Placental villous stroma as a model system for myofibroblast differentiation

Different subtypes of myofibroblasts have been described according to their cytoskeletal protein patterns. It is quite likely that these different subtypes represent distinct steps of differentiation. We propose the human placental stem villi as a particularly suitable model to study this differentiation process. During the course of pregnancy, different types of placental villi develop by differentiation of the mesenchymal stroma surrounding the fetal blood vessels. In order to characterise the differentiation of placental stromal cells in the human placenta, the expression patterns of the cytoskeletal proteins vimentin, desmin, alpha- and gamma-smooth muscle actin, pan-actin, smooth muscle myosin, and the monoclonal antibody GB 42, a marker of myofibroblasts, were investigated on placental tissue of different gestational age (7th-40th week of gestation). Proliferation patterns were assessed with the proliferation markers MIB 1 and PCNA. Additionally, dipeptidyl peptidase IV distribution was studied in term placenta and the ultrastructure of placental stromal cells was assessed by electron microscopy. Different subpopulations of extravascular stromal cells were distinguished according to typical co-expression patterns of cytoskeletal proteins. Around the fetal stem vessels in term placental villi they were arranged as concentric layers with increasing stage of differentiation. A variable layer of extravascular stromal cells lying beneath the trophoblast expressed vimentin (V) or vimentin and desmin (VD). They were mitotically active. The next layer co-expressed vimentin, desmin, and alpha-smooth muscle actin (VDA). More centrally towards the fetal vessels, extravascular stromal cells co-expressed vimentin, desmin, alpha- and gamma-smooth muscle actin, and GB 42 (VDAG). Cells close to the fetal vessels additionally co-expressed smooth muscle myosin (VDAGM). Ultrastructurally, V cells resembled typical mesenchymal cells. VD cells corresponded to fibroblasts, while VDA and VDAG cells developed features of myofibroblasts. Cells of the VDAGM-type revealed a smooth muscle cell-related ultrastructure. In earlier stages of pregnancy, stromal cell types with less complex expression patterns prevailed. The media smooth muscle cells of the fetal vessels showed a mixture of different co-expression patterns. These cells were separated from extravascular stromal cells by a layer of collagen fibres. The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessels in the centre of the stem villi. The spatial distribution of the various stages of differentiation suggests that human placental villi could be a useful model for the study of the differentiation of myofibroblasts.     

Echocardiography can detect cloth cover tears in fully covered Starr-Edwards valves: a long-term clinical and echocardiographic study

Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (approximately 10%) and large muscular artery (approximately 30% to 50%) SMCs was positive for smoothelin. In general, smoothelin and desmin were coexpressed in the same SMCs, but expression of desmin appeared to be less abundant. However, the majority of SMCs in these blood vessels were smoothelin- and desmin negative but expressed vimentin, whereas alpha-smooth muscle actin (alpha-SMA) was present in all SMCs. The SMCs in the media of small muscular arteries were positive for smoothelin and desmin (> 95%), whereas the vimentin-positive SMC type was scarce. Smoothelin was absent in capillaries, pericytic venules, and small veins but was occasionally observed in the SMCs of large veins. Thus, the distribution of smoothelin in the SMCs of the vascular system appears to be limited to blood vessels that are capable of pulsatile contraction. In atherosclerotic femoral arteries, smoothelin-positive cells were detected in the media, the atheromatous plaque, and the intimal thickening. Smoothelin-positive cells were present primarily at the luminal portion of advanced lesions. The presence of a considerable number of such smoothelin-positive cells at that location may indicate that these plaques are no longer expanding.     

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

The expression of keratins, vimentin, neurofilament proteins, smooth muscle actin, neuron-specific enolase, and synaptophysin in tumors of the specific glands in the canine anal region

