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A case of multifocal epithelioid angiosarcoma of the femur, tibia, fibula, and astragalus in a 54-year-old man is reported. The tumor was composed of nests and cords of malignant cells with epithelioid morphology, with foci of vascular differentiation, necrosis, and hemorrhage. By immunohistochemistry, the neoplastic cells showed positivity for endothelial cell markers (CD31, CD34, factor VIII-related antigen, and Ulex europaeus agglutinin I), epithelial markers (cytokeratins and epithelial membrane antigen), and vimentin. The authors' findings point out the need for a panel of antibodies for the careful search of histologic features of vascular differentiation to correctly diagnose vascular bone tumors with epithelioid features, especially in evaluating small core biopsy specimens in which a sheetlike rather than obviously vasoformative architecture may lead to an erroneous diagnosis of metastatic carcinoma.

Matrix metalloproteinases (MMPs) have been implicated in the formation of microvessels during angiogenesis, but their role in vascular regression is poorly understood. The rat aorta model of angiogenesis was used to study the function of MMPs at different stages of the angiogenic process. Gelatin zymography and Western analysis demonstrated production of MMP-2 and MMP-9 by aortic outgrowths in serum-free collagen gel culture. MMP-2 was found in both culture medium and collagen gel, whereas MMP-9 was predominantly associated with the gel. MMP expression increased gradually during the angiogenic growth phase and stayed high when vessels regressed and collagen lysed around the aortic rings. The MMP inhibitors, batimastat and marimastat, blocked formation of microvessels when added to the culture medium at the beginning of the experiment. They, however, stabilized the microvessels and prevented vascular regression after the angiogenic growth phase. This effect was observed also under conditions of angiogenic stimulation by basic fibroblast growth factor. MMP inhibitor-mediated stabilization of microvessels was associated with inhibition of collagen lysis and accumulation of collagen fibrils in the subendothelial space. This study demonstrates that MMPs promote microvessel formation during the early stages of angiogenesis, but also contribute to the reabsorption of the neovasculature in the later stages of this process. The time-dependent divergent effects of MMPs on microvessel growth and survival may influence the in vivo activity of MMP inhibitors used to treat angiogenesis-dependent disorders.

Background: Pleural adhesions are frequently encountered in patients undergoing reduction pneumoplasty. We evaluated the impact that pleural adhesions had on the surgical technique and outcome of thoracoscopic reduction pneumoplasty., Methods: 59 operated patients were divided into 2 groups depending on the presence (group A) or absence (group B) of pleural adhesions., Results: At inter-group comparison (A versus B) a significant difference was found for mean duration of operation (128+/-55 min versus 73+/-33 min; p<0.005), morbidity (14 versus 9 patients; p<0.05), and hospital stay (14.1+/-11.8 days versus 12.0+/-7.4 days; p<0.001). Complications occurred less frequently in the last 29 patients than in the first 30 patients (11 versus 24; p<0.03). At histopathologic analysis subpleural (p<0.005) and interstitial fibrosis (p<0.001), and interstitial granulomas (p<0.012) were more frequent in group A specimens. At six months dyspnea index, six-minute-walk test, FEV1, FVC, PaO2, and prednisone and oxygen independence improved significantly in both groups. However FEV1 increased less in group A (1.20+/-0.2L vs 1.31+/-0.3L; p < 0.01)., Conclusions: Pleural adhesions may be associated with increased morbidity and less improvement in FEV1 but they do not contraindicate thoracoscopic reduction pneumoplasty.

A case of solitary fibrous tumor (SFT) of the perithyroidal soft tissue is reported. Due to its proximity to the upper aspect of the right thyroid lobe, its slow growth and its ultrasound features, the tumor was confused with a thyroid nodule. Two fine needle aspirations (FNA) gave only scant non diagnostic material. For these and cosmetic reasons together with the fact that ultrasound exam detected two solid nodules in the left lobe, a thyroidectomy was decided. Examination of the surgical specimen showed a nodule that was well demarcated and, although entirely extrathyroidal, bulged from the thyroid profile in a niche of the upper medial aspect of the right lobe. On histologic, immunohistochemical, and ultrastructural examination the tumor fulfilled the criteria for a diagnosis of solitary fibrous tumor. Thus, SFT must be considered in the differential diagnosis of suspected thyroid nodules mainly when FNA gives only scanty non diagnostic material which is unusual in thyroid hyperplastic nodules.

