. Skin fibrosis was assessed by H&E and Masson staining. Flow cytometry was used to determine IL-17A, IFN-γ, tumor necrosis factor-β, IL-10 and IL-12 levels in serum samples and T cell subsets in murine spleen. Additionally, gene expression levels of cytokines and fibrosis markers in skin samples were measured by reverse transcription-quantitative PCR. Immunofluorescence staining was performed to track UC-MSC localization and lymphocyte cell infiltration in vivo. UC-MSC treatment exerted an anti-fibrotic role in bleomycin-induced SSc mice, as confirmed by histological improvement, decreased collagen synthesis, and reduced collagen-1α1, collagen-1α2, fibronectin-1 and α-smooth muscle actin gene expression levels. The results indicated that UC-MSC treatment only had a limited systematic effect on cytokine production in serum samples and T cell activation in the spleen. By contrast, T helper (Th)17 cell infiltration and activation in skin were efficiently inhibited after UC-MSC infusion, as evidenced by the decreased IL-17A and retinoic acid-related orphan receptor γt gene expression as well as IL-17A production. UC-MSC administration significantly ameliorated bleomycin-induced skin fibrosis and collagen formation primarily by eliminating local inflammation and Th17 cell activation in the skin; however, the systemic inhibitory effect of UM-MSCs on cytokines was less profound.

Tumor Necrosis Factor Alpha and Interleukin 1 Beta Suppress Myofibroblast Activation Via Nuclear Factor Kappa B Signaling in 3D-Cultured Mitral Valve Interstitial Cells

Mitral valve disease is a major cause of cardiovascular morbidity throughout the world. Many different mitral valve pathologies demonstrate a pronounced degree of fibrotic remodeling, often accompanied by an inflammatory state. Mitral valve fibrosis is mediated by valvular interstitial cells (VICs), which reside in the valve leaflets and show a tendency to differentiate into myofibroblast-like cells during disease conditions. In this study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) on mitral VICs, since these pro-inflammatory cytokines have been shown to exert pleiotropic effects on various cell types in other fibrotic disorders. Using biomimetic three-dimensional culture systems, we demonstrated that TNF-α and IL-1β suppress myofibroblast differentiation in mitral VICs, as evidenced by gene and protein expression of alpha smooth muscle actin and smooth muscle 22 alpha. Addition of TNF-α and IL-1β also inhibited mitral VIC-mediated contraction of collagen gels. Furthermore, inhibition of NF-κB, which is downstream of TNF-α and IL-1β, reversed these effects. These results reveal targetable pathways that could enable the development of pharmaceutical treatments for alleviating fibrosis during mitral valve disease.

Ni and TiO2 nanoparticles cause adhesion and cytoskeletal changes in human osteoblasts.

Titanium-based alloys have established a crucial role in implantology. As material deteriorates overtime, nanoparticles of TiO2 and Ni are released. This study is focused on the impact of TiO2 and Ni nanoparticles with size of 100 nm on cytoskeletal and adhesive changes in human physiological and osteoarthritic osteoblasts. The impact of nanoparticles with concentration of 1.5 ng/mL on actin and tubulin expression and gene expression of FAK and ICAM-1 was studied. The cell size and actin expression of physiological osteoblasts decreased in presence of Ni nanoparticles, while TiO2 nanoparticles caused increase in cell size and actin expression. Both cell lines expressed more FAK as a response to TiO2 nanoparticles. ICAM-1 gene was overexpressed in both cell lines as a reaction to both types of nanoparticles. The presented study shows a crucial role of Ni and TiO2 nanoparticles in human osteoblast cytoskeletal and adhesive changes, especially connected with the osteoarthritic cells. Graphical abstract.

