Desmin-positive Cells Research Articles

Liver sinusoidal endothelial cells represents an important blood clearance system in pigs

BackgroundNumerous studies in rats and a few other mammalian species, including man, have shown that the sinusoidal cells constitute an important part of liver function. In the pig, however, which is frequently used in studies on liver transplantation and liver failure models, our knowledge about the function of hepatic sinusoidal cells is scarce. We have explored the scavenger function of pig liver sinusoidal endothelial cells (LSEC), a cell type that in other mammals performs vital elimination of an array of waste macromolecules from the circulation.Results125I-macromolecules known to be cleared in the rat via the scavenger and mannose receptors were rapidly removed from the pig circulation, 50% of the injected dose being removed within the first 2–5 min following injection. Fluorescently labeled microbeads (2 μm in diameter) used to probe phagocytosis accumulated in Kupffer cells only, whereas fluorescently labeled soluble macromolecular ligands for the mannose and scavenger receptors were sequestered only by LSEC. Desmin-positive stellate cells accumulated no probes. Isolation of liver cells using collagenase perfusion through the portal vein, followed by various centrifugation protocols to separate the different liver cell populations yielded 280 × 107 (range 50–890 × 107) sinusoidal cells per liver (weight of liver 237.1 g (sd 43.6)). Use of specific anti-Kupffer cell- and anti-desmin antibodies, combined with endocytosis of fluorescently labeled macromolecular soluble ligands indicated that the LSEC fraction contained 62 × 107 (sd 12 × 107) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors.ConclusionsWe show here for the first time that pig LSEC, similar to what has been found earlier in rat LSEC, represent an effective scavenger system for removal of macromolecular waste products from the circulation.

Expression of CD10 in Malignant Müllerian Mixed Tumors and Adenosarcomas: An Immunohistochemical Study

CD10 has been demonstrated to be positive in endometrial stromal sarcoma (ESS) and thus is useful in establishing the diagnosis, but its expression in malignant müllerian mixed tumor (MMMT) and müllerian adenosarcoma remains to be clarified. In this study, 12 cases of MMMT (9 uterine, 2 tubal, and 1 metastatic), 6 cases of müllerian adenosarcoma (three corporeal, two cervical, and one tubal), and 7 cases of primary uterine sarcomas had their tissues examined immunohistochemically for expression of CD10, desmin, myoglobin, α-smooth muscle actin (SMA), and cytokeratin. Of the primary uterine sarcomas, two were primary rhabdomyosarcomas (one cervical and one corporeal), two were ESSs, two were high-grade leiomyosarcomas, and one was a high-grade endometrial sarcoma. Sarcomatous components in all cases of MMMT and müllerian adenosarcoma, as well as all uterine sarcomas, were positive for CD10, showing moderate to marked staining intensity with varying distribution except in one MMMT, which showed weak and very focal staining. In four MMMTs, three adenosarcomas, and one rhabdomyosarcoma, myoglobin- and/or desmin-positive rhabdomyoblastic cells were positive for CD10. The immunoreactivity for CD10 showed the same distribution for α-SMA and myoglobin in three and two MMMTs, respectively. In five cases of MMMT, carcinomatous components were focally positive for CD10, and in two cases small populations of round or short spindle cells in sarcomatous components were positive for CD10, α-SMA, and cytokeratin (CAM5.2). These results indicate that CD10 expression is not restricted to ESS but can be positive in MMMT and müllerian adenosarcoma as well as in a variety of uterine tumors including high-grade leiomyosarcoma and rhabdomyosarcoma. CD10 expression might be one of the characteristics of müllerian system-derived neoplastic mesenchymal cells.

Demonstration of intermediate filaments in sheep ovary

Distribution patterns of intermediate filaments, vimentin, smooth muscle actin, and desmin were studied in the ovine ovary using an immunohistochemical method. Vimentin was consistently expressed in follicular cells of primary, secondary, and antral follicles. Stromal cells of cortex and medulla and those surrounding the corpus luteum were immunostained with the anti-vimentin antibody. Endothelial cells lining blood vessels showed strong anti-vimentin positivity. Smooth muscle cells positive for smooth muscle actin were incorporated in the cortical region in the theca interna and externa and formed incomplete spheres around large antral follicles. In atretic follicles, the presence of positive smooth muscle cells inside follicular spaces were related with capillaries. Corpora lutea were surrounded by a layer of positive smooth muscle cells. In the hilus, smooth muscle actin was localized in the wall of blood vessels. In cortical regions, desmin-positive cells were randomly distributed and occasionally formed ill-defined clusters around tertiary follicles, but not around secondary follicles, whereas desmin was expressed in the medullary region in blood vessels. The distribution pattern of intermediate filaments in sheep ovary suggests a possible role of smooth muscle cells in the mechanism of ovulation.

Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model.

Tissue-engineered vascular autografts (TEVAs) were made by seeding 4-6 x 10(6) of mixed cells obtained from femoral veins of mongrel dogs onto tube-shaped biodegradable polymer scaffolds composed of a polyglycolid acid (PGA) nonwoven fabric sheet and a copolymer of L-lactide and caprolactone (n = 4). After 7 days, the inferior vena cavas (IVCs) of the same dogs were replaced with TEVAs. After 3, 4, 5, and 6 months, angiographies were performed, and the dogs were sacrificed. The implanted TEVAs were examined both grossly and immunohistologically. The implanted TEVAs showed no evidence of stenosis or dilatation. No thrombus was found inside the TEVAs, even without any anticoagulation therapy. Remnants of the polymer scaffolds were not observed in all specimens, and the overall gross appearance similar to that of native IVCs. Immunohistological staining revealed the presence of factor VIII positive nucleated cells at the luminal surface of the TEVAs. In addition, lesions were observed where alpha-smooth muscle actin and desmin positive cells existed. Implanted TEVAs contained a sufficient amount of extracellular matrix, and showed neither occlusion nor aneurysmal formation. In addition, endothelial cells were found to line the luminal surface of each TEVA. These results strongly suggest that "ideal" venous grafts with antithrombogenicity can be produced.

The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells.

Bone marrow stem cells (BMC) develop into hematopoietic and mesenchymal lineages but have not been known to differentiate into glomerular cells. To investigate whether such differentiation is possible, a search was made for donor glomerular cells in lethally irradiated C57BL/6j (B6) mice given transplants of BMC from syngeneic mice transgenic for green fluorescence protein (GFP) ([GFP-->B6] mice). After the recipients of donor BMC manifested GFP-positive cells in their glomeruli, the numbers of such cells increased markedly, in a time-dependent manner, from 2 wk to 24 wk after bone marrow transplantation. Immunohistochemical analyses revealed that most GFP-positive cells in the glomeruli were neither macrophages nor T cells. With the use of a laser-scanning confocal microscope, GFP-positive cells were observed within the mesangium of [GFP-->B6] mice. Furthermore, indirect immunofluorescence assays demonstrated that desmin-positive cells in the glomeruli of these chimeric mice were also positive for GFP. Among glomerular cells isolated from [GFP-->B6] mice 24 wk after bone marrow transplantation and then cultured, the majority of cells (approximately 84%) stained for desmin and approximately 60% of the desmin-positive cells expressed GFP. In addition, these GFP-positive cells in the cultures contracted in response to angiotensin II stimulation. These results suggest that bone marrow-derived cells may have the potential to differentiate into glomerular mesangial cells.

Proliferating Bile Duct Epithelial Cells Are a Major Source of Connective Tissue Growth Factor in Rat Biliary Fibrosis

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-beta1) and thus a potential target for antifibrotic treatment strategies. CTGF is up-regulated in disorders such as atherosclerosis, scleroderma, and fibrosis of kidneys and lungs. We investigated the temporospatial expression patterns of CTGF and TGF-beta1 mRNA in rat livers with acute fibrogenesis (after a single dose of CCl(4)) and with advanced fibrosis (6 weeks after complete bile duct occlusion). Multiprobe ribonuclease protection assay revealed increasing TGF-beta1 and CTGF mRNA levels 6 hours after injection of CCl(4), with peak levels after 72 hours. In biliary fibrosis TGF-beta1 and CTGF mRNA levels increased fourfold and sevenfold, respectively (P: < 0.001). In situ hybridization combined with cell-specific markers revealed CTGF transcripts in desmin-positive cells after a single dose of carbon tetrachloride, whereas no transcripts were found in normal livers. In biliary fibrosis, however, proliferating bile duct epithelial cells were the predominant source of CTGF mRNA. We conclude that in rat liver fibrogenesis CTGF is up-regulated in close association with TGF-beta1 and that, contrary to a previous report, not solely hepatic stellate cells but activated bile duct epithelial cells are the main source of this profibrogenic factor.

