Dupuytren's disease is a chronic, progressive fibroproliferative condition of the hand fascia which results in digital contraction. So far, treatments do not directly interfere with the (myo)fibroblasts that are responsible for the formation of the collagen-rich cords and its contraction. Here we investigated whether verteporfin (VP) is able to inhibit the activation and subsequent differentiation of DD nodular fibroblasts into myofibroblasts. Fibroblasts were isolated from nodules of 7 Dupuytren patients. Cells are treated (1) for 48 h with 5 ng/ml transforming growth factor β1 (TGF-β1) followed by 48 h with/without 250 nM VP in the absence of TGF-β1, or treated (2) for 48 h with TGF-β1 followed by 48 h with/without VP in the presence of TGF-β1. mRNA levels were measured by means of Real-Time PCR, and proteins were visualized by means of Western blotting and/or immunofluorescence. Quantitative data were statistically analyzed with GraphPad Prism using the paired t-test. We found that fibroblasts activated for 48 h with TGF-β1 show a decrease in mRNA levels of COL1A1, COL3A1, COL4A1, PLOD2, FN1EDA, CCN2 and SERPINE1 when exposed for another 48 h with VP, whereas no decrease is seen for ACTA2, YAP1, SMAD2 and SMAD3 mRNA levels. Cells exposed for an additional 48 h with TGF-β1, but now in the presence of VP, are not further activated anymore, whereas in the absence of VP the cells continue to differentiate into myofibroblasts. Collagen type I, fibronectin-extra domain A, α-smooth muscle actin, YAP1, Smad2 and Smad3 protein levels were attenuated by both VP treatments. We conclude that VP has strong anti-fibrotic properties: it is able to halt the differentiation of fibroblasts into myofibroblasts, and is also able to reverse the activation status of fibroblasts. The decreased protein levels of YAP1, Smad2 and Smad3 in the presence of VP explain in part the strong anti-fibrotic properties of VP. Verteporfin is clinically used as a photosensitizer for photodynamic therapy to eliminate abnormal blood vessels in the eye to attenuate macular degeneration. The antifibrotic properties of VP do not rely on photo-activation, as we used the molecule in its non-photoinduced state.

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFβ1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFβ or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFβ and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4(SMK-1)). Loss of PP4(SMK-1) hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4(SMK-1) by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4(SMK-1). Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4(SMK-1) crucial for many of DAF-16's physiological roles.

Extracellular matrix stiffening is an active player in the development of fibrosis. YAP is a mechanosensitive transcriptional co-activator that associates with Smad transcription factors, the signal transducers of the TGFβ1 cascade. Here, we studied the mechanisms that govern activation of YAP in response to substrate stiffness and TGFβ1 exposure in human fibroblasts. YAP nuclear accumulation after TGFβ1 exposure was found to be dependent on actin polymerization, together with myosin II activity. TGFβ1-induced activation of Smad2/3 resulted in association with YAP and nuclear accumulation of YAP/ Smad complexes. Smad3 knockdown antagonized nuclear accumulation of YAP, which could be mimicked by treatment with verteporfin, a known inhibitor of YAP function. Moreover, independent of stiffness, verteporfin blocked nuclear accumulation of YAP, Smad2 and Smad3, and resulted in decreased expression of signature myofibroblast genes. Our data indicate that ECM stiffening promotes pro-fibrotic TGFβ1 signaling by inducing YAP/Smad cross-talk. Harnessing the clinically approved drug verteporfin may be a promising strategy in targeting YAP/Smad-mediated fibrogenesis.

Fibrosis is involved in approximately 45% of all deaths in developed countries and available pharmacological treatments are scarce, if at all. Fibrosis is the result of a chronic pathological tissue repair process which can affect different organs of the body, leading to organ failure over time. In fibrotic diseases, healthy tissue is replaced by scar tissue mainly consisting of collagen type I, due to the imbalance between the synthesis and degradation. The increased deposition of extracellular matrix is driven by the activated fibroblast, the so-called myofibroblast.Only a few antifibrotic drugs are available, which is mainly due to the discordance of translation between pre-clinical studies and human clinical trials. In this thesis, we first present an optimized format of in vitro macromolecular crowding with a reduced culture time and minimal effect on myofibroblast phenotype, and tested the efficacy of four antifibrotic drugs (Imatinib, Omipalisib, Nintedanib and Galunisertib). Furthermore, we used the combination of in vitro and ex vivo models to evaluate its predictive power regarding three antifibrotic drugs (Imatinib, Pirfenidone and Galunisertib) in the context of animal models and clinical trials of kidney fibrosis. Finally, we describe the in vitro antifibrotic effect of a repurposed FDA-approved drug (Verteporfin) in Dupuytren’s disease fibroblasts, as well as a pilot study for establishing an ex vivo model for antifibrotic drug testing for the same disease.