Relationship between tenascin and α-smooth muscle actin expression in the developing human small intestinal mucosa

The expression of tenascin (Tn) and -smooth muscle actin (-SMA) was analyzed in the developing and adult human small intestine by means of double immunofluorescent staining with specific antibodies. By 7 weeks of gestation, the gut anlage has a simple tubular shape and is formed of a stratified undifferentiated epithelium surrounded by a poorly organized mesenchyme. Both Tn and -SMA were found exclusively at the periphery of the tissue, corresponding to the presumptive muscularis propria. By 9 weeks, villus rudiments had formed but Tn and -SMA remained restricted to the muscularis propria. Tn was first detected in the mesenchyme at 11 weeks. By 13 weeks, a preferential distribution of Tn in the subepithelial region of the mesenchyme was readily observed while -SMA was still absent. From this stage to 20 weeks, Tn gradually concentrated in this region that, as determined by -SMA detection, corresponded to the future muscularis mucosa area. As shown by double staining of Tn and -SMA, deposition of Tn also preceded the appearance of the other -SMA-expressing cells in the mucosa. These observations suggest that Tn could have a role in the differentiation of intestinal contractile cells.     

The role of α-smooth muscle actin in confirming the microinvasion of laryngeal squamous cell carcinoma

BackgroundThe diagnosis of microinvasive laryngeal squamous cell carcinoma (LSCC) is not always straightforward and sometimes can be very challenge in daily clinical practice. The focus lies in the confirmation of microinvasion. Cancer-associated fibroblasts (CAFs), as the major element of reactive tumor stroma, are believed to participate actively in the growth and invasion of tumor cells.ObjectivesTo evaluate the diagnostic role of α-smooth muscle actin (α-SMA) labelling CAFs in microinvasive LSCC.MethodsA total of 81 laryngeal biopsy specimens were retrieved, including 41 cases of microinvasive LSCC with depth of invasion no more than 3 mm, 20 laryngeal squamous intraepithelial lesion (SIL), and 20 benign pseudoepitheliomatous hyperplasia (PEH). All cases were stained for immunohistochemistry, using antibody against the α-SMA antigen. The correlation between the presence of CAFs in microinvasive LSCC and tumor histological characteristics was investigated.ResultsImmunoreactivity of α-SMA was detected in twenty-nine microinvasive LSCC (29/41, 71%), while no reactivity was observed in laryngeal SIL (0/20, 0%), and rarely in PEH (2/20, 10%). The α-SMA expression pattern in stroma of microinvasive LSCC was significantly different from that of SIL (χ² = 26.966, p = 0.000) and PEH (χ² = 19.838, p = 0.000). In addition, there seemed to be a certain correlation between the histological characteristics of microinvasive LSCC and the presence of interstitial CAFs.ConclusionsThis study highlights the practical role of utilizing α-SMA in the pathological diagnosis of microinvasive LSCC, with emphasis on variable histomorphologic features of microinvasive LSCC.     

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.     

Value of α-smooth muscle actin and glial fibrillary acidic protein in predicting early hepatic fibrosis in chronic hepatitis C virus infection

Introduction

α-Smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. The reports concerning GFAP expression in human liver are still conflicting. The aim of the study is investigation the utility of GFAP compared to α-SMA as an indicator of early activated HSCs, in predicting fibrosis in chronic hepatitis C (CHC) patients.

Material and methods

With immunohistochemistry and a semi-quantitative scoring system, the expressions of α-SMA and GFAP on HSCs in liver biopsies from patients with pure CHC (n = 34), hepatitis C virus-induced cirrhosis (n = 24), mixed CHC/schistosomiasis (n = 11) and normal controls (n = 10) were analysed.

Results

The immunoreactivity of α-SMA and GFAP in perisinusoidal, periportal and pericentral areas was assessed. α-Smooth muscle actin and GFAP-positive HSCs were significantly increased in all diseased groups compared with normal controls. In pure CHC with or without cirrhosis, perisinusoidal α-SMA-positive HSCs were predominant in relation to GFAP-positive cells. On the other hand, GFAP-positive cells were predominant in the group of schistosomiasis as compared with the other diseased groups. It was noticed that expression of GFAP on perisinusoidal HSCs in CHC patients sequentially decreased with the progression of fibrosis.

