Dupuytren's Disease Is Mediated by Insufficient TGF-ß1 Release and Degradation.

Dupuytren's disease (DD) is a fibroproliferative disorder affecting the palmar fascia, causing functional restrictions of the hand and thereby limiting patients' daily lives. The disturbed and excessive myofibroblastogenesis, causing DD, is mainly induced by transforming growth factor (TGF)-ß1. But, the extent to which impaired TGF-ß1 release or TGF-ß signal degradation is involved in pathologically altered myofibroblastogenesis in DD has been barely examined. Therefore, the complex in which TGF-ß1 is secreted in the extracellular matrix to elicit its biological activity, and proteins such as plasmin, integrins, and matrix metalloproteinases (MMPs), which are involved in the TGF-ß1 activation, were herein analyzed in DD-fibroblasts (DD-FBs). Additionally, TGF-ß signal degradation via caveolin-1 was examined with 5-fluoruracil (5-FU) in detail. Gene expression analysis was performed via Western blot, PCR, and immunofluorescence analyses. As a surrogate parameter for disturbed myofibroblastogenesis, 𝛼-smooth-muscle-actin (𝛼-SMA) expression was evaluated. It was demonstrated that latency-associated peptide (LAP)-TGF-ß and latent TGF-ß-binding protein (LTBP)-1 involved in TGF-ß-complex building were significantly upregulated in DD. Plasmin a serinprotease responsible for the TGF-ß release was significantly downregulated. The application of exogenous plasmin was able to inhibit disturbed myofibroblastogenesis, as measured via 𝛼-SMA expression. Furthermore, a reduced TGF-ß1 degradation was also involved in the pathological phenotype of DD, because caveolin-1 expression was significantly downregulated, and if rescued, myofibroblastogenesis was also inhibited. Therefore, our study demonstrates that a deficient release and degradation of TGF-ß1 are important players in the pathological phenotype of DD and should be addressed in future research studies to improve DD therapy or other related fibrotic conditions.

Evaluation of pro-angiogenic properties of an inorganic silica gel fibre fleece.

Hard-to-heal wounds represent an increasing health and economic burden on society. At present, therapy options for hard-to-heal wounds are often unsatisfactory, and the development of more effective wound treatments is urgently needed. We have shown that orthosilicic acid-releasing silica fibre fleece (SIFIB), via its pronounced anti-inflammatory properties, exhibited a significantly enhanced effect on wound closure kinetics in a porcine wound model in vivo. In this present study, we have examined in vitro the impact of the pro-angiogenic potential of SIFIB. Using an in vitro angiogenesis assay we describe for the first time how an inorganic biodegradable silica-based material significantly improved endothelial microvessel-like structure formation. We further demonstrate that the molecular mechanism of this pro-angiogenic activity of SIFIB is based on a significantly increased and tumour necrosis factor (TNF)α-dependent VEGF protein expression. In conclusion, due to its positive effects on angiogenesis, our results further indicate that decomposition products of silica-based biodegradable inorganic materials might represent very relevant therapeutic components of modern wound dressings for the treatment of hard-to-heal wounds.

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro.

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu(2+)) can dissociate from their physiological carrier proteins. Reduced by the body's own antioxidants, the resultant Cu(1+) might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu(1+)-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu(2+) ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe(2+)) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu(1+)-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Tumorigenic effects of human mesenchymal stromal cells and fibroblasts on bladder cancer cells.

Background: Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells. Methods: We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays. Results: Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFß-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities. Conclusions: The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy.

Comparison of compositional MRI techniques to quantify the regenerative potential of articular cartilage: a preclinical minipig model after osteochondral defect treatments with autologous mesenchymal stromal cells and unseeded scaffolds.

