Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream ...effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.
Until recently, skeletal myoblasts (SkMBs) have been the most widely used cells in basic research and clinical trials of cell based therapy for cardiac repair and regeneration. Although SkMB ...engraftment into the post-infarcted heart has been consistently found to improve cardiac contractile function, the underlying therapeutic mechanisms remain still a matter of controversy and debate. This is basically because SkMBs do not attain a cardiac-like phenotype once homed into the diseased heart nor they form a contractile tissue functionally coupled with the surrounding viable myocardium. This issue of concern has generated the idea that the cardiotropic action of SkMBs may depend on the release of paracrine factors. However, the paracrine hypothesis still remains ill-defined, particularly concerning the identification of the whole spectrum of cell-derived soluble factors and details on their cardiac effects. In this context, the possibility to genetically engineering SkMBs to potentate their paracrine attitudes appears particularly attractive and is actually raising great expectation. Aim of the present review is not to cover all the aspects of cell-based therapy with SkMBs, as this has been the object of previous exhaustive reviews in this field. Rather, we focused on novel aspects underlying the interactions between SkMBs and the host cardiac tissues which may be relevant for directing the future basic and applied research on SkMB transplantation for post ischemic cardiac dysfunction.
The permeability transition pore (PTP) is a mitochondrial channel whose opening causes the mitochondrial membrane potential (ΔΨ) collapse that leads to apoptosis. Some ubiquinone analogues have been ...demonstrated previously to modulate the PTP open-closed transition in isolated mitochondria and thought to act through a common PTP-binding site rather than through oxidation-reduction reactions. We have demonstrated recently both in vitro and in vivo that the ubiquitous free radical scavenger and respiratory chain coenzyme Q10 (CoQ10) prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. On this basis, we hypothesized that the antiapoptotic property of CoQ10 could be independent of its free radical scavenging ability and related to direct inhibition of PTP opening. In this study, we have verified this hypothesis by evaluating the antiapoptotic effects of CoQ10 in response to apoptotic stimuli, serum starvation, antimycin A, and ceramide, which do not generate free radicals, in comparison to control, free radical-generating UVC irradiation. As hypothesized, CoQ10 dramatically reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli. This was accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and caspase 9 activation. Because these events are consequent to mitochondrial PTP opening, we suggest that the antiapoptotic activity of CoQ10 could be related to its ability to prevent this phenomenon.
Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms ...underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.
To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation.
Cultured rabbit keratocytes were ...irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single-pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 microM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay.
Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser-generated free radicals. ATP assay also confirmed that it raised cellular energy levels.
The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death.
The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I–R) during aortic surgery and the effect of vitamin E pretreatment were investigated.
Two ...groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes.
Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment.
Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I–R.
Bechelli C, Zecchi Orlandini S, Colafranceschi M. Scanning electron microscope study on the efficacy of root canal wall debridement of hand versus LightspeedTM instrumentation. International ...Endodontic Journal, 32, 484–493 1999.
Aim The aim of this in vitro study was to compare the efficacy of root canal wall debridement following hand versus LightSpeedTM instrumentation.
Methodology Twenty recently extracted single‐rooted teeth were paired and randomly placed into two treatment groups of 10 teeth each. In group 1, a step‐back instrumentation without initial coronal flaring with stainless steel Hedstroem files was used; group 2 was instrumented with Ni‐Ti LightSpeedTM instru‐ments. Both groups had the same irrigation regimen: 2.5% NaOCl and a 15% EDTA solution. The teeth were then decoronated and each root split longitudinally into two halves to be examined using the scanning electron microscope (SEM). The presence of superficial debris and smear layer was evaluated by a standardized grading system, and the resulting scores submitted to nonparametric statistics.
Results Under the conditions of this study, the removal of superficial debris was generally excellent with both canal preparation techniques. Both techniques resulted in variable presence of residual smear layer, with a canal wall covered by smear layer as the predominant characteristic. Generally, the amount of smear layer was greater in the apical than in the middle third of the root, however, this difference was statistically significant (P 0.005) only in hand‐instrumented teeth. The use of LightSpeedTM instruments was associated with significantly more (P 0.05) smear layer presence in the middle region of the root when compared with hand instrumentation. In addition, less smear layer was present in the apical region following LightSpeedTM instrumentation than stainless steel hand files, but this difference was not statistically significant. Differences in debridement between the two halves of the same root were more evident with LightSpeedTM than manual instrumentation, however, there was no statistical significance.
Conclusions It may be inferred that the choice between hand and LightSpeedTM instrumentation should be based on factors other than the amount of root canal debridement, which does not vary significantly according to the instruments used.
Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better ...characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.
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