Conclusions

Glial fibrillary acidic protein could represent a more useful marker than α-SMA of early activation of HSCs in CHC patients and seems to be an early indicator of hepatic fibrogenesis.     

Collagen type IV,laminin, α-smooth muscle actin (αSMA), α1 and α6 integrins expression in the liver with metastases from malignant gastrointestinal tumours

Basement membrane proteins and integrins can profoundly affect the biological behaviour of metastasic tumour cells. Using light and ultrastructural immunohistochemistry, we showed the presence of alterations in the occurrence of collagen type IV and laminin, and the expression of α1 and α6 integrin chains in the livers of patients with metastases from gastric, colorectal and pancreatic cancers. The myofibroblast-like cells in the metastatic stroma were studied. Parallel expressions of α-SMA, collagen type IV and α1 integrin chain, and appearance of laminin and α6 integrin chain immunoreactivity in the extratumoral liver tissue were markedly increased in sinusoids associated with metastases. Furthermore, ultrastructural immunohistochemistry detected tumour cells adhered to amorphous laminin deposits in the metastases. Laminin occurrence in liver sinusoids was visible as fine amorphous deposits in the space of Disse. The similarity between α-SMA-positive stromal cells in metastatic stroma and hepatic stellate cells (HSCs) was established by the presence of lipid droplets in their cytoplasm. The immune deposits of α1 and α6 integrin chains were observed on the hepatocyte microvilli and on the membrane of sinusoidal endothelial cells. These findings suggest that metastatic cells produce stimuli that induce HSCs activation and sinusoidal changes. In addition, the enhanced parallel expression of αSMA, collagen type IV, laminin and of α1 and α6 integrin chains in sinusoids associated with metastases, might potentiate the further dissemination of tumour cells in new liver areas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Beneficial effect of all-trans retinoic acid (ATRA) on glomerulosclerosis rats via the down-regulation of the expression of α-smooth muscle actin: A comparative study between ATRA and benazepril

Although ATRA is a potent renoprotective agent, relatively little is known regarding the mechanisms of its action. The present study was designed to further elucidate the mechanisms of ATRA's action to GS rats and compare that with the beneficial effect of benazepril. Male SD rats weighting 160 to 200 g were used in this study. GS was induced by unilateral nephrectomy and intravenous injection of adriamycin (6 mg/kg). They were divided randomly 20 ones per group into GS group, GS treated with ATRA (20 mg/kg/day) group, and GS treated with benazepril (10 mg/kg/day) group. The other 20 ones were taken as sham-operation group, injected normal saline into caudal vein. 12 weeks later, all rats were subjected to sacrifice. As expected, the GS group exhibited significant lower serum TP and Alb, and higher BUN, Cr and proteinuria than those of the sham group. Administration of ATRA or benazepril did ameliorate these above disorders of biochemical parameters in GS rats. Extensive renal damage was observed in the GS group, such as mononuclear infiltration, mesangial proliferation, focal segment glomerular sclerosis, and tubulointerstitial fibrosis. The pathological changes in both ATRA and benazepril group were alleviated remarkably. Semiquantitative GSI was used to evaluate the degree of GS in all groups. GSI was significantly higher in the GS group than in sham group. GSI decreased from 21.9 ± 6.7 in the GS group to 6.9 ± 2.8 in the ATRA group and 7.0 ± 2.7 in benazepril group respectively. However, no significant difference in GSI between rats treated with ATRA and rats treated with benazepril was found. RT-PCR analysis revealed the renal expression of α-SMA mRNA was induced substantially in GS group as compared to sham group, which could be offset completely by ATRA or benazepril administration. However, expression level of α-SMA mRNA in GS rats treated with ATRA was identical to that in GS rats treated with benazepril. We also examined immunohistochemical staining for renal α-SMA, TGF-β1, Col IV, and FN in this model. Weak staining was observed in some glomerulus, mesangial cells, and tubular interstitium of sham rats. Staining was markedly enhanced in the majority of glomerulus, mesangial cells, and tubular interstitium of untreated GS rats. Compared with untreated GS animals, intensity and extent of staining for renal α-SMA, TGF-β1, Col IV, and FN were markedly reduced in glomerulus, mesangial cells, and tubular interstitium of GS rats treated with either ATRA or benazepril. However, no significant differences existed between ATRA and benazepril with respect to the glomerular and tubulointerstitial staining scores. Interestingly, our data documented some differences of therapeutic capacities between ATRA and benazepril. In comparison with benazepril, ATRA exerted no improvement in hypoproteinemia, but more significant decrease in serum Cr level in GS rats. The reasons leading to these variations are unclear. Whatever they are, the properties of down-regulate inflammatory/proliferative programs may make ATRA an attractive potential candidate for future therapeutic use in kidney disease.     