Background: The field of orthopedics seeks effective, safer methods for evaluating articular cartilage regeneration. Despite various treatment innovations, non-invasive, contrast-free full quantitative assessments of hyaline articular cartilage's regenerative potential using compositional magnetic resonance (MR) sequences remain challenging. In this context, our aim was to investigate the effectiveness of different MR sequences for quantitative assessment of cartilage and to compare them with the current gold standard delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC) measurements. Methods: We employed ex vivo imaging in a preclinical minipig model to assess knee cartilage regeneration. Standardized osteochondral defects were drilled in the proximal femur of the specimens (n=14), which were divided into four groups. Porcine collagen scaffolds seeded with autologous adipose-derived stromal cells (ASC), autologous bone marrow stromal cells (BMSC), and unseeded scaffolds (US) were implanted in femoral defects. Furthermore, there was a defect group which received no treatment. After 6 months, the specimens were examined using different compositional MR methods, including the gold standard dGEMRIC as well as T1, T2, T2*, and T1ρ techniques. The statistical evaluation involved comparing the defect region with the uninjured tibia and femur cartilage layers and all measurements were performed on a clinical 3T MR Scanner. Results: In the untreated defect group, we observed significant differences in the defect region, with dGEMRIC values significantly lower (404.86±64.2 ms, P=0.018) and T2 times significantly higher (44.24±2.75 ms, P<0.001). Contrastingly, in all three treatment groups (ASC, BMSC, US), there were no significant differences among the three regions in the dGEMRIC sequence, suggesting successful cartilage regeneration. However, T1, T2*, and T1ρ sequences failed to detect such differences, highlighting their lower sensitivity for cartilage regeneration. Conclusions: As expected, dGEMRIC is well suited for monitoring cartilage regeneration. Interestingly, T2 imaging also proved to be a reliable cartilage imaging technique and thus offers a contrast agent-free alternative to the former gold standard for subsequent in vivo studies investigating the cartilage regeneration potential of different treatment modalities.

Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence.

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-ß signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-ß signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-ß signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Illuminating the effect of beneficial blue light and ROS-modulating enzymes in Dupuytren's disease.

Dupuytren's disease (DD) is a fibroproliferative disorder of the palmar aponeurosis, which is characterized by a compound myofibrogenesis and evidenced by an increased expression of α-smooth muscle actin (α-SMA). In Dupuytren's tissue, higher levels of reactive oxygen species (ROS) are documented, stimulating the proliferation and differentiation of myofibroblasts. Our preliminary study demonstrates that α-SMA-expression is significantly inhibited by blue light irradiation in DD. The objective of this study was to investigate the beneficial effect of blue light irradiation and to elucidate the influence of ROS on myofibrogenesis in the pathogenesis of DD. Therefore, an in-vitro model of human DD fibroblasts was used. DD fibroblasts and control fibroblasts isolated from carpal tunnel syndrome (CTS) were daily irradiated with 40 J/cm2 (λ = 453 nm, 38 mW/cm2). Protein expression of ROS-modulating enzymes (Catalase, NOX4, SOD1, MnSOD) and α-SMA were determined, and additionally analysed after a pharmacological inhibition of the TGF-ß1-signaling with SB431542. Furthermore, the protein expression of α-SMA as surrogate parameter for myofibrogenesis was evaluated after applying different concentrations of long-lasting ROS. It could be determined that the beneficial blue light irradiation, which inhibited myofibrogenesis, is mediated by a significant inhibition of catalase protein expression. This effect should be accompanied with an increased intracellular ROS level. Proof of evidence was an H2O2-application on DD fibroblasts, also leading to a decreased myofibrogenesis. Furthermore, it could be demonstrated that endogenous MnSOD was significantly downregulated in resting DD fibroblasts. If DD fibroblasts were treated with the pharmacological inhibitor SB431542, myofibrogenesis was inhibited, but MnSOD expression was simultaneously elevated, which ought to affect ROS level by raising intracellular H2O2 amount. Blue light irradiation as well as the pharmacological action of SB431542 in consequence mediates their beneficial effect on disturbed myofibrogenesis in DD by further increasing ROS level. The present study demonstrates the importance of intracellular ROS homeostasis in DD and illuminates the beneficial effect of blue light as a promising therapy option for DD.

Impact of growth factor content on proliferation of mesenchymal stromal cells derived from adipose tissue.

Autologous adipose tissue (AT) transfer has gained widespread acceptance and is used for a broad variety of regenerative clinical indications. It is assumed that the successful outcome of AT transfer essentially depends on the amount of autocrine-generated growth factors (GF). It is supposed that several GF enhance and improve the anatomic and functional integration of the transplanted AT grafts at the site of implantation. In the present study we have investigated for the first time the correlation between the concentration of GF of freshly isolated AT and the proliferation and migration capacity of mesenchymal stroma cells (MSCs) derived from the respective AT sample. We here show that the proliferation and migration capacity of MSCs strongly depends on the GF content of the AT the cells were isolated from but in an inversely proportional manner. The lower the GF content of an AT sample was, the higher was the proliferation and migration capacity of the respective MSC population contained in the AT and vice versa. Furthermore, we found that supplementation with recombinant GFs only in the case of AT samples with low but not with higher growth factor contents led to a significant enhancement of proliferation and migration of the AT-resident MSCs. As we further show, this inefficiency of GFs to enhance MSC proliferation and migration in AT samples with high GF contents indicates a GF-mediated negative feedback mechanism leading to an impaired GF signaling in MSC obtained from those AT samples. Our results might explain why the successful use of AT grafting is frequently limited by low and unpredictable survival rates, and we suggest to use the knowledge of GF content of harvested AT as a predictive clinical parameter for risk assessment of the therapeutic outcome of autologous AT transfer.