Myocardin-Related Transcription Factor A (MRTF-A) Regulates the Balance between Adipogenesis and Osteogenesis of Human Adipose Stem Cells

Previous studies have demonstrated that myocardin-related transcription factor A (MRTF-A) generates a link between the dynamics of the actin cytoskeleton and gene expression with its coregulator, serum response factor (SRF). MRTF-A has also been suggested as a regulator of stem cell differentiation. However, the role of MRTF-A in human mesenchymal stem cell differentiation remains understudied. We aimed to elucidate whether MRTF-A is a potential regulator of human adipose stem cell (hASC) differentiation towards adipogenic and osteogenic lineages. To study the role of MRTF-A activity in the differentiation process, hASCs were cultured in adipogenic and osteogenic media supplemented with inhibitor molecules CCG-1423 or CCG-100602 that have been shown to block the expression of MRTF-A/SRF-activated genes. Our results of image-based quantification of Oil Red O stained lipid droplets and perilipin 1 staining denote that MRTF-A inhibition enhanced the adipogenic differentiation. On the contrary, MRTF-A inhibition led to diminished activity of an early osteogenic marker alkaline phosphatase, and export of extracellular matrix (ECM) proteins collagen type I and osteopontin. Also, quantitative Alizarin Red staining representing ECM mineralization was significantly decreased under MRTF-A inhibition. Image-based analysis of Phalloidin staining revealed that MRTF-A inhibition reduced the F-actin formation and parallel orientation of the actin filaments. Additionally, MRTF-A inhibition affected the protein amounts of α-smooth muscle actin (α-SMA), myosin light chain (MLC), and phosphorylated MLC suggesting that MRTF-A would regulate differentiation through SRF activity. Our results strongly indicate that MRTF-A is an important regulator of the balance between osteogenesis and adipogenesis of hASCs through its role in mediating the cytoskeletal dynamics. These results provide MRTF-A as a new interesting target for guiding the stem cell differentiation in tissue engineering applications for regenerative medicine.

MicroRNA Regulatory Pathways in the Control of the Actin-Myosin Cytoskeleton.

MicroRNAs (miRNAs) are key modulators of post-transcriptional gene regulation in a plethora of processes, including actin–myosin cytoskeleton dynamics. Recent evidence points to the widespread effects of miRNAs on actin–myosin cytoskeleton dynamics, either directly on the expression of actin and myosin genes or indirectly on the diverse signaling cascades modulating cytoskeletal arrangement. Furthermore, studies from various human models indicate that miRNAs contribute to the development of various human disorders. The potentially huge impact of miRNA-based mechanisms on cytoskeletal elements is just starting to be recognized. In this review, we summarize recent knowledge about the importance of microRNA modulation of the actin–myosin cytoskeleton affecting physiological processes, including cardiovascular function, hematopoiesis, podocyte physiology, and osteogenesis.

4121 The beneficial, anti-fibrotic effects of chemokine receptor 2 and 5 antagonists on fat-exposed mouse primary hepatic stellate cells (pHSCs)