Eight canine tumors originating from specific glandular structures in the anal region, as well as metastatic tumor tissue of two of these cases (case Nos. 7, 8), were immunohistochemically analyzed using various monoclonal antibodies (MoAbs) directed against human keratin types, vimentin, neurofilament proteins, and alpha-smooth muscle actin. These tumors also were stained for the broad-spectrum neuroendocrine markers neuron-specific enolase (NSE) and synaptophysin. In histologically normal canine anal structures, alpha-smooth muscle actin and NSE antibodies stained basally localized (probably myoepithelial) cells in the anal glands and the anal sac glands. NSE staining also was present in a limited number of luminal cells in both anal glands and anal sac glands. Synaptophysin labeling was not observed in any of these glandular structures. Histologically, the tumors were differentiated into well- and moderately differentiated perianal gland tumors (n = 5) and carcinomas without perianal gland differentiation (n = 3), corresponding to the so-called apocrine carcinomas of the anal region. Immunohistochemically, the perianal gland tumors could be differentiated from the carcinomas by marked differences in staining pattern with the various keratin MoAbs, particularly MoAbs directed against human keratin types 7 and 18. The keratin-staining characteristics of the carcinomas suggest a glandular luminal cell origin. Metastases of the carcinomas showed loss of some keratin-staining characteristics as compared with the primary tumor. Staining for NSE was only observed in solitary cells and small cell clusters in the carcinomas and their metastases, whereas the alpha-smooth muscle actin antibody did not react with the carcinoma cells. None of the tumors stained for neurofilament proteins or synaptophysin. An unequivocal neuroendocrine nature of the carcinomas could not be substantiated by our immunohistochemical study, although the presence of a population of neuroendocrine cells within these neoplasms seems likely. Because the immunohistochemical features of the carcinomas with respect to various keratin MoAbs and NSE are similar to those of the anal glands and the anal sac glands, both these glands might be considered as site of origin of these carcinomas.     

Production and characterisation of polyclonal antisera against feline IgE

To confirm the characteristics of the stromal cells of Wharton's jelly, we investigated the morphological changes in these cells during each trimester of pregnancy. We evaluated the cytoskeletal features of these cells by examining immunohistochemically the localization of one of the contractile proteins, alpha-smooth muscle actin (ASMA). After the second trimester, the stromal cells of Wharton's jelly were stained with ASMA antibody, exhibited the ultrastructural characteristics of the myofibroblasts, and began to express numerous microfilaments in the cytoplasm. Postembedding immunogold labeling detected immunoreactivity for ASMA on these microfilaments. The finding indicated that the stromal cells of Wharton's jelly undergo a time-dependent maturation involving the differentiation of myofibroblasts during the last 6 months of pregnancy. These cells possess a contractile function that may help to protect the umbilical vessels from compression, considering that ASMA was detected in the microfilamentous bundles.     

Contractile cells of the kidney in primary glomerular disorders: an immunohistochemical study using an anti-alpha-smooth muscle actin monoclonal antibody

Mesangial cells of the renal glomerulus are thought to have contractile properties, resembling those of smooth muscle cells. Since actin synthesis in mesangial cells is increased in selected animal models of glomerulonephritis, we evaluated the expression of alpha-smooth muscle actin (ASMA), the principal actin isoform found in smooth muscle cells, in biopsy specimens from patients with primary glomerular disorders and in control tissues. Normal glomeruli and glomeruli in acute tubulointerstitial disorders showed few or no ASMA-positive cells in the glomeruli. In contrast, ASMA expression in mesangial cells was increased in minimal change disease, focal segmental glomerulosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, and immunoglobulin A nephropathy. In membranoproliferative glomerulonephritis and cryoglobulinemic glomerulonephritis both mesangial and capillary loop ASMA-positive cells were observed with a segmental distribution. In addition, ASMA-positive interstitial cells were seen in many biopsy specimens and often were increased in number in biopsy specimens showing early interstitial fibrosis and tubular atrophy. We conclude that ASMA synthesis in mesangial cells is upregulated in a variety of glomerular disorders, frequently associated with increased cell proliferation and mesangial matrix production. This phenotypic change may be an indicator of mesangial cell activation after injury and may have important pathophysiologic consequences.     

Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function

We investigated the effect of transforming growth factor-beta1 (TGF-beta1) on the expression of calponin-h1, alpha-smooth muscle actin (alpha-SMA), and extracellular matrix (ECM) components in a cultured human Ito cell line, LI90. The TGF-beta1 treatment stimulated productions of hyaluronic acid and laminin, and significantly decreased the secretion of hepatocyte growth factor in LI90 cells. The functional characteristics of LI90 cells were compatible with those of human-activated Ito cells that are known as pericyte-like mesenchymal liver cells. TGF-beta1 induced a slight growth-inhibition of LI90 cells. TGF-beta1 enhanced the expressions of both alpha-SMA and calponin-h1 at the protein level, while tumor necrosis factor-alpha and interleukin-1alpha did not affect the expressions of these cytoskeletal proteins on LI90 cells. The addition of TGF-beta1 to LI90 cells resulted in a significant increase of calponin-h1 mRNA levels, but not calponin-h2. These data suggest that the expression of calponin-h1 is controlled at the level of mRNA under the coordinate regulation together with alpha-SMA as the process of perpetuation of activated Ito cells promoted by TGF-beta1. The identification of smooth muscle features promoted by TGF-beta1 support the hypothesis that the activation of Ito cells coincides with their contractile behavior, indicating that these cells may be important in vasoregulation during liver injury and fibrosis.     