A comparative study of the epiligament of the medial collateral and anterior cruciate ligaments in the human knee: Immunohistochemical analysis of CD 34, α-smooth muscle actin and vascular endothelial growth factor in relation to epiligament theory

BackgroundThis study evaluated and compared the expression of VEGF, CD34, and α-SMA in the anterior cruciate ligaments and medial collateral ligaments in healthy human knees in order to enrich the epiligament theory regarding ligament healing after injury.MethodsSamples from the mid-substance of the anterior cruciate ligament and the medial collateral ligament of 12 fresh knee joints were used. Monoclonal antibodies against CD34, α-SMA, and VEGF were used for immunohistochemical analysis. Photomicrographs were analyzed using the ImageJ software.ResultsThe epiligament of the anterior cruciate ligament showed slightly higher expression of CD34, α-SMA, and VEGF than the epiligament of the medial collateral ligament. Overall, among the tested markers, α-SMA expression was most pronounced in anterior cruciate ligament epiligament images and CD34 dominated in medial collateral ligament epiligament images. The intensity of DAB staining for CD34, α-SMA, and VEGF was higher in vascular areas of the epiligament than in epiligament connective tissue.ConclusionsThe results illustrate that CD34, α-SMA, and VEGF are expressed in the human epiligament. The differences between the epiligament of the investigated ligaments and the fact that CD34, α-SMA, and VEGF, which are known to have a definite role in ligament healing, are predominantly expressed in the main vascular part of the ligament–epiligament complex enlarge the existing epiligament theory. Future investigations regarding better ligament healing should not overlook the epiligament tissue.     

CD34<Superscript>+</Superscript> fibrocytes, α-smooth muscle antigen-positive myofibroblasts,and CD117 expression in the stroma of invasive squamous cell carcinomas of the oral cavity,pharynx, and larynx

We investigated tumor-free mucosa and squamous cell carcinomas of the oral cavity, the pharynx, and larynx with respect to the presence of stromal CD34⁺ fibrocytes and -smooth muscle antigen (SMA)-positive myofibroblasts. Additionally, stromal expression of CD117 was analyzed. A total of 39 squamous cell carcinomas were assessed immunohistochemically. In all cases investigated, CD34⁺ fibrocytes were found in the tumor-free stroma, whereas -SMA-positive myofibroblasts were lacking. Areas of lymphocytic infiltration disclosed a focal reduction of CD34⁺ fibrocytes. CD117 expression was absent from the tumor-free stroma. Of 39 squamous cell carcinomas, 33 were free of stromal CD34⁺ fibrocytes, and, in 31 carcinomas, stromal -SMA-positive myofibroblasts occurred at least focally. CD117-positive stromal spindle cells were found in 25 carcinomas. Compared with tumor-free mucosa, the number of tissue mast cells was significantly increased in carcinomas. We conclude that stromal remodeling induced by invasive carcinomas is characterized by a loss of CD34⁺ fibrocytes and subsequent gain of -SMA-positive myofibroblasts. The diagnostic impact of this finding is, however, limited by the fact that chronic inflammation may also be accompanied by a focal loss of CD34⁺ fibrocytes.     

