Epigenetic-based differentiation therapy for Acute Myeloid Leukemia.

Despite the development of novel therapies for acute myeloid leukemia, outcomes remain poor for most patients, and therapeutic improvements are an urgent unmet need. Although treatment regimens promoting differentiation have succeeded in the treatment of acute promyelocytic leukemia, their role in other acute myeloid leukemia subtypes needs to be explored. Here we identify and characterize two lysine deacetylase inhibitors, CM-444 and CM-1758, exhibiting the capacity to promote myeloid differentiation in all acute myeloid leukemia subtypes at low non-cytotoxic doses, unlike other commercial histone deacetylase inhibitors. Analyzing the acetylome after CM-444 and CM-1758 treatment reveals modulation of non-histone proteins involved in the enhancer-promoter chromatin regulatory complex, including bromodomain proteins. This acetylation is essential for enhancing the expression of key transcription factors directly involved in the differentiation therapy induced by CM-444/CM-1758 in acute myeloid leukemia. In summary, these compounds may represent effective differentiation-based therapeutic agents across acute myeloid leukemia subtypes with a potential mechanism for the treatment of acute myeloid leukemia.

HDACi vorinostat protects muscle from degeneration after acute rotator cuff injury in mice.

Aims: Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. Methods: HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential. Results: Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy. Conclusion: The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury.

Loss of the matrix metalloproteinase-10 causes premature features of aging in satellite cells.

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

Uso del sildenafilo oral en el tratamiento del linfangioma orbitario / Use of Oral Sildenafil in the Treatment of Orbital Lymphangioma

Los linfangiomas orbitarios son malformaciones vasculares benignas, de crecimiento lento, abortivas y no funcionales, que se presentan principalmente en la primera década de la vida. Las opciones terapéuticas en estos casos presentan una resolutividad limitada, algunos tratamientos suelen ser agresivos y provocar daños del aparato visual. Se presenta un caso de una paciente femenina de 6 años de edad atendida por proptosis del ojo izquierdo a la que se le realizó el diagnóstico clínico-imagenológico de linfangioma de la órbita, con el objetivo de mostrar el resultado alcanzado en el manejo de la misma mediante el uso del sildenafilo por vía oral, modalidad terapéutica en estudio a nivel mundial en el tratamiento de estas afecciones. El tratamiento con sildenafilo en el linfangioma orbitario demostró ser eficaz en la mejoría del cuadro clínico y por imágenes. Durante el tratamiento no se reportaron reacciones adversas(AU)

Orbital lymphatic malformations are benign, slow-growing, abortive, nonfunctional vascular malformations that occur mainly in the first decade of life. Therapeutic options in these cases present limited resolution, some treatments are usually aggressive and cause damage to the visual apparatus. We present a case of a 6-year-old female patient treated for proptosis of the left eye. The clinical-imaging diagnosis of lymphangioma of the orbit was made to show the results achieved in its treatment through the use of oral sildenafil, a therapeutic modality under study worldwide in the treatment of these conditions. The treatment with sildenafil in orbital lymphangioma proved to be effective in the improvement of the clinical and imaging picture. No adverse reactions were reported during treatment(AU)

Deficiency of MMP-10 Aggravates the Diseased Phenotype of Aged Dystrophic Mice.

Matrix metalloproteinases (MMPs) have been implicated in the progression of muscular dystrophy, and recent studies have reported the role of MMP-10 in skeletal muscle pathology of young dystrophic mice. Nevertheless, its involvement in dystrophin-deficient hearts remains unexplored. Here, we aimed to investigate the involvement of MMP-10 in the progression of severe muscular dystrophy and to characterize MMP-10 loss in skeletal and cardiac muscles of aged dystrophic mice. We examined the histopathological effect of MMP-10 ablation in aged mdx mice, both in the hind limb muscles and heart tissues. We found that MMP-10 loss compromises survival rates of aged mdx mice, with skeletal and cardiac muscles developing a chronic inflammatory response. Our findings indicate that MMP-10 is implicated in severe muscular dystrophy progression, thus identifying a new area of research that could lead to future therapies for dystrophic muscles.

