Human Amniotic membrane modulates collagen production and deposition in vitro
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ABSTRACT: Pathological fibrosis, an excessive accumulation of collagen, compromises tissue architecture, impedes its function and represents a medical complication. Properties of the amniotic membrane have demonstrated potential to regulate fibrotic processes. RNA sequencing analysis evaluated the effects of dehydrated human amnion/chorion membrane (DHACM) on fibrotic events using in vitro macromolecular crowding to mimic a physiologically relevant microenvironment. Functional enrichment with Reactome pathway analysis indicated that the function of a subset of these dysregulated genes was associated with the composition and function of the extracellular matrix. Further use of macromolecular crowding in vitro was used to evaluate the effects of DHACM and a lyophilized human amnion/chorion membrane (LHACM) on type 1 collagen production and deposition under profibrotic conditions (addition of TGFβ1). In the continued presence of TGFβ1, LHACM and DHACM regulated the intracellular production and extracellular deposition of type 1 collagen. In addition, results suggest that the effect on collagen deposition could in part be modulated through the regulation of proteins responsible for the extracellular post-translational modifications of collagen. These observations may support the use of DHACM and LHACM in the regulation collagen production/deposition in profibrotic environments.
Project description:We compared fetal membrane tissue from preterm labor deliveries to fetal tissue from preterm labor with preterm prelabor rupture of membranes (PPROM) deliveries to further explore the concept that spontaneous preterm birth can result from the initializing of two separate but overlapping pathological events. Chorioamnion, separated into amnion and chorion, was collected from gestationally age-matched cases and controls within 15 minutes of birth, and analyzed using RNA sequencing. In our study, transcriptome analysis of preterm fetal membranes revealed distinct differentially expressed genes for PPROM, separate from preterm labor. This study is the first to report transcriptome data that reflects the individual pathophysiology of amnion and chorion tissue from PPROM deliveries.
Project description:We performed scRNAseq on embryonic and extra-embryonic mesoderm cells from mouse embryo at Mid/Late Streak stage (Embryonic day 7.25) as well as on Late Bud stage (Embryonic day 7.75) microdissected amnion, chorion and allantois. We used transgenic embryos (Brachyury-Cre; mTmG) in which all mesoderm cells are converted to membrane-GFP upon Cre-mediated recombination and the rest express membrane Tomato. For all the samples, we identified clusters related to the localization in the tissue or the structures. Those results provide an atlas of early stages of morphogenesis of the maternal-fetal interface.
Project description:Mice deficient in the BMP-effector, Smad5 (Smad5 KO), develop severe defects in embryonic morphogenesis as well as a delay in amnion-chorion separation, important extraembryonic tissues. After closure of the proamniotic canal, a remarkable ectopic primitive streak-like aggregate develops in the amnion of these mutants. We investigated the earliest steps of mutant amnion misdifferentiation by RNAseq of single Control (Ctrl) and Smad5 KO amnion samples collected before the appearance of the aggregate. The transcriptome analysis revealed two separate sets of non-squamous amnion defects. One set of mutants (KO-SetA) robustly overexpressed streak mesoderm-related genes conform former analyses (Pereira et al., 2012). The other set overexpressed extraembryonic ectoderm markers suggestive of chorionic inclusion in amnion (KO-SetB). Tetraploid chimera analyses confirmed that SMAD5 deficiency in the epiblast can result in two distinct sets of amnion defects: one with impaired anterior amnion expansion and differentiation, and another with inclusion of chorionic extraembryonic ectoderm in the space normally occupied by amnion.
Project description:Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasty (TKA). Current surgical and pharmacologic treatment options are limited. Therefore, an in vitro model for joint myofibroblastogenesis is valuable to investigate the arthrofibrotic process and identify diagnostic biomarkers and treatment options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissue from knees of four patients undergoing primary TKA for osteoarthritis and characterized primary outgrowth cells from these tissues in the absence and presence of transforming growth factor beta 1 (TGFβ1), a pro-myofibroblastic cytokine. Light microscopy of knee outgrowth cells revealed spindle-shaped cells while immunofluorescence (IF) established staining for the fibroblast-specific antigen TE-7 and Vimentin, which are characteristics of fibroblasts. These fibroblasts differentiate readily into myofibroblasts as highlighted by enhanced alpha smooth muscle actin (ACTA2) mRNA and protein expression and increased collagen mRNA (i.e., collagen type 1 (COL1A1) and collagen type 3 (COL3A1)) expression and collagenous matrix deposition in the presence of TGFβ1. Of note, these studies also revealed that knee-derived fibroblasts are more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stem cells. Importantly, while outgrowth fibroblasts isolated from four patients and three anatomical regions exhibit similar gene expression profiles, these knee fibroblasts form a unique gene expression cluster within the fibroblast niche as revealed by RNA-sequencing analysis. In conclusion, our study provides a fibroblast/myofibroblast model of outgrowth knee cells derived from patients undergoing primary TKA that can be employed to assess myofibroblast-related processes and test novel pharmacological strategies in vitro for arthrofibrosis.
