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

Matrix and cell phenotype differences in Dupuytren's disease

BACKGROUND: Dupuytren's disease is a fibroproliferative disease of the hand and fingers, which usually manifests as two different phenotypes within the same patient. The disease first causes a nodule in the palm of the hand, while later, a cord develops, causing contracture of the fingers. RESULTS: We set out to characterize the two phenotypes by comparing matched cord and nodule tissue from ten Dupuytren's patients. We found that nodule tissue contained more proliferating cells, CD68-positive macrophages and α-smooth muscle actin (α-SMA)-positive myofibroblastic cells. qPCR analysis showed an increased expression of COL1A1, COL1A2, COL5A1, and COL6A1 in nodule tissue compared to cord tissue. Immunohistochemistry showed less deposition of collagen type I in nodules, although they contained more fibronectin, collagen type V, and procollagen 1. Lower collagen levels in nodule were confirmed by HPLC measurements of the Hyp/Pro ratio. PCOLCE2, an activator of BMP1, the main enzyme cleaving the C-terminal pro-peptide from procollagen, was also reduced in nodule. Cord tissue not only contained more collagen I, but also higher levels of hydroxylysylpyridinoline and lysylpyridinoline residues per triple helix, indicating more crosslinks. CONCLUSIONS: Our results clearly show that in Dupuytren's disease, the nodule is the active disease unit, although it does not have the highest collagen protein levels. The difference in collagen type I deposition compared to mRNA levels and procollagen 1 levels may be connected to a decrease in procollagen processing

Microsphere-Based Rapamycin Delivery, Systemic Versus Local Administration in a Rat Model of Renal Ischemia/Reperfusion Injury

The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method

Increased Liver Uptake and Reduced Hepatic Stellate Cell Activation with a Cell-Specific Conjugate of the Rho-kinase Inhibitor Y27632

Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC. Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl(4) model in mice. Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl(4)-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. We conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver

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

HSC-gezielte Hemmung der Rho-kinase senkt den Portaldruck mit nur geringen systemischen Nebenwirkungen

Hintergrund: Der erhöhte Portaldruck in der Leberzirrhose ist zum Teil auf einen erhöhten intrahepatischen Widerstand zurückzuführen. Die Rho-kinase vermittelt eine Kontraktion der hepatischen Sternzellen (HSCs), die zur Erhöhung des intrahepatischen Widerstandes führt. Die Hemmung der Rho-kinase senkt den Portaldruck, löst aber eine systemische Hypotension aus (Gastroenterology 2006). Deshalb ist eine HSC-gezielte Hemmung der Rho-kinase notwendig. Mannose-6-Phosphat-modifiziertes Human-Serum-Albumin (M6P-HSA) bindet spezifisch an aktivierte HSCs (Hepatology 1999). Der Rho-kinase-Inhibitor (Y-27632) wurde an M6P-HSA konjugiert und dessen Effekte wurden in zirrhotischen Ratten untersucht. Methoden: Es wurden zirrhotische CCl4-behandelte und gallengangsligierte (BDL) Ratten mit Sham-operierten Ratten verglichen. Der Rho-kinase-Inhibitor wurde in zwei verschiedenen Dosen i.v. injiziert (0,05mg/kg und 0,1mg/kg). Nach 3h wurden der Portaldruck und der mittlere arterielle Druck (MAD) invasiv gemessen. Mittels der Mikrosphären-Technik wurde die Organperfusion untersucht. Ergebnisse: Beide Zirrhose-Modelle zeigten eine portale Hypertension. In diesen Tieren war der Portaldruck (CCL4:15,1±1,4; BDL: 18,4±1,6mmHg) aufgrund des hohen intrahepatischen Widerstandes und des niedrigen splanchnischen Widerstandes erhöht. Der niedrige MAD (CCL4:108±5,8; BDL:101±8,0mmHg) war mit einem verminderten systemischen Widerstand und einem erhöhten Herz-Zeit-Volumen verknüpft. Der M6P-HSA konjugierte Rho-kinase-Inhibitor senkte in vivo bei beiden Dosen den Portaldruck (CCL4 (0,05mg):12,8±1,9; (0,1mg): 6,6±0,7 und BDL (0,05mg): 13,0±2,0; (0,1mg): 13,7±1,8mmHg). Dieser Effekt ist auf einen verminderten intrahepatischen Widerstand zurück zu führen. Der splanchnische Widerstand blieb nach der gezielten Rho-kinase Hemmung unverändert und es konnten nur geringe systemische Effekte gemessen werden (MAD CCL4 (0,05mg: 95,0±5,7; (0,1mg): 77,8±9,8; BDL (0,05mg): 82,0±3,0, (0,1mg): 78,8±2,2mmHg). Diskussion: Die HSC-gezielte Hemmung der Rho-kinase ist ein möglicher therapeutischer Ansatz zur Senkung des Portaldrucks in der Leberzirrhose. Die Konjugation des Rho-kinase-Inhibitors mit M6P-HSA sollte als eine Therapieoption beim Menschen evaluiert werden

