The Protective Effect of Carotenoids, Polyphenols, and Estradiol on Dermal Fibroblasts under Oxidative Stress

Skin ageing is influenced by several factors including environmental exposure and hormonal changes. Reactive oxygen species (ROS), which mediate many of the effects of these factors, induce inflammatory processes in the skin and increase the production of matrix metalloproteinases (MMPs) in dermal fibroblasts, which leads to collagen degradation. Several studies have shown the protective role of estrogens and a diet rich in fruits and vegetables on skin physiology. Previous studies have shown that dietary carotenoids and polyphenols activate the cell’s antioxidant defense system by increasing antioxidant response element/Nrf2 (ARE/Nrf2) transcriptional activity and reducing the inflammatory response. The aim of the current study was to examine the protective effect of such dietary-derived compounds and estradiol on dermal fibroblasts under oxidative stress induced by H2O2. Human dermal fibroblasts were used to study the effect of H2O2 on cell number and apoptosis, MMP-1, and pro-collagen secretion as markers of skin damage. Treatment of cells with H2O2 led to cell death, increased secretion of MMP-1, and decreased pro-collagen secretion. Pre-treatment with tomato and rosemary extracts, and with estradiol, reversed the effects of the oxidative stress. This was associated with a reduction in intracellular ROS levels, probably through the measured increased activity of ARE/Nrf2. Conclusions: This study indicates that carotenoids, polyphenols, and estradiol protect dermal fibroblasts from oxidative stress-induced damage through a reduction in ROS levels.

Deleterious Role of Th9 Cells in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology. Immune disorders play an important role in IPF pathogenesis. Here, we show that Th9 cells differentiate and activate in the lung tissue of patients with IPF and bleomycin (BLM)-induced lung fibrosis mice. Moreover, we found that Th9 cells promote pulmonary fibrosis in two ways. On the one hand, Th9 cells promote fibroblast differentiation, activation, and collagen secretion by secreting IL-9. On the other hand, they promote differentiation of Th0 cells into Th2 cells by secreting IL-4. Th9 cells and Th2 cells can promote each other, accelerating the Th1/Th2 imbalance and eventually forming a positive feedback of pulmonary fibrosis. In addition, we found that neutralizing IL-9 in both preventive and therapeutic settings ameliorates bleomycin-induced pulmonary fibrosis. Furthermore, we identified several critical signaling pathways involved in the effect of neutralizing IL-9 on pulmonary fibrosis by proteomics study. From an immunological perspective, we elucidated the novel role and underlying mechanism of Th9 cells in pulmonary fibrosis. Our study suggested that Th9-based immunotherapy may be employed as a treatment strategy for IPF.

Cytokine-Mediated Alterations of Human Cardiac Fibroblast’s Secretome

Fibroblasts contribute to approximately 20% of the non-cardiomyocytic cells in the heart. They play important roles in the myocardial adaption to stretch, inflammation, and other pathophysiological conditions. Fibroblasts are a major source of extracellular matrix (ECM) proteins whose production is regulated by cytokines, such as TNF-α or TGF-β. The resulting myocardial fibrosis is a hallmark of pathological remodeling in dilated cardiomyopathy (DCM). Therefore, in the present study, the secretome and corresponding transcriptome of human cardiac fibroblasts from patients with DCM was investigated under normal conditions and after TNF-α or TGF-β stimulation. Secreted proteins were quantified via mass spectrometry and expression of genes coding for secreted proteins was analyzed via Affymetrix Transcriptome Profiling. Thus, we provide comprehensive proteome and transcriptome data on the human cardiac fibroblast’s secretome. In the secretome of quiescent fibroblasts, 58% of the protein amount belonged to the ECM fraction. Interestingly, cytokines were responsible for 5% of the total protein amount in the secretome and up to 10% in the corresponding transcriptome. Furthermore, cytokine gene expression and secretion were upregulated upon TNF-α stimulation, while collagen secretion levels were elevated after TGF-β treatment. These results suggest that myocardial fibroblasts contribute to pro-fibrotic and to inflammatory processes in response to extracellular stimuli.