Background: We previously reported that the intimal endothelium of the rat aorta switches to a microvascular phenotype during angiogenesis in vitro. The microvessels formed by the rat aortic endothelium are coated with pericytes. The purpose of this study was to evaluate the relation of the pericytes to the angiogenic process and to identify the site of origin of these cells in the aortic wall., Experimental Design: Rings of rat aorta were cultured in collagen gel under serum-free conditions. The formation of a pericyte coating around aorta-derived microvessels was evaluated by counting pericytes and microvessels in the living cultures. Pericytes and endothelial cells were studied by immunohistochemistry, lectin labeling, electron microscopy, 3H-thymidine labeling followed by autoradiography, and time-lapse video microscopy. The capacity of aortic smooth muscle cells to differentiate into pericytes was studied by coculturing intimal- or medial-derived smooth muscle cells with endothelial cells in a collagen gel overlay assay that induced reorganization of endothelial cells into microvessels., Results: Microvessels during the early stages of angiogenesis were composed primarily of endothelial cells. As vascular proliferation decreased, the microvessels became coated with pericytes. The pericytes migrated from the root to the tip of the microvessels using the endothelium as a surface for attachment, proliferation, and contact guidance. The pericytes were continuous with the myointimal endothelial cells of the cultured aorta. Pericytes and myointimal cells were positive for alpha-smooth muscle actin and vimentin and were actively engaged in DNA synthesis. Treatment of the cultures with heparin caused a marked reduction in the number of pericytes. Smooth muscle cells isolated from the intimal aspect of the rat aorta migrated toward the endothelium and differentiated into pericytes when cocultured with microvessels formed by isolated endothelial cells in a collagen gel overlay assay. Conversely, smooth muscle cells isolated from the deep layers of the media had no significant endothelial tropism and failed to differentiate into pericytes., Conclusions: This study demonstrates that the rat aorta contains a subpopulation of intimal/subintimal smooth muscle cells that differentiate into pericytes during angiogenesis in vitro. These cells have a distinct endothelial tropism and respond to endothelial cues by contributing to the differentiation and maturation of microvessels. Smooth muscle cells of rat aortic intimal/subintimal origin can be used as a source of pericytes for the in vitro assembly of histotypic microvessels.

Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epitheloid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.

Background: Previous data suggest that paracrine interactions between fibroblasts and endothelial cells modulate the formation of granulation tissue during wound healing. The study of these interactions in vivo is hindered by the interference of serum and other cell types. To overcome this limitation, we developed a serum-free in vitro model in which microvessels were cocultured with fibroblasts in a 3-dimensional collagen gel., Experimental Design: Microvascular networks were obtained by culturing rat aortic endothelial cells between two layers of collagen. Microvessels were cultured in serum-free medium with or without fibroblasts embedded in the collagen. The cultures were studied by phase contrast microscopy, conventional electron microscopy, and by light and electron immunohistochemistry. The role of endothelial cells and the endothelial-derived peptide endothelin-1 in the transformation of fibroblasts into myofibroblasts was studied by evaluating fibroblast alpha-smooth muscle actin expression and collagen contraction. The angiogenic properties of fibroblasts were investigated with the rat aorta assay., Results: Microvessels cultured without fibroblasts degenerated within 3 to 4 days. Fibroblasts stabilized the microvessels greatly prolonging their life span. This effect was associated with an increased deposition of subendothelial extracellular matrix. Both fibroblasts and fibroblast-conditioned medium stimulated angiogenesis in the rat aorta assay. Endothelial cells and endothelin-1 enhanced the expression of alpha-smooth muscle actin in fibroblasts and stimulated fibroblast-mediated collagen contraction., Conclusions: This study demonstrates that endothelial-fibroblast interactions can be studied in vitro under serum-free conditions. Our results suggest that paracrine mechanisms operating between endothelial cells and fibroblasts play a key role in the formation and contraction of granulation tissue during wound healing. We propose that fibroblasts stimulate angiogenesis and stabilize the neovascular endothelium which in turn promotes the morphological and functional transformation of fibroblasts into myofibroblasts.

