Cardiovascular diseases are often considered to be a predominantly male health problem, and it has been suggested that testosterone exerts deleterious effects on cardiovascular function; however, few experimental studies support this suggestion. Moreover, the cellular and molecular mechanism(s) underlying vascular responses to testosterone is unknown. The present study has investigated the acute effects of testosterone on porcine coronary artery smooth muscle at the tissue and cellular levels. Contractile studies demonstrated that testosterone or dihydrotestosterone (a nonaromatizable metabolite) relaxed these arteries by an endothelium-independent mechanism involving potassium efflux. Direct evidence from patch-clamp studies confirmed that testosterone opened K(+) channels in single coronary myocytes, and further analysis identified this protein as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel. Moreover, inhibiting BK(Ca) channel activity significantly attenuated testosterone-induced coronary relaxation. These findings indicate that testosterone relaxes porcine coronary arteries predominantly by opening BK(Ca) channels in coronary myocytes, and this response may be associated with accumulation of cGMP. This novel mechanism may provide a better understanding of testosterone-induced vasorelaxation reported in recent experimental and early clinical studies.
Transforming growth factor-beta (TGF-β) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-β is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-β signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-β-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-β-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a ‘therapeutic’ regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.
We report an experimental approach to study the mechanosensitivity of cell-cell contact upon mechanical stimulation in suspended cell-doublets. The doublet is placed astride an hourglass aperture, and a hydrodynamic force is selectively exerted on only one of the cells. The geometry of the device concentrates the mechanical shear over the junction area. Together with mechanical shear, the system also allows confocal quantitative live imaging of the recruitment of junction proteins (e.g., E-cadherin, ZO-1, occludin, and actin). We observed the time sequence over which proteins were recruited to the stretched region of the contact. The compressed side of the contact showed no response. We demonstrated how this mechanism polarizes the stress-induced recruitment of junctional components within one single junction. Finally, we demonstrated that stabilizing the actin cortex dynamics abolishes the mechanosensitive response of the junction. Our experimental design provides an original approach to study the role of mechanical force at a cell-cell contact with unprecedented control over stress application and quantitative optical analysis.
The cytoskeletal mechanics and cell mechanical properties play an important role in cellular behaviors. In this study, in order to provide comprehensive insights into the relationship between different cytoskeletal components and cellular elastic moduli, we built a phasemodulated surface acoustic wave microfluidic device to measure cellular compressibility and a microfluidic micropipette-aspiration device to measure cellular Young's modulus. The microfluidic devices were validated based on experimental data and computational simulations. The contributions of structural cytoskeletal actin filament and microtubule to cellular compressibility and Young's modulus were examined in MCF-7 cells. The compressibility of MCF-7 cells was increased after microtubule disruption, whereas actin disruption had no effect. In contrast, Young's modulus of MCF-7 cells was reduced after actin disruption but unaffected by microtubule disruption. The actin filaments and microtubules were stained to confirm the structural alteration in cytoskeleton. Our findings suggest the dissimilarity in the structural roles of actin filaments and microtubules in terms of cellular compressibility and Young's modulus. Based on the differences in location and structure, actin filaments mainly contribute to tensile Young's modulus and microtubules mainly contribute to compressibility. In addition, different responses to cytoskeletal alterations between acoustophoresis and micropipette aspiration demonstrated that micropipette aspiration was better at detecting the change from actin cortex, while the response to acoustophoresis was governed by microtubule networks.
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