Investigation of changes in skeletal muscle α-actin expression in normal and pathological human and mouse hearts

We have developed a quantitative antibody-based assay to measure the content of skeletal muscle α-actin relative to cardiac α-actin. We found 21 ± 2% skeletal muscle α-actin content in normal heart muscle of adult man and mouse. In end stage failing heart 53 ± 5% of striated actin was skeletal muscle α-actin and in samples of inter-ventricular septum from patients with hypertrophic obstructive cardiomyopathy (HOCM) skeletal muscle α-actin was 72 ± 2% of sarcomeric actin. Thin filaments containing actin isolated from normal and HOCM heart muscle were functionally indistinguishable when studied by quantitative in vitro motility assay. We also found elevated skeletal muscle α-actin (60 ± 7%) in a mouse model of dilated cardiomyopathy.     

Long-term depolarization regulates the α1s subunit of skeletal muscle Ca2+ channels and the amplitude of L-type Ca2+ currents

The effects of long-term depolarization on the level of alpha1s and on L-type Ca2+ currents of skeletal muscle were investigated. Long-term depolarization (14 h) caused a 50% decrease of alpha1s, revealed with the Western blot technique. This decline was prevented by preincubation with the Ca2+ channel blocker nifedipine. Electrophysiological experiments using the voltage-clamp technique were performed to measure the actions of long-term depolarization on Ca2+ currents and charge movement. A progressive decline in the amplitude of the Ca2+ currents by depolarizations lasting 0.5-14 h was observed. Similar to Western blot results, the fall in current amplitude was prevented by nifedipine, and it depended on external Ca2+. The nonlinear charge mobilized by step pulses was also significantly reduced (50%) by long-term depolarization. It is suggested that alpha1s subunit is down-regulated by long-term depolarization by a very stringent mechanism and that, in this process, Ca2+ ions permeating through L-type channels play a key role. A new role for the L-type Ca2+ current in skeletal muscle fibers in which the channels are self-regulated is proposed.     

Colonic MUC2 mucin regulates the expression and antimicrobial activity of β-defensin 2

In this study we identified mechanisms at the colonic mucosa by which MUC2 mucin regulated the production of β-defensin in a proinflammatory milieu but functionally protected susceptible bacteria from its antimicrobial effects. The regulator role of MUC2 on production of β-defensin 2 in combination with the proinflammatory cytokine interleukin-1β (IL-1β) was confirmed using purified human colonic MUC2 mucin and colonic goblet cells short hairpin RNA (shRNA) silenced for MUC2. In vivo, Muc2^−/− mice showed impaired β-defensin mRNA expression and peptide localization in the colon as compared with Muc2^+/− and Muc2^+/+ littermates. Importantly, purified MUC2 mucin abrogated the antimicrobial activity of β-defensin 2 against nonpathogenic and enteropathogenic Escherichia coli. Sodium metaperiodate oxidation of MUC2 removed the capacity of MUC2 to stimulate β-defensin production and MUC2's inhibition of defensin antimicrobial activity. This study highlights that a defective MUC2 mucin barrier, typical in inflammatory bowel diseases, may lead to deficient stimulation of β-defensin 2 and an unbalanced microbiota that favor the growth of β-defensin-resistant microbes such as Clostridium difficile.     

Role of α2-adrenoceptors in the lateral parabrachial nucleus in the control of body fluid homeostasis