We describe here a modified nonenzymatic method for the isolation of rat aortic endothelial cells with vasoformative properties. Aortic rings placed on plastic or gelatin-coated surfaces generated outgrowths primarily composed of endothelial cells. Prompt removal of aortic explants after endothelial migration minimized fibroblast contamination. However, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers. This potential pitfall was avoided by weeding out fibroblasts with the rounded tip of a bent glass pipette. Primary endothelial colonies free of fibroblasts were segregated in cloning rings, trypsin-treated, and transferred to gelatin-coated dishes. Endothelial cells were cultured in MCDB 131 growth medium containing 10% fetal bovine serum, endothelial cell growth supplement, and heparin. Using this technique, pure endothelial cell strains were obtained from single aortic rings. Confluent endothelial cells formed a contact-inhibited monolayer with typical cobblestone pattern. The endothelial cells were positive for Factor VIII-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplicifolia-isolectin-B4. Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-Palade bodies and caveolae, and were connected by tight junctions. In addition, they reorganized into a network of microvascular cords and tubes when overlaid with a second layer of collagen and formed microvascular sprouts in response to fibroblast-conditioned medium. This isolation procedure yields stable strains of vasoformative endothelial cells, which can be used to study aortic endothelium-related angiogenesis and its mechanisms.

The autocrine role of basic fibroblast growth factor (bFGF) in angiogenesis was studied in the rat aortic ring-collagen gel model using serum-free culture conditions. Immunohistochemical staining of the rat aorta showed bFGF in the cytoplasm of endothelial and smooth muscle cells. Aortic rings mechanically injured during the dissection procedure released bFGF, which was demonstrated in the conditioned medium by slot and Western blot analysis. bFGF-containing aorta-conditioned medium and purified bFGF increased both the number and length of microvessels sprouting from the explants. This effect was particularly evident during the second week of culture, when the release of endogenous bFGF was minimal. Neutralizing anti-bFGF antibodies induced a 40% reduction of angiogenesis. Regression of microvessels, which regularly occurred toward the end of the second week, was prevented by purified bFGF. These data support the idea that bFGF released by vascular cells plays an important role in the autoregulation of angiogenesis after injury.

The purpose of this study was to investigate the vasoformative behavior in vitro of the native intimal endothelium of the rat aorta. To visualize the intimal surface directly, thoracic aortas were everted using a procedure that sequestered adventitial cells and possible remnant microvessels of periaortic soft tissues inside the aortic tube. Everted aortas embedded in collagen gel and cultured under serum-free conditions generated branching microvessels by a process of sprouting from the aortic intima. The newly formed microvessels originated from patches of activated intimal endothelial cells, which had survived the mechanical damage of the eversion procedure. Activated endothelial cells crawled over each other and engaged in lumen formation forming bilayers or multilayers of cells which became the source of sprouting histotypic microvessels. The endothelium of the newly formed microvessels was positive for factor VIII-related antigen and was partially surrounded by periendothelial cells which expressed alpha-smooth muscle actin. The results of this study indicate that the intimal endothelium of the rat aorta has considerable functional plasticity and can switch to a vasoformative phenotype in response to changes in the surrounding extracellular matrix environment.

The time-course of damage and repair in the bronchial epithelium of the mouse was studied morphometrically after a single inhalation of trichloroethylene (TCE). Mice were exposed for 30 min to 500, 1000, 2000, 3500, or 7000 ppm of TCE under controlled conditions and sacrificed after 2 and 24 h, and 2, 5, and 7 days. At all TCE concentrations, the damage consisted of dilation of endoplasmic reticulum and was selective for nonciliated cells throughout the bronchial tree. Damage was quantitatively evaluated by counting the percentage of vacuolated cells. Injury was maximal 24 h after exposure and was significantly (p less than 0.05) higher in bronchi with a diameter smaller than 0.4 mm (67.0% +/- 8.5%) in comparison with larger bronchi (34.0% +/- 13.2%). Cell turnover was studied by autoradiography after [3H]thymidine injection. Following TCE inhalation, the percentage of [3H]thymidine labeled cells, which in the controls was virtually zero, increased to a maximum of 13.5% +/- 6.8% (average at all doses) 48 h after treatment and then rapidly decreased to baseline values at 7 days (0.2% +/- 0.1%). Repair of some cells occurred after 24 h, and at 48 h the percentage of vacuolated cells decreased and became significantly related (p less than 0.01) to the administered TCE dose (2.4% +/- 1.4% and 21.2% +/- 6.0% average in the 500 and 7000 ppm groups, respectively). Repair of bronchial epithelium took place by both recovery of less damaged cells and proliferation of residual nonciliated cells, leading to restoration of the normal ratio between ciliated and nonciliated cells.