Differential effect of thyroid-stimulating hormone (TSH) on intracellular free calcium and cAMP in cells transfected with the human TSH receptor

The major part of research dealing with the biophysical and biochemical properties of airway smooth muscle is based on the assumption that the cells constituting the tissue are homogenous. For striated muscle this has been shown untenable. In recent years almost every property of vascular smooth muscle has been also demonstrated to be heterogeneous. This realization has been late in arriving on the airway smooth muscle research scene. Our own studies have shown that mechanical properties are, in quantitative terms, heterogeneously distributed down the airways and that contractility, for example, in extrapulmonary and intrapulmonary airways differs markedly. Another indication of heterogeneity is derived from studies of the biochemical properties of airway smooth muscle cells (ASMCs) in culture. Dramatic changes in phenotype expression were found with days in culture. Just after isolation from the tissue, the cells were of contractile type and contained mature isoforms of contractile, regulatory and cytoskeletal proteins. After the fourth day in culture the cellular phenotype changed such that contractile filaments diminished rapidly with smooth muscle isoforms being replaced by non-muscle isoforms. The cell assumed secretory or synthetic properties and commenced proliferating rapidly. It is possible that similar changes in phenotype could occur in vivo in cells undergoing hypertrophy or hyperplasia. Thus, a thickened medial layer of the type seen in the walls of airways from asthmatic airways is not necessarily one endowed with increased contractility and, in fact, the latter may be subnormal. Finally, using the so-called motility assay, we studied the velocity of translation of actin filaments by myosin molecules obtained from antigen-sensitized and control airway smooth muscle. We found no change in maximum velocity of actin translation. This was under conditions where the myosin light chain (MLC) was fully phosphorylated. However, in these tissues we found heterogeneity in myosin light chain kinase (MLCK) content which, we inferred, accounted for the difference in shortening velocity between control and sensitized muscle strips in vitro.     

Role of tyrosine phosphorylation in excitation-contraction coupling in vascular smooth muscle

Increasingly it is recognized that tyrosine phosphorylation plays an important part in the regulation of function in differentiated contractile vascular smooth muscle. Tyrosine kinases and phosphatases are present in large amounts in vascular smooth muscle and have been reported to influence a number of processes crucial to contraction, including ion channel gating, calcium homeostasis and sensitization of the contractile process to [Ca2+]i. This review summarizes current understanding regarding the role of tyrosine phosphorylation in excitation-contraction coupling in blood vessels.     

Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer

Urokinase plasminogen activator (uPA) is a serine proteinase involved in degradation of the extracellular matrix during cancer invasion. uPA is up-regulated in breast cancer, and high levels of uPA in tumor extracts are strongly associated with poor prognosis. Like several other matrix proteinases, uPA is in some types of cancer, including breast cancer, expressed by stromal cells. The present study was undertaken to determine the identity of the uPA-expressing stromal cells in breast cancer tissue. By in situ hybridization, a positive signal for uPA mRNA was in 26 of 28 ductal and four of five lobular carcinomas demonstrated in stromal cells adjacent to nests of cancer cells, whereas only one ductal carcinoma showed a positive reaction in the epithelial component itself. The positive stromal cells were found in both the peripheral and central parts of the tumors. Stromal cells surrounding carcinoma in situ lesions were uPA mRNA positive in a few cases, and no signal was observed in the neighboring nonmalignant tissue. Cell identification was done by immunostaining with Ab to markers for the following cell types: myoepithelial cells, myofibroblasts, smooth muscle cells, macrophages, endothelial cells, and epithelial cells. The only one of these cell types that had a distribution similar to the uPA mRNA-expressing cells was myofibroblasts, recognized as extravascular alpha-smooth muscle actin-positive and cytokeratin-negative cells. On adjacent sections, colocalization was found of cells positive for uPA mRNA and cells positive for alpha-smooth muscle actin and negative for cytokeratin. We concluded that the uPA mRNA-expressing cells are myofibroblasts. The myofibroblasts have previously been found to be abundant in breast cancer tissue. They primarily originate by differentiation of fibroblasts, probably induced by cytokines released from the cancer cells. The present findings suggest that the myofibroblasts, through production of uPA, play an active role in breast cancer invasion.     