The impact of non-toxic blue light (453 nm) on cellular antioxidative capacity, TGF-ß1 signaling, and myofibrogenesis of human skin fibroblasts.

Studies have demonstrated that blue light induces biological effects, such as cell death, and inhibition of proliferation and differentiation. Since blue light at longer wavelength (>440 nm) exerts less injurious effects on cells than at shorter wavelengths, (400-440 nm), we have investigated the impact of non-toxic (LED) blue light at 453 nm wavelength on human skin fibroblasts (hsFBs). We found that besides its decreasing effects on the proliferation rate, repeated blue light irradiations (80 J/cm2) also significantly reduced TGF-ß1-induced myofibrogenesis as shown by diminished α-SMA and EDA-FN expression accompanied by reduced protein expression and phosphorylation of ERK 1/2, SMAD 2/3, and p38-key players of TGF-ß1-induced myofibrogenesis. In parallel, catalase protein expression, intracellular FAD concentrations as well as NADP+/NADPH ratio were reduced, whereas intracellular reactive oxygen species (ROS) were increased. We postulate that as a molecular mechanism downregulation of catalase and photoreduction of FAD induce intracellular oxidative stress which, in turn, affects the signaling factors of myofibrogenesis leading to a lower rate of α-SMA and EDA-FN expression and, therefore, myofibroblast formation. In conclusion, blue light even at longer wavelengths shows antifibrotic activity and may represent a suitable and safe approach in the treatment of fibrotic skin diseases including hypertrophic scarring and scleroderma.

MMP-7 Serum and Tissue Levels Are Associated with Poor Survival in Platinum-Treated Bladder Cancer Patients.

Chemotherapy resistance is a main cause of therapeutic failure and death in bladder cancer. With the approval of immune checkpoint inhibitors, prediction of platinum treatment became of great clinical importance. Matrix metalloproteinase-7 (MMP-7) was shown to be involved in cisplatin resistance. Therefore, tissue and circulating MMP-7 levels were evaluated in 124 bladder cancer patients who received postoperative platinum-based chemotherapy. Tissue MMP-7 levels were analyzed by immunohistochemistry in 72 formalin-fixed, paraffin-embedded chemo-naïve tumor samples, while MMP-7 serum concentrations were determined in 132 serum samples of an independent cohort of 52 patients. MMP-7 tissue and serum levels were correlated with clinicopathological and follow-up data. MMP-7 gene expression was determined by RT-qPCR in 20 urothelial cancer cell lines and two non-malignant urothelial cell lines. MMP-7 was overexpressed in RT-112 and T-24 cells by stable transfection, to assess its functional involvement in platinum sensitivity. High MMP-7 tissue expression and pretreatment serum concentrations were independently associated with poor overall survival (tissue HR = 2.296, 95%CI = 1.235-4.268 and p = 0.009; serum HR = 2.743, 95%CI = 1.258-5.984 and p = 0.011). Therefore, MMP-7 tissue and serum analysis may help to optimize therapeutic decisions. Stable overexpression in RT-112 and T-24 cells did not affect platinum sensitivity.

Blue light irradiation and its beneficial effect on Dupuytren's fibroblasts.

Dupuytren's contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm2, continuous radiance, spot size 10-12 cm2) as well as the molecular mechanism mediating this effect. It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren's fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren's disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.

Blue light (λ=453 nm) nitric oxide dependently induces ß-endorphin production of human skin keratinocytes in-vitro and increases systemic ß-endorphin levels in humans in-vivo.

ß-Endorphin exerts a broad spectrum of physiological activity on mood, immune functions, pain management, reward effects, and behavioral stability. ß-Endorphin is produced in certain neurons within the central and peripheral nervous system but also in the skin, especially in response to ultraviolet radiation. In the present study we have investigated the impact of visible blue light at λ = 453 nm (BL) on ß-endorphin production of primary human skin keratinocytes (hKC) in-vitro as well as on systemic ß-endorphin formation of whole-body exposed subjects in-vivo. We found that BL irradiation significantly enhanced both keratinocytic ß-endorphin production of hKC cultures as well as systemic ß-endorphin concentrations in light exposed healthy subjects. Interestingly, in hKC cultures elevated ß-endorphin formation was paralleled by significantly increased levels of non-enzymatically generated nitric oxide (NO), whereas elevated systemic ß-endorphin values of BL-exposed subjects were accompanied by enhanced systemic concentration of bioactive NO-derivates. These findings point to a pivotal role of NO in the molecular mechanism of the observed BL-induced effects, and indeed, exogenously applied NO was able to significantly enhance ß-endorphin production in hKC cultures. Thus, our finding of BL-induced increases in systemic ß-endorphin concentration in-vivo can be plausibly explained by an event sequence comprising 1.) BL-driven non-enzymatic formation of NO in the exposed skin tissue, 2.) systemic distribution of cutaneously produced NO in the form of bioactive nitroso compounds, 3.) a subsequent NO-dependent induction of ß-endorphin synthesis in epidermal keratinocytes, and 4.) probably also a NO-dependent modulation of ß-endorphin synthesis in specialized neurons within the central and peripheral nervous system.

