Tacrolimus induces a pro-fibrotic response in donor-derived human proximal tubule cells dependent on common variants of the CYP3A5 and ABCB1 genes.

BACKGROUND: Common genetic variants of the enzymes and efflux pump involved in tacrolimus disposition have been associated with calcineurin inhibitor nephrotoxicity, but their importance is unclear because of the multifactorial background of renal fibrosis. This study explores the pro-fibrotic response of tacrolimus exposure in relation to the differential capacity for tacrolimus metabolism in proximal tubule cells (PTCs) with a variable (pharmaco)genetic background. METHODS: PTCs were obtained from protocol allograft biopsies with different combinations of CYP3A5 and ABCB1 variants and were incubated with tacrolimus within the concentration range found in vivo. Gene and protein expression, CYP3A5 and P-glycoprotein function, and tacrolimus metabolites were measured in PTC. Connective tissue growth factor (CTGF) expression was assessed in protocol biopsies of kidney allograft recipients. RESULTS: PTCs produce CTGF in response to escalating tacrolimus exposure, which is approximately 2-fold higher in cells with the CYP3A5*1 and ABCB1 TT combination in vitro. Increasing tacrolimus exposure results in relative higher generation of the main tacrolimus metabolite {13-O-desmethyl tacrolimus [M1]} in cells with this same genetic background. Protocol biopsies show a larger increase in in vivo CTGF tissue expression over time in TT vs. CC/CT but was not affected by the CYP3A5 genotype. CONCLUSIONS: Tacrolimus exposure induces a pro-fibrotic response in a PTC model in function of the donor pharmacogenetic background associated with tacrolimus metabolism. This finding provides a mechanistic insight into the nephrotoxicity associated with tacrolimus treatment and offers opportunities for a tailored immunosuppressive treatment.

Galectin-3 interferes with tissue repair and promotes cardiac dysfunction and comorbidities in a genetic heart failure model.

Galectin-3, a biomarker for heart failure (HF), has been associated with myocardial fibrosis. However, its causal involvement in HF pathogenesis has been questioned in certain models of cardiac injury-induced HF. To address this, we used desmin-deficient mice (des-/-), a model of progressive HF characterized by cardiomyocyte death, spontaneous inflammatory responses sustaining fibrosis, and galectin-3 overexpression. Genetic ablation or pharmacological inhibition of galectin-3 led to improvement of cardiac function and adverse remodeling features including fibrosis. Over the course of development of des-/- cardiomyopathy, monitored for a period of 12 months, galectin-3 deficiency specifically ameliorated the decline in systolic function accompanying the acute inflammatory phase (4-week-old mice), whereas a more pronounced protective effect was observed in older mice, including the preservation of diastolic function. Interestingly, the cardiac repair activities during the early inflammatory phase were restored under galectin-3 deficiency by increasing the proliferation potential and decreasing apoptosis of fibroblasts, while galectin-3 absence modulated macrophage-fibroblast coupled functions and suppressed both pro-fibrotic activation of cardiac fibroblasts and pro-fibrotic gene expression in the des-/- heart. In addition, galectin-3 also affected the emphysema-like comorbid pathology observed in the des-/- mice, as its absence partially normalized lung compliance. Collectively galectin-3 was found to be causally involved in cardiac adverse remodeling, inflammation, and failure by affecting functions of cardiac fibroblasts and macrophages. In concordance with this role, the effectiveness of pharmacological inhibition in ameliorating cardiac pathology features establishes galectin-3 as a valid intervention target for HF, with additive benefits for treatment of associated comorbidities, such as pulmonary defects. Schematic illustrating top to bottom, the detrimental role of galectin-3 (Gal3) in heart failure progression: desmin deficiency-associated spontaneous myocardial inflammation accompanying cardiac cell death (reddish dashed border) is characterized by infiltration of macrophages (round cells) and up-regulation of Lgals3 (encoding secretable galectin-3, green) and detrimental macrophage-related genes (Ccr2 and Arg1). In this galectin-3-enriched milieu, the early up-regulation of profibrotic gene expression (Tgfb1, Acta2, Col1a1), in parallel to the suppression of proliferative activities and a potential of senescence induction by cardiac fibroblasts (spindle-like cells), collectively promote des-/- cardiac fibrosis and dysfunction establishing heart failure (left panel). Additionally, galectin-3+ macrophage-enrichment accompanies the development of emphysema-like lung comorbidities. In the absence of galectin-3 (right panel), the effect of macrophage-fibroblast dipole and associated events are modulated (grey color depicts reduced expression or activities) leading to attenuated cardiac pathology in the des-/-Lgals3-/- mice. Pulmonary comorbidities are also limited.

LRG1 facilitates corneal fibrotic response by inducing neutrophil chemotaxis via Stat3 signaling in alkali-burned mouse corneas.

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-ß (TGF-ß) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Limiting angiogenesis to modulate scar formation.

