Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling

[Abstract] Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.Dr Barallobre-Barreiro is a British Heart Foundation Intermediate Fellow (FS/19/33/34328). Drs Mayr and Shah are British Heart Foundation Chair Holders (CH/16/3/32406 and CH/1999001/11735, respectively) and received support from the British Heart Foundation Center for Vascular Regeneration With Edinburgh/Bristol (RM/17/3/33381). Dr Doménech’s work was supported by Project PI16/02049 integrated in the National Plan for Scientific Research, Development and Technological Innovation, 2013–2016, and funded by the ISCIII–General Subdirection of Assessment and Promotion of Research–European Regional Development Fund. Dr Merkely’s work was funded by the National Research, Development and Innovation Fund (NVKP_16-1–2016-0017) and the Thematic Excellence Program of the Ministry for Innovation and Technology (2020-4.1.1.-TKP2020), Hungary. Dr Radovits is supported by the National Research, Development and Innovation Office of Hungary (K134939)British Heart Foundation; FS/19/33/34328British Heart Foundation; CH/16/3/32406British Heart Foundation; CH/1999001/11735British Heart Foundation Center for Vascular Regeneration; RM/17/3/33381Hungría. Ministry for Innovation and Technology; NVKP_16-1–2016-0017Hungría. Ministry for Innovation and Technology; 2020-4.1.1.-TKP2020Hungría. National Research, Development and Innovation Office; K13493

