Satellite cells produce neural chemorepellent semaphorin 3A upon muscle injury

Regenerative mechanisms that regulate intramuscular motor innervation. including configuration of the neuromuscular connections are thought to reside in the spatiotemporal expression of axon‐guidance molecules. Our previous studies proposed a heretofore unexplored role of satellite cells as a key source of a secreted neural chemorepellent semaphorin 3A (Sema3A) expression. In order to verify this concept, there is still a critical need to provide direct evidence to show the up‐regulation of Sema3A protein in satellite cells in vivo upon muscle injury. The present study employed a Sema3A/MyoD double‐immunohistochemical staining for cryo‐sections prepared from cardiotoxin injected gastrocnemius muscle of adult mouse lower hind‐limb. Results clearly demonstrated that Sema3A expression was up‐regulated in myogenic differentiation‐positive satellite cells at 4–12 days post‐injury period, the time that corresponds to the cell differentiation phase characterized by increasing myogenin messenger RNA expression. This direct proof encourages a possible implication of satellite cells in the spatiotemporal regulation of extracellular Sema3A concentrations, which potentially ensures coordinating a delay in neurite sprouting and re‐attachment of motoneuron terminals onto damaged muscle fibers early in muscle regeneration in synchrony with recovery of muscle‐fiber integrity.     

Time‐coordinated prevalence of extracellular HGF,FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon‐guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early‐differentiated satellite cells up‐regulate a secreted neural chemorepellent Sema3A upon in vivo muscle‐crush injury. The study also revealed that Sema3A expression is up‐regulated in primary satellite‐cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)‐β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time‐course of extracellular HGF, FGF2 and TGF‐β3 concentrations after crush‐injury of Gastrocnemius muscle in the rat lower hind‐limb, using a combination of a non‐homogenization/non‐spin extraction of extracellular wound fluids and enhanced chemiluminescence–Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2–8 days post‐crush, whereas active TGF‐β3 increases after 12 days, providing a better understanding of the time‐coordinated levels of HGF, FGF2 and TGF‐β3 that drive regulation of Sema3A expression during regenerative intramuscular moto‐neuritogenesis.     

Preliminary time‐course study of antiinflammatory macrophage infiltration in crush‐injured skeletal muscle

Muscle damage induces massive macrophage infiltration of the injury site, in which activated pro‐inflammatory and anti‐inflammatory phenotypes (currently classified as M1 and M2, respectively) have been documented as distinct functional populations predominant at different times after the conventional acute injury by intramuscular injection of snake venoms (cardiotoxin, notexin) or chemicals (bupivacaine hydrochloride, barium chloride). The present study employed a muscle‐crush injury model that may better reflect the physiologic damage and repair processes initiated by contusing a gastrocnemius muscle in the lower hind‐limb of adult mice with hemostat forceps, and examined the time‐course invasion of M1 and M2 macrophages during muscle regeneration by immunocytochemistry of CD197 and CD206 marker proteins. CD197‐positive M1 macrophages were observed exclusively at 1–4 days after crush followed by the alternative prevalence of CD206‐positive M2 at 7 days of myogenic differentiation, characterized by increasing levels of myogenin messenger RNA expression. Preliminary PCR analysis showed that M2 may produce hepatocyte growth factor (HGF) in culture, providing additional benefit to understanding that M2 populations actively promote regenerative myogenesis (muscle fiber repair) and moto‐neuritogenesis (re‐attachment of motoneuron terminals onto damaged fibers) through their time‐specific infiltration and release of growth factor at the injury site early in muscle regeneration.     

Adipocytes suppress differentiation of muscle cells in a co‐culture system

The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co‐culture system. The fusion index of C2C12 myoblasts co‐cultured with 3T3‐L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin‐1 and MuRF‐1 was higher in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes than the control. These results suggest that 3T3‐L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3‐L1 adipocytes induced the expression and secretion of IL‐6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co‐cultured with 3T3‐L1 cells in medium containing IL‐6‐neutralizing antibody than the control. Taken together, there is a possibility that adipocyte‐induced IL‐6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Leucine regulates slow‐twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow‐twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow‐twitch muscle fibers and mitochondrial function‐related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho‐AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow‐twitch muscle fibers and mitochondrial function‐related gene expression. Finally, we showed that Sirt1 was required for leucine‐induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow‐twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Histopathological findings,phenotyping of inflammatory cells,and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

Background

The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.

