Calponin Expression in Renal Tubulointerstitial Fibrosis Induced in Rats by Cisplatin

Renal tubulointerstitial fibrosis is the common feature of chronic renal failure, regardless of its etiology. Myofibroblasts play important roles in progression of the fibrosis and are characterized by expressions of various cytoskeletons such as vimentin, desmin and α-smooth muscle actin (α-SMA). To pursue the characteristics of the cells, we immunohistochemically investigated the relationship between calponin (a marker of terminal smooth muscles) expression and myofibroblasts in cisplatin-induced rat renal tubulointerstitial fibrosis. Calponin-expressing interstitial cells increased with fibrosis and reacted simultaneously to vimentin or α-SMA (a marker of well-differentiated myofibroblasts) but not desmin or Thy-1 (a marker of myofibroblasts at the early stage). The present study shows that calponin may be expressed transiently in relatively well-developed myofibroblasts in rat renal fibrosis. Calponin could become a marker for myofibroblast development in chronic renal toxicity in rats.     

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.     

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.     

Intestinal smooth muscle cells locally enhance stem cell factor (SCF) production against gastrointestinal nematode infections

Smooth muscle cells can produce stem cell factor (SCF) in the normal state for the preservation of mast cells, but it is still unknown whether smooth muscle cells can enhance SCF production in response to the pathological stimuli. The present study showed that smooth muscle cells in mast cell-increased regions around worm cysts of intestinal nematodes significantly enhanced SCF gene expression compared with mast cell non-increased regions in same sample. SCF gene expression in mast cell non-increased regions in nematode-infected mice showed almost the same level as in non-infected control groups. These results indicate that smooth muscle cells can locally enhance SCF gene expression, and may have a role in local immunological reactions as growth factor-producing cells.     

Histological and Immunohistochemical Evaluation of Canine Ovary

In the present study, 15 canine ovaries without morphological lesions were examined histologically and immunohistochemically by using a large number of proteins including AE1/AE3, cytokeratin7 (CK7), CK13, CK20, vimentin, desmin, alpha smooth muscle actin (alphaSMA), calponin, S100, Neurofilaments, Inhibinalpha, placental alkaline phosphatase (PLAP) and neuron-specific enolase. Ovarian structures observed in this study included surface epithelium (SE), cortical tubules (CT), tunica albuginea (TA), stromal cells (SC), internal endocrine cells (IE), rete ovarii (RO) and fallopian tubes (FT). SE, CT, RO and FT were broadly immunoreactive for desmin. Besides AE1/AE3 and vimentin, desmin was also closely linked to these structures. Rete ovarii forming a reticular structure showed a positive reaction to S100. Surface epithelium was immunoreactive for PLAP at a significantly high level. In conclusion, these results indicate a specific segment of immunoreactivity as well as the broad range of immunoreactivity in canine ovary. The distinct patterns of immunoreactive for various kinds of proteins will play an important role in facilitating their identification and discrimination even in a normal canine ovary with a complex structure.     

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.     

Anti-emetic drug maropitant induces intestinal motility disorder but not anti-inflammatory action in mice

Maropitant is a neurokinin 1 receptor (NK1R) antagonist that is clinically used as a new anti-emetic drug for dogs. Substance P (SP) and its receptor NK1R are considered to modulate gastrointestinal peristalsis. In addition, SP works as an inflammatory mediator in gastrointestinal diseases. Aim of this study is to clarify the effects of maropitant on intestinal motility and inflammation in mice. Ex vivo examination of luminal pressure-induced intestinal motility of whole intestine revealed that maropitant (0.1–10 µM) increased frequency of contraction, decreased amplitude of contraction and totally inhibited motility index in a concentration-dependent manner. We measured intestinal transit in vivo by measuring transportation of orally administered luminal content labeled with phenol red. Our results demonstrated that maropitant (10 mg/kg, SC) delayed intestinal transit. Geometric center value was significantly decreased in maropitant-treated mice. Anti-inflammatory effects of maropitant against leukocytes infiltration into the intestinal smooth muscle layer in post-operative ileus (POI) model mice were measured by immunohistochemistry. In POI model mice, a great number of CD68-positive macrophages or MPO-stained neutrophils infiltrated into the inflamed muscle region of the intestine. However, in the maropitant treated mice, the infiltration of leukocytes was not inhibited. The results indicated that maropitant has ability to induce disorder of intestinal motility in mice, but has no anti-inflammatory action in the mouse of a POI model. In conclusion, in mice, maropitant induces disorder of intestinal motility in vivo.     