Project description:Fibrosis is an uncontrolled wound healing process resulting from tissue injury, inflammation and fibroblast activation, finally leading to accumulation of extracellular matrix (ECM) components in tissues and to organ failure. The underlying mechanisms of fibrosis have been intensely studied and it has become clear that multiple pathways and their crosstalk regulate fibrotic diseases with a few central players, including TGFβ1. Recently, YAP and TAZ were implicated in fibrosis as one of the essential components of its pathogenesis. YAP/TAZ are known to crosstalk with the TGFβ pathway in the nucleus and regulate its activity on transcriptional level by binding to Smad2/3/4 complexes. We hypothesized that factors, such as GPCR ligands which regulate YAP/TAZ, might modulate the profibrotic responses to TGFβ1. We performed gene expression microarray in NHDF treated with TGFβ1, LPA, the combination of TGFβ1/LPA or vehicle. The aim was to have a broader look at transcriptional signatures induced by the individual treatments and in the combination of TGFβ1 and LPA.
Project description:TGFβ1 is a profibrotic mediator that contributes to a broad spectrum of pathologies, including pulmonary fibrosis (PF). However, the secretome of TGFβ1-stimulated primary human normal lung (NL) fibroblasts has not been well characterized. Using fluorescent 2-dimensional gel electrophoresis (2D-PAGE) and differential gel electrophoresis (DIGE), we identified 37 differentially secreted proteins in the conditioned media of TGFβ1-activated NL fibroblasts and generated a protein-protein association network of the TGFβ1 secretome using STRING. Functional enrichment revealed that biological processes and pathways characteristics of PF were enriched. Using the DrugBank database, we determined that 32 of the secreted proteins are targets of known experimental, investigational and approved drugs. Additionally, by comparing the TGFβ1 secretome of NL fibroblasts to proteomic biomarkers from biological fluids of systemic sclerosis (SSc) patients, we identified 11 overlapping proteins. Together our data validate the TGFβ1-induced secretome of NL fibroblasts as a valid in vitro model that reflects SSc biomarkers and identifies potential therapeutic targets for SSc-PF.
Project description:Endometriosis (EMs) is a common infertility-related disease in women of reproductive age. Impaired endometrial decidualization is one of the most important factors contributing to the embryo implantation failure EMs patients. However, the exact mechanism remains unclear. Previous studies have shown collagen I deposition in the eutopic endometrium of EMs patients, which may lead to impaired endometrial decidualization. The level of collagen I in eutopic endometrium of EMs was analyzed. We performed a proteomic analysis of ectopic endometrial stromal cell-derived extracellular vesicles (EMs-EVs) and extracellular vesicles derived from endometrial stromal cells in the endometrium of control patients (CTL-EVs). An endometrial transcriptional profiles of EMs patients and normal controls in the mid-secretory phase of menstrual cycle was compared. Endometrial stromal cells (ESCs) were stimulated with chloroquine or rapamycin to evaluate the association between autophagy and collagen I. The expression of PKM2 in EMs-EVs and serum extracellular vesicles from EMs patients were examined by western blotting. PKM2 was overexpressed or knockdown in ESCs to investigate the level of autophagy and collagen I. ESCs were treated with ectopic ESC-derived extracellular vesicles with highly expressed PKM2 protein, and the potential molecular mechanisms were further confirmed through western blotting and immunohistochemical analysis. We found that endometrial collagen I expression during the mid-secretory phase was increased in the EMs group. We demonstrated that autophagy is defective in eutopic endometrial stromal cells (ESCs) of EMs patients. In ESCs, pharmacological inhibition of autophagy by chloroquine (CQ) promoted collagen I deposition. Ectopic endometrial stromal cell-derived extracellular vesicles (EMs-EVs) inhibited autophagy of ESCs and promoted collagen I deposition in vivo and in vitro. Mechanistically, EMs-EVs encapsulating PKM2 impair eutopic endometrial autophagy via Akt/mTOR signaling pathway. Together, we demonstrated that EMs-EVs encapsulating PKM2 impaired endometrial autophagy inducing collagen I deposition in EMs, which provided a potential target for therapeutic implications.
Project description:Explored the potential role of mTOR in regulating the extra cellular matrix of TGF-b1-stimulated IPF fibroblasts grown under macromolecular crowding conditions in the presence of either CZ415 or rapamycin
Project description:Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by fragile skin forming blisters that heal invariably with scars. It is due to mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils connecting the cutaneous basement membrane to the dermis. Identical COL7A1 mutations often result in inter- and intra-familial disease variability, suggesting that additional modifiers contribute to RDEB course. Here, we studied a monozygotic twin pair with RDEB presenting markedly different phenotypic manifestations, while expressing similar amounts of collagen VII. Genome-wide expression analysis in twins' fibroblasts showed differential expression of genes associated with TGF-β pathway inhibition. In particular, decorin, a skin matrix component with anti-fibrotic properties, was found to be more expressed in the less affected twin. Accordingly, fibroblasts from the more affected sibling manifested a profibrotic and contractile phenotype characterized by enhanced α-smooth muscle actin and plasminogen activator inhibitor 1 expression, collagen I release and collagen lattice contraction. These cells also produced increased amounts of proinflammatory cytokines interleukin 6 and monocyte chemoattractant protein-1. Both TGF-β canonical (Smads) and non-canonical (MAPKs) pathways were basally more activated in the fibroblasts of the more affected twin. The profibrotic behaviour of these fibroblasts was suppressed by decorin delivery to cells. Our data show that the amount of type VII collagen is not the only determinant of RDEB clinical severity, and indicate an involvement of TGF-β pathways in modulating disease variability. Moreover, our findings identify decorin as a possible anti-fibrotic/inflammatory agent for RDEB therapeutic intervention. Primary fibroblast cultures from biopsies from two twins affected by recessive dystrophic epidermolysis bullosa were analyzed. Each hybridization was performed in biological triplicate and in technical duplicate.