Angiogenesis, the process of new blood vessel formation from existing blood vessels, is a key aspect of virtually every repair process. During wound healing an extensive, but immature and leaky vascular plexus forms which is subsequently reduced by regression of non-functional vessels. More recent studies indicate that uncontrolled vessel growth or impaired vessel regression as a consequence of an excessive inflammatory response can impair wound healing, resulting in scarring and dysfunction. However, in order to elucidate targetable factors to promote functional tissue regeneration we need to understand the molecular and cellular underpinnings of physiological angiogenesis, ranging from induction to resolution of blood vessels. Especially for avascular tissues (e.g. cornea, tendon, ligament, cartilage, etc.), limiting rather than boosting vessel growth during wound repair potentially is beneficial to restore full tissue function and may result in favourable long-term healing outcomes.

4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice.

Silicosis is caused by exposure to crystalline silica (CS). We have previously shown that blocking 4-1BB signaling attenuated CS-induced inflammation and pulmonary fibrosis. However, the cells that express 4-1BB, which plays a vital role in promoting fibrosis, are still unknown. In this study, we demonstrated that the expression of 4-1BB is elevated in alveolar macrophages (AMs) in the lungs of CS-injured mice. CS exposure also markedly enhanced the expression of 4-1BB in macrophage-like, MH-S cells. In these cells, activation of the 4-1BB signaling with an agonist antibody led to upregulated secretion of pro-fibrotic mediators. Consistently, blocking 4-1BB downstream signaling or genetic deletion of 4-1BB alleviated pro-fibrotic responses in vitro, while treatment with a 4-1BB fusion protein promoted pro-fibrotic responses. In vivo experiments showed that blocking 4-1BB signaling decreased the expressions of pro-fibrotic mediators and fibrosis. These data suggest that 4-1BB signaling plays an important role in promoting AMs-mediated pro-fibrotic responses and pulmonary fibrosis. Our findings may provide a potential molecular target to reduce CS-induced fibrotic responses in occupational lung disease.

Sodium Houttuyfonate Alleviates Post-infarct Remodeling in Rats via AMP-Activated Protein Kinase Pathway.

With the chronic ischemia persisting after acute myocardial infarction, the accompanying low-degree inflammation and subsequent fibrosis result in progression of cardiac remodeling and heart failure. Recently, Sodium Houttuyfonate (SH), a pure compound extracted from Houttuynia cordata, has been confirmed exerting anti-inflammatory and anti-fibrotic effects under diseased situations. Here, we aimed to investigate whether SH could reverse the cardiac remodeling post-myocardial infarction by alleviating cardiac inflammation and fibrosis. Left anterior descending coronary artery of adult male Sprague-Dawley rats was ligated to elicit myocardial infarction. Low and high dose of SH was administered by oral gavage for four consecutive weeks post-myocardial infarction. Long-term SH treatment decreased heart rate, heart weight/ body weight (HW/BW), and left ventricle weight/body weight (LVW/BW), reduced cardiac expression of brain natriuretic peptide (BNP), improved left ventricular heart function, and ameliorated the histopathological changes caused by myocardial infarction. Western blotting revealed the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-ß (TGF-ß), collagen I, and collagen III of the infarcted ventricle were reduced by SH treatment. Meanwhile, we found that SH treatment post-myocardial infarction activated AMP-activated protein kinase (AMPK) and suppressed nuclear factor-κB p65 (NF-κB p65). Furthermore, on H9C2 cells induced hypoxic injury with cobalt chloride (CoCl2), the reduction of inflammatory cytokines (IL-6, TNF-α, and TGF-ß), activation of AMPK, and suppression of NF-κB p65 were also observed by SH treatment. However, transfection of H9C2 with AMPKα siRNA blunted the suppression of NF-κB p65 and inflammatory cytokines (IL-6, TNF-α, and TGF-ß) by SH post-hypoxia. Taken together, these findings suggested that long-term administration of SH post-myocardial infarction reduced cardiac inflammatory and fibrotic responses, and reversed cardiac remodeling process. The underlying mechanism may be activating AMPK and suppressing NF-κB pathway.

High-water-content and resilient PEG-containing hydrogels with low fibrotic response.

Hydrogels such as those based on polyethylene glycol (PEG) are broadly used in biomedicine where high water contents, robust mechanical properties such as resilience and favorable interactions with the body are often simultaneously desirable. However, the mechanical properties of conventional hydrogels often degrade rapidly after swelling or with increasing water content, limiting their potential in many applications. Here we describe a new class of PEG-containing hydrogels that remain highly resilient after maximum swelling. We achieved the hydrogels by incorporating reversible "dual" hydrogen bonding into highly coiled, elastic PEG networks. These hydrogels, due to their high water content and high mechanical resilience, can form highly permeable, yet durable and easy-to-handle cell delivery devices without any additional structural support. In addition, optimization of chemical composition resulted in hydrogels with superior bio-inertness, inducing much less fibrosis upon subcutaneous implantation in mice than a polyhydroxyethylmethacrylate (PHEMA) hydrogel control. STATEMENT OF SIGNIFICANCE: Hydrogels such as polyethylene glycol (PEG)-based ones are broadly used in the biomedical world. Examples include wound dressings, tissue scaffolds, medical implants, biosensors and drug or cell delivery devices. In many of these applications, robust mechanical property, high water content (or facile mass transfer) and favorable interactions with the body are often simultaneously desirable. However, the mechanical property of hydrogels often degrades rapidly after swelling or with increasing water content. Here we report a new class of PEG-based hydrogels that simultaneously possess high water content, high mechanical resilience and low fibrotic response upon subcutaneous implantation in mice. These hydrogels may therefore find broad applications in biomedicine.

