Pericellular fibronectin is required for RhoA-dependent responses to cyclic strain in fibroblasts

To test the hypothesis that the pericellular fibronectin matrix is involved in mechanotransduction, we compared the response of normal and fibronectin-deficient mouse fibroblasts to cyclic substrate strain. Normal fibroblasts seeded on vitronectin in fibronectin-depleted medium deposited their own fibronectin matrix. In cultures exposed to cyclic strain, RhoA was activated, actin-stress fibers became more prominent, MAL/MKL1 shuttled to the nucleus, and mRNA encoding tenascin-C was induced. By contrast, these RhoA-dependent responses to cyclic strain were suppressed in fibronectin knockdown or knockout fibroblasts grown under identical conditions. On vitronectin substrate, fibronectin-deficient cells lacked fibrillar adhesions containing alpha5 integrin. However, when fibronectin-deficient fibroblasts were plated on exogenous fibronectin, their defects in adhesions and mechanotransduction were restored. Studies with fragments indicated that both the RGD-synergy site and the adjacent heparin-binding region of fibronectin were required for full activity in mechanotransduction, but not its ability to self-assemble. In contrast to RhoA-mediated responses, activation of Erk1/2 and PKB/Akt by cyclic strain was not affected in fibronectin-deficient cells. Our results indicate that pericellular fibronectin secreted by normal fibroblasts is a necessary component of the strain-sensing machinery. Supporting this hypothesis, induction of cellular tenascin-C by cyclic strain was suppressed by addition of exogenous tenascin-C, which interferes with fibronectin-mediated cell spreading.

Observation of ventilation-induced Spo(2) oscillations in pigs: first step to noninvasive detection of cyclic recruitment of atelectasis?

High arterial partial oxygen pressure (Pao(2)) oscillations within the respiratory cycle were described recently in experimental acute lung injury. This phenomenon has been related to cyclic recruitment of atelectasis and varying pulmonary shunt fractions. Noninvasive detection of Spo(2) (oxygen saturation measured by pulse oximetry) as an indicator of cyclic collapse of atelectasis, instead of recording Pao(2) oscillations, could be of clinical interest in critical care. Spo(2) oscillations were recorded continuously in three different cases of lung damage to demonstrate the technical feasibility of this approach. To deduce Pao(2) from Spo(2), a mathematical model of the hemoglobin dissociation curve including left and right shifts was derived from the literature and adapted to the dynamic changes of oxygenation. Calculated Pao(2) amplitudes (derived from Spo(2) measurements) were compared to simultaneously measured fast changes of Pao(2), using a current standard method (fluorescence quenching of ruthenium). Peripheral hemoglobin saturation was capable to capture changes of Spo(2) within each respiratory cycle. For the first time, Spo(2) oscillations due to cyclic recruitment of atelectasis within a respiratory cycle were determined by photoplethysmography, a technology that can be readily applied noninvasively in clinical routine. A mathematic model to calculate the respective Pao(2) changes was developed and its applicability tested.

Preclinical Evaluation of Radiolabeled DOTA-Derivatized Cyclic Minigastrin Analogs for Targeting Cholecystokinin Receptor Expressing Malignancies

Targeting of cholecystokinin receptor expressing malignancies such as medullary thyroid carcinoma is currently limited by low in vivo stability of radioligands. To increase the stability, we have developed and preclinically evaluated two cyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-minigastrin analogs radiolabeled with (111)In and (68)Ga.

Cyclic alternating pattern in narcolepsy patients and healthy controls after partial and total sleep deprivation

To investigate the regulation NREM sleep at baseline and in morning recovery sleep after partial and total sleep deprivation (SD) in narcolepsy-cataplexy (NC) using cyclic alternating pattern (CAP).

Stochastic amplitude-modulated stretching of Rabbit flexor digitorum profundus tendons reduces stiffness compared to Cyclic Loading but does not affect Tenocyte Metabolism

Background It has been demonstrated that frequency modulation of loading influences cellular response and metabolism in 3D tissues such as cartilage, bone and intervertebral disc. However, the mechano-sensitivity of cells in linear tissues such as tendons or ligaments might be more sensitive to changes in strain amplitude than frequency. Here, we hypothesized that tenocytes in situ are mechano-responsive to random amplitude modulation of strain. Methods We compared stochastic amplitude-modulated versus sinusoidal cyclic stretching. Rabbit tendon were kept in tissue-culture medium for twelve days and were loaded for 1h/day for six of the total twelve culture days. The tendons were randomly subjected to one of three different loading regimes: i) stochastic (2 – 7% random strain amplitudes), ii) cyclic_RMS (2–4.42% strain) and iii) cyclic_high (2 - 7% strain), all at 1 Hz and for 3,600 cycles, and one unloaded control. Results At the end of the culture period, the stiffness of the “stochastic” group was significantly lower than that of the cyclic_RMS and cyclic_high groups (both, p < 0.0001). Gene expression of eleven anabolic, catabolic and inflammatory genes revealed no significant differences between the loading groups. Conclusions We conclude that, despite an equivalent metabolic response, stochastically stretched tendons suffer most likely from increased mechanical microdamage, relative to cyclically loaded ones, which is relevant for tendon regeneration therapies in clinical practice.

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.