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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.

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.

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.