Inflammation of the cardiac coronary artery in ICR mice

Inflammation of the cardiac coronary artery in ICR mice is occasionally observed in toxicity studies; however, this has not been well explored histologically. Herein, we investigated the detailed histology of the associated lesions in 6–8-week-old ICR mice. Coronary artery inflammation in the right ventricular wall was observed in 10 of 142 mice (7.0%). Histopathological examination revealed hypertrophy of the vascular smooth muscle cells and perivascular infiltration of macrophages in mild cases. In moderate to marked cases, single-cell necrosis of vascular smooth muscle cells, hemorrhage of the tunica media, and fibrinoid necrosis of the vessel wall were observed, in addition to the changes seen in mild cases. Electron microscopic examination of moderate cases revealed a discontinuous internal elastic lamina suggestive of rupture, and vascular smooth muscle cells beneath the elastic lamina showed degeneration and necrosis. These findings suggest that the lesions developed as a rupture of the internal elastic lamina and necrosis of vascular smooth muscle cells, while leaked plasma components caused vascular and perivascular inflammation. In ICR mice, dystrophic calcinosis (DCC) is known to occur rarely in the right ventricle. DCC is defined as focal calcification in necrotic myocardial fibers, the pathogenesis of which is considered to involve ectopic calcification. Since calcification was not observed in any part of the heart, including the inflammation region, the pathophysiology of cardiac arterial inflammation seen in our ICR mice was considered to differ from that of DCC.     

Porcine stress syndrome and PSE meat: clinical symptoms, pathogenesis, etiology and animal rights aspects

A review is given about the clinical symptoms, pathogenesis and aetiology of the porcine stress syndrome, furthermore aspects of animal welfare are discussed. The current breeding programmes of pig industry in Germany in many cases include animals with a mutation of the ryanodine-receptor (RYR-1)-gene--homozygous or heterozygous. This situation is the result of an intensive breeding of pigs during the last decades with the intention of increased lean carcass content and corresponding proceeds. The homozygous pigs are more stress susceptible (porcine stress syndrome) and produce meat of poor quality (PSE), which is also the case to some extend in heterozygous animals. The clinical symptoms of this muscle disease are characterised by a deficit of oxygen and a rapid glycolysis accompanied by a production of lactic acid and acidosis primarily in II B white muscle fibres. There is no doubt that a very close causal relation exists between the mutation of the RYR-1 and the porcine stress syndrome as well as the poor meat quality. The present knowledge of this disease, the genetic background, the physiology and pathophysiology of the mutation of the RYR-1 leads to the imperative conclusion to eliminate this mutated RYR-1 by selection of healthy pigs, which has been done successfully in other countries with important pig production. This conclusion is also supported by simple economic reasons because fertility, reproduction and daily weight gain are significantly reduced in stress susceptible pigs. Furthermore, it should be emphasised that regular breeding with the mutated RYR-1 is also a matter of animal welfare. The evident correlation between the mutated RYR-1 and the porcine stress syndrome, which includes degeneration of the muscle, pain and even life threatening malignant hyperthermia, can easily lead to the accusation in the public that diseased animals are used for pig meat production. Consequently, the authors would like to urge the breeding companies and the responsible authorities to discuss the problem with the intention to finish the current breeding programmes using animals with the mutated RYR-1 within a reasonable period of time.     

低温环境下禁食对小鼠自发运动及腓肠肌中PGC-lα、ERRα表达的影响

应用旷场试验测定不同时长4℃冷暴露下采食和禁食C57BL/6小鼠的自发运动,并结合骨骼肌中PGC-lα与ERRα表达水平,探讨低温环境下禁食对C57BL/6小鼠自发运动的影响。结果显示,低温环境下禁食对C57BL/6小鼠的自发运动并无显著性影响,但焦虑情绪12 h差异显著(P<0.05),探索行为8 h和12 h差异极显著(P<0.01);腓肠肌中PGC-1α表达在4,8 h时差异极显著(P<0.01),12 h差异显著(P<0.05);ERRα表达在4,6 h时差异极显著(P<0.01)。结果表明,低温环境下禁食对C57BL/6小鼠自发运动未出现明显影响,而对小鼠焦虑情绪、探索行为和腓肠肌中PGC-lα、ERRα表达存在不同程度的影响,相比于禁食而言,冷刺激对小鼠机体的影响更大。     

Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits.