Immunohistochemical analysis of development of desmin-positive hepatic stellate cells in mouse liver.

Development of desmin-positive hepatic stellate cells was studied in mice using double immunofluorescent techniques and in vitro cultures with special attention given to their cell lineages. Several studies recently reported on the presence of cells that are immunologically reactive with both antidesmin and anticytokeratin antibodies in young fetal rat livers, and suggested the possibility that these cells give rise to hepatocytes and hepatic stellate cells. At early stages of mouse liver development, stellate cells with desmin-positive filaments were scattered in the liver parenchyma. However, the stellate cells definitely differed from hepatoblasts and hepatocytes in terms of their morphology and expression of desmin and hepatoblast and hepatocyte-specific E-cadherin in the liver. Fetal hepatoblasts and hepatocytes did not react with antidesmin antibodies, nor did desmin-positive stellate cells express E-cadherin in vivo and in vitro. Thus it is likely that desmin-positive stellate cells and hepatoblasts belong to different cell lineages. In the fetal liver, the desmin-positive stellate cells surrounded blood vessels, and extended their processes to haematopoietic cells and megakaryocytes. Many, but not all, hepatoblasts and hepatocytes were observed to be associated with the stellate cells. At fetal stages, cellular processes positive for desmin in the stellate cells were also thick compared with those in the adult liver, in which desmin-positive stellate cells lay in Disse's space and were closely associated with all hepatocytes. These developmental changes in the geography of desmin-positive cells in the liver parenchyma and their morphology may be associated with their maturation and interactions with other cell types.

Uptake and Killing of Leptospira interrogans and Borrelia burgdorferi , Spirochetes Pathogenic to Humans, by Reticuloendothelial Cells in Perfused Rat Liver

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled cells of Leptospira interrogans serovar icterohaemorrhagiae, the agent of leptospirosis, or with Borrelia burgdorferi IRS, the agent of Lyme disease. Significant (P<0.0001) differences in the liver uptake of L. interrogans and of B. burgdorferi were observed, the uptakes being 37.4%+/-2.3% for L. interrogans and 60.5%+/-3.1% for B. burgdorferi. Leptospires, in contrast to borreliae, were recovered from the livers when liver samples were cultured in growth medium. Leptospires but not borreliae were recovered in bile within 30 min of infusion. The association of leptospires and borreliae with reticuloendothelial cells of the liver was demonstrated by immunohistochemistry. Leptospires and borreliae were found to be associated with vimentin-positive cells and not with desmin-positive cells. Few leptospires but no borreliae were also seen associated with vimentin- and desmin-negative cells, suggesting the presence of leptospires outside the sinusoidal spaces, in the liver parenchyma.

Cellular Origins of Ultraviolet Radiation-induced Corneal Tumours in the Grey, Short-tailed South American Opossum (Monodelphis domestica)

Corneal tumours were induced in almost 100% of grey, short-tailed South American opossums (Monodelphis domestica) exposed three times weekly to ultraviolet radiation (UVR) for periods of a year or more. Five tumours, representing the morphological spectrum of UVR-induced corneal tumours (two fibrosarcomas, one malignant fibrous histiocytoma, one putative haemangiosarcoma, and one squamous cell carcinoma overlying a sarcoma), were assayed immunohistochemically for reactivity with antibodies against the intermediate filaments vimentin, smooth muscle actin (α isoform), muscle-specific actins (α and γ isoforms), desmin and cytokeratin, and with antibodies against the vascular endothelial marker von Willebrand factor. The squamous cell carcinoma was cytokeratin-positive. Other tumours were cytokeratin-negative and vimentin-positive. Three tumours had scattered individual cells and groups of cells immunoreactive with antibodies against smooth muscle actin and muscle-specific actins; two tumours (a fibrosarcoma and the malignant fibrous histiocytoma) had small numbers of desmin-positive cells. The putative haemangiosarcoma contained two populations of neoplastic cells, von Willebrand factor-positive vascular endothelial cells and smooth muscle actin-positive spindle cells. It was concluded (1) that UVR-induced corneal tumours may be composed of cells derived from resident epithelial cells, immigrant vascular endothelial cells, or fibroblast-like cells of unknown origin, and (2) that such tumours may contain more than one neoplastic cell type.

