scholarly journals Murine Precision-cut Intestinal Slices as a Potential Screening Tool for Antifibrotic Drugs

2020 ◽  
Vol 26 (5) ◽  
pp. 678-686 ◽  
Author(s):  
Raditya Iswandana ◽  
Bao Tung Pham ◽  
Su Suriguga ◽  
Theerut Luangmonkong ◽  
Louise A van Wijk ◽  
...  
Keyword(s):  
Growth Factor ◽  
Screening Tool ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Plasminogen Activator Inhibitor 1 ◽  
Intestinal Fibrosis ◽  
Fibrotic Process ◽  
The Impact ◽  
Pai 1 ◽  
Antifibrotic Drugs

Abstract Background Intestinal fibrosis is a hallmark of Crohn’s disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. Methods Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1α1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor β (TGF-β) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. Results Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-β1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1α1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. Conclusions Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis.

scholarly journals Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

2012 ◽  
Vol 303 (1) ◽  
pp. F11-F20 ◽  
Author(s):  
Jiandong Zhang ◽  
Jie Wu ◽  
Chunyan Gu ◽  
Nancy A. Noble ◽  
Wayne A. Border ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Transforming Growth Factor Β ◽  
Diabetic Patients ◽  
Ang Ii ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10−7 M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10−7 M alone similarly and significantly induced transforming growth factor-β1, plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β1 via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.

scholarly journals ID2033 Precision-cut tissue slices as a novel ex-vivo model for evaluating the efficacy of potential drugs

2017 ◽  
Vol 4 (S) ◽  
pp. 63
Author(s):  
Raditya Iswandana ◽  
Bao Tung Pham ◽  
Theerut Luangmonkon ◽  
Henricus A.M. Mutsaers ◽  
Peter Olinga
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Ex Vivo Model ◽  
Tissue Slices ◽  
Cancer Disease ◽  
Fibrotic Process ◽  
Pai 1

Background: Recently, we developed a novel model for drug screening by culturing ex-vivo precision-cut tissue slices (PCTS). The tissue slice consists of multiple cell types still in their normal matrix environment and structure provides numerous advantages compare to other models. Our objective was to use this model to investigate the effect of various potential compounds. In this study precision-cut intestinal slices (PCIS) were used to evaluate some transforming growth factor (TGF- β) and platelet-derived growth factor (PDGF) pathway inhibitors. TGF-β and PDGF are key cytokines in fibrotic and cancer diseases and are the main targets for treatment. Methods: Murine PCIS were cultured for 48 h in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using a variety of markers including (pro)collagen 1a1 (Col1a1), heat shock protein 47 (Hsp47), fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1). The effects of potential drugs mainly inhibiting the TGFb pathway i.e. valproic acid, tetrandrine, pirfenidone, SB203580 and LY2109761 as well as compounds mainly acting on the PDGF pathway i.e. imatinib, sorafenib and sunitinib were assessed in the model at maximum non-toxic concentrations. Results: Murine PCIS remained viable for 48 h and the onset of fibrosis was observed during culture, as demonstrated by an increased expression of, amongst others, Hsp47, Fn2 and Pai-1. Furthermore, TGFb1 stimulated fibrogenesis while PDGF had no effect. Regarding the tested antifibrotics, pirfenidone, LY2109761 and sunitinib had the most pronounced impact on fibrogenesis, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1a1, Hsp47, Fn2 and Pai-1 following treatment. Moreover, LY2109761 significantly reduced fibronectin protein expression in the presence of TGFb1. Conclusion: PCIS can successfully be used to test drug efficacy. Using the model we demonstrated that tetrandrine, pirfenidone, LY2109761 and sunitinib showed potential antifibrotic effects on a gene level, warranting further evaluation of these compounds for the treatment of fibrosis disease. By using tissue extracted from patient, PCIS could also be a promising model to screen drug for personalized treatment in fibrotic and cancer disease.

Differential Mechanisms of Plasminogen Activator Inhibitor-1 Gene Activation by Transforming Growth Factor-β and Tumor Necrosis Factor-α in Endothelial Cells

2001 ◽  
Vol 86 (12) ◽  
pp. 1563-1572 ◽  
Author(s):  
Yan Chen ◽  
Joanne Sloan-Lancaster ◽  
David Berg ◽  
Mark Richardson ◽  
Brian Grinnell ◽  
...  
Keyword(s):  
Map Kinases ◽  
Transforming Growth Factor ◽  
Gene Activation ◽  
Plasminogen Activator Inhibitor ◽  
Transforming Growth Factor Β ◽  
Promoter Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tnf Α ◽  
Factor Α ◽  
Pai 1

SummaryPlasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (SERPIN) specific for tissue-type and urokinase-like plasminogen activators. High plasma PAI-1 activity is a risk factor for thrombotic diseases. Due to the short half-life of PAI-1, regulation of PAI-1 gene expression and secretion of active PAI-1 into the blood stream is important for hemostatic balance. We have investigated transcriptional control of PAI-1 gene expression in bovine aortic endothelial cells (BAECs) and human cell lines using PAI-1 5’ promoter-luciferase reporter assays. Contrary to the cytokine-induced up-regulation of PAI-1 mRNA and protein levels, we found that only transforming growth factor-β (TGF-β) was efficient in inducing PAI-1 promoter activation. Tissue necrosis factor-α (TNF-α) induced a small luciferase activity with the 2.5 kb PAI-1 promoter, but not with the PAI-800/4G/5G and p3TP-lux promoters. Next we investigated whether a lack of response to TNF-α was due to deficient signaling pathways. BAECs responded to TNF-α with robust NFκB promoter activation. TGF-β activated the p38 MAP kinase, while TNF-α activated both the SAPK/JNK and p38 MAP kinases. The ERK1/2 MAP kinases were constitutively activated in BAECs. BAEC therefore responded to TNF-α stimulation with activation of the MAP kinases and the NFκB transcriptional factors. We further measured the messenger RNA stability under the influence by TGF-β and TNF-α and found no difference. PAI-1 gene activation by TNF-α apparently is yet to be defined for the location of the response element and/or the signaling pathway, while TGF-β is the most important cytokine for PAI-1 transcriptional activation through its 5’ proximal promoter.

TGF-β- and CTGF-mediated fibroblast recruitment influences early outward vein graft remodeling

2007 ◽  
Vol 293 (1) ◽  
pp. H482-H488 ◽  
Author(s):  
Zhihua Jiang ◽  
Peng Yu ◽  
Ming Tao ◽  
Chessy Fernandez ◽  
Cristos Ifantides ◽  
...  
Keyword(s):  
Growth Factor ◽  
Vein Graft ◽  
Transforming Growth Factor ◽  
Mechanical Load ◽  
Wall Stress ◽  
Transforming Growth Factor Β ◽  
Tissue Growth ◽  
Vein Grafts ◽  
Circumferential Wall Stress ◽  
The Impact

Luminal shearing forces have been shown to impact both geometric remodeling and the development of intimal hyperplasia. Less well studied is the influence of intramural wall stresses on vessel growth and adaptation. Using a vein graft-fistula configuration to isolate the impact of circumferential wall stress, we identify the reorganization of adventitial myofibroblasts as the dominant histological event that limits early outward remodeling of vein grafts in response to elevated wall stress. We hypothesize that increased production of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) induces recruitment of myofibroblasts, promotes adventitial reorganization, and limits early outward remodeling in response to increased intramural wall stress. Vein grafts with a distal arteriovenous fistula in the neck of rabbits were constructed, resulting in a fourfold differential in circumferential wall stress. Using this model, we demonstrate 1) elevated wall stress augments the production of TGF-β and CTGF, 2) increased TGF-β expression and CTGF expression are correlated with the enhanced differentiation from fibroblasts to myofibroblasts, as evidenced by the significant increase in the α-actin-positive cells in adventitia, and 3) the levels of TGF-β, CTGF, and α-actin are inversely correlated with the magnitude of outward remodeling of the graft wall. Increased wall stress after vein graft implantation appears to induce a TGF-β- and CTGF-mediated recruitment of adventitial fibroblasts and a conversion to a myofibroblast phenotype. Although important in the maintenance of wall stability in the face of an increased mechanical load, this adventitial adaptation limits early outward remodeling of the vein conduit and may prove deleterious in maintaining long-term vein graft patency.

scholarly journals Transforming growth factor-beta and matrix protein expression in acute and chronic rejection of human renal allografts.

1995 ◽  
Vol 6 (2) ◽  
pp. 286-294
Author(s):  
F S Shihab ◽  
T Yamamoto ◽  
C C Nast ◽  
A H Cohen ◽  
N A Noble ◽  
...  
Keyword(s):  
Growth Factor ◽  
Acute Rejection ◽  
Transforming Growth Factor Beta ◽  
Chronic Rejection ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Statistical Significance ◽  
Tgf Beta ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

Because the increased tissue expression of TGF-beta underlies fibrosis in many diseases, it was hypothesized that sustained elevated transforming growth factor (TGF)-beta overexpression might be responsible for fibrosis in chronic rejection of the renal allograft. To test this hypothesis, biopsies were obtained from 5 patients with acute rejection, 5 patients with chronic rejection, 10 normal individuals, and 10 patients with kidney disease. The tissues were examined by immunofluorescence for the three TGF-beta isoforms (1, 2, and 3) and the two matrix proteins induced by TGF-beta that serve as markers of fibrosis: fibronectin extradomain A positive (EDA+) and plasminogen activator inhibitor-1 (PAI-1). The tubulointerstitium from all cases of acute rejection and chronic rejection showed highly significant increases in immunostaining for the three TGF-beta isoforms (P < 0.001), fibronectin EDA+ (P < 0.005), and PAI-1 (P < 0.001). In the glomeruli, only TGF-beta 1 expression achieved statistical significance (P < 0.005) in acute rejection, whereas in chronic rejection, all three TGF-beta isoforms (p < 0.001) in addition to fibronectin EDA+ (p < 0.001) and PAI-1 (p < 0.001) were elevated. There was both cellular and matrix staining of the TGF-beta isoforms. In striking contrast, control kidney tissues were negative or only weakly positive. Because TGF-beta was present both in acute and in chronic rejection but not in control tissues and because acute rejection episodes are a good predictor for chronic rejection, these results suggest that TGF-beta may play a role in the pathogenesis of fibrosis in chronic rejection.

Sign in / Sign up

Export Citation Format

Share Document