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.</p

Biochemical and biomechanical regulation of the myofibroblast phenotype:focus on Hippo and TGFβ signaling

The extracellular matrix (ECM) is a dynamic collection of proteins and carbohydrates, which are constantly synthesized, modified, and degraded by cells. In chronic disorders such as fibrosis, ECM synthesis and degradation are out of balance, and myofibroblasts alter the composition and cross-linking of the matrix. The replacement of normal functional ECM with collagenous scar tissue causes distortion of tissue architecture by contraction of the connective tissue. Understanding how myofibroblasts transduce biomechanical and biochemical cues to modulate the regulation of gene transcription is of paramount importance for the development of anti-fibrotic therapeutics. The aim of this thesis was to elucidate how biomechanical and biochemical cues regulate the myofibroblast phenotype. To this end, we focused on three main areas of research. First, we uncovered an important role for YAP signaling in myofibroblasts and the fibroproliferative disorder Dupuytren disease. We provide novel insights in how YAP regulates the myofibroblast phenotype, by interacting with Smad transcription factors. Second, we found that the spectrin cytoskeleton does not seem to be involved in fibroblast mechanosensing and the formation of a myofibroblast phenotype. Finally, we show how vitamin C acts as important co-factor for functioning of myofibroblasts. In conclusion, our findings do not only provide a better understanding of the myofibroblast phenotype and fibrosis, but also provide support for changes in general laboratory practice and tools for the possible treatment of fibrosis

Signaling in fibrosis: TGF-β, 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 TGF-β, Wnt, and more recently YAP/TAZ signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, Wnt-stabilized β-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-β, 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

alpha II-spectrin and beta II-spectrin do not affect TGF beta 1-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 II- and II-spectrin are required for the phenotypic properties of adult human dermal (myo)fibroblasts. Knockdown of II- or II-spectrin in fibroblasts did not affect cell adhesion, cell size and YAP nuclear/cytosolic localization. We further investigated whether II- and II-spectrin play a role in the phenotypical switch from fibroblasts to myofibroblasts under the influence of the pro-fibrotic cytokine TGF1. Knockdown of spectrins did not affect myofibroblast formation, nor did we observe changes in the organization of SMA 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 II- or II-spectrin. In fact, fibroblasts stimulated with TGF1 demonstrated significantly lower endogenous mRNA levels of II- and II-spectrin. Taken together, despite the diverse roles of spectrins in a variety of other cells, II- and II-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

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 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

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