Results

The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.

Conclusions

The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.     

Effects of transforming growth factor‐β1 treatment on muscle regeneration and adipogenesis in glycerol‐injured muscle

Transforming growth factor (TGF)‐β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF‐β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF‐β1 on muscle regeneration and adipogenesis in glycerol‐injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF‐β1 was either co‐injected with glycerol, as an ‘early treatment’ group, or injected at day 4 after glycerol, as a ‘late treatment’ group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol‐injured group (without TGF‐β1 treatment). Moreover, the Oil red O‐positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol‐injured group. Furthermore, TGF‐β1 treatment increased endomysial fibrosis and induced immunostaining of α‐smooth muscle actin. The greater inhibitory effects of early TGF‐β1 treatment than that of late TGF‐β1 treatment during regeneration in glycerol‐injured muscle suggest a more potent effect of TGF‐β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF‐β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies.     

HGF/c-Met signaling regulates early differentiation of placental trophoblast cells

Depletion of hepatocyte growth factor (HGF) or mesenchymal-epithelial transition factor (c-Met) in mice leads to fetal lethality and placental maldevelopment. However, the dynamic change pattern of HGF/c-Met signaling during placental development and its involvement in the early differentiation of trophoblasts remain to be elucidated. In this study, using in situ hybridization assay, we elaborately demonstrated the spatial-temporal expression of Hgf and c-Met in mouse placenta from E5.5, the very early stage after embryonic implantation, to E12.5, when the placental structure is well developed. The concentration of the soluble form of c-Met (sMet) in maternal circulation peaked at E10.5. By utilizing the induced differentiation model of mouse trophoblast stem cells (mTSCs), we found that HGF significantly promoted mTSC differentiation into syncytiotrophoblasts (STBs) and invasive parietal trophoblast giant cells (PTGCs). Interestingly, sMet efficiently reversed the effect of HGF on mTSC differentiation. These findings indicate that HGF/c-Met signaling participates in regulating placental trophoblast cell fate at the early differentiation stage and that sMet acts as an endogenous antagonist in this aspect.     

Tissue-specific expression of the NOD-like receptor protein 3 in BALB/c mice

Activation of the innate immune system requires recognition of pathogen-associated molecular patterns, such as NOD-like receptors. The NOD-like receptor protein 3 (NLRP3) inflammasome is involved in induction of the pro-inflammatory cytokine, IL-1β, and subsequent inflammatory responses. NLRP3 inflammasome plays important roles in the inflammatory and innate immune responses associated with autoimmune/inflammatory syndrome. However, analysis of the tissue distribution and expression profiles in BALB/c mice is still incomplete. In this study, we investigated the tissue distribution and expression pattern of NLRP3 in BALB/c mice to further elucidate its function in innate immunity in this commonly used laboratory animal model. NLRP3 mRNA expression levels and tissue distribution of the protein were investigated by real-time quantitative PCR and immunohistochemical analyses, respectively. NLRP3 mRNA expression was higher in the kidney and inguinal lymph nodes than in other tissues. Cytoplasmic expression of NLRP3 was detected in the epithelial reticular cells of the spleen and thymus, lymphocytes in the inguinal lymph nodes, cardiac muscle cells, cerebral cortex neurons, alveolar macrophages, renal tubule cells and liver sinusoidal endothelial cells. The results of this study will assist investigators in interpreting site-specific functions and roles of NLRP3 in inflammatory responses.     

Mechano-biology of resident myogenic stem cells: Molecular mechanism of stretch-induced activation of satellite cells