Laminin α4 and Integrin α6 Are Upregulated in Regenerating dy/dy Skeletal Muscle: Comparative Expression of Laminin and Integrin Isoforms in Muscles Regenerating after Crush Injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin α2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and “preactivated” myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin α1 was not detectable in skeletal muscle. Laminin α2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin α2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin α chains in early myogenic differentiation, such as laminin α4 and α5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin β1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin α3 was not expressed in uninjured or regenerating muscle, while integrin α6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin α6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin α6 occurred on some newly formed myotubes. Integrin α7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin α7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin α7 negative. No marked difference was observed in integrin α7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin α4 and integrin α6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin α4 and integrin α6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin α2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Diffuse-type giant cell tumor: clinicopathologic and immunohistochemical analysis of 50 cases with extraarticular disease.

The clinical and pathologic features of 50 cases of diffuse-type tenosynovial giant cell tumor (D-TGCT), also known as extraarticular pigmented villonodular tenosynovitis (PVNTS), are presented. Patients' ages ranged from 4 to 76 years (median, 41 yrs), with a slight female predominance (28 women, 22 men). By definition, all lesions presented as predominant soft tissue masses, with or without an associated articular component. Tumor sites included the wrist (9 cases), knee (8 cases), thigh and foot (6 cases each), finger (5 cases), ankle (3 cases), hand, elbow, toes, buttock, paravertebral region (2 cases each), lower leg, sacrococcygeal area, and retroperitoneum; 27 cases were described as entirely extraarticular. Tumors showed infiltrative margins and, in most cases, a sheet-like growth pattern. Striking variation in the number of osteoclast-like giant cells, foamy cells, amount of hemosiderin, and in the degree of stromal hyalinization were responsible for a wide morphologic spectrum. In addition to the predominant histiocyte-like cells, we identified in most cases a subpopulation of large dendritic, desmin-positive cells showing characteristic, but potentially misleading, cytologic features, including abundant eosinophilic cytoplasm, large vesicular nuclei, paranuclear eosinophilic inclusions, and occasional nuclear inclusions. Follow-up information was available for 24 patients, with a duration ranging from 6 months to 30 years (mean, 55 mos). Local recurrence occurred in eight cases (33%), between 4 months and 6 months after surgery (median, 15 mos) and was repeated in five cases; recurrence did not appear to correlate with morphologic parameters. Six cases showed atypical histologic features and four of these contained areas of sarcomatous change. Among the latter, one of three cases with available follow up developed pulmonary metastases and died after 35 months. In addition, one histologically benign lesion gave rise, after two local recurrences, to inguinal and iliac lymph node metastases. Despite this exceedingly uncommon event, we think most cases of D-TGCT are best regarded as benign but locally aggressive neoplasms with significant recurrent potential and should be treated, when possible, by wide excision. Atypical features such as increased mitotic activity, necrosis, spindling of the mononucleate cells, and cytologic atypia are not indicative of malignancy when present individually. This study also confirms the existence of malignant tenosynovial giant cell tumors, some of which are characterized by aggressive behavior.

Angiomatoid “malignant” fibrous histiocytoma: A clinicopathologic study of 158 cases and further exploration of the myoid phenotype

