We developed an assay using Idiopathic pulmonary fibrosis (IPF) and normal primary fibroblasts. The IPF‐fibroblast‐conditioned matrix (CM) induced fibroblast to myofibroblast transition (FMT) with transforming growth factor beta (TGFβ) and signal transducer and activator of transcription 3 (STAT3) pathway activation in normal cells. This assay demonstrated differences in drug activities for newly approved IPF drugs (nintedanib and pirfenidone) at clinically relevant concentrations.
Abstract Nonwoven fiber mats of poly(ε-caprolactone) (PCL) and PCL blended with poly(ethylene oxide) (PEO) were generated by electrospinning. Differential scanning calorimetry, scanning electron microscopy, and gravimetric measurement confirm the removal of PEO after immersion in water, as well as an increase in the PCL crystallinity. The reorganization of PCL resulted in the macroscopic alteration of the electrospun mat, decreasing the peak pore diameter up to a factor of 3 while only minimally affecting the fiber diameter. This technique was used to create electrospun PCL scaffolds with similar fiber diameters but different pore diameters to examine the effect of pore diameter on cell growth. Human Dermal Fibroblasts (HDF) were seeded into multiple samples using a perfusion seeding technique to guarantee successful cell deposition. Fluorescence analysis at 7, 14, and 21 days found that cells proliferated at a faster rate on scaffolds with peak pore diameters greater than 6 μm, as determined by mercury porosimetry. Cell conformation was also found to change as the peak pore diameter grew from 12 to 23 μm; cells began aligning along single fibers instead of attaching to multiple fibers. Knowledge of the effect of void architecture on cell proliferation and conformation could lead to the development of more effective scaffolds for tissue engineering.
Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet‐derived growth factor (PDGF)‐dependent manner. We also identified B‐cell lymphoma‐extra large (Bcl‐xL) as a key survival factor in PDGF‐activated human and mouse fibroblasts. Bcl‐xL was also up‐regulated in senescent cholangiocytes. In vitro , inhibition of Bcl‐xL by the small molecule Bcl‐2 homology domain 3 mimetic, A‐1331852, or Bcl‐xL‐specific small interfering RNA induced apoptosis in PDGF‐activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl‐xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout ( Mdr2 −/− ) mice with A‐1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis‐inducing growth factors and cytokines, decrease of α‐smooth muscle actin–positive ASFs, and finally in a significant reduction of liver fibrosis. Conclusion: Bcl‐xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl‐xL‐specific inhibitor, A‐1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (H epatology 2018;67:247‐259).
Background: Thrombin promotes cardiac fibroblast proliferation and fibrosis via protease-activated receptor (PAR-1). PAR-4 is reportedly absent in cardiac fibroblasts. In smooth muscle cells, PAR-4 expression is also low but increases upon hyperglycemia and contributes to vascular remodelling in diabetic mice. We examined if PAR-4 is a glucose-responsive gene with remodelling-related functions in cardiac fibroblasts. Methods and results: Cardiac PAR-4 increased in mice with streptozotocin- or diabetogenic diet (DD)-induced diabetes. PAR-4 mRNA and protein were detectable in cardiac fibroblasts from chow-fed mice and increased in high (HG, 25 mM) vs. low glucose (LG; 5.5 mM) cultures. Conversely PAR-4 mRNA was higher in fibroblasts from DD-fed mice but reduced in LG cultures. Cardiac fibroblasts in HG culture responded more strongly to thrombin or PAR-4 activating peptide in terms of migration (wound-scratch assay), remodelling-associated gene expression ( interleukin 6, alpha smooth muscle actin) and oxidative stress (dihydroethidium fluorescence). Conclusion: PAR-4 is expressed in mouse cardiac fibroblasts and is dynamically regulated by extracellular glucose in vitro and diabetes in vivo, thereby impacting on fibroblast functions relevant for cardiac remodelling. These findings add further evidence for the usefulness of the recently developed PAR-4 antagonists in clinical settings. (C) 2017 Elsevier B.V. All rights reserved.
Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing vascular grafts, the in vitro biocompatibility of poly(epsilon-caprolactone) (PCL), recently Suggested for several clinical applications, was evaluated ill this study using L929 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture oil PCL films: adhesion, proliferation, morphology, LDH release and mitochondrial function. Since topography and other Surface characteristics of materials play ail essential part in cell adhesion, PCL membranes with either smooth or rough surface were prepared, characterized and used to carry out cell cultures. During short culture times, PCL produced a significant stimulation of mitochondrial activity evaluated by reduction of the MTT reagent. The results provide evidences of good adhesion, growth, viability, morphology and mitochondrial activity of cells on PCL films. Therefore, it can be Concluded that PCL is a Suitable Land biocompatible material as a scaffold for vascular graft development. (C) 2004 Elsevier Ltd. All rights reserved.
Cancer‐associated fibroblasts ( CAF ) are a major constituent of the pancreatic cancer microenvironment and that the meaning is as intended. Pancreatic cancer cells can induce normal fibroblasts to convert into CAF and, reciprocally, CAF promote tumor invasions and proliferations. The mechanism of the conversion from normal fibroblasts ( NF ) to CAF remains unclear. Micro RNA are short non‐coding RNA involved in the post‐transcription gene regulation, which have been defined as an imperative controller in tumor invasions, proliferations and colony formations. Microvesicles ( MV ) have been proved to be an important mediator of intercellular communication and can selectively transport secreted micro RNA from a donor cell into a recipient cell. In this study, we isolated primary pancreatic fibroblasts from wild type C57 mice and co‐cultured them with pancreatic cancer cell lines, Bx PC ‐3 and SW 1990, and observed the conversion from NF to CAF , or at least CAF ‐like cells. This phenomenon could also be replicated in primary fibroblasts treated with MV separated from a cancer cell media. We identified that miR‐155 was upregulated in PaC‐derived MV and we confirmed that normal fibroblasts could convert into CAF after MV containing miR‐155 had been taken up. TP 53 INP 1 is a target of miR‐155 in fibroblasts and a downregulation of TP 53 INP 1 protein levels could contribute to the fibroblasts' activation. These results indicated that pancreatic cancer cells might reprogram normal adjacent fibroblasts into CAF by means of secreted MV containing miR‐155. Targeting the circulating micro RNA might be a potential therapy for malignant tumors. In this study, we discovered that microvesicles, containing miR‐155, might be released by pancreatic cancer cells and incorporated by co‐cultivated primary fibroblasts. TP53INP1 might be a target gene of miR‐155 in fibroblasts, which may mediate proliferation and the activation of normal fibroblasts and to manifest the characteristics of cancer‐associated fibroblasts.
Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive desmoplastic stromal response. Fibroblast activation protein-alpha (FAP) is best known for its presence in stromal cancer-associated fibroblasts (CAFs). Our aim was to assess whether FAP expression was associated with the prognosis of patients with PDAC and to investigate how FAP expressing CAFs contribute to the progression of PDAC. Methods: FAP expression was immunohistochemically assessed in 48 PDAC specimens. We also generated a fibroblastic cell line stably expressing FAP, and examined the effect of FAP-expressing fibroblasts on invasiveness and the cell cycle in MiaPaCa-2 cells (a pancreatic cancer cell line). Results: Stromal FAP expression was detected in 98 % (47/48) of the specimens of PDAC, with the intensity being weak in 16, moderate in 19, and strong in 12 specimens, but was not detected in the 3 control noncancerous pancreatic specimens. Patients with moderate or strong FAP expression had significantly lower cumulative survival rates than those with negative or weak FAP expression (mean survival time; 352 vs. 497 days, P = 0.006). Multivariate analysis identified moderate to strong expression of FAP as one of the factors associated with the prognosis in patients with PDAC. The intensity of stromal FAP expression was also positively correlated to the histological differentiation of PDAC (P < 0.05). FAP-expressing fibroblasts promoted the invasiveness of MiaPaCa-2 cells more intensively than fibroblasts not expressing FAP. Coculture with FAP-expressing fibroblasts significantly activated cell cycle shift in MiaPaCa-2 cells compared to coculture with fibroblasts not expressing FAP. Furthermore, coculture with FAP expressing fibroblasts inactivated retinoblastoma (Rb) protein, an inhibitor of cell cycle progression, in MiaPaCa-2 cells by promoting phosphorylation of Rb. Conclusions: The present in vitro results and the association of FAP expression with clinical outcomes provide us with a better understanding of the effect of FAP-expressing CAFs on the progression of PDAC.