Satellite cells, resident myogenic stem cells found between the basement membrane and the sarcolemma in postnatal skeletal muscle, are normally quiescent in adult muscles. But when muscle is injured, exercised, overused or mechanically stretched, these cells are activated to enter the cell cycle, divide, differentiate, and fuse with the adjacent muscle fiber. In this way, satellite cells are responsible for regeneration and work-induced hypertrophy of muscle fibers. Therefore, a mechanism must exist to translate mechanical changes in muscle tissue into chemical signals that can activate satellite cells. This mechanism has not been clearly delineated. Recent in vivo studies and studies of satellite cells and single muscle fibers in culture demonstrated the essential role of hepatocyte growth factor (HGF) and nitric oxide (NO) radical in the activation pathway. These experiments also showed that mechanically stretching cultured satellite cells or living skeletal muscles stimulates satellite cell activation. This is achieved by rapid release of HGF from its tethering in the extracellular matrix and its presentation to the c-met receptor. HGF release has been shown to depend on NO radical production by nitric oxide synthase (NOS) in satellite cells and/or muscle fibers, and relies on the subsequent upregulation of matrix metalloproteinase (MMP) activity (possibly achieved by its nitrosylation). These results suggest that the activation mechanism is a cascade of molecular events including calcium-calmodulin formation, NOS activation, NO radical production, MMP activation, HGF release and HGF binding to c-met. An understanding the 'mechano-biology' of satellite cell activation is essential when planning procedures that could enhance muscle growth and repair. This is particularly important for meat-animal agriculture and in human health, disease and aging.     

干扰MSTN对牛骨骼肌卫星细胞增殖分化的影响

为研究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌卫星细胞增殖与成肌分化的影响,本试验以牛骨骼肌卫星细胞体外诱导成肌分化模型为对象,以前期设计合成3个干扰RNA（si-MSTN-1、si-MSTN-2、si-MSTN-3）并对其进行干扰效果筛选为基础,将干扰效果极显著的si-MSTN-2（si-MSTN）转染牛骨骼肌卫星细胞,通过EdU染色法检测干扰MSTN对牛骨骼肌卫星细胞增殖的影响;进一步对干扰MSTN的牛骨骼肌卫星细胞进行体外成肌诱导分化,通过肌管形成状态和分化标志因子综合分析干扰MSTN对牛骨骼肌卫星细胞分化的影响：首先通过显微镜观察牛骨骼肌卫星细胞分化时期的肌管形成状态,然后利用实时荧光定量PCR和Western blotting技术检测牛骨骼肌卫星细胞分化标志因子MyoG和MyHC在mRNA和蛋白水平的表达情况。结果显示,干扰MSTN后,牛骨骼肌卫星细胞中EdU阳性细胞率极显著增加（P < 0.01）,说明下调MSTN表达极显著促进了牛骨骼肌卫星细胞的增殖;牛骨骼肌卫星细胞诱导分化后形成的肌管数量和直径均呈现增大趋势,牛骨骼肌卫星细胞成肌分化标志因子MyHC在mRNA和蛋白水平的表达均极显著高于对照组（P < 0.01）,说明下调MSTN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰MSTN可以显著促进牛骨骼肌卫星细胞的增殖及成肌分化过程。本试验结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究提供了参考。     

Mechano-biology of skeletal muscle hypertrophy and regeneration: Possible mechanism of stretch-induced activation of resident myogenic stem cells

In undamaged postnatal muscle fibers with normal contraction and relaxation activities, quiescent satellite cells of resident myogenic stem cells are interposed between the overlying external lamina and the sarcolemma of a subjacent mature muscle fiber. When muscle is injured, exercised, overused or mechanically stretched, these cells are activated to enter the cell proliferation cycle, divide, differentiate, and fuse with the adjacent muscle fiber, and are responsible for regeneration and work-induced hypertrophy of muscle fibers. Therefore, a mechanism must exist to translate mechanical changes in muscle tissue into chemical signals that can activate satellite cells. Recent studies of satellite cells or single muscle fibers in culture and in vivo demonstrated the essential role of hepatocyte growth factor (HGF) and nitric oxide (NO) radical in the activation pathway. These experiments have also reported that mechanically stretching satellite cells or living skeletal muscles triggers the activation by rapid release of HGF from its extracellular tethering and the subsequent presentation to the receptor c-met. HGF release has been shown to rely on calcium-calmodulin formation and NO radical production in satellite cells and/or muscle fibers in response to the mechanical perturbation, and depend on the subsequent up-regulation of matrix metalloproteinase (MMP) activity. These results indicate that the activation mechanism is a cascade of events including calcium ion influx, calcium-calmodulin formation, NO synthase activation, NO radical production, MMP activation, HGF release and binding to c-met. Better understanding of 'mechano-biology' on the satellite cell activation is essential for designing procedures that could enhance muscle growth and repair activities in meat-animal agriculture and also in neuromuscular disease and aging in humans.     

Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro

It has previously been shown that mechanical stretch induces activation of cultured quiescent satellite cells by rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c‐met receptor. The present study provides evidence that the stretch activation activity varies according to the origin of satellite cells from back and leg skeletal muscles in vitro. Satellite cells were isolated from three muscle groups, back (BK), upper hind limb (UL) and lower hind limb (LL) muscles, of adult male rats and stretch activation activities were compared. In response to stretch, lower hind limb satellite cells showed significantly greater response than upper hind limb and back muscles (LL > UL > BK). Immunoblots of stretched culture media revealed a higher HGF‐releasing capacity of lower hind limb satellite cells than back muscle satellite cells. In addition, lower hind limb satellite cells exhibited a greater activation activity in response to exogenous HGF added to culture media than compared to satellite cells from back and upper hind limb (LL > UL > BK). The increased ability to release HGF and the increased cellular responsiveness might account for higher stretch activation activities of lower hind limb satellite cells. Electrophoretic analysis of myosin heavy chain isoforms verified a higher content of slow muscle fibers in lower limb muscles (LL > UL > BK), suggesting a difference in stretch‐induced activation activity between satellite cells associated with fast and slow muscle fibers.     

干扰核心蛋白聚糖对牛骨骼肌卫星细胞增殖分化的影响

为探究肌肉生长抑制素(MSTN)对牛骨骼肌生长发育的作用机制,本研究以前期MSTN^+/-蒙古牛与野生蒙古牛腿臀肌肌肉组织定量蛋白质组学与磷酸化蛋白质组学筛选获得的表达差异倍数较大的核心蛋白聚糖(DCN)为靶标,以实验室前期分离培养的牛骨骼肌卫星细胞及建立的体外诱导成肌分化模型为对象,通过对设计合成的3个DCN siRNA干扰效果的筛选,将干扰效果最显著的si-DCN-2(si-DCN)转染牛骨骼肌卫星细胞。采用实时荧光定量PCR和Western blotting方法检测增殖期(GM)牛骨骼肌卫星细胞中增殖标志因子Pax7和MyoD的mRNA水平及蛋白水平的表达变化,以及使用EdU染色的方法检测干扰DCN对细胞增殖的影响。对转染DCN siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化,通过显微镜观察牛骨骼肌卫星细胞分化第3天(DM3)的肌管形成状态,同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MyHC的mRNA水平及蛋白水平的表达变化,并对DM3期肌管MyHC进行免疫荧光染色,以研究干扰DCN对细胞分化的影响。结果显示,干扰DCN表达后,增殖期牛骨骼肌卫星细胞中Pax7和MyoD的mRNA水平及蛋白水平都显著或极显著上调(P<0.05;P<0.01),且EdU阳性细胞率显著增加(P<0.05),表明干扰DCN表达显著促进了牛骨骼肌卫星细胞的增殖。干扰DCN表达后,牛骨骼肌卫星细胞分化第3天诱导形成的肌管直径呈现增大趋势,检测成肌分化标志因子MyoG在mRNA和蛋白水平的表达分别极显著和显著高于对照组(P<0.01;P<0.05),MyHC在mRNA水平显著降低(P<0.05),但在蛋白水平上极显著升高(P<0.01),免疫荧光结果显示,下调DCN后肌管融合指数显著高于对照组(P<0.05),说明干扰DCN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰DCN可以显著促进牛骨骼肌卫星细胞的增殖和成肌分化过程。研究结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究奠定了基础。     

Plasticity of endometrial epithelial and stromal cells—A new approach towards the pathogenesis of equine endometrosis