Características epidemiológicas, clínicas y oftalmológicas de pacientes con queratoconjuntivitis seca / Epidemiological, clinical and ophthalmologic characteristics of patients with keratoconjunctivitis Sicca

Introducción: La queratoconjuntivitis seca es una enfermedad de causa multifactorial, que afecta la producción de lágrimas y la superficie ocular, a la vez que ocasiona malestar, visión borrosa e inestabilidad de la película lagrimal. Métodos: Se realizó un estudio descriptivo y transversal de 50 pacientes con diagnóstico de queratoconjuntivitis seca, atendidos en el Centro Oftalmológico del Hospital General Docente Dr. Juan Bruno Zayas Alfonso de Santiago de Cuba, desde enero hasta julio de 2020. Objetivo: Caracterizar a los pacientes con queratoconjuntivitis seca según variables epidemiológicas clínicas y oftalmológicas. Resultados: En la serie predominaron las féminas (66,0 %), los pacientes mayores de 60 años (40,0 %), la lectura excesiva como factor de riesgo (42,0 %), la irritación, la fatiga ocular y la visión borrosa como criterios diagnósticos, además de la disminución del tiempo de rotura de la película lagrimal en 68,0 % de la muestra, entre otros resultados. Conclusión: La queratoconjuntivitis seca, desde el punto de vista clínico y epidemiológico fue similar a lo descrito en la bibliografía especializada en cuanto a los grupos de edades, sexo y principales síntomas descritos.

Introduction: The keratoconjunctivitis sicca is a disease of multifactorial cause that affects the production of tears and the ocular surface, at the same time that causes discomfort, blurred vision and instability of the lacrimal thin layer. Methods: A descriptive and cross-sectional study of 50 patients with diagnosis of keratoconjunctivitis sicca, assisted in the Ophthalmolgic Center of Dr. Juan Bruno Zayas Alfonso Teaching General Hospital in Santiago de Cuba, was carried out from January to July, 2020. Objective: To characterize patients with keratoconjunctivitis sicca according to clinical, epidemiologic and ophthalmologic variables. Results: In the series there was a prevalence of the females (66.0 %), patients over 60 years (40.0 %), excessive reading as risk factor (42.0 %), irritation, ocular fatigue and blurred vision as diagnostic criteria, besides the decrease of the lacrimal thin layer break up time in 68.0 % of the sample, among other results. Conclusion: The keratoconjunctivitis sicca was similar to that described in the specialized literature review as for the age groups, sex and main described symptoms from the clinical and epidemiologic points of view.

Local Preirradiation of Infarcted Cardiac Tissue Substantially Enhances Cell Engraftment.

The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen+ iPSCs in the infarcted heart, due to their ease of use and detection. We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.

Muscular and Tendon Degeneration after Achilles Rupture: New Insights into Future Repair Strategies.

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians' knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved.

Blefaroplastia: resultados del empleo de dos técnicas en el Centro Oftalmológico de Santiago de Cuba / Blepharoplasty: results of using two techniques in the Ophthalmologic Center from Santiago de Cuba

Introducción: La blefaroplastia produce resultados clínicos y estéticos favorables en los pacientes aquejados por dermatocalasis. Objetivo: Evaluar los resultados de los procedimientos quirúrgicos convencional y con láser de CO2 en estos afectados. Métodos: Se realizó un estudio descriptivo, prospectivo y trasversal de 300 pacientes con dermatocalasis, atendidos en el Servicio de Oculoplastia del Centro Oftalmológico del Hospital General Docente Dr Juan Bruno Zayas Alfonso de Santiago de Cuba, desde enero de 2015 hasta diciembre de 2016. Resultados: Predominaron el grupo etario de 40 a 59 años, el sexo femenino y la afectación en los párpados superiores, sin diferencias significativas en cuanto a las técnicas quirúrgicas empleadas; se observó un mayor número de complicaciones en los pacientes operados de manera convencional, con más frecuencia del sangrado. En general los pacientes evolucionaron satisfactoriamente y los que presentaron limitaciones del campo visual superior se recuperaron sin dificultad luego de la intervención quirúrgica. Conclusiones: La blefaroplastia con láser de CO2 constituye una mejor y más calificada opción terapéutica para la corrección de la dermatocalasis(AU)

Introduction: Blepharoplasty produces favorable clinical and cosmetic results in the patients that are suffering from dermatochalasis. Objective: To evaluate the results of the conventional and CO2 laser surgical procedures in these affected patients.Methods: A descriptive, prospective and cross-sectional study of 300 patients with dermatochalasis, assisted in the Oculoplasty Service of Dr Juan Bruno Zayas Alfonso Teaching General Hospital Ophthalmologic Center was carried out in Santiago de Cuba, from January, 2015 to December, 2016. Results: There was a prevalence of the 40 to 59 age group, female sex and upper lids disorder, without significant differences concerning the surgical techniques that were used; a higher number of complications in patients operated with a conventional way was observed, with more frequency of bleeding. In general the patients had a favorable clinical course and those that presented limitations of the upper visual field recovered without difficulty after the surgical intervention. Conclusions: Blepharoplasty with CO2 laser constitutes a better and more qualified therapeutic option for the correction of dermatochalasis(AU)

