After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2-to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-1O5MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of [35Slmethi-onine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that (g) SMCF is in fact MCP-1 and (it) MCP-1 is induced by MM-LDL.An important early event in atherogenesis is an increased recruitment of monocytes into the arterial subendothelium (1)(2)(3)(4). Previous studies have shown that endothelial cells (EC) (5, 6) and smooth muscle cells (SMC) (7,8) in culture constitutively produce a chemotactic factor that acts on monocytes but not neutrophils. Graves and colleagues (9) demonstrated that the monocyte chemotactic activity in the supernatants from a number of tumor cell lines was inhibited by an antibody made against baboon smooth muscle cell chemotactic factor (anti-SMCF) (10). Additionally they demonstrated a strong concordance with immunoprecipitation of a protein with an apparent molecular mass of 14.4 kDa. A human glioma cell line, U-1OSMG, constitutively expresses monocyte chemotactic activity. The protein responsible for this activity has been purified, sequenced, and named monocyte chemotactic protein 1 (MCP-1) (11, 12). Graves and colleagues did not test their antibody against the chemotactic activity secreted by the U-1O5MG cell line, but based on limited primary sequence data from the baboon smooth muscle chemotactic protein, they concluded that their protein was homologous to MCP-1.We have recently demonstrated that low density lipoprotein (LDL) that has been minimally modified (MM-LDL) is indistinguishable from native LDL by the LDL receptor, is not recognized by the scavenger receptor, and induces EC to secrete high levels of monocyte chemotactic activity, whereas native LDL does not (13). We have also shown that MM-LDL induces EC to produce colony-stimulating factors, including monocyte colony-stimulating factor (M-CSF), w...

Oxidized lipoproteins have been identified in atherosclerotic plaques and in early lesions in humans as well as in animals. There is accumulating evidence that such oxidized lipoproteins have an important role in atherosclerosis. Treatment of endothelial cells with altered lipoproteins stimulates monocyte binding as well as the production of chemotactic factors for monocytes. Both these findings could be relevant to the accumulation of monocytes-macrophages in the arterial wall during the early stages of lesion development. We now report that treatment of endothelial cells (EC) with modified low-density lipoproteins obtained by mild iron oxidation or by prolonged storage, results in a rapid and large induction of the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (M-CSF) and granulocyte CSF (G-CSF). These growth factors affect the differentiation, survival, proliferation, migration and metabolism of macrophages/granulocytes, and G-CSF and GM-CSF also affect the migration and proliferation of EC. Because EC and macrophages are important in the development of atherosclerosis, the expression of the CSFs by these cells could contribute to the disease.

The pathogenesis of syphilis reflects invasive properties of Treponemapallidum, but the actual mode oftissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema plangedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.The mechanisms by which Treponema pallidum subsp. pallidum causes the diverse clinical manifestations of syphilis and establishes latent infection are not well understood.

Human aortic endothelial cells (EC) and smooth muscle cells (SMC) were isolated and used to form a multilayer of EC-SMC separated by a layer of collagen. SMC and/or collagen layers exerted minimal effects on Na' transport but impeded the transport of LDL. The presence of an endothelial monolayer markedly reduced the transport of Na' and LDL. When monocytes were presented to the complete coculture, in the absence of added chemoattractant, one monocyte entered the subendothelial space for every one to three EC present. In contrast, neither collagen nor SMC plus collagen nor EC plus collagen induced comparable monocyte migration. Despite massive migration of monocytes into the coculture, no significant alteration in Na+ transport was observed. LDL transport into the preparation during massive monocyte migration increased modestly, but this was far less than the amount of LDL transported in the absence of an endothelial monolayer. We conclude that (a) the endothelial monolayer was the principal permeability barrier, (b) a substantial migration of monocytes occurred in the absence of added chemoattractant when both EC and SMC were present in the coculture, (c) endothelial barrier function was largely maintained after monocyte migration; and (d) these experiments indicate the need to study all three cell types (monocytes, EC, and SMC) together to understand the complex interactions that occur between these cells.

Minimally modified low density lipoprotein (MM-LDL), de-