OBJECTIVES/GOALS: Non-alcoholic steatohepatitis (NASH) is a leading cause of cirrhosis in the world for which no anti-fibrotic therapies exist. We hypothesized that BMS-22 and maraviroc (MVC), chemokine receptor 2 (CCR2) and 5 (CCR5) antagonists, respectively, would diminish the fibrogenic activity of "fat-exposed" murine pHSCs. METHODS/STUDY POPULATION: pHSCs were isolated from livers of 6 week old male mice following 4 weeks on a NASH-inducing choline-deficient high fat diet (CDAHFD, “fat-exposed”) or standard diet (SD) and passaged in vitro. Early passage (6-12) pHSCs were plate-adhered and TGF-b-treated (10ng/mL) to maximally activate their pro-fibrogenic genes, collagen 1α1 (Col1A1), tissue inhibitor of metalloproteinase 1 (TIMP1), or α-smooth muscle actin (ACTA2). CDAHFD and SD pHSCs were then treated for 48 hours with increasing doses of BMS-22 or MVC (range: 0.3-120ng/mL) to determine (1) the degree of attenuation of the pro-fibrogenic response as measured by qPCR of fibrogenic genes (Col1A1, TIMP1,ACTA2); (2) enhancement of a fibrolytic response as measured by qPCR of matrix metalloproteinases (MMP) 2, 9 and 13 genes; and (3) pHSC migration using the scratch assay. Cell viability and CCR2 and CCR5 gene expression in response to escalating doses of antagonists were also measured. RESULTS/ANTICIPATED RESULTS: Plate- and TGF-b activated CDAHFD pHSCs had a 2-fold greater, dose-dependent attenuation of their pro-fibrogenic activity in response to BMS-CCR2-22 and MVC, when compared with plate- and TGF-b activated SD pHSCs, as measured by reductions in collagen 1α1 (Col1A1) and α-smooth muscle actin (ACTA2) gene expression. TIMP1 gene expression was unaffected by drug treatment for 48 hours. Cell viability was not affected up to doses of 30ng/mL of each drug. pHSCs also demonstrated a dose-dependent increase in CCR2, CCR5 and MMP-9 gene expression in response to surface receptor antagonism. Migration assays comparing CDAHFD and SD pHSCs in response to escalating doses of MVC and BMS-22 are ongoing and expected to demonstrate a significantly decreased migratory capacity of CDAHFD pHSCs than SD pHSCs in response to therapy, reflecting the increased susceptibility of the “fat-exposed” pHSCs to anti-fibrotic therapy than normal pHSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-fibrotic drugs that dampen pro-fibrogenic activities of “fat-exposed” pHSCs are urgently needed. CCR2 and CCR5 antagonists, BMS-22 and MVC, respectively, can selectively dampen the pro-fibrogenic response of fat-exposed pHSCs, and must be considered for future trials in human NASH. CONFLICT OF INTEREST DESCRIPTION: Dr. Jill Smith has a patent licensing agreement with Immune Therapeutics, Inc.

Intervertebral disc herniation effects on multifidus muscle composition and resident stem cell populations.

AbsractBackgroundParaspinal muscles are crucial for vertebral stabilization and movement. These muscles are prone to develop fatty infiltration (FI), fibrosis, and atrophy in many spine conditions. Fibro‐adipogenic progenitors (FAPs), a resident muscle stem cell population, are the main contributors of muscle fibrosis and FI. FAPs are involved in a complex interplay with satellite cells (SCs), the primary myogenic progenitor cells within muscle. Little is known about the stem cell composition of the multifidus. The aim of this study is to examine FAPs and SCs in the multifidus in disc herniation patients. Multifidus muscle samples were collected from 10 patients undergoing decompressive spine surgery for lumbar disc herniation. Hamstring muscle was collected from four patients undergoing hamstring autograft ACL reconstruction as an appendicular control. Multifidus tissue was analyzed for FI and fibrosis using Oil‐Red‐O and Masson's trichrome staining. FAPs and SCs were visualized using immunostaining and quantified with fluorescence‐activated cell sorting (FACS) sorting. Gene expression of these cells from the multifidus were analyzed with reverse transcription‐polymerase chain reaction and compared to those from hamstring muscle. FI and fibrosis accounted for 14.2%± 7.4% and 14.8%±4.2% of multifidus muscle, respectively. The multifidus contained more FAPs (11.7%±1.9% vs 1.4%±0.2%; P<.001) and more SCs (3.4%±1.6% vs 0.08%±0.02%; P=.002) than the hamstring. FAPs had greater α Smooth Muscle Actin (αSMA) and adipogenic gene expression than FAPs from the hamstring. SCs from the multifidus displayed upregulated expression of stem, proliferation, and differentiation genes.ConclusionThe multifidus in patients with disc herniation contains large percentages of FAPs and SCs with different gene expression profiles compared to those in the hamstring. These results may help explain the tendency for the multifidus to atrophy and form FI and fibrosis as well as elucidate potential approaches for mitigating these degenerative changes by leveraging these muscle stem cell populations.