The role of NOX4 in skin fibroblasts

Differentiation of fibroblasts to myofibroblasts consists of a phenotypic conversion to α-smooth muscle actin (α-SMA)-positive, highly contractile cells, which secrete proteins of the extracellular matrix (ECM). This differentiation process is crucial for physiological wound healing, as myofibroblasts are responsible for the restoration of damaged tissue and wound closure. Exaggerated, abnormal myofibroblasts activity is associated with the development of fibrosis. TGF-β is the most potent growth factor involved in fibroblasts-to-myofibroblasts conversion. Several studies indicated that the H2O2-generating NADPH oxidase 4 (NOX4) mediates TGF-β-dependent myofibroblasts differentiation during the development of pathological fibrosis in different organs. The profibrotic role of NOX4 has intensively been studied, but its role in physiological skin wound healing has not been described. The hypothesis of this thesis was that NOX4 regulates physiological skin wound healing. In order to investigate this hypothesis, both in vivo and in vitro approaches were applied. Skin wounds were created on the NOX4 KO and WT mice and the time of wound closure, wound size, epithelization, and contraction were determined. Immunohistochemical staining was performed to quantify α-SMA, cellular infiltration, and collagen content in the wounds. This in vivo study did not show any influence of NOX4 deletion on skin wound repair. In vitro, NOX4 KO fibroblasts were compared to WT fibroblasts in terms of proliferation capacity, cell viability and myofibroblast characteristics following TGF-β2 simulation. NOX4 KO fibroblasts differentiated into myofibroblasts similarly to WT fibroblasts, as confirmed by up-regulation of α-SMA expression, stress fibers formation, and increased contraction capabilities. To confirm this phenomenon, we used different genetic systems, where NOX4 is inactive, namely p22phox-deficient mouse fibroblasts, human fibroblasts treated with siRNA directed against NOX4, and human fibroblasts isolated from a patient with p22phox mutation. Upon stimulation with TGF-β1/2, they all exhibited up-regulation of α-SMA and formation of stress fibers, without any significant difference, when compared to their respective control cells. Nevertheless, TGF-β1/2 robustly induced NOX4 expression in both human and mouse fibroblasts. Thus, in order to examine the role of TGF-β-induced NOX4 expression, RNA sequencing was performed, using primary fibroblasts from NOX4 KO and WT mice. This approach confirmed previous results: TGF-β2 treatment led to increased expression of typical myofibroblastic markers and initiation of pathways associated with a cytoskeletal organization and ECM structure, in both WT and NOX4 KO fibroblasts. However, RNA sequencing revealed significant up-regulation of Ucp2 and Hddc3 and down-regulation of Islr in TGF-β-stimulated NOX4 KO fibroblasts. Ucp2 is an inner mitochondrial protein widely studied especially for its role in glucose metabolism and stress response, while Hddc3 and Islr functions are mostly unknown. Thus, fibroblast metabolic parameters were examined. NOX4 KO fibroblasts showed increased mitochondrial staining, while TGF-β2-stimulated NOX4 KO fibroblasts exhibited upregulated glycolysis. For the first time, this study documented NOX4-mediated upregulation of Ucp2 expression and the role of NOX4 in the regulation of cellular metabolism. Altogether, this study provides a strong argument that NOX4 does not play a key role in physiological skin wound healing and myofibroblast differentiation, however, it indicates a novel function of NOX4 in stress-response and metabolism of fibroblasts.La différenciation des fibroblastes en myofibroblastes consiste en une conversion phénotypique des fibroblastes en cellules positives pour le marqueur “α-smooth muscle actin” (α-SMA), hautement contractiles et qui sécrètent des protéines de la matrice extracellulaire. Ce processus de différenciation est crucial pour la cicatrisation physiologique des plaies, car les myofibroblastes sont responsables de la restauration des tissus endommagés et de la fermeture des plaies. Une activité exagérée et anormale des myofibroblastes est associée au développement de fibroses. Le TGF-β est le facteur de croissance le plus puissant impliqué dans la conversion des fibroblastes en myofibroblastes. Plusieurs études indiquent que la NADPH oxydase 4 (NOX4), génératrice de H2O2, contrôle la différenciation des myofibroblastes lors de fibroses de nombreux organes. Le rôle pro-fibrotique de NOX4 a été intensivement étudié, mais son rôle dans la cicatrisation physiologique des plaies cutanées n'a pas été décrit. L'hypothèse de cette thèse était que NOX4 régule la cicatrisation physiologique des plaies cutanées. Afin d'étudier cette hypothèse, des approches in vivo et in vitro ont été appliquées. Des plaies cutanées ont été créées sur des souris NOX4 KO et WT et le temps de fermeture de la plaie, la taille de la plaie, l'épithélisation et la contraction ont été mesurés. Une coloration immunohistochimique a été réalisée pour quantifier l'α-SMA, l'infiltration cellulaire et la teneur en collagène des plaies. Cette étude in vivo n'a montré aucune influence de la délétion de NOX4 sur la réparation des plaies cutanées. In vitro, les fibroblastes NOX4 knockout (KO) ont été comparés aux fibroblastes sauvages (WT) en termes de capacité de prolifération, de viabilité cellulaire et de caractéristiques myofibroblastiques suite à la simulation TGF-β2. Les fibroblastes NOX4 KO se sont différenciés en myofibroblastes de la même manière que les fibroblastes WT, tant au niveau de la surexpression de α-SMA, de la formation de fibres de stress et de l'augmentation des capacités contractiles. Afin de confirmer cette observation, nous avons utilisé différents systèmes génétiques où NOX4 est inactivé, à savoir des fibroblastes de souris déficientes en p22phox, des fibroblastes humains traités avec des siRNA dirigés contre NOX4 et des fibroblastes humains isolés de patients porteurs d’une mutation p22phox. Lors de la stimulation avec TGF-β1/2, tous ont présenté une surexpression de α-SMA et la formation de fibres de stress de façon similaire à leurs cellules témoins respectives. Malgré tout, le TGF-β1/2 induit fortement l'expression de NOX4 dans les fibroblastes humains et murins. Ainsi, afin d'examiner le rôle de la surexpression de NOX4 induite par le TGF-β, un séquençage d'ARN a été réalisé en utilisant des fibroblastes primaires de souris NOX4 KO et WT. Cette approche a confirmé les résultats précédents : le traitement par TGF-β2 induit une surexpression de marqueurs myofibroblastiques et des voies associées à l'organisation du cytosquelette ainsi qu’à la structure de la ME dans les fibroblastes tant WT que NOX4 KO. Cependant, le séquençage de l'ARN a révélé une surexpression s de Ucp2 et Hddc3 dans les fibroblastes NOX4 KO. Ucp2 est une protéine de la paroi interne des mitochondries largement étudiée, notamment pour son rôle dans le métabolisme du glucose et la réponse au stress, tandis que la fonction de Hddc3 est en grande partie inconnue. Ainsi, les paramètres métaboliques des fibroblastes ont été examinés. Les fibroblastes NOX4 KO ont une plus grande quantité de mitochondries, tandis que les fibroblastes NOX4 KO stimulés par le TGF-β2 ont une glycolyse accrue. Cette étude montre pour la première fois, une surexpression d'Ucp2 régulée par par NOX4 et un rôle de NOX4 dans la régulation du métabolisme cellulaire. Dans l'ensemble, cette étude fournit un argument solide selon lequel NOX4 ne joue pas un rôle clé dans la cicatrisation physiologique des plaies cutanées et la différenciation des myofibroblastes, cependant, elle indique une nouvelle fonction de NOX4 dans la réponse au stress et le métabolisme des fibroblastes

NADPH oxidase 4 is dispensable for skin myofibroblast differentiation and wound healing

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-β) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix (ECM) and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-β-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-β in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-β treatment, Nox4 did not influence skin myofibroblast differentiation although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism