Search

Myofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca2+](i), contribute to myofibroblast contraction, we analysed both [Ca2+](i) and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (similar to 100 seconds) [Ca2+](i) oscillations that control small (similar to 400 nm) and weak (similar to 100 pN) contractions. Whereas depletion of [Ca2+](i) reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho-and ROCK-mediated Ca2+-independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho- and ROCK-dependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and high-frequency contractions mediated by [Ca2+](i). The joint action of both contraction modes can result in macroscopic tissue contractures of similar to 1 cm per month.

Lipocalin-2 (also known as NGAL) levels are elevated in obesity and diabetes yet relatively little is known regarding effects on the heart. We induced pressure overload (PO) in mice and found that lipocalin-2 knockout (LKO) mice exhibited less PO-induced autophagy and NLRP3 inflammasome activation than Wt. PO-induced mitochondrial damage was reduced and autophagic flux greater in LKO mice, which correlated with less cardiac dysfunction. All of these observations were negated upon adenoviral-mediated restoration of normal lipocalin-2 levels in LKO. Studies in primary cardiac fibroblasts indicated that lipocalin-2 enhanced priming and activation of NLRP3-inflammasome, detected by increased IL-1β, IL-18 and Caspase-1 activation. This was attenuated in cells isolated from NLRP3-deficient mice or upon pharmacological inhibition of NLRP3. Furthermore, lipocalin-2 induced release of HMGB1 from cells and NLRP3-inflammasome activation was attenuated by TLR4 inhibition. We also found evidence of increased inflammasome activation and reduced autophagy in cardiac biopsy samples from heart failure patients. Overall, this study provides new mechanistic insight on the detrimental role of lipocalin-2 in the development of cardiac dysfunction.

A novel imaging method employing the different kinetics of the energetic states of fluorescent markers is presented, where the contrast depends on the triplet kinetics. An attractive feature of triplet states is their long lifetime, rendering them highly sensitive to the immediate environment of their fluorescent host molecules. They typically have a lifetime of the order of ~10us to 10ms, which is 3-6 orders of magnitude longer than the lifetimes of fluorescent singlet states. The concept exploits characteristic variations of population of these states for different modulated excitations. It is fully compatible with low time- resolution detection by a CCD camera and does not impose any constraints on the concentration or the fluorescence brightness of the sample molecules to be measured. As first proof of principle, we demonstrate how triplet parameters of various fluorophores in different environments can be exploited for imaging. As control, the triplet parameters have been measured by FCS experiments. This concept opens for imaging of fluorophore markers in biological samples, as a novel contrast method for visualizing subtle changes in the immediate microenvironment of the fluorophore markers.

Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment (∼2-fold), spreading (∼2.5-fold) and proliferation (∼1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 ± 5%) stretched cells on our PDMS surface coating was ∼7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells. © 2008 Elsevier Ltd. All rights reserved.

Myofibroblasts are reparative connective tissue cells that contribute to the reconstruction of injured tissue by secreting new extracellular matrix and by exerting high contractile force. Deregulation of these activities results in tissue contracture and development of fibrosis which makes the myofibroblast an important target for anti-fibrotic therapies. Two principle factors drive the development of myofibroblasts from different precursor cells and guarantee maintenance of the contractile phenotype: mechanical stress and transforming growth factor beta (TGFβ1). In this mini-review, we recapitulate the current understanding (1) of how myofibroblasts feel stress using specialized matrix adhesions, (2) of the level of stress that is required to induce their development and (3) of how myofibroblast mechanical activity can have a direct influence on the level of TGFβ1 activation. From these findings it emerges that the specific matrix adhesion structures of myofibroblasts are promising targets to modulate myofibroblast differentiation and activity. © 2008 Elsevier Ltd. All rights reserved.