Wnt pathway in Dupuytren disease:connecting profibrotic signals

A role of Wnt signaling in Dupuytren disease, a fibroproliferative disease of the hand and fingers, has not been fully elucidated. We examined a large set of Wnt pathway components and signaling targets and found significant dysregulation of 41 Wnt-related genes in tissue from the Dupuytren nodules compared with patient-matched control tissue. A large proportion of genes coding for Wnt proteins themselves was downregulated. However, both canonical Wnt targets and components of the noncanonical signaling pathway were upregulated. Immunohistochemical analysis revealed that protein expression of Wnt1-inducible secreted protein 1 (WISP 1), a known Wnt target, was increased in nodules compared with control tissue, but knockdown of WISP I using small interfering RNA (siRNA) in the Dupuytren myofibroblasts did not confirm a functional role. The protein expression of noncanonical pathway components Wnt5A and VANGL2 as well as noncanonical coreceptors Ror2 and Ryk was increased in nodules. On the contrary, the strongest downregulated genes in this study were 4 antagonists of Wnt signaling (DKK1, FRZB, SFRP1, and WIF1). Downregulation of these genes in the Dupuytren tissue was mimicked in vitro by treating normal fibroblasts with transforming growth factor beta 1 (TGF-beta 1), suggesting cross talk between different profibrotic pathways. Furthermore, siRNA-mediated knockdown of these antagonists in normal fibroblasts led to increased nuclear translocation of Wnt target p-catenin in response to TGF-beta 1 treatment. In conclusion, we have shown extensive dysregulation of Wnt signaling in affected tissue from Dupuytren disease patients. Components of both the canonical and the noncanonical pathways are upregulated, whereas endogenous antagonists are downregulated, possibly via interaction with other profibrotic pathways

HSC gezielte Hemmung der Rho-kinase senkt den Portaldruck mit geringen systemischen Nebenwirkungen

Hintergrund: Der erhöhte Portaldruck (PP) in der Leberzirrhose beruht auf einen erhöhten intrahepatischen Widerstand (HR). Die Hemmung der Rho-kinase in hepatischen Sternzellen (HSC) kann den HR senken und damit auch den PP. Allerdings löst die systemische Rho-kinase Hemmung auch eine systemische Hypotension aus (Gastroenterol. 2006). Deshalb ist eine HSC gezielte Hemmung der Rho-kinase wünschenswert. Mannose–6-Phosphat-modifiziertes Human-Serum-Albumin (M6P-HSA) bindet spezifisch an aktivierte HSC (Hepatol. 1999). Der Rho-kinase Inhibitor (Y–27632) wurde an M6P-HSA konjugiert und bei zirrhotischen Ratten untersucht. Methoden: Es wurden CCl4 behandelte und gallengangsligierte (BDL) Ratten verwendet und mit Sham operierten Ratten verglichen. Der Rho-kinase Inhibitor wurde in zwei Dosen injiziert (0,05 und 0,1mg/kg). Nach 3h wurden der PP, der mittlere arterielle Druck (MAP) und die Organperfusion mittels der Mikrosphären Technik untersucht. Die Rho-kinase Aktivität und Expression wurde in verschiedenen Organen mittels Western Blot nachgewiesen. Ergebnisse: Das M6P-HSA konjugierte Y–27362 senkte in vivo den PP in beiden Zirrhosemodellen. Dieser Effekt war auf einen verminderten HR zurückzuführen. Der splanchnische Widerstand blieb unverändert und es konnten nur geringe systemische Effekte gemessen werden. Die spezifische Rho-kinase Hemmung senkte dessen Enzymaktivität und Expression nur in der Leber. In extrahepatischen Geweben konnte keine Hemmung gezeigt werden. Diskussion: Die HSC-gezielte Hemmung der Rho-kinase ist ein möglicher therapeutischer Ansatz zur Senkung des PP bei der Leberzirrhose. Die Konjugation des Rho-kinase Inhibitors mit M6P-HSA sollte daher als eine Therapieoption beim Menschen für die Behandlung der portalen Hypertension evaluiert werden

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 profibro tic transforming growth factor-beta 1, 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-beta 1-stimulated dermal fibroblasts decreased the formation of contractile smooth muscle alpha-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 alpha-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-beta 1-induced myofibroblast differentiation and primary Dupuytren myofibroblasts