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Summary Age‐related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age‐related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age‐related increase in muscle stiffness drives YAP/TAZ‐mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

Scope Bacterial infection induces mucus overproduction, contributing to acute exacerbations and lung function decline in chronic respiratory diseases. A diet enriched in apples may provide protection from pulmonary disease development and progression. We examined whether phloretin, an apple polyphenol, inhibits mucus synthesis and secretion induced by the predominant bacteria associated with chronic respiratory diseases. Methods and results We analyzed expression of the mucus constituent mucin 5AC (MUC5AC) in FVB/NJ mice and NCI-H292 epithelial cells. Nontypeable Haemophilus influenzae (NTHi)-infected mice developed increased MUC5AC mRNA, which a diet containing phloretin inhibited. In NCI-H292 cells, NTHi, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa increased MUC5AC mRNA, which phloretin inhibited. Phloretin also diminished NTHi-induced MUC5AC protein secretion. NTHi-induced increased MUC5AC required toll-like receptor 4 (TLR4) and NADH oxidase 4 (NOX4) signaling and subsequent activation of the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway. Phloretin inhibited NTHi-induced TLR4/NOX4 and EGFR/MAPK signaling, thereby preventing increased MUC5AC mRNA. EGFR activation can also result from increased EGFR ligand synthesis and subsequent ligand activation by matrix metalloproteinases (MMPs). In NCI-H292 cells, NTHi increased EGFR ligand and MMP1 and MMP13 mRNA, which phloretin inhibited. Conclusions In summary, phloretin is a promising therapeutic candidate for preventing bacterial-induced mucus overproduction. This article is protected by copyright. All rights reserved.

Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung.

MK-852, a cyclic heptapeptide, is a potent platelet fibrinogen receptor antagonist. When administered to normal healthy male subjects by 1- and 4-hour constant rate intravenous infusions, it provides a generally well-tolerated and reversible means of inhibition of platelet function. At infusion rates of 1 microgram/kg/min for 1 hour and 0.44 microgram/kg/min for 4 hours, respectively, MK-852 extended baseline bleeding time by greater than 2.2-fold and 2.6-fold, inhibited ADP-induced platelet aggregation by 76% and 69%, and inhibited collagen-induced platelet aggregation by 65% and 67%, respectively. The pharmacokinetics of MK-852 include an elimination half-life of approximately 2 hours, total clearance of about 150 ml/min, and volume of distribution of about 18 liters. Examination of the relationship between MK-852 whole-blood concentration in vitro and inhibition of platelet aggregation showed an EC50 of about 55 ng/ml and a Hill coefficient of 1.55. The infusions were generally well tolerated, with no study drug-related changes in blood counts or biochemical profiles.

Objective To examine the mechanism of interleukin-1 (IL-1)–induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. Methods The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor κB (NF-κB) were used to assess the role of each pathway in IL-1–mediated induction of MMP-13. Results We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-κB translocation. These results suggest that both NF-κB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal–regulated kinase pathway) and does not require JNK or NF-κB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. Conclusion These studies demonstrate, for the first time, that p38, JNK, and NF-κB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type–specific.

A free lignocaine index was developed on the basis of measurements of plasma lignocaine and its principle binding protein, alpha 1-acid glycoprotein (AAG) in 80 samples from 16 patients admitted to the coronary care unit and given prophylactic lignocaine therapy. The free drug fraction, fu, of lignocaine was determined by equilibrium dialysis and its relationship to AAG and total lignocaine concentration (T) defined by multiple linear regression analysis as l/fu = 1.45 + 0.023 (AAG) -0.129 (T) (multiple r = 0.872, P less than 0.001). This relationship was used to calculate the 'free lignocaine index' as fu X T and compared with the observed value obtained by equilibrium dialysis of 178 samples from 41 separate subjects who received lignocaine after suspected myocardial infarction. There was a highly significant relationship (r = 0.933, n = 178, P less than 0.001) between the observed and predicted values. We conclude that the free drug index may be useful in rapidly assessing the unbound (free) concentration of lignocaine in plasma.