Topical application of a commercially available formulation of vitamin C stabilized by vitamin E and ferulic acid reduces tissue viability and protein synthesis in ex vivo human normal skin.

Aqueous formulations of vitamin C stabilized by vitamin E and ferulic acid at low pH effectively protect skin against reactive oxygen species-induced damage. However, the effects of these formulations on human skin have not clearly been described. The aim of this study was to investigate whether topical application of two commercially available formulations of vitamin C alter human skin using an ex vivo model. Human skin explants were topically treated on alternate days with commercially available formulation 1 (15% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline or formulation 2 (20% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline. Only saline was applied to control skin explants. Topical formulation 1 at 100%, 50%, or 10%, but not formulation 2 at 100%, 50%, or 10%, reduced the viability of ex vivo human skin compared to the control after 7, 10, and 13days. In addition, compared to the control, ex vivo human skin treated with formulation 1 at 50%, but not formulation 2 at 50%, also decreased mRNA levels of actin and ribosomal protein L10 and gene expression of extracellular matrix components after 10days. Furthermore, after 10days, topical application of formulation 1 at 50%, but not formulation 2 at 50%, decreased the protein expression of proliferating cellular nuclear antigen, lysyl oxidase, β-actin, and glyceraldehyde-3-phosphate dehydrogenase compared to the control. Topical formulation 1, but not formulation 2, may reduce the viability of and protein synthesis in ex vivo human skin. Those effects might be due to action of vehicle of formulation 1 on ex vivo human skin.

Cytoskeletal Protein Zyxin Inhibits the Activity of Genes Responsible for Embryonic Stem Cell Status

Zyxin is a cytoskeletal LIM domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we found that Zyxin downregulation in Xenopus laevis embryos reduced the expression of numerous genes regulating cell differentiation but enhanced that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2 pou5f3.3 and vent2.1/2. For pou5f3 family genes (mammalian OCT4 orthologs), we showed that this effect was the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, Zyxin interfered with the formation of these complexes, thereby stimulating pou5f3 mRNAs degradation. In addition, in zebrafish embryos and human HEK293 cells, Zyxin downregulation increased the mRNA levels of the pluripotency genes KLF4, NANOG and OCT4. Our findings indicate that Zyxin may play a role as a switching hub coupling cell morphogenetic movements, differentiation and the embryonic stem cell status.

Functions of beta2-adrenergic receptor in human periodontal ligament cells.

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. Test-tube Lab Research. Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. NA Laryngoscope, 2020.

The microenvironment and cytoskeletal remodeling in tumor cell invasion.

The physical cues of tumor microenvironment (TME) contribute greatly to the initiation and progression of cancer. Tumor tissues usually become stiffer than healthy tissues with more aligned fibers and changed porosity. In recent years, numerous studies attempted to investigate whether biophysical cues from the surrounding environment affect the biophysical, biochemical, and biological behavior of cells and consequently attribute to the development of cancer. Here, we review recent advances of our understanding of these physical cues in terms of extracellular matrix (ECM) stiffness and topography (alignment and porosity). We discuss the underlying mechanisms of changes in TME physical parameters. Then, we summarize how cancer cells sense the mechanical signals, transfer them to the downstream signaling pathways, and finally translate them to different cellular behaviors. Specifically, we discuss the role of mechanical changes of ECM in cancer cell stiffness, actin cytoskeleton organization, gene and protein expressions, the migration of cancer cells, and their response to specific treatments. We then review different methods which have been successfully utilized to model ECM physical properties. This review paper concluded with the limitations of current studies which followed by some insights into clarifying the therapeutic potential of ECM mechanical properties to target and control the development of cancer.