In smooth muscle tissue, two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been identified. The purpose of our study was to clarify whether smooth muscle MHC mRNA expression reflects the physiological and functional state of the muscle. We studied the expression pattern of MHC mRNAs, using the S1-nuclease mapping procedure, in functionally and morphologically changeable organs; the ductus arteriosus (DA) during development (25 and 29 days of gestation, and from 3-day-old neonates) and uteri from virgin, day-10 pregnant (P10) and day-29 pregnant (P29) rabbits. The results demonstrated that SM2 expression was greater in the fetal DA than in the fetal aortic and pulmonary arteries, but that it decreased significantly following closure of DA. In the gravid uterus, SM1 expression was significantly (P<0.05) strong compared to other MHC mRNAs from virgin to P10 rabbits. During pregnancy, NMB expression showed a tendency to increase until P10, and after P10, SM2 expression increased dramatically and NMB expression decreased to give almost a mirror image of the SM2 expression. Smooth muscle type (SM1, SM2) was significantly (P<0.05) strong compared to non-muscle type expression (NMA, NMB) at P29. These data suggest that smooth muscle MHC mRNA, especially SM2 expression reflects the physiological and functional state of the smooth muscle.     

Beta 3-adrenergic agonist up-regulates uncoupling proteins 2 and 3 in skeletal muscle of the mouse

Chronic stimulation of the beta3-adrenergic receptor (AR) in obese animals resulted in a reduced adiposity associated with an increased expression of thermogenic uncoupling protein (UCP)1 in adipose tissues. In this study, the mRNA expression of newly cloned UCP isoforms (UCP2 and UCP3) were examined in obese yellow KK and C57BL control mice. UCP2 mRNA was found in all tissues examined, with higher levels in adipose tissues and skeletal muscle of the obese mice. UCP3 mRNA was expressed in skeletal muscle, heart and brown adipose tissue similarly in the two mouse strains. Daily injection of a selective beta3-adrenergic agonist, CL316,243 (0.1 mg/kg), for 10 days resulted in a marked reduction of white fat pad weight and 1.8-4.8-fold increase in the mRNA levels of UCP2 and UCP3 in skeletal muscle of obese mice. No noticeable change in the UCP2 and 3 mRNA levels was found in brown and white adipose tissues. It was also found that CL316,243 injection produced a marked and sustained elevation of the plasma free fatty acid level. These results, together with our previous findings of the fatty acid-induced UCP expression in a myocyte cell line in vitro, suggest that the beta3-AR agonist-induced UCP expression in skeletal muscle may be mediated through the elevated plasma free fatty acids. It was also suggested that anti-obesity effect of beta3-AR agonists is attributable to increased thermogenesis not only by UCP1 but also by UCP2 and UCP3.     

Response of equine airway smooth muscle to acetylcholine and electrical stimulation in vitro

Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contractile response to electrical stimulation was obtained at 18 V, 25 Hz, and 0.5 ms. Atropine (10(-6)M) or tetrodotoxin (3 x 10(-6)M) blocked the contraction, indicating that the contractile response was attributable to the release of neurotransmitter from cholinergic nerves. Cumulative concentration-response curves to acetylcholine (10(-9)M through 10(-4)M) were determined. Isometric contractile force increased as acetylcholine concentration increased. There was a significant (P less than 0.05) difference in the 50% effective dose for acetylcholine in tracheal smooth muscle and bronchial smooth muscle. The mean (+/- SD) contractile response to maximal electrical stimulus was 89% (+/- 7.4%) of that in response to 10(-4)M acetylcholine in tracheal smooth muscle and was 68% (+/- 10.4%) of the response to 10(-4)M acetylcholine in bronchial smooth muscle.     

Galectin-7 overexpression destroys airway epithelial barrier in transgenic mice

When the integrity of airway epithelium is destroyed, the ordered airway barrier no longer exists and increases sensitivity to viral infections and allergens, leading to the occurrence of airway inflammation such as asthma. Here, we found that galectin-7 transgenic(+) mice exhibited abnormal airway structures as embryos and after birth. These abnormalities included absent or substantially reduced pseudostratified columnar ciliated epithelium and increased monolayer cells with irregular arrangement and widening of intercellular spaces. Moreover, airway tissue from galectin-7 transgenic(+) mice showed evidence of impaired cell–cell junctions and decreased expression of zonula occludens-1(ZO-1) and E-cadherin. When treated with respiratory syncytial virus (RSV) or ovalbumin (OVA), galectin-7 transgenic(+) mice developed substantially increased bronchial epithelial detachment and apoptosis, airway smooth muscle and basement membrane thickening, and enhanced airway responsiveness. We found that Galectin-7 localized in the cytoplasm and nucleus of bronchial epithelial cells, and that increased apoptosis was mediated through mitochondrial release of cytochrome c and upregulated JNK1 activation and expression of caspase-3 in galectin-7 Tg(+) mice. These findings suggested that Galectin-7 causes airway structural defects and destroys airway epithelium barrier, which predispose the airways to RSV or OVA-induced epithelial apoptosis, injury, and other asthma responses.     