Central α₂-adrenoceptors and the pontine lateral parabrachial nucleus (LPBN) are involved in the control of sodium and water intake. Bilateral injections of moxonidine (α₂-adrenergic/imidazoline receptor agonist) or noradrenaline into the LPBN strongly increases 0.3 M NaCl intake induced by a combined treatment of furosemide plus captopril. Injection of moxonidine into the LPBN also increases hypertonic NaCl and water intake and reduces oxytocin secretion, urinary sodium, and water excreted by cell-dehydrated rats, causing a positive sodium and water balance, which suggests that moxonidine injected into the LPBN deactivates mechanisms that restrain body fluid volume expansion. Pretreatment with specific α₂-adrenoceptor antagonists injected into the LPBN abolishes the behavioral and renal effects of moxonidine or noradrenaline injected into the same area, suggesting that these effects depend on activation of LPBN α₂-adrenoceptors. In fluid-depleted rats, the palatability of sodium is reduced by ingestion of hypertonic NaCl, limiting intake. However, in rats treated with moxonidine injected into the LPBN, the NaCl palatability remains high, even after ingestion of significant amounts of 0.3 M NaCl. The changes in behavioral and renal responses produced by activation of α₂-adrenoceptors in the LPBN are probably a consequence of reduction of oxytocin secretion and blockade of inhibitory signals that affect sodium palatability. In this review, a model is proposed to show how activation of α₂-adrenoceptors in the LPBN may affect palatability and, consequently, ingestion of sodium as well as renal sodium excretion.     

Differential expression of α-enolase in the normal and pathological cardiac growth

Objectives

It was found that α-enolase was dramatically up-regulated in the hypertrophic hearts of SHR in our previous study. The purposes of this study were to examine the expression pattern of α-enolase in pre- and postnatal myocardium of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, and to explore the relationship between the overexpression of α-enolase and left ventricular hypertrophy.

Methods

HE staining was used for the measurement of cardiac hypertrophy. Immunohistochemical technique was used to evaluate the location of α-enolase. The expressions of α-enolase in the left cardiac ventricles at different development times were examined by Real-time RT-PCR and Western blot.

Results

Cardiac hypertrophy was found in SHR rats at 4 weeks of age and remained up to 24 weeks of age. The signals of α-enolase protein were strong and existed extensively in hypertrophic myocardium in SHR, while in the normal myocardium of WKY, the signals were scarcely found and weak. The levels of α-enolase mRNA and protein in SHR and WKY hearts during fetal stage and newborn stage were similar, while from 4 weeks of age to 24 weeks of age, accompanied by the cardiac hypertrophy, the levels of α-enolase mRNA and protein in left ventricle of SHR were significantly higher than that in WKY.

Conclusions

The expressions of α-enolase in the left ventricle of the rats during normal and pathological cardiac development were different. This phenomenon provides the potential clues to understanding pathophysiological mechanisms in cardiac hypertrophy of SHR.     

Pulmonary epithelial expression of human α1-antitrypsin in transgenic mice results in delivery of α1-antitrypsin protein to the interstitium

α₁-Antitrypsin (α₁AT) therapy is used as a treatment for α₁AT deficiency. It has also been proposed as a therapy for cigarette smoke-induced emphysema, although the efficacy of such therapy is as yet unproven. Moreover, the optimal route of delivery of α₁AT to the lung interstitium, the crucial locus of action, is unknown. We created transgenic mice with expression of the human α₁AT gene directed by a human surfactant protein C (SpC) promoter fragment or a rat Clara cell 10-kDa protein (CC10) promoter fragment in order to examine the ability of pulmonary epithelial cell expression of α₁AT to deliver protein to the interstitium, and to produce a model that would allow studies on the efficacy of α₁AT in preventing lung damage after cigarette smoke exposure. Four transgenic lines were studied. In situ hybridization and light microscopic immunohistochemistry showed that two CC10 driven lines expressed human α₁AT in type II alveolar cells and airway epithelial cells; α₁AT expression was seen in the alveolar parenchyma in two SpC driven lines, and in small airway epithelium in one of the SpC lines. Electron microscopic immunochemistry showed the presence of the human α₁AT protein in the interstitium in all lines. Mean levels of human protein varied from 0.37 to 2.9 μg/g lung protein and serum levels from 0.72 to 1.3 μg/ml, compared to normal human serum α₁AT levels of 2–5 mg/ml. We conclude that transgene-mediated expression of α₁AT in pulmonary epithelial cells results in diffuse expression of the transgene in the alveolar parenchyma and reproducibly leads to transfer of protein to the interstitium. The present model is, however, limited by low levels of protein production; limited protein production may be a problem in other forms of gene therapy in which relatively large amounts of extracellular protein are needed in the lung for a therapeutic effect. Received: 5 August 1998 / Accepted: 25 January 1999