Mesenchymal stem cell applications on the chronic liver disease

Objective The aim was to evaluate the effect of application of bone marrow mesenchymal stem cells (BMMSCs) after transplantation into mice with carbon tetrachloride-induced chronic liver. Background Chronic disease causes hepatocyte fibrosis, with subsequent morbidity and mortality. Stem cells have emerged an alternative therapy for liver fibrosis, which can be harvested from different sources, such as mesenchymal stem cells (MSCs). Materials and methods This study was conducted on 55 mice and 18 human bone marrow samples. Primarily, liver fibrosis was induced in five mice, and after the fourth week of CCl4induction, they were killed, and liver biopsy was done to estimate fibrosis occurrence. The remaining 50 mice were classified as 25 mice injected with 0.9% saline to be used as control (group I) and 25 mice were infused intravenously with 1 × 106 human BMMSC (group II) after continuous CCl4administration in both groups. After 4 weeks of MSCs infusion, serum alanine transaminase, aspartate transaminase, albumin, and total bilirubin were determined for all 50 mice. Moreover, liver histology was performed to determine the fibrosis degree as well as α-fetoprotein immunohistochemistry, and RT-PCR was done for α-smooth muscle actin expression. Human BMMSCs were collected and isolated by bone marrow culture and evaluated by flow cytometric characterization of MSCs using CD45-negative and CD90-positive cell markers. Results MSCs-treated group revealed significant lower alanine transaminase, aspartate transaminase, and albumin; higher total bilirubin levels; decreased histopathological fibrosis; diffuse α-fetoprotein immunostaining; and lower α-smooth muscle actin gene expression when compared with the control group. Conclusion MSCs could be used as a therapeutic tool in end-stage liver disease.

Gremlin-1 potentiates the dedifferentiation of VSMC in early stages of atherosclerosis

Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to pro-atherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. ResultsComparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22-alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4–6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. ConclusionOur data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.

A novel NADPH oxidase inhibitor targeting Nox4 in TGFβ-induced lens epithelial to mesenchymal transition

Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGFβ-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3 μM of GLX7013114, and then treated with 200 pg/ml of TGF-β2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGFβ-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGFβ-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGFβ and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGFβ-induced gene expression of α-smooth muscle actin (αSMA), collagen 1a and fibronectin. GLX7013114, given that it appears to block aspects of TGFβ-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies.

Molecular effects of Microcystin-LA in tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus) is a freshwater phytoplanktivorous fish species reported to accumulate and tolerate large amounts of cyanotoxins such as microcystins (MCs). The present study aimed to investigate molecular responses to the acute exposure of Nile tilapia to the Microcystin-LA analogue (MC-LA). Thus, the specimens were sublethally exposed to 1000 μg kg−1 of MC-LA for 12, 24, 48, and 96 h. Gene expression of PP1, PP2A, GST, GPX and actin was analyzed by quantitative PCR. The protein abundance profile of PP2A was determined by immunoblotting, while the integrity of its biological function was assessed by a phosphatase enzymatic assay. PP2A activity was significantly and strongly reduced by MC-LA. A resulting feedback mechanism significantly increased PP2A gene expression and protein abundance in all assessed times. However, a recovery of that phosphatase activity was not observed. In this study, the observed increase in GPX gene expression was the only response that could be directly related to the unknown factors associated to the fish survival to such high dose exposure.

Normalisation of circulating adiponectin levels in obese pregnant mice prevents cardiac dysfunction in adult offspring