Galectin-3 mediates pulmonary vascular remodeling in hypoxia-induced pulmonary arterial hypertension.

Pulmonary vascular adventitia serves as a key regulator of pulmonary vascular remodeling in the pathogenesis of pulmonary arterial hypertension (PAH). Excessive proliferation and differentiation of pulmonary adventitial fibroblasts (PAFs) are proven to be crucial in the pathogenesis of PAH. Galectin-3 (Gal-3) is known as a key fibroblasts activating factor which is involved in the fibrogenesis of several diseases, such as pulmonary fibrosis, vascular fibrosis, and heart failure. Therefore, we seek to investigate the potential role of Gal-3 in regulating PAF cells in the pathogenesis of PAH. Gal-3 plasma concentration was significantly higher in PAH patients. Gal-3 was upregulated in pulmonary artery adventitia of hypoxia-induced PAH rats. Inhibition of Gal-3 with N-Acetyl-D-lactosamine (N-Lac) ameliorated PAH and pulmonary vascular remodeling. Gal-3 can stimulate the proliferation, differentiation, and collagen synthesis of PAFs, which was reversed by N-Lac. Transforming growth factor ß1 increased Gal-3 expression in PAFs, whereas N-Lac significantly suppressed transforming growth factor ß1-induced proliferation, differentiation, and collagen synthesis of PAFs. Gal-3 serves as a critical regulator in the pathogenesis of PAH by regulating the proliferation, differentiation, and extracellular matrix deposition synthesis of PAFs. Inhibition of Gal-3 may represent a novel therapeutic target for PAH treatment.

TGF-ß1 Improves Biomechanical Strength by Extracellular Matrix Accumulation Without Increasing the Number of Tenogenic Lineage Cells in a Rat Rotator Cuff Repair Model.

BACKGROUND: Transforming growth factor ß1 (TGF-ß1) positively regulates the tenogenic marker genes scleraxis ( Scx) and tenomodulin ( Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-ß1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. HYPOTHESIS: TGF-ß1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-ß1 (100 ng) or phosphate-buffered saline. The effects of TGF-ß1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [ Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro-computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell-related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. RESULTS: The TGF-ß1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell-related marker immunoreactivity were not affected by exogenously administrated TGF-ß1 at all time points. In the TGF-ß1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased, whereas the expression of MMP-9 and MMP-13 significantly decreased at 2 and 4 weeks postoperatively. CONCLUSION: TGF-ß1 enhances formation of tough fibrous tissues at the healing site by inhibiting MMP-9 and MMP-13 expression to increase collagen accumulation but without the growth of tenogenic lineage cells. CLINICAL RELEVANCE: These findings suggest that TGF-ß1 could be used for enhancing biomechanical strength after RC surgical repair.

Primary mouse lung fibroblasts help macrophages to tackle Mycobacterium tuberculosis more efficiently and differentiate into myofibroblasts up on bacterial stimulation.

Keeping with their classical role in wound healing, fibroblasts of the lung take part in the resolution of tubercular granulomas. They are totally absent in nascent granulomas, but surround necrotizing granulomas, and are the majority of cells in healed granulomas. Lung fibroblasts may become infected with Mycobacterium tuberculosis (Mtb). Two previous studies suggested an immunomodulatory effect of fibroblasts on infected macrophages. In the present study, we looked at the role of primary mouse lung fibroblasts on naive or activated mouse bone marrow macrophages infected with Mtb and the effect of infection on fibroblast properties. We observed that with fibroblasts in the vicinity, infected naive macrophages restricted the bacterial growth, while activated macrophages turned more bactericidal with concomitant increase in nitrite production. Neutralizing IL-1α in fibroblast supernatant reduced the nitrite production by infected macrophages. Secretion of IL-6 and MCP-1 was down-regulated, while TNF-α was up-regulated in infected naive macrophages. In infected activated macrophages, the secretion of IL-6 was up-regulated, while that of MCP-1 and TNF-α was unaffected. The 'fibroblast effects' were enhanced when the fibroblasts too were infected. Mtb induced IL-1 secretion and pro-fibrotic responses by fibroblasts. Mtb-induced myofibroblast conversion was blocked by rapamycin suggesting cell signalling via mTOR.