A histological study of the hph-1 mouse mutant: an animal model of phenylketonuria and infantile hypertrophic pyloric stenosis

AIM: To quantify the chronological sequence of changes in the morphology and immunoreactivity for neurotransmitters in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria. METHOD: Thirty specimens of pylorus from hph-1 mice and age/sex matched controls (age range: 10-180 days) were examined using conventional histology and immunohistochemistry for a variety of antigens: protein gene product 9.5, a pan neuronal marker; vasoactive intestinal polypeptide; nitric oxide synthase two antigens coalesced to the same inhibitory neurons in humans; substance P, a potent excitatory neurotransmitter; and calcitonin gene related peptide, a neurotransmitter implicated in the somatic afferent innervation of the stomach. The changes in the morphology of the muscle layers were quantified and statistically analysed for each age group (10, 20, 40, 90 and 180 days). RESULTS: Between 10 and 90 days of age, all muscle layers of the hph-1 mice were hypertrophied, for example, 10 days, hph-1 longitudinal muscle mean diameter = 3.4, control = 1.8; hph-1 circular muscle width = 11.5, control = 4.7. The hph-1 mice were significantly smaller during this period (40 days, hph-1 weight = 10 g, control = 25 g). There was no change in the pattern of expression of the antigens examined within the hph-1 mice compared with the controls. CONCLUSION: Hph-1 mice develop a transient smooth muscle hypertrophy of the pylorus attended by gastric distension and failure to gain weight. These changes resolve as the pyloric muscle hypertrophy resolves.     

Effects of smooth and rough Pasteurella haemolytica lipopolysaccharides on plasma cyclic-nucleotides and free fatty acids in calves

The present study examined the potency of smooth or rough Pasteurella haemolytica lipopolysaccharide infusion (LPS, 24 ng kg-1 min-1 for 500 min) on plasma cyclic-nucleotides and several free fatty acids (FFA) in calves. Both smooth or rough LPS increased plasma cAMP immediately to its maximum at 1 h of infusion, whereas plasma cGMP levels rose slowly and peaked 12 h later. The increases in cAMP levels were more prolonged for smooth LPS than rough LPS. The maximum plasma cAMP rise coincided with increases of several plasma FFA. Rough LPS increased plasma oleic greater than palmitic greater than stearic greater than linoleic acids in the second hour and reached their steady state levels between 2 h of infusion and 5 h post-infusion. Thereafter, oleic acid remained maximally elevated, while stearic acid decreased and other FFA returned to baseline. Smooth LPS had no effects on palmitic and stearic acids, but elevated oleic acid in an essentially similar manner to rough LPS and increased linoleic acid initially at 5 h, followed by decreases throughout post-infusion. These results demonstrate that endotoxemia produces early marked elevations in plasma cAMP, a gradual rise in plasma cGMP and disproportionate increases in several plasma FFA. The data also demonstrate that smooth and rough LPS differ in their abilities to increase plasma cAMP and FFA and these may be attributed to differences in their in vivo mechanisms of action. The study suggests that cAMP and cGMP may mediate actions of endotoxin at the cellular level and that differences exist in release and/or utilization of each FFA at different stages of endotoxemia.     

Elevated level of microRNA-210 at the initiation of muscular regeneration in acetic acid-induced non-ischemic skeletal muscular injury in mice

The alteration in microRNA-210 level, a hypoxia-inducible microRNA, is not well known in non-ischemic tissue injury. In this study, we characterized the histopathological time course of acetic acid-induced skeletal muscle injury as a non-ischemic tissue injury model and investigated the expression of microRNA-210, hypoxia-inducible factor 1α, and growth factors using quantitative polymerase chain reaction analysis. After a single intramuscular dose of 3% (v/v) acetic acid to C57BL/6J mice, focal coagulative necrosis of muscle fibers was noted from 3 h after dosing and infiltration of F4/80 and Galectin-3 positive M2 macrophage was noted at 1 d after dosing. Muscular regeneration was initiated from 3 d, when M2 macrophage infiltration was most prominent, till 14 d after dosing. Hif1α and Hgf expression increased from 3 h onwards, and microRNA-210 level increased after 3 d after the treatment. However, no clear elevation in the levels of Igf1 or Vegf was observed. The infiltrative macrophages and regenerative muscle fibers were positive for hypoxia-inducible factor 1α, microRNA-210, and hepatocyte growth factor as assessed by immunohistochemistry or in situ hybridization. In this study, dominant infiltration of M2 macrophages at muscular necrosis and subsequent regeneration after a single intramuscular injection of acetic acid in mice were observed. The increase in hif1α level was observed just after the muscular injury in this non-ischemic tissue injury model, and the elevation in microRNA-210 level was noted at the initiation of tissue regeneration, indicating its effects on tissue protection and repair.