Background/objectivesAdiponectin concentrations are low in obese pregnant women. Restoring normal adiponectin concentrations by infusion in obese pregnant mice prevents placental dysfunction, foetal overgrowth and metabolic syndrome in the offspring. We hypothesised that normalising maternal adiponectin in obese late pregnant dams prevents cardiac dysfunction in the adult offspring.Subjects/methodsPregnant female mice with diet-induced obesity were infused with adiponectin (0.62 μg g−1 day−1, n = 24) or saline (n = 22) over days 14.5–18.5 of pregnancy (term = day 19.5). Control dams ate standard chow and received saline (n = 22). Offspring were studied at 3 and 6 months of age.ResultsMaternal obesity impaired ventricular diastolic function, increased cardiomyocyte cross-sectional area and upregulated cardiac brain natriuretic peptide (Nppb) and α-skeletal actin (Acta1) gene expression in adult male offspring, compared to control offspring. In adult female offspring, maternal obesity increased Nppb expression, decreased end-diastolic volume and caused age-dependent diastolic dysfunction but not cardiomyocyte hypertrophy. Maternal obesity also activated cardiac Akt and mechanistic target of rapamycin (mTOR) signalling in male, but not in female, offspring and inhibited cardiac extracellular signal-regulated kinase 1/2 (ERK1/2) in both sexes. Normalising maternal circulating adiponectin concentrations by infusing obese dams with adiponectin prevented offspring diastolic dysfunction and ventricular dilation and normalised cardiac Akt-mTOR signalling irrespective of sex. Maternal adiponectin infusion also reduced cardiac Nppb expression and increased ERK1/2 signalling in offspring of obese dams. Adiponectin infusion did not prevent cardiomyocyte hypertrophy but reduced ventricular wall thickness in male offspring and increased collagen content in female offspring of obese dams, compared to controls.ConclusionsLow maternal adiponectin levels in obese mice in late pregnancy are mechanistically linked to in utero programming of cardiac dysfunction in their offspring. Interventions enhancing endogenous adiponectin secretion or signalling in obese pregnant women could prevent the development of cardiac dysfunction in their children.

Administration of a selective prostaglandin E2 receptor agonist attenuates renal fibrosis in vitro and in vivo

Renal fibrosis plays a pivotal role in the development and progression of chronic kidney disease (CKD), which affects 10% of the adult population. Previously, it has been demonstrated that the cyclooxygenase‐2 (COX‐2)/prostaglandin (PG) system influences the progression of renal injury. PGE2 interacts with four different G‐protein‐coupled PGE2 EP receptors, known as EP1–EP4, and plays a role in several biological functions. Previous studies show that the EP2 receptor is involved in progression of lung fibrosis. In this study, we investigated the effect of an EP2 agonist (butaprost) on renal fibrogenesis on a cellular, renal tissue and organismal level using well‐established in vivo and in vitro models as well as a recently developed human model of renal fibrosis. This model is ideal to study multicellular pathological processes, e.g. fibrosis, directly in human tissue since cellular diversity and organ architecture is maintained in the slices. Butaprost (50 μM) reduced TGF‐β‐induced fibronectin (FN) expression, Smad2/3 phosphorylation and epithelial‐mesenchymal transition in MDCK cells. In addition, treatment with 4 mg/kg butaprost attenuated the development of fibrosis in mice that underwent unilateral ureteral obstruction (UUO) surgery, as illustrated by a significant reduction in the gene and protein expression of α‐smooth muscle actin, FN and collagen 1A1. More importantly, a similar anti‐fibrotic effect of butaprost was observed in human precision‐cut kidney slices exposed to TGF‐β. The mechanism of action of butaprost appeared to be a direct effect on TGF‐β/Smad signaling, which was independent of the cAMP/PKA pathway. In conclusion, this study demonstrates that stimulation of the EP2 receptor effectively mitigates renal fibrogenesis in various fibrosis models. These findings warrant further research into the clinical application of butaprost, or other EP2 agonists, for the inhibition of renal fibrosis.Support or Funding Information Sources of support: This work was kindly supported by Lundbeckfonden, grant number R231‐2016‐2344 as well as the Danish Council for Independent Research, grant number 6110‐00231B, Aarhus University Research Foundation, grant number AUFF‐E‐2015‐FLS‐8‐69 and Hildur and Dagny Jacobsens Foundation grant number 1295716‐1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.