Angiomatoid “malignant” fibrous histiocytoma (AMFH) has been considered to be a low-grade sarcoma of childhood, and, with its fibrous pseudocapsule, angiomatoid change, dense lymphoplasmacytic response, and proliferation of spindled or round cells, has been classified as a fibrohistiocytic neoplasm. We wanted to study the clinicopathologic and immunophenotypic features of a large number of these tumors and to especially further explore their myoid differentiation. Cases coded as AMFH from 1979 to 1995 were retrieved from the Soft Tissue Registry of the AFIP. Only cases that met the criteria for AMFH by light microscopy were included, a total of 158 cases. Immunohistochemistry was obtained on 98 cases. Clinical history on 92% of all cases revealed a gender ratio of 1.3 females: males, age range of 2 to 71 years, median size of 2.0 cm, and a distribution of extremities > trunk > head and neck, with 66% lesions occurring in areas of normal lymphoid tissue. All tumors with available margins were well-circumscribed. Eighty percent of cases had some degree of lymphoplasmacytic infiltration; 50% cases had pseudovascular spaces filled with blood. Fifty-two percent had predominantly round cell morphology; 48% had a predominantly spindle cell pattern. Desmin positivity was noted in 51% cases and occurred in both predominantly round cell and spindle cell tumors. Most of the desmin-positive cases with adjacent lymphoid infiltrate (67%) showed scattered similar, desmin-positive cells in the surrounding lymphoid infiltrate, adjacent to the tumor. Muscle-specific and smooth-muscle actins were seen in 14% cases. Heavy-caldesmon was strongly positive in 3%, and calponin was focally positive in 73% and extensively positive in 12% cases. MyoD1, myoglobin, and myogenin (myf4) were negative in all tumors studied. Forty-five percent of cases were positive for CD99; 52% of these had round cell morphology. Fifteen percent of cases were positive for KP-1. All tumors were positive for vimentin and negative for CD21, CD35, S100 protein, CD34, keratins 8 18 , and lysozyme. Clinical follow-up on 86 patients indicated that only 1 patient was alive with a local nodal metastasis (1% frequency of metastasis) within 1 year, and 2 others had local recurrence, all over a mean follow-up period of 6 years. The myoid, primarily myofibroblastic, phenotype of these lesions is supported by desmin, calponin, and occasional actin positivity. The occasional heavy-caldesmon and smooth muscle actin additionally suggest rare smooth muscle phenotype; however, lack of skeletal muscle markers indicate no relationship of AMFH to skeletal muscle tumors. The resemblance of these lesions to lymph nodes, clinically and morphologically, the finding of similar desmin positive cells in the adjacent lymphoid infiltrate, and the fact that 66% cases were found in sites of normal lymphoid tissue raise the possibility that some of these lesions may arise from or be related to myoid cells of lymphoid tissue. AMFH has an almost invariably benign behavior, but the 1% metastatic rate warrants its classification as low-grade “malignant.” The predominantly round cell, CD99-positive and desmin positive AMFH cases, respectively, should not be confused with Ewing's sarcoma/PNET or rhabdomyosarcoma, respectively.

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle α-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3–0.5 % of cells were found to be desmin-positive and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.

Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats

BackgroundlAims: Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-de-rived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor β subunit in a rat model of chronic cholestasis. Methods: Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor β subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver. Results: In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor β subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells. Conclusions: These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.

Role of glomerular epithelial cell-derived heat shock protein 47 in experimental lipid nephropathy

Heat shock protein 47 (hsp47) is a collagen-specific stress protein and is shown to be involved in the synthesis/assembly of various collagens as a molecular chaperone. This study was undertaken to investigate the possible role of hsp47 in dietary-induced hypercholesterolemic rat kidneys, which showed glomerular hypercellularity with expansion of mesangial matrix. Dietary-induced hypercholesterolemia was induced in male Wistar rats by giving 2% cholesterol diet for four months. Immunohistochemistry was used for localization of protein products for collagens (types I, III, and IV). alpha-smooth muscle actin, vimentin, desmin, and ED-1, a macrophage/monocyte marker, and hsp47 in control and hypercholesterolemic rat kidneys. Compared with the control, increased accumulation of collagens was accompanied with increased expression of hsp47 in hypercholesterolemic rat kidneys, with predominant expression in the glomeruli. By double immunostaining, desmin-positive glomerular epithelial cells were found to be the main source of hsp47 in hypercholesterolemic rat kidneys. From these results, it is concluded that induced expression of hsp47 by phenotypically altered glomerular epithelial cells might play a role in the excessive assembly/synthesis of collagens and could thereby contribute to the glomerulosclerosis found in dietary-induced hypercholesterolemic rat kidneys.

Morphological characteristics of lipid accumulation in liver-constituting cells of acid lipase deficiency rats (Wolman's disease model rats).

