Project description:Whole genome expression analyses of autologous, paired eutopic and ectopic endometrial samples obtained during proliferative and secretory phases of menstrual cycles from eighteen (n=18) fertile women suffering from confirmed stage 3 (moderate) and stage 4 (severe) ovarian endometriosis were performed using whole human genome oligo microarray Agilent paltform (Cat. No. G4112F). In the present study, genome-wide expression analysis of autologous, paired eutopic and ectopic endometrial samples obtained during proliferative (n=13) and secretory (n=5) phases of menstrual cycle from fertile women (n=18) suffering from moderate (stage 3; n=8) or severe (stage 4; n=10) endometrioma was performed by using Agilent single color oligo microarray platform (G4112, 4X44K). Thus eighteen (18) eutopic (shown as EU) and eighteen (18) ectopic (shown as EC) samples from eighteen (18) subjects with confirmed menstrual phase (proliferative and secretory) and severity stages (stage 3 and stage 4) were studied.
Project description:Whole genome expression analyses of autologous, paired eutopic and ectopic endometrial samples obtained during proliferative and secretory phases of menstrual cycles from eighteen (n=18) fertile women suffering from confirmed stage 3 (moderate) and stage 4 (severe) ovarian endometriosis were performed using whole human genome oligo microarray Agilent paltform (Cat. No. G4112F).
Project description:The aim of the study was to investigate the effects of autologous equine serum (AES) incubated for 24 h and autologous conditioned serum (ACS) on inflamed equine chondrocyte pellets in vitro.
Project description:Liver fibrosis is a reversible wound-healing response to liver injury and hepatic stellate cells (HSCs) are central cellular players that mediate hepatic fibrogenesis. However, the molecular mechanisms that govern this process remain unclear. Here, we reveal a novel cistromic circuit in HSCs comprising the vitamin D receptor (VDR) and SMAD transcription factors that restrains the intensity of hepatic fibrogenesis. Ligand-activated VDR suppresses TGFβ1-induced pro-fibrotic gene expression in HSCs. Administration of a vitamin D analogue, calcipotriol, diminishes the fibrotic response in a mouse model of liver fibrosis, while VDR knockout mice spontaneous develop extensive hepatic fibrosis by age 6 months. Using ChIP-Seq, we find that the anti-fibrotic properties of VDR are due to crosstalk with SMAD, mediated by their co-occupancy of DNA-binding sites on pro-fibrotic genes. Specifically, SMAD binding potentiates local chromatin accessibility to enhance VDR recruitment at the same cis-regulatory elements, which reciprocally antagonizes the interaction between SMAD3 and chromatin and limits the assembly of transcriptional activation complexes at fibrotic genes, a process that is enhanced by the presence of VDR agonists. These results not only establish this coordinated VDR/SMAD cistromic circuit as a master regulator of hepatic fibrogenesis, but also support VDR as a potential drug target to ameliorate liver fibrosis. Identification of VDR, SMAD3 and H3 binding sites in human stellate LX2 cells that were pre-treated with calcipotriol (100nM) for 16 hrs (where calcipotriol treatment is indicated) followed by incubation of calcipotriol (100nM) or TGFβ1 (1ng/ml) for another 4 hours (where indicated).
Project description:We employed single-cell combinatorial indexing RNA-seq (sci-RNA-seq), a scRNA-seq technology with high throughput, high sample multiplexing capacity and low costs, to decipher the molecular events involved in mouse kidney fibrogenesis. With the hypothesis that different types of kidney insults may lead to distinct cellular injury responses, we leveraged sci-RNA-seq to profile mouse kidneys collected from two mouse kidney fibrogenesis models, unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO), at multiple stages. We described an atlas of kidney fibrogenesis (available at http://humphreyslab.com/SingleCell/) with a total of 309,666 cells profiled from 11 biological conditions and 24 samples in one experiment. We discovered that uni-IRI and UUO produced two types of early-stage injured PT cells with different transcriptomic signature. Further investigation on the two cell states highlighted their distinct mechanisms of metabolic regulation. Analysis of other structures of TECs revealed a common cellular response to injury and repair. In addition, we described the heterogeneity within kidney stroma and the dynamics of cell-cell communications in kidney fibrogenesis.