Cataract induction in lenses cultured with transforming growth factor-beta

PURPOSE: Anterior subcapsular cataracts are characterized by the appearance of opaque plaques of abnormal cells. Distinctive spindle-shaped cells containing alpha-smooth muscle actin are present and are associated with wrinkling of the overlying lens capsule. Accumulations of extracellular matrix, including type I collagen, also are found. The authors previously reported that transforming growth factor-beta (TGF-beta) induces similar aberrant morphologic changes in lens epithelial explants. More recently, they identified alpha-smooth muscle actin in explants cultured with TGF-beta. The aim of this study was to determine whether TGF-beta induces comparable cataractous changes in whole lenses and to examine the effects of this treatment on the transparency of the lens. METHODS: Whole lenses from 21-day-old rats were cultured in defined serum-free medium with TGF-beta 2 or without added growth factors for 5 days. Lenses were then photographed and prepared for histology and immunolocalization. RESULTS: Lenses cultured with TGF-beta developed distinct anterior opacities just beneath the lens capsule. Histologically, clumps of abnormal cells corresponded with these opacities. Spindle-shaped cells, which contained alpha-smooth muscle actin, were present, and the overlying capsule was often wrinkled. The clumps contained accumulations of type I collagen, laminin, and heparan sulphate proteoglycan. In contrast, lenses cultured without growth factors remained transparent, retained normal lens morphology, and did not accumulate alpha-smooth muscle actin or type I collagen. CONCLUSIONS: These results show that TGF-beta induces whole lenses to form opacities that contain morphologic and biochemical markers for subcapsular cataract.     

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.     

Cystic pulmonary metastases of endometrial stromal sarcoma of the uterus, mimicking lymphangiomyomatosis: a case report with immunohistochemistry of HMB45

A case of endometrial stromal sarcoma (ESS) showed cystic pulmonary metastases mimicking lymphangiomyomatosis (LAM). A 58-year-old female, who had undergone total hysterectomy for low-grade ESS 16 years previously, had repeated bouts of pneumothorax. Multiple thin-walled cysts in the peripheral lung were revealed by radiological examinations. In an open-lung biopsy specimen, cystic lesions were surrounded by layers of spindle-shaped cells of varying thickness that resembled LAM. However, in addition to subtle histologic differences from LAM, HMB45 (antimelanoma antibody) showed positive in LAM (n = 3), but was negative in ESS (n = 2) and the cystic lesions of this case. Using myogenic markers (desmin and alpha-smooth muscle actin), metastatic ESS could be immunohistochemically differentiated from mesenchymal cystic hamartoma (n = 1). HMB45 immunohistochemistry is useful in the differential diagnosis of cystic pulmonary lesions.     

Angiogenesis in wound healing and tumor metastasis

Formation of new blood vessels is essential for several physiological and pathological events, e.g. embryogenesis, wound healing and tumor growth and metastasis. In order to increase the insight into the mechanisms of angiogenesis we have visualized the different components of the microvasculature in human wounds and tumors by immunohistochemistry on the light and electronmicroscopic level. For this purpose, antibodies recognizing distinct markers for human endothelial cells, pericytes and basal lamina were used on freshly frozen or paraformaldehyde-fixed tissue samples. In terms of efficacy, the PAL-E antigen is highly specific for blood vessel endothelium. Its sensitivity is less than other endothelial markers, such as von Willebrand factor and CD 31, as it is not expressed in arterioles. Within the context of the microvasculature alpha-smooth muscle actin and the HMW-MAA chondroitin sulphate proteoglycan are useful markers for pericytes. Type IV Collagen and Laminin can be visualized consistently in the microvascular basal lamina. During the formation of granulation tissue in wound healing a heterogeneity of the expression of endothelial and pericyte markers is found. In the least matured zone in granulation tissue of decubitus lesions and experimental skin wounds microvessels already contained both endothelial cells and pericytes, suggesting a role for both cell types in the early steps of angiogenesis. Regarding the tumor microvasculature, antibodies to von Willebrand factor often failed to stain capillaries, that did show expression of the other endothelial markers studied. Broad staining in pericytes was found for the HMW-MAA chondroitin sulphate proteoglycan. In contrast, these cells only locally expressed alpha-smooth muscle actin. Staining of the basal lamina components Type IV Collagen and Laminin within tumors was not restricted to the microvasculature. Therefore, antibodies recognizing endothelial markers, particularly PAL-E and BMA 120, are preferable as tools to visualize the tumor microvasculature. In accordance with the situation in granulation tissue of wound healing the broad presence of pericytes in the microvasculature of human tumor suggests an involvement of this cell type in tumor angiogenesis. Recent immunohistochemical studies on human tumor lesions indicated that a high number of microvessels adjacent to the tumor as a measure of tumor angiogenesis is an unfavorable prognostic factor in cutaneous melanoma, mammary carcinoma and non-small cell pulmonary carcinoma. This new application of immunohistochemistry represents a valuable, clinically relevant adjunct to the repertoire of the surgical pathologist.     

