Inhibition of tyrosine kinase receptor signaling attenuates fibrogenesis in an ex vivo model of human renal fibrosis.

Poor translation from animal studies to human clinical trials is one of the main hurdles in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of murine and human origins to obtain PCKS. Control slices and slices treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, and gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation and reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 µM, whereas high concentrations (1 and 5 µM) elicited possible nonselective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.

αII-spectrin and ßII-spectrin do not affect TGFß1-induced myofibroblast differentiation.

Mechanosensing of fibroblasts plays a key role in the development of fibrosis. So far, no effective treatments are available to treat this devastating disorder. Spectrins regulate cell morphology and are potential mechanosensors in a variety of non-erythroid cells, but little is known about the role of spectrins in fibroblasts. We investigate whether αII- and ßII-spectrin are required for the phenotypic properties of adult human dermal (myo)fibroblasts. Knockdown of αII- or ßII-spectrin in fibroblasts did not affect cell adhesion, cell size and YAP nuclear/cytosolic localization. We further investigated whether αII- and ßII-spectrin play a role in the phenotypical switch from fibroblasts to myofibroblasts under the influence of the pro-fibrotic cytokine TGFß1. Knockdown of spectrins did not affect myofibroblast formation, nor did we observe changes in the organization of αSMA stress fibers. Focal adhesion assembly was unaffected by spectrin deficiency, as was collagen type I mRNA expression and protein deposition. Wound closure was unaffected as well, showing that important functional properties of myofibroblasts are unchanged without αII- or ßII-spectrin. In fact, fibroblasts stimulated with TGFß1 demonstrated significantly lower endogenous mRNA levels of αII- and ßII-spectrin. Taken together, despite the diverse roles of spectrins in a variety of other cells, αII- and ßII-spectrin do not regulate cell adhesion, cell size and YAP localization in human dermal fibroblasts and are not required for the dermal myofibroblast phenotypical switch.

YAP1 Is a Driver of Myofibroblast Differentiation in Normal and Diseased Fibroblasts.

Dupuytren disease is a fibrotic disorder characterized by contraction of myofibroblast-rich cords and nodules in the hands. The Hippo member Yes-associated protein 1 (YAP1) is activated by tissue stiffness and the profibrotic transforming growth factor-ß1, but its role in cell fibrogenesis is yet unclear. We hypothesized that YAP1 regulates the differentiation of dermal fibroblasts into highly contractile myofibroblasts and that YAP1 governs the maintenance of a myofibroblast phenotype in primary Dupuytren cells. Knockdown of YAP1 in transforming growth factor-ß1-stimulated dermal fibroblasts decreased the formation of contractile smooth muscle α-actin stress fibers and the deposition of collagen type I, which are hallmark features of myofibroblasts. Translating our findings to a clinically relevant model, we found that YAP1 deficiency in Dupuytren disease myofibroblasts resulted in decreased expression of ACTA2, COL1A1, and CCN2 mRNA, but this did not result in decreased protein levels. YAP1-deficient Dupuytren myofibroblasts showed decreased contraction of a collagen hydrogel. Finally, we showed that YAP1 levels and nuclear localization were elevated in affected Dupuytren disease tissue compared with matched control tissue and partly co-localized with smooth muscle α-actin-positive cells. In conclusion, our data show that YAP1 is a regulator of myofibroblast differentiation and contributes to the maintenance of a synthetic and contractile phenotype, in both transforming growth factor-ß1-induced myofibroblast differentiation and primary Dupuytren myofibroblasts.

A genome-wide association meta-analysis implicates Hedgehog and Notch signaling in Dupuytren's disease.

Dupuytren's disease (DD) is a highly heritable fibrotic disorder of the hand with incompletely understood etiology. A number of genetic loci, including Wnt signaling members, have been previously identified. Our overall aim was to identify novel genetic loci, to prioritize genes within the loci for functional studies, and to assess genetic correlation with associated disorders. We performed a meta-analysis of six DD genome-wide association studies from three European countries and extensive bioinformatic follow-up analyses. Leveraging 11,320 cases and 47,023 controls, we identified 85 genome-wide significant single nucleotide polymorphisms in 56 loci, of which 11 were novel, explaining 13.3-38.1% of disease variance. Gene prioritization implicated the Hedgehog and Notch signaling pathways. We also identified a significant genetic correlation with frozen shoulder. The pathways identified highlight the potential for new therapeutic targets and provide a basis for additional mechanistic studies for a common disorder that can severely impact hand function.

Verteporfin ameliorates fibrotic aspects of Dupuytren's disease nodular fibroblasts irrespective the activation state of the cells.

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 ß1 (TGF-ß1) followed by 48 h with/without 250 nM VP in the absence of TGF-ß1, or treated (2) for 48 h with TGF-ß1 followed by 48 h with/without VP in the presence of TGF-ß1. 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-ß1 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-ß1, 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.

Fibrosis and cancer: A strained relationship.

Tumors are characterized by extracellular matrix (ECM) deposition, remodeling, and cross-linking that drive fibrosis to stiffen the stroma and promote malignancy. The stiffened stroma enhances tumor cell growth, survival and migration and drives a mesenchymal transition. A stiff ECM also induces angiogenesis, hypoxia and compromises anti-tumor immunity. Not surprisingly, tumor aggression and poor patient prognosis correlate with degree of tissue fibrosis and level of stromal stiffness. In this review, we discuss the reciprocal interplay between tumor cells, cancer associated fibroblasts (CAF), immune cells and ECM stiffness in malignant transformation and cancer aggression. We discuss CAF heterogeneity and describe its impact on tumor development and aggression focusing on the role of CAFs in engineering the fibrotic tumor stroma and tuning tumor cell tension and modulating the immune response. To illustrate the role of mechanoreciprocity in tumor evolution we summarize data from breast cancer and pancreatic ductal carcinoma (PDAC) studies, and finish by discussing emerging anti-fibrotic strategies aimed at treating cancer.

Collagen cross-linking mediated by lysyl hydroxylase 2: an enzymatic battlefield to combat fibrosis.

The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.

Ascorbic acid promotes a TGFß1-induced myofibroblast phenotype switch.

l-Ascorbic acid (AA), generally known as vitamin C, is a crucial cofactor for a variety of enzymes, including prolyl-3-hydroxylase (P3H), prolyl-4-hydroxylase (P4H), and lysyl hydroxylase (LH)-mediated collagen maturation. Here, we investigated whether AA has additional functions in the regulation of the myofibroblast phenotype, besides its function in collagen biosynthesis. We found that AA positively influences TGFß1-induced expression of COL1A1, ACTA2, and COL4A1 Moreover, we demonstrated that AA promotes αSMA stress fiber formation as well as the synthesis and deposition of collagens type I and IV Additionally, AA amplified the contractile phenotype of the myofibroblasts, as seen by increased contraction of a 3D collagen lattice. Moreover, AA increased the expression of several TGFß1-induced genes, including DDR1 and CCN2 Finally, we demonstrated that the mechanism of AA action seems independent of Smad2/3 signaling.

Signaling in Fibrosis: TGF-ß, WNT, and YAP/TAZ Converge.

Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myofibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-ß, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-ß1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, WNT-stabilized ß-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myofibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-ß, WNT, and YAP/TAZ pathways in the development of organ fibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of fibrotic disorders.