Project description:Aberrant expression of master phenotype regulators by lung fibroblasts may play a central role in idiopathic pulmonary fibrosis (IPF). Interrogating IPF fibroblast transcriptome datasets, we identified Forkhead Box F1 (FOXF1), a DNA-binding protein required for lung development, as a candidate actor in IPF. Thus, we determined FOXF1 expression levels in fibroblasts cultured from normal or IPF lungs in vitro, and explored FOXF1 functions in these cells using transient and stable loss-of-function and gain-of-function models. FOXF1 mRNA and protein were expressed at higher levels in IPF compared with controls. In normal lung fibroblasts, FOXF1 repressed key fibroblast functions such as proliferation, survival, and expression of collagen-1 (COL1) and actin related protein 2/3 complex, subunit 2 (ARPC2). ARPC2 knockdown mimicked FOXF1 overexpression with regard to proliferation and COL1 expression. FOXF1 expression was induced by the antifibrotic mediator prostaglandin E2 (PGE2). Ex vivo, FOXF1 knockdown conferred CCL-210 lung fibroblasts the ability to implant and survive in uninjured mouse lungs. In IPF lung fibroblasts, FOXF1 regulated COL1 but not ARPC2 expression. In conclusion, FOXF1 functions and regulation were consistent with an antifibrotic role in lung fibroblasts. Higher FOXF1 levels in IPF fibroblasts may thus participate in a compensatory response to fibrogenesis. Lung fibroblasts derived from 4 different IPF patients (P313, P355, P375 and P426) were transiently transfected with pcfoxf1 or control pcDNA3.1-constructs. Total RNAs were extracted 24 h after transfection and hybridized on microarrays. One color experiment with 2 experimental conditions: pcfoxf1 and pcDNA3.1
Project description:Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P<0.05). Intravital confocal microscopy performed immediately after HIFD showed improved microvascular perfusion. Notably, HIFD also resulted in immediate enhancement of parenchymal labeling with the fluorescent dye Hoechst 33342. HIFD also associated with a significant reduction in the accumulation of renal leukocytes, including proinflammatory T cells. Additionally, HIFD significantly reduced peritubular capillary erythrocyte congestion and improved histologic scores of tubular injury 4 days after IRI. Taken together, these results indicate that HIFD performed after establishment of AKI rapidly restores microvascular perfusion and small molecule accessibility, with improvement in overall renal function.
Project description:Liver fibrosis is a reversible wound-healing response to liver injury and hepatic stellate cells (HSCs) are central cellular players that mediate hepatic fibrogenesis. However, the molecular mechanisms that govern this process remain unclear. Here, we reveal a novel cistromic circuit in HSCs comprising the vitamin D receptor (VDR) and SMAD transcription factors that restrains the intensity of hepatic fibrogenesis. Ligand-activated VDR suppresses TGFβ1-induced pro-fibrotic gene expression in HSCs. Administration of a vitamin D analogue, calcipotriol, diminishes the fibrotic response in a mouse model of liver fibrosis, while VDR knockout mice spontaneous develop extensive hepatic fibrosis by age 6 months. Using ChIP-Seq, we find that the anti-fibrotic properties of VDR are due to crosstalk with SMAD, mediated by their co-occupancy of DNA-binding sites on pro-fibrotic genes. Specifically, SMAD binding potentiates local chromatin accessibility to enhance VDR recruitment at the same cis-regulatory elements, which reciprocally antagonizes the interaction between SMAD3 and chromatin and limits the assembly of transcriptional activation complexes at fibrotic genes, a process that is enhanced by the presence of VDR agonists. These results not only establish this coordinated VDR/SMAD cistromic circuit as a master regulator of hepatic fibrogenesis, but also support VDR as a potential drug target to ameliorate liver fibrosis.
Project description:In regenerative medicine, histocompatibility of pluripotent stem cells is required to solve the problem of immunorejection after therapeutic transplantation. In this study, we show that autologous germline stem cells (GSCs), often called spermatogonial stem cells, could be derived by testis biopsy from individual mice and that GSCs subsequently could be dedifferentiated into autologous germline-derived pluripotent stem (gPS) cells. The establishment of GSCs by testicular biopsy in the mouse model can prove the principle of human clinical application to derive autologous GSCs to generate patient-specific pluripotent cells for regenerative medicine.