Evaluation of Pirfenidone and Nintedanib in a Human Lung Model of Fibrogenesis

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease with a poor prognosis and increasing incidence. Pirfenidone and nintedanib are the only approved treatments for IPF but have limited efficacy and their mechanisms of action are poorly understood. Here we have examined the effects of pirfenidone and nintedanib in a human model of lung fibrogenesis, and compared these with the putative anti-fibrotic compounds Lipoxin A4 (LXA4), and senicapoc, a KCa3.1 ion channel blocker.Methods: Early fibrosis was induced in cultured human lung parenchyma using TGFβ1 for 7 days, ± pirfenidone, nintedanib, or LXA4. Pro-fibrotic responses were examined by RT-PCR, immunohistochemistry and soluble collagen secretion.Results: Thirty six out of eighty four IPF and fibrosis-associated genes tested were significantly upregulated by TGFβ1 in human lung parenchyma with a ≥0.5 log2FC (n = 32). Nintedanib (n = 13) reduced the mRNA expression of 14 fibrosis-associated genes including MMPs (MMP1,−4,−13,−14), integrin α2, CXCR4 and PDGFB, but upregulated α-smooth muscle actin (αSMA). Pirfenidone only reduced mRNA expression for MMP3 and −13. Senicapoc (n = 11) previously attenuated the expression of 28 fibrosis-associated genes, including αSMA, several growth factors, collagen type III, and αV/β6 integrins. Pirfenidone and nintedanib significantly inhibited TGFβ1-induced fibroblast proliferation within the tissue, but unlike senicapoc, neither pirfenidone nor nintedanib prevented increases in tissue αSMA expression. LXA4 was ineffective.Conclusions: Pirfenidone and nintedanib demonstrate modest anti-fibrotic effects and provide a benchmark for anti-fibrotic activity of new drugs in human lung tissue. Based on these data, we predict that the KCa3.1 blocker senicapoc will show greater benefit than either of these licensed drugs in IPF.

Deficiency of myeloid prolyl hydroxylase domain proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling

Background Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. Methods Myeloid specific PHD knockout (PHDko) mice were fed high cholesterol diet for 6–12 weeks to induce atherosclerosis. Plaque parameters, e.g. plaque size and macrophage content, were analyzed. Bulk and single cell RNA sequencing was performed on PHD2 BMDMs and plaque macrophages, respectively. Results Aortic root plaque size was augmented 2.6fold in PHD2cko, and 1.4-fold in PHD3ko, but not in PHD1ko mice compared to controls. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, confirmed these findings and were validated by siRNA silencing. Human plaque BNIP3 mRNA associated with plaque necrotic core, suggesting similar adverse effects. Further, PHD2cko plaques displayed enhanced fibrosis, independent of macrophage MMP activity, collagen secretion or proliferation and of SMC collagen production, or proliferation. Rather, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. Nichenet in silico analysis of macrophage-fibroblast communication predicted SPP1 signaling as regulator, in line with enhanced plaque SPP1 protein content, and SPP1 mRNA in TREM2-foamy plaque macrophages, but not in neutrophils. Conclusion Myeloid PHD2cko and PHD3ko enhanced plaque growth, macrophage apoptosis, and PHD2cko activated paracrine collagen secretion by fibroblasts. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO, Leducq

Novel Mg-Incorporated Micro-Arc Oxidation Coatings for Orthopedic Implants Application

In this study, Ti-6Al-4V alloy samples were processed by micro-arc oxidation (MAO) in phytic acid (H12Phy) electrolytes with the addition of different concentrations of EDTA-MgNa2 (Na2MgY) and potassium hydroxide (KOH). The surface characterization and cytocompatibility of MAO-treated samples were evaluated systematically. H12Phy is a necessary agent for MAO coating formation, and the addition of Na2MgY and KOH into the electrolytes increases the surface roughness, micropore size and Mg contents in the coatings. The MAO coatings are primarily composed of anatase, rutile, MgO and Mg3(PO4)2. Magnesium (Mg) ions in the electrolytes enter into MAO coatings by diffusion and electromigration. The MAO coatings containing 2.97 at% Mg show excellent cell viability, adhesion, proliferation, alkaline phosphatase activity, extracellular matrix (ECM) mineralization and collagen secretion, but the cytocompatibility of the MAO coatings containing 6.82 at% Mg was the worst due to the excessively high Mg content. Our results revealed that MAO coatings with proper Mg contents improve the cytocompatibility of the Ti-6Al-4V alloys and have large potential in orthopedic applications.