Blefaroplastia: resultados del empleo de dos técnicas en el Centro Oftalmológico de Santiago de Cuba / Blepharoplasty: results of using two techniques in the Ophthalmologic Center from Santiago de Cuba

Introducción: La blefaroplastia produce resultados clínicos y estéticos favorables en los pacientes aquejados por dermatocalasis. Objetivo: Evaluar los resultados de los procedimientos quirúrgicos convencional y con láser de CO2 en estos afectados. Métodos: Se realizó un estudio descriptivo, prospectivo y trasversal de 300 pacientes con dermatocalasis, atendidos en el Servicio de Oculoplastia del Centro Oftalmológico del Hospital General Docente Dr Juan Bruno Zayas Alfonso de Santiago de Cuba, desde enero de 2015 hasta diciembre de 2016. Resultados: Predominaron el grupo etario de 40 a 59 años, el sexo femenino y la afectación en los párpados superiores, sin diferencias significativas en cuanto a las técnicas quirúrgicas empleadas; se observó un mayor número de complicaciones en los pacientes operados de manera convencional, con más frecuencia del sangrado. En general los pacientes evolucionaron satisfactoriamente y los que presentaron limitaciones del campo visual superior se recuperaron sin dificultad luego de la intervención quirúrgica. Conclusiones: La blefaroplastia con láser de CO2 constituye una mejor y más calificada opción terapéutica para la corrección de la dermatocalasis

Introduction: Blepharoplasty produces favorable clinical and cosmetic results in the patients that are suffering from dermatochalasis. Objective: To evaluate the results of the conventional and CO2 laser surgical procedures in these affected patients. Methods: A descriptive, prospective and cross-sectional study of 300 patients with dermatochalasis, assisted in the Oculoplasty Service of Dr Juan Bruno Zayas Alfonso Teaching General Hospital Ophthalmologic Center was carried out in Santiago de Cuba, from January, 2015 to December, 2016. Results: There was a prevalence of the 40 to 59 age group, female sex and upper lids disorder, without significant differences concerning the surgical techniques that were used; a higher number of complications in patients operated with a conventional way was observed, with more frequency of bleeding. In general the patients had a favorable clinical course and those that presented limitations of the upper visual field recovered without difficulty after the surgical intervention. Conclusions: Blepharoplasty with CO2 laser constitutes a better and more qualified therapeutic option for the correction of dermatochalasis

Cuerpo extraño intraorbitario de gran tamaño en un niño / Intraorbitary foreign body of considerable size in a child

Se describe el caso clínico de un niño de 11 años de edad, procedente de un distrito de Timor Oriental, que recibió un trauma en el ojo derecho al caer de un árbol y enclavársele un fragmento de una rama en la órbita de ese ojo, por lo cual fue trasladado de urgencia al Hospital Nacional Guido Valadares de Dili, donde fue ingresado y se decidió operar inmediatamente para extraer el cuerpo extraño intraorbitario -- cuya longitud era de 10,5 cm --. El paciente evolucionó con buen estado general y una agudeza visual de 6/18 en el ojo dañado, de modo que al quinto día se le dio el alta hospitalaria.

The case report of a 11 years child is described, coming from a Timor Oriental district who suffered from a trauma in the right eye when falling from a tree and a fragment of branch was introduced in the orbit of that eye, reason why he was transferred as an emergency to Guido Valadares of Dili National Hospital, where he was admitted and it was decided to operate immediately to extract the strange intraorbitary body--which measured 10,5 cm--. The patient had a good general clinical course and a visual acuity of 6/18 in the damaged eye, so by the fifth day he was discharged from the hospital(AU)

Infiltration of plasma rich in growth factors enhances in vivo angiogenesis and improves reperfusion and tissue remodeling after severe hind limb ischemia.

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia.

Functional MMP-10 is required for efficient tissue repair after experimental hind limb ischemia.