Human iPSC-derived iMSCs improve bone regeneration in mini-pigs.

Autologous bone marrow concentrate (BMC) and mesenchymal stem cells (MSCs) have beneficial effects on the healing of bone defects. To address the shortcomings associated with the use of primary MSCs, induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been proposed as an alternative. The aim of this study was to investigate the bone regeneration potential of human iMSCs combined with calcium phosphate granules (CPG) in critical-size defects in the proximal tibias of mini-pigs in the early phase of bone healing compared to that of a previously reported autograft treatment and treatment with a composite made of either a combination of autologous BMC and CPG or CPG alone. iMSCs were derived from iPSCs originating from human fetal foreskin fibroblasts (HFFs). They were able to differentiate into osteoblasts in vitro, express a plethora of bone morphogenic proteins (BMPs) and secrete paracrine signaling-associated cytokines such as PDGF-AA and osteopontin. Radiologically and histomorphometrically, HFF-iMSC + CPG transplantation resulted in significantly better osseous consolidation than the transplantation of CPG alone and produced no significantly different outcomes compared to the transplantation of autologous BMC + CPG after 6 weeks. The results of this translational study imply that iMSCs represent a valuable future treatment option for load-bearing bone defects in humans.

Molecular- and microarray-based analysis of diversity among resting and osteogenically induced porcine mesenchymal stromal cells of several tissue origin.

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches in bone-healing disorders. Although bone marrow-derived MSCs are most frequently used, the knowledge that many other adult tissues represent promising sources for potent MSCs has gained acceptance. In the present study, the osteogenic differentiation potential of porcine skin fibroblasts (FBs), as well as bone marrow- (BMSCs), adipose tissue- (ASCs) and dental pulp-derived stromal cells (DSCs) were evaluated. However, additional application of BMP-2 significantly elevated the delayed osteogenic differentiation capacity of ASC and FB cultures, and in DSC cultures the supplementation of platelet-rich plasma increased osteogenic differentiation potential to a comparable level of the good differentiable BMSCs. Furthermore, microarray gene expression performed in an exemplary manner for ASCs and BMSCs revealed that ASCs and BMSCs use different gene expression patterns for osteogenic differentiation under standard media conditions, as diverse MSCs are imprinted dependent from their tissue niche. However, after increasing the differentiation potential of ASCs to a comparable level as shown in BMSCs, a small subset of identical key molecules was used to differentiate in the osteogenic lineage. Until now, the importance of identified genes seems to be underestimated for osteogenic differentiation. Apparently, the regulation of transmembrane protein 229A, interleukin-33 and the fibroblast growth factor receptor-2 in the early phase of osteogenic differentiation is needed for optimum results. Based on these results, bone regeneration strategies of MSCs have to be adjusted, and in vivo studies on the osteogenic capacities of the different types of MCSs are warranted. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

The performance of an orthosilicic acid-releasing silica gel fiber fleece in wound healing.

In the present work, we have examined the impact of an inorganic orthosilicic acid-releasing spun fiber fleece (SIFIB) on wound closure in a porcine wound model in vivo as well as on wound healing-relevant parameters in vitro. In vivo SIFIB was completely bio-degradable and had no negative effects on wound closure or the wound healing process. In the in vitro experiments, SIFIB had no negative effects on proliferation of human skin fibroblast (FB) and endothelial cell (EC) cultures but strongly retarded the growth of the human monocyte cell line THP-1, and effectively inhibited human skin keratinocyte (KC) proliferation, which based on significantly enhanced KC differentiation. Furthermore, SIFIB exhibited strong anti-inflammatory properties, which based on SIFIB-dependent inhibition of expression and activity of NF-кB and/or concomitant enhanced expression of IкB, a NF-кB-inhibiting protein. Additionally, SIFIB significantly inhibited TGFß-induced fibroblast differentiation and collagen synthesis as well as effectively reduced TGF-ß synthesis of activated fibroblasts. We have demonstrated wound healing-relevant biological activities of a silica-based bio-degradable inorganic material, which might represent a new therapeutic tool in the treatment of chronic wounds.