3D-Printed Scaffolds from Alginate/Methyl Cellulose/Trimethyl Chitosan/Silicate Glasses for Bone Tissue Engineering

Alginate-based hydrogel inks are commonly used in printing due to their biocompatibility, biodegradation, and cell adhesion. In the present work, 3D printing of hydrogels comprising alginate/methyl cellulose (MC)/trimethyl chitosan (TMC) and silicate glasses was investigated. It was found that TMC increased the stability of the scaffolds after immersion in normal saline solution in comparison with alginate/MC 3D constructs. The stability also remained after the incorporation of pure silicate glasses or bioactive glasses. Immersion in simulated body fluid (SBF) resulted in the formation of hydroxyapatite in all samples. Scanning electron microscopy (SEM) analysis revealed a good cell adhesion of pre-osteoblasts on all scaffold compositions, cell viability assessment displayed a proliferation increase up to seven days in culture, and alkaline phosphatase (ALP) activity was similar in all scaffold compositions without significant differences. Total collagen secretion by the pre-osteoblasts after 7 days in culture was significantly higher in scaffolds containing silicate glasses, demonstrating their ability to promote extracellular matrix formation. In conclusion, 3D-printed porous scaffolds based on alginate/methyl cellulose/TMC are promising candidates for bone tissue engineering applications.

Glomerular Mesangial Cell pH Homeostasis Mediates Mineralocorticoid Receptor-Induced Cell Proliferation

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na+/H+-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO3− transporters and were not dependent on HCO3−. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease.

Proteomic basis of modulation of post ischemic fibrosis by MSC exosomes

Following an ischemic event, there is activation of fibroblasts leading to scar formation. It is critical to limit the pro-fibrotic remodeling and activate the reparative, remodeling phase to limit cardiac diastolic dysfunction. Mesenchymal stem cell (MSC) exosomes offer significant protection against ischemia-related systolic dysfunction. Here we studied if MSC exosomes would offer protection against pro-fibrotic events in mouse hearts subjected to acute ischemia (1 hr. left coronary artery occlusion [LCA]) or chronic ischemia (7 days LCA occlusion). Following acute ischemia, there was activation of inflammatory signals, more in the peri-infarct than in the infarct area, in the saline (vehicle)-treated mice. At the same time, there was expression of cardiac remodeling signals (vimentin, collagens-1 and -3, and fibronectin), more in the infarct area. Treatment with MSC exosomes before LCA ligation suppressed inflammatory signals during acute as well as chronic ischemia. Further, exosome treatment promoted pro-reparative cardiac ECM remodeling, in both infarct and peri-infarct areas by suppressing fibronectin secretion and by modulating collagen secretion to reduce fibrotic scar formation through altered cellular signaling pathways. Proteomics study revealed intense expression of IL-1b and activation of pro-fibrotic signals in the saline-treated hearts and their suppression in MSC exosome-treated hearts. To our knowledge, this is the first report on the infarct and peri-infarct area proteomics of ischemic mice hearts to explain MSC exosome-mediated suppression of scar formation in the ischemic mouse hearts.

Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood.Methods and ResultsImmunohistochemistry analysis showed accumulation of CD14+ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68+ and mannose-R+ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14+ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-β signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14+ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14+ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-β, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14+ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-β signalling pathway with SD208 (TGF-β receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-β-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion.ConclusionsOur findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14+ monocytes and CD14+ pulmonary macrophages in SSc and highlighted the capability of CD14+ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14+ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.