We studied the role of matrix metalloproteinase-10 (MMP-10) during skeletal muscle repair after ischemia using a model of femoral artery excision in wild-type (WT) and MMP-10 deficient (Mmp10(-/-)) mice. Functional changes were analyzed by small animal positron emission tomography and tissue morphology by immunohistochemistry. Gene expression and protein analysis were used to study the molecular mechanisms governed by MMP-10 in hypoxia. Early after ischemia, MMP-10 deficiency resulted in delayed tissue reperfusion (10%, P < 0.01) and in increased necrosis (2-fold, P < 0.01), neutrophil (4-fold, P < 0.01), and macrophage (1.5-fold, P < 0.01) infiltration. These differences at early time points resulted in delayed myotube regeneration in Mmp10(-/-) soleus at later stages (regenerating myofibers: 30 ± 9% WT vs. 68 ± 10% Mmp10(-/-), P < 0.01). The injection of MMP-10 into Mmp10(-/-) mice rescued the observed phenotype. A molecular analysis revealed higher levels of Cxcl1 mRNA (10-fold, P < 0.05) and protein (30%) in the ischemic Mmp10(-/-) muscle resulting from a lack of transcriptional inhibition by MMP-10. This was further confirmed using siRNA against MMP-10 in vivo. Our results demonstrate an important role of MMP-10 for proper muscle repair after ischemia, and suggest that chemokine regulation such as Cxcl1 by MMP-10 is involved in muscle regeneration.

The CXCR4/SDF1 axis improves muscle regeneration through MMP-10 activity.

The CXCR4/SDF1 axis participates in various cellular processes, including cell migration, which is essential for skeletal muscle repair. Although increasing evidence has confirmed the role of CXCR4/SDF1 in embryonic muscle development, the function of this pathway during adult myogenesis remains to be fully elucidated. In addition, a role for CXCR4 signaling in muscle maintenance and repair has only recently emerged. Here, we have demonstrated that CXCR4 and stromal cell-derived factor-1 (SDF1) are up-regulated in injured muscle, suggesting their involvement in the repair process. In addition, we found that notexin-damaged muscles showed delayed muscle regeneration on treatment with CXCR4 agonist (AMD3100). Accordingly, small-interfering RNA-mediated silencing of SDF1 or CXCR4 in injured muscles impaired muscle regeneration, whereas the addition of SDF1 ligand accelerated repair. Furthermore, we identified that CXCR4/SDF1-regulated muscle repair was dependent on matrix metalloproteinase-10 (MMP-10) activity. Thus, our findings support a model in which MMP-10 activity modulates CXCR4/SDF1 signaling, which is essential for efficient skeletal muscle regeneration.

MMP-10 is required for efficient muscle regeneration in mouse models of injury and muscular dystrophy.

Matrix metalloproteinases (MMPs), a family of endopeptidases that are involved in the degradation of extracellular matrix components, have been implicated in skeletal muscle regeneration. Among the MMPs, MMP-2 and MMP-9 are upregulated in Duchenne muscular dystrophy (DMD), a fatal X-linked muscle disorder. However, inhibition or overexpression of specific MMPs in a mouse model of DMD (mdx) has yielded mixed results regarding disease progression, depending on the MMP studied. Here, we have examined the role of MMP-10 in muscle regeneration during injury and muscular dystrophy. We found that skeletal muscle increases MMP-10 protein expression in response to damage (notexin) or disease (mdx mice), suggesting its role in muscle regeneration. In addition, we found that MMP-10-deficient muscles displayed impaired recruitment of endothelial cells, reduced levels of extracellular matrix proteins, diminished collagen deposition, and decreased fiber size, which collectively contributed to delayed muscle regeneration after injury. Also, MMP-10 knockout in mdx mice led to a deteriorated dystrophic phenotype. Moreover, MMP-10 mRNA silencing in injured muscles (wild-type and mdx) reduced muscle regeneration, while addition of recombinant human MMP-10 accelerated muscle repair, suggesting that MMP-10 is required for efficient muscle regeneration. Furthermore, our data suggest that MMP-10-mediated muscle repair is associated with VEGF/Akt signaling. Thus, our findings indicate that MMP-10 is critical for skeletal muscle maintenance and regeneration during injury and disease.

Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

beta-Catenin promotes self-renewal of skeletal-muscle satellite cells.

