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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.

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.

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.

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 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.

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.

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.