Three cases of peritoneal cystic mesothelioma are reported. All patients were women who had undergone previous abdominal surgery for unrelated conditions. Tumors consisted of solitary and multiple cystic masses involving the abdominal and pelvic peritoneum. The cysts focally infiltrated the muscularis externa of the small intestine in case 1, the outer muscular layer of the uterus in case 2, and the omental tissue in case 3. These findings give morphologic support to the borderline clinical behavior of this tumor that often recurs and support the hypothesis that previous surgery may play a role in its pathogenesis.

The occurrence of occult thyroid carcinoma at autopsy was examined in 507 consecutive autopsies performed over one-year in subjects without clinical evidence of thyroid cancer, from different regions of Italy, including areas of endemic goiter. We found 54 (10.65%) occult thyroid carcinomas. In 37 cases the histologic pattern was of the papillary type, with diameter ranging between 176 and 6000 microns, 12 of these cases showed a typical papillary pattern, 6 had a marked fibrosis, 2 had a cystic pattern, one showed a lymphoid stroma, and 17 had a follicular pattern. The remaining 17 cases were medullary carcinomas, with a diameter ranging from 50 to 1600 microns. The percentage of occult thyroid carcinomas reported in the present study may constitute real value of the occurrence of this tumor in the Italian population.

Foam cells, either myogenic or macrophagic, are commonly detected in experimental and human fibro-atheromatous plaques. Their role in human atherosclerosis is not yet understood. This paper reports on a preliminary autoradiographic study combined with ultrastructural observations in the human fibro-atheromatous plaque. Most of the autoradiographic silver grains appeared on foam cells and monocyte-like cells, thus suggesting a local proliferation of these cells.

We have examined, by electron microscopy, the effect of glycosylation on binding, uptake, and transport of albumin-gold complexes in capillaries of isolated perfused rat lungs. Lungs were perfused for 2 min at 4 degrees with native and glycosylated albumin-coated gold particles. After the excess probe was washed out for 5 min the lungs were fixed immediately or perfused for 30 and 60 min at 37 degrees with a isoosmotic buffer before fixation. The localization and density of the probes over different microvascular endothelial compartments were determined by electron microscopy. We found that both native and glycosylated albumin were bound to the surface of the pulmonary microvascular endothelium, preferentially over the stomatal diaphragms of luminal smooth-walled vesicles. In both BSA and glycosylated BSA perfusions at 37 degrees, most of the probe was internalized and accumulated in lysosomes and only a few gold particles were observed in the extravascular compartment even after 60 min perfusion. However, binding, internalization, and transport of the protein-gold complexes was enhanced by nonenzymatic glycosylation of albumin.

This study was undertaken to detect diagnostic ultrastructural changes in the alveolar walls in biopsies of patients with primary pulmonary hypertension (PPH). Open lung biopsies from 8 patients referred to the Hospital of the University of Pennsylvania were available for study. By light microscopy 4 patients were diagnosed as having plexogenic pulmonary arteriopathy, 2 thromboembolic arteriopathy and 2 veno-occlusive (PVOD) disease. In 4 patients the diagnosis of PPH was further confirmed at autopsy. Changes were observed in the basement membrane of the alveolar capillaries in 6 of the patients. These consisted in marked thickening and duplication of the basement membrane of alveolar capillaries in the 4 patients with the plexogenic form of pulmonary hypertension. Patients with PVOD instead showed uniform thickening of the basement membrane. No changes were detected in the 2 biopsies of patients with the thromboembolic form of PPH. We conclude that ultrastructural studies contribute significantly to the precise characterization of pathologic changes in the lungs of patients with PPH.