Satellite cells are the resident stem cells of adult skeletal muscle. As with all stem cells, how the choice between self-renewal or differentiation is controlled is central to understanding their function. Here, we have explored the role of beta-catenin in determining the fate of myogenic satellite cells. Satellite cells express beta-catenin, and expression is maintained as they activate and undergo proliferation. Constitutive retroviral-driven expression of wild-type or stabilised beta-catenin results in more satellite cells expressing Pax7 without any MyoD -- therefore, adopting the self-renewal pathway, with fewer cells undergoing myogenic differentiation. Similarly, preventing the degradation of endogenous beta-catenin by inhibiting GSK3beta activity also results in more Pax7-positive-MyoD-negative (Pax7(+)MyoD(-)) satellite-cell progeny. Consistent with these observations, downregulation of beta-catenin using small interfering RNA (siRNA) reduced the proportion of satellite cells that express Pax7 and augmented myogenic differentiation after mitogen withdrawal. Since a dominant-negative version of beta-catenin had the same effect as silencing beta-catenin using specific siRNA, beta-catenin promotes self-renewal via transcriptional control of target genes. Thus, beta-catenin signalling in proliferating satellite cells directs these cells towards the self-renewal pathway and, so, contributes to the maintenance of this stem-cell pool in adult skeletal muscle.

Control of Myf5 activation in adult skeletal myonuclei requires ERK signalling.

Myf5 plays a central role in determination of the myogenic lineage, yet the signalling pathways that control its activation remain unclear. In adult muscle, Myf5 is expressed in satellite cells and muscle spindles but not by myonuclei. However, Myf5 expression is activated in myonuclei in response to muscle denervation. This can be modelled in culture using Myf5nlacZ/+ mice, allowing signalling pathways controlling Myf5 to be readily examined. We found that mitogen-rich medium induces activation of the Myf5 locus through calcium, which interacts with calmodulin to promote calcineurin and calmodulin kinase. Calcineurin activates NFAT to control Myf5 activation, while p38/JNK activity prevents activation by this route. Calmodulin kinase however, acts predominately through ERK signalling to activate Myf5. Interestingly, we found that IGF-1 can substitute for mitogen-rich medium and activates Myf5 through calcium, PI3K and ERK pathways. Together these observations show that Myf5 activation in adult muscle is accomplished by a complex signalling pathway, and provides candidates that can be examined for their role in Myf5 regulation during development.

Effects of a low molecular weight heparin, bemiparin, and unfractionated heparin on hemostatic properties of endothelium.

Human endothelial cells synthesize and secrete a variety of molecules involved in fibrinolysis and coagulation. The effects of a low molecular weight heparin, Bemiparin, and unfractionated heparin (UFH) were compared on plasminogen activator inhibitor-1 (PAI-1), tissue-plasminogen activator (t-PA), tissue factor (TF), tissue factor pathway inhibitor (TFPI) release, and PAI-1 gene expression by human umbilical vein endothelial cells (HUVEC). Cell cultures were supplemented with Bemiparin or UFH at 1 or 10 U/mL. Culture media samples were obtained before the addition of the drugs and 2, 6, and 24 hours afterward to measure the antigen levels of TF, TFPI, t-PA, and PAI-1. RNA was obtained to study the endothelial expression of PAI-1 by reverse transcriptase-polymerase chain reaction (RT-PCR). Bemiparin at 1 U/mL resulted in a decreased messenger RNA (mRNA) PAI-1 expression, which remained unaltered when UFH had been added. PAI-1 levels increased after the cultures had been supplemented with either Bemiparin or UFH at both doses. UFH induced an increase in t-PA either at 1 or 10 U/mL. Both doses of UFH, but not Bemiparin, induced an important increase in TF secretion. An increase in the TFPI levels was seen with UFH at 1 U/mL. The decrease in PAI-1 gene expression observed with a therapeutic dose of Bemiparin might confer this drug interesting profibrinolytic properties. The fact that Bemiparin, in contrast with UFH, does not induce an increase in TF could give this drug another positive feature.

Regulation by nitric oxide of endotoxin-induced tissue factor and plasminogen activator inhibitor-1 in endothelial cells.

The increase in nitric oxide (NO) production in lipopolysaccharide (LPS)-induced sepsis is thought to contribute to the development of shock. However, NO could also play an antithrombotic role. Little is known about the modulating effect of NO on the endothelial overexpression and production of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) occurring in endotoxemia. We analyzed the effect of N(G)-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases, and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, on the expression and synthesis of TF and PAI-1 by LPS-challenged human umbilical vein endothelial cells (HUVEC): L-NAME enhanced the increase in TF mRNA and antigen levels (P <0.05) observed in LPS-treated HUVEC; SNAP down-regulated the LPS-induced TF increment (p <0.05). However, no effects of NO on regulation of the LPS-dependent increase in PAI-1 could be seen. Thus, NO could play an antithrombotic role in sepsis by down-regulating the endothelial overexpression and production of TF.