Increased release of serotonin from rat primary isolated adult cardiac myofibroblasts.
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ABSTRACT:
SUBMITTER: Tarbit E
PROVIDER: S-EPMC8514503 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Previous studies have extensively demonstrated the effect of endothelin-1 (ET-1), angiotensin II (Ang II), and TGF-β1 on the stimulation of collagen type I expression in cardiac myofibroblasts. However, the role of pro-remodeling peptides in the transcriptional regulation of the collagen promoter remains unclear. Thus, the purpose of this study was to investigate the net regulatory effects of pro-remodeling peptides on collagen type I promoter activity. Constructs of various lengths (300 bp, 1.1 kbp, 1.7 kbp, 2.3 kbp and 3.5 kbp) of the rat collagen α1(I) promoter were transfected into cardiac myofibroblasts in vitro and promoter activity was measured using chloramphenicol acetyl transferase (CAT) assays. Reduced promoter activity occurred across all treatments in myofibroblasts transfected with the 1.7 kbp construct. ET-1 was unable to increase promoter activity with constructs 300, 1.1, and 1.7 kbp, but induced promoter activity in cells with the 2.3 kbp construct. Additionally, while a combination of pro-remodeling peptides induced promoter activity across constructs, the resultant increase in the 2.3 and 3.5 kbp constructs were comparable to that observed from ET-1 treatment alone. Lastly, cells transfected with the entire promoter sequence had the lowest promoter activity. This data suggests that the collagen promoter is tightly regulated and that pro-remodeling factors produce an overall net effect on collagen expression, rather than additive.
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Project description:Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.
| S-EPMC3892894 | biostudies-literature
Project description:BackgroundThe hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.ResultsWith the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.ConclusionsOur results indicate that FuGENE6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.
| S-EPMC29065 | biostudies-literature
Project description:Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4-5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in primary culture. In conclusion, this straightforward and reproducible method of PMF culture, can be used to identify new markers of PMFs at different stages of differentiation, to compare their phenotype with those of HSC-MFs and ultimately determine their progenitors and specific functions in liver wound-healing.
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Project description:Serotonin (5-HT) is member of a family of indolamine molecules that participate in a wide variety of biological processes. Despite its important role in the regulation of local blood systems, little is known about the physiological function of 5-HT in reproductive organs, its functional implications, and its role in the reproduction of mammals. In the present work, we evaluated the localization and distribution of 5-HT (using histochemical analysis of indolamines) and different components of the serotoninergic system in rat testes. We detected local synthesis and degradation through immunofluorescence and western blot analyses against the TPH1, MAOA, 5-HTT, and VMAT1 serotonin transporters. We also identified the localization and distribution of the 5-HT1B, 5-HT2A, and 5-HT3A receptors. RT-PCR results showed the presence of the Tph1, Maoa, Slc6a4, and Htr3a genes in testes and in the brain stem (Tph1 was used as a negative control). High-performance liquid chromatography was used to determine the presence of 5-HT and the activity of tryptophan hydroxylase in testes homogenates in vitro. Our observations suggest that TPH1 activity and local 5-HT synthesis befall in rat testes. We propose that 5-HT could participate in the regulation of testosterone synthesis and in the spermatogenesis process via local serotoninergic system. However, more studies are needed before concluding that rat testes, or those of other mammals, contain an active form of tryptophan hydroxylase and produce 5-HT.
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Project description:Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo's cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3's impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200-380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100-500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3's cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3's protection is a cardiomyocyte phenomenon and targets mitochondria.
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Project description:Connexin 43 expression (Cx43) is increased in cardiac fibroblasts (CFs) following myocardial infarction. Here, potential mediators responsible for increasing Cx43 expression and effects of differential CF phenotype on cardiac myocyte (CM) function were investigated. Stimulating adult rat CFs with proinflammatory mediators revealed that interleukin 1β (IL-1β) significantly enhanced Cx43 levels through the IL-1β pathway. Additionally, IL-1β reduced mRNA levels of the myofibroblast (MF) markers: (i) connective tissue growth factor (CTGF) and (ii) α smooth muscle actin (αSMA), compared to control CFs. A co-culture adult rat CM:CF model was utilised to examine cell-to-cell interactions. Transfer of calcein from CMs to underlying CFs suggested functional gap junction formation. Functional analysis revealed contraction duration (CD) of CMs was shortened in co-culture with CFs, while treatment of CFs with IL-1β reduced this mechanical effect of co-culture. No effect on action potential rise time or duration of CMs cultured with control or IL-1β-treated CFs was observed. These data demonstrate that stimulating CFs with IL-1β increases Cx43 and reduces MF marker expression, suggesting altered cell phenotype. These changes may underlie the reduced mechanical effects of IL-1β treated CFs on CD of co-cultured CMs and therefore have an implication for our understanding of heterocellular interactions in cardiac disease.
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Project description:The blood testis barrier (BTB) is often studied with isolated immature Sertoli cells (SCs), transepithelial resistance (TER) measurements and FITC dextran diffusion assays. Recently, it was found that even in the absence of SCs, only few immune cells enter the seminiferous tubules. Thus, in this study, we evaluated the testicular immunological barrier (TIB) in vitro by transmigration of macrophages through SCs with and without peritubular cells (PCs) and with or without matrigel (MG). Primary PCs were isolated from adult rat testis and kept in mono- or co-cultures with the conditionally reprogrammed primary adult Sertoli cell line (PASC1) from rat that has been recently generated by our group. Rat monocytes isolated from fresh blood were differentiated into M0 macrophages, and after polarization to M1 or M2 macrophages characterized by gene expression of CXCL11 and TNF-α for M1, or CCL17 and CCL22 for M2. Transmigration of LeukoTracker-labeled M0, M1, and M2 macrophages through mono- and co-cultures of PCs/SCs with and without MG demonstrated that SCs are the main constituent of the TIB in vitro with only a negligible contribution of PCs or MG. Moreover, M2 macrophages showed less migration activity compared to M0 or M1. Treatment of SCs with testosterone (T) showed positive effects on the barrier in contrast to negative effects by interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α). The new transmigration model is suitable to evaluate transmigration of macrophages through a barrier consisting of testicular cells and can be applied to study the integrity of testicular barriers with respect to immunological aspects.
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Project description:Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.
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Project description:We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-β1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.
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