Lysosomal acid lipase is a hydrolase essential for the intracellular degradation of cholesteryl esters and triglycerides. In the laboratory, rats with congenital deficiency of lysosomal acid lipase and marked accumulation of cholesteryl ester, cholesterol free and triglyceride in livers (Wolman's disease rat or Yoshida rat) that corresponded to human Wolman's disease were found and maintained. The morphological characteristics of accumulated lipids in the livers of affected rats were examined also. Many small lipid droplets and lipid crystals were found in the cytoplasms of hepatocytes and ED1-positive and ED2-positive foamy Kupffer's cells, respectively. Electron microscopically, many electron-lucent lipid droplets with limiting membrane were found in hepatocytes. Foamy Kupffer's cells had many multivesicular bodies with limiting membrane, which contained crivilinear bodies, lipid droplets and crystal clefts. At areas of aggregation of foamy Kupffer's cells forming islets, there were many desmin-positive Ito cells. Small lipid droplets with limiting membrane were also found in the cytoplasm of Ito cells and endothelial cells. These findings, which were obtained by morphological methods, indicated that triglyceride and both cholesteryl ester and free cholesterol accumulated in lipolysosomes mainly in hepatocytes and Kupffer's cells, respectively, and suggest that lysosomal acid lipase could participate in dissolution of the membrane.

Low-Grade Endometrial Stromal Sarcoma with Intracardiac Extension: Evolution of Extensive Smooth Muscle Differentiation and Usefulness of Immunohistochemistry for Its Recognition and Distinction from Intravenous Leiomyomatosis

This case, a rare example of low-grade endometrial stroma sarcoma with extensive smooth muscle differentiation which extended to the inferior vena cava and cardiac chambers closely resembling intravenous leiomyomatosis grossly and microscopically, illustrates the importance of extensive sectioning and the usefulness of immunohistochemistry. Although spindle cell components arranged in interlacing bundles consistent with smooth muscle differentiation were recognizable in the primary tumor (on retrospective review), extensive smooth muscle differentiation in the recurrent tumors masked prototypical morphologic features of stromal sarcoma and only small neoplastic stromal components were preserved in limited areas, leading to initial failure to distinguish the lesion from intravenous leiomyomatosis. The immunophenotyping disclosed two distinct cell populations in the tumor: i.e. vimentin-positive and smooth muscle marker negative stromal cells, and vimentin-negative spindle-shaped desmin-positive smooth muscle cells. Our observation suggests that the predominance of a smooth muscle component in such a tumor can be misleading and does not always warrant a diagnosis of intravenous leiomyomatosis, nor does it predict a benign clinical course. This case also provides an insight into the relationship of the endometrial stroma and myometrium, and their cell of origin and the histogenesis of endometrial stromal sarcoma.

Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor.

Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen-coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium-forming vascular structures. Desmin-positive cells surrounded hepatocytes. Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF, and transforming growth factor-alpha (TGF-alpha) enhance these phenomena. HGF plus EGF elicited maximal response. TGF-beta1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-beta1. Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure. The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%. These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate. (HEPATOLOGY 1999;29:90-100.)

Development of approaches to improve cell survival in myoblast transfer therapy.

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle-derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.

Defect repair after somite removal in avian embryos is not true regeneration.

The question of regeneration after experimental somite extirpation has been controversial in the literature. While all workers agree that repair of the defects occurs, results concerning the extent and mechanism of this process, as well as the origin of the cells filling the defect, show great discrepancies. Our approach towards a re-examination of this question involved microsurgical removal of individual somites in 2-day-chick embryos in combination with grafting of quail somites and lateral plate. We show that the defect in the paraxial mesoderm is filled within a day after extirpation and that the reconstituting cells are derived only from the cranial and caudal somites, but not from the contralateral somites or from the lateral plate. There are no indications of an increase of proliferation in the neighbouring somites. In order to examine the differentiation capacities of the cells that fill the defect, we used immunohistochemistry and in situ-hybridization. We show that the cells in the defect are mesenchymal in morphology and express Pax-1 and Twist. There are a few desmin-positive cells in the defect that can be shown to derive from adjacent somites. An epithelial dermomyotome and myotome are absent at the operation site. Neural crest cells do not participate in the reconstitution of the defect. We conclude that cells in the defect either already have or adopt a ventral somitic (sclerotomal) identity, whereas derivatives with dorsal identity are absent from the defect except for a few individual cells.