Equine endometrosis, a frequent cause of subfertility, is characterized by periglandular fibrosis, and no treatment exists. Endometrial biopsies not only contain diseased glands, but also contain healthy glands and stroma. Myoepithelial (ME) and myofibroblastic (MF) markers are calponin, smooth muscle actin (SMA), desmin and glial fibrillary acidic protein (GFAP). Epithelial vimentin expression indicates epithelial to mesenchymal transition (EMT). The aim of this immunohistochemical study was to investigate whether biopsies with endometrosis express MF and ME markers and vimentin. Compared to healthy areas, significantly higher percentages of endometrotic glands were lined by calponin‐ and vimentin‐positive epithelial cells, whereas periglandular fibrosis contained significantly higher percentages of stromal cells positive for vimentin, desmin and SMA and significantly less calponin‐positive stromal cells. The rare GFAP expression was restricted to endometrotic glands. Of these, the most frequent features of endometrotic glands were higher percentages of SMA‐ and vimentin‐positive stromal cells and the prominent epithelial calponin staining that occurred in 100%, 93% and 95% of examined biopsies. Results indicate plasticity of equine endometrial epithelial and stromal cells. Particularly, endometrotic glands show evidence for ME differentiation and EMT. The different expression of MF markers between stromal cells from healthy and endometrotic areas suggests functional differences. The characteristic changes in the expression of SMA, vimentin and calponin between endometrotic glands and healthy areas can be helpful to confirm early stages of endometrosis. The characterization of cellular differentiation may help to decipher the pathogenesis of endometrosis and could lead to therapeutic strategies.     

In vitro effects of lidocaine on the contractility of equine jejunal smooth muscle challenged by ischaemia‐reperfusion injury

Reasons for performing study: Post operative ileus (POI) in horses is a severe complication after colic surgery. A commonly used prokinetic drug is lidocaine, which has been shown to have stimulatory effects on intestinal motility. The cellular mechanisms through which lidocaine affects smooth muscle activity are not yet known. Objectives: To examine the effects of lidocaine on smooth muscle in vitro and identify mechanisms by which it may affect the contractility of intestinal smooth muscle. Hypothesis: Ischaemia and reperfusion associated with intestinal strangulation can cause smooth muscle injury. Consequently, muscle cell functionality and contractile performance is decreased. Lidocaine can improve basic cell functions and thereby muscle cell contractility especially in ischaemia‐reperfusion‐challenged smooth muscle. Methods: To examine the effects of lidocaine on smooth muscle function directly, isometric force performance was measured in vitro in noninjured and in vivo ischaemia‐reperfusion injured smooth muscle tissues. Dose‐dependent response of lidocaine was measured in both samples. To assess membrane permeability as a marker of basic cell function, release of creatine kinase (CK) was measured by in vitro incubations. Results: Lidocaine‐stimulated contractility of ischaemia‐reperfusion injured smooth muscle was more pronounced than that of noninjured smooth muscle. A 3‐phasic dose‐dependency was observed with an initial recovery of contractility especially in ischaemia‐reperfusion injured smooth muscle followed by a plateau phase where contractility was maintained over a broad concentration range. CK release was decreased by lidocaine. Conclusion: Lidocaine may improve smooth muscle contractility and basic cell function by cellular repair mechanisms which are still unknown. Improving contractility of smooth muscle after ischaemia‐reperfusion injury is essential in recovery of propulsive intestinal motility. Potential relevance: Characterisation of the cellular mechanisms of effects of lidocaine, especially on ischaemia‐reperfusion injured smooth muscle, may lead to improved treatment strategies for horses with POI.     

The Endometrium of Cycling Cows Contains Populations of Putative Mesenchymal Progenitor Cells

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1–5) and late luteal phases (days 13–18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD‐117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin‐embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c‐KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.     

Characterization, expression and function of c-Met in canine spontaneous cancers

Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. To better define the potential role of Met dysregulation in canine cancer, the canine Met, hepatocyte growth factor (HGF) and HGF activator were cloned. Inappropriate expression of Met was present in canine tumour cell lines derived from a wide variety of cancers. Furthermore, both HGF and HGF activator were also expressed in several of these cell lines, providing evidence of a possible autocrine loop of Met activation. Stimulation of tumour cell lines with recombinant human HGF induced Met autophosphorylation, as well as activation of the downstream signalling elements Gab‐1, Akt and Erk1/2. Scattering of tumour cells and migration across a defect occurred in response to HGF stimulation. The Met inhibitor PHA665752 blocked both HGF‐induced phosphorylation of canine Met and HGF‐mediated cell cycling, scattering and migration. These studies provide evidence that Met dysregulation may play a role in the biology of canine cancer and lay the groundwork for future studies employing Met inhibitors.