Immunolocalization of three novel smooth muscle-specific proteins in salivary gland pleomorphic adenoma: assessment of the morphogenetic role of myoepithelium

Myoepithelial cells of salivary glands have a complex cytoskeletal immunophenotype. To elaborate the smooth muscle phenotype of salivary gland myoepithelium and to assess its contribution to the histogenesis of pleomorphic adenomas, we evaluated the immunohistochemical expression of three novel monoclonal antibodies (MAbs) to alpha smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chains (SMMH), and calponin in formalin-fixed tissues of 65 pleomorphic adenomas (51 contained surrounding normal salivary gland as well). Different cell types within the pleomorphic adenomas were classified as inner tubular epithelial cells, myoepithelium-like cells (juxtatubular, cuboidal, and spindle), modified myoepithelium (myxoid, chondroid, hyaline), and transformed myoepithelium (solid epithelioid, squamous, basaloid-cribriform). Periacinar and periductal myoepithelial cells of all of the 51 normal salivary glands were diffusely stained by all of the 3 MAbs, whereas all of the acinar/ductal epithelial cells were entirely negative. Of 65 pleomorphic adenomas, 61 (94%) reacted to all of the 3 MAbs. None of the smooth muscle markers stained the inner-tubular epithelial cells. Both alpha-SMA and SMMH were essentially limited to the myoepithelium-like cells, whereas modified and transformed myoepithelia lacked these myofilaments. Calponin was found in 64 (98%) of the tumors, reacting to almost all of the myoepithelium-like cells, to 60% of the modified myoepithelium, and to 30% of the transformed myoepithelium. We found the expression of these smooth muscle-specific proteins in the neoplastic myoepithelium to be associated with morphologic differentiation. Alpha-SMA and SMMH are only expressed in better differentiated neoplastic myoepithelium. Calponin is the most sensitive marker of neoplastic myoepithelium, and its identification in different cell types of pleomorphic adenomas denotes a major histogenetic role of myoepithelial cells.     

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.     

Relationship of angiographic finding with neovascular structure detected by immunohistochemical staining of alpha-smooth muscle actin in small hepatocellular carcinoma

To elucidate the relationship between angiographic features and histological findings, an immunohistological study of alpha-smooth muscle actin was performed in 106 patients with small hepatocellular carcinoma. Arterial dominance or portal blood paucity were found in 73 patients (68.9%) on digital subtraction angiography, 88 (83.0%) on computerized tomographic arterial portography and 87 (82.1%) on carbon dioxide-enhanced ultrasonography. Among 73 patients with hypervascularity on angiography, 57 (78.1%) had thick-walled, nuclei-rich and slender-shaped vessels (type II), eight (11.0%) had thin-walled, nuclei-poor and oval-shaped vessels (type I) and the remaining eight had a mixed type of II and I. Conversely, among 33 patients without hypervascularity, five (15.2%) had a type II, 21 (63.6%) had a type I, five had a mixed type and two had no positive vessel. Tumour size, histological classification and amount of non-triadal vessels were also associated with the angiographic appearance of the tumours. Among varied aspects of the cancer including tumour size, tumour multiplicity, microscopic portal invasion, histological classification, amount of alpha-smooth muscle actin-positive vessels and shape of alpha-smooth muscle actin-positive vessels, multivariate logistic regression analysis demonstrated that the shape of alpha-smooth muscle actin-positive vessels was solely associated with angiographic hypervascularity independently (P<0.0001). Although the existence of non-triadal vessels characterized hepatocellular carcinoma, angiographic hypervascularity was closely associated with the type II vessel. A morphological change of non-triadal vessel from type I to type II was considered to occur in an early stage of hepatocellular carcinoma.