This work reviews the problems associated with the diagnosis of well differentiated carcinomas of the thyroid (follicular and papillary), which anatomically and clinically can show characteristics not readily distinguishable from those found in thyroid hyperplasia and adenomas. Some features of atypical adenomas and Hurthle cell tumors are detailed, in particular the borderline malignancy of the latter. We have examined the histological parameters useful in diagnosis of follicular carcinoma (cellular polymorphism and size variability with increased and atypical mitoses, invasion of the capsule and vessels, metastasis to lymph nodes and distant organs) and of papillary carcinoma (true papillae, large and crowded nuclei displaying a "ground glass" appearance with grooves and cytoplasmic inclusions and psammoma bodies). The limitations of these parameters are discussed with emphasis on frozen section examination when the lack of time limits examination to few and small tissue fragments. Invasion of the capsule and vessels is not easily detected and the "ground glass" nucleus and presence of grooves are not evident. The biological behavior of well differentiated carcinomas is discussed and, although unpredictable and variable in the single case, is generally that of a slow growing tumor. It is partially influenced by the age of the patient, size and stage of the tumor, invasion of the capsule and vessels and metastatic spread. We have observed that the biological behavior, apparently different in the follicular and papillary forms, appears identical for both tumors when a large number of cases are analyzed. Mention is made of the various surgical choices (total thyroidectomy, subtotal thyroidectomy, lobectomy, different surgical procedures which take into account the various risk factors). No statistically significant differences in recurrences and metastatic spread are obtained by electing more or less aggressive surgery with or without extensive dissection of cervical lymph nodes.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].

A group of 14 uremic patients on intermittent hemodialysis was admitted to a cross-over double-blind trial in order to evaluate serum and muscle carnitine levels before and after 60 days of L-carnitine oral (2 g/day) treatment. The morphology of muscle fragments was studied by light and electron microscopy. Symptoms (asthenia, cramps) occurring during hemodialysis were also recorded. After L-carnitine treatment the blood and muscle levels of the metabolite increased simultaneously to reduced asthenia and cramps. Morphological examination of the muscle of 13 of 14 patients did not reveal any pre- or posttreatment pathological alterations. The presence of nemaline rods was morphologically diagnosed in only one case and was no longer observed at the second biopsy at 2 months of L-carnitine treatment.

We have studied the kinetics of smooth muscle cell growth in the tunica media of human arteries obtained from 12 patients affected by obstructive vascular disease, and one case operated because of a trauma. The studies were carried out on arterial samples by autoradiography after in vitro labelling with 3H-thymidine. Different patterns of radioactive nucleotide uptake were detected. On nine elastic and muscular arteries without pathological changes it was demonstrated that the turnover of smooth muscle cells in human medias was very low, and similar to that found in experimental animals. The medias under atherosclerotic plaques showed variable levels of tritium uptake. Although the number of cases surveyed was limited, a possible explanation is that the different levels of uptake could depend on the different evolutive stages of the atherosclerotic process. It cannot, however, be excluded that the proliferative activity of the smooth muscle cells may be related to focal damages of the tunica media.

By applying at the ultrastructural level the silver methenamine impregnation method to glutaraldehyde-osmium fixed pancreatic tissue of Snell-Bagg mice it was found that two islet cell types show deposition of reduced silver on their secretory granules: B cells and another cell type. Histochemical analyses indicate that very likely the osmium dependent argentaffinity is due to S-S groups. After alkaline treatment the reactivity of B cells is lost, while that of the other cells remains unaffected. It is suggested that the third cell type stores the pancreatic somatostatin, an S-S containing polypeptide hormone.

Human arterial biopsies were studied by morphometrical, cytochemical, autoradiographic and ultrastructural methods with the aim of determining whether intimal lesions in atherosclerosis are characterized by definite cytological patterns. The results have shown: 1. a strong association between fibro-atheromatous plaques and foam and monocyte-like cells; 2. very few foam cells in diffuse intimal thickening and pearly-white fibrous plaques; 3. macrophages and foam cells were the only cytotypes consistently labelled with 3H-TdR and presumably proliferating in the fibro-atheromatous plaques.

Electron microscopy and autoradiography with 3H-thymidine have been employed to study the cellular dynamic of intimal thickening occurring in the rabbit carotid artery after selective endothelial injury. The results obtained suggest that migration of SMC of the tunica media rather than proliferation is the key event in the development of the intimal thickening.