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

Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal (‘control’) supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by a-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of d-aspartic acid (% d-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively;

Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs:The Scar-in-a-Jar System Revisited

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

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen -chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen -chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted

Interleukin-1 beta Attenuates Myofibroblast Formation and Extracellular Matrix Production in Dermal and Lung Fibroblasts Exposed to Transforming Growth Factor-beta 1

One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ). TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β) can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase), and matrix metalloproteinases (MMPs). We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1), the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, -2, -9 and -14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future anti-fibrotic treatment regimes

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

Further evidence of the involvement of the Wnt signaling pathway in Dupuytren's disease

Genetic background plays an important role in the development of Dupuytren's disease. A genome-wide association study (GWAS) showed that nine loci are associated with the disease, six of which contain genes that are involved in Wnt signaling (WNT2, WNT4, WNT7B, RSPO2, SFRP4, SULF1). To obtain insight in the role of these genes, we performed expression studies on affected and unaffected patient's tissues. Surgically obtained nodules and cords from eight Dupuytren's patients were compared to patient-matched control tissue (unaffected transverse palmar fascia). The Wnt-related genes found in the GWAS, the classical Wnt-downstream protein beta-catenin, as well as (myo) fibroblast markers were analyzed using real-time qPCR and immunohistochemical stainings for mRNA levels and protein levels, respectively. The collagen-coding genes COL1A1 and COL3A1 were highly upregulated on mRNA level, both in cords and nodules. Three Wnt-related genes were found to be differently regulated compared to control tissue: WNT2 was downregulated in nodules, WNT7B was upregulated in nodules, and SFRP4 was upregulated in nodules and cords. Immunohistochemistry revealed significantly less staining of Wnt2 in cords, but significantly more staining for Wnt7b in nodules. There was significantly more staining of alpha-SMA in nodules and cord and beta-catenin in nodules than in control tissue. We found differences in expression, both at mRNA and protein level, in several Wnt-related genes found earlier to be associated with Dupuytren's disease. Of these, Wnt7b was upregulated and found in close association with both alpha-SMA and beta-catenin expressing cells, making it a candidate pro-fibrotic mediator in Dupuytren's disease

Intestinal stenosis in Crohn's disease shows a generalized upregulation of genes involved in collagen metabolism and recognition that could serve as novel anti-fibrotic drug targets

Background and Aims: Crohn's disease (CD) can be complicated by intestinal fibrosis. Pharmacological therapies against intestinal fibrosis are not available. The aim of this study was to determine whether pathways involved in collagen metabolism are upregulated in intestinal fibrosis, and to discuss which drugs might be suitable to inhibit excessive extracellular matrix formation targeting these pathways. Methods: Human fibrotic and non-fibrotic terminal ileum was obtained from patients with CD undergoing ileocecal resection due to stenosis. Genes involved in collagen metabolism were analyzed using a microfluidic low-density TaqMan array. A literature search was performed to find potential anti-fibrotic drugs that target proteins/enzymes involved in collagen synthesis, its degradation and its recognition. Results: mRNA expression of collagen type I (COL1A1, 0.76 ± 0.28 versus 37.82 ± 49.85, p = 0.02) and III (COL3A1, 2.01 ± 2.61 versus 68.65 ± 84.07, p = 0.02) was increased in fibrotic CD compared with non-fibrotic CD. mRNA expression of proteins involved in both intra- and extracellular post-translational modification of collagens (prolyl- and lysyl hydroxylases, lysyl oxidases, chaperones), collagen-degrading enzymes (MMPs and cathepsin-K), and collagen receptors were upregulated in the fibrosis-affected part. A literature search on the upregulated genes revealed several potential anti-fibrotic drugs. Conclusion: Expression of genes involved in collagen metabolism in intestinal fibrosis affected terminal ileum of patients with CD reveals a plethora of drug targets. Inhibition of post-translational modification and altering collagen metabolism might attenuate fibrosis formation in the intestine in CD. Which compound has the highest potential depends on a combination anti-fibrotic efficacy and safety, especially since some of the enzymes play key roles in the physiology of collagen

Glimpses into the molecular pathogenesis of Peyronie's disease

Peyronie's disease (PD) is a fibroproliferative disease of the penis. Since little is known about the molecular pathogenesis of PD, we compared the biochemical make-up of PD plaques with normal tunica albuginea to clarify pathological processes in the scarred tissue. Protein and mRNA levels were measured in plaques and in unaffected pieces of the tunica albuginea. We investigated the presence of myofibroblasts, the deposition of collagens, and some key elements of Wnt and YAP1 signaling at protein level. The expression of 45 genes, all related to collagen homeostasis and extracellular matrix proteins, was quantified. In plaques, more myofibroblasts were present, and we observed an activation of Wnt signaling and YAP1 signaling. Increased levels of the collagens types I and III confirm the fibrotic nature of plaques. The mRNA ratio of collagen types III, IV, and VI to type I was increased. The expression of lysyl hydroxylase 3 was higher, whereas a decreased expression level was seen for fibronectin and cathepsin K. The biochemical composition of plaques was different from unaffected tunica albuginea: the relative and absolute abundance of various extracellular matrix proteins were changed, as well as the quality of collagen and the level of the collagen-degrading enzyme cathepsin K