Localisation of mRNA for JE/MCP-1 and its receptor CCR2 in atherosclerotic lesions of the ApoE knockout mouse.

MCP-1 has potent chemotactic activity for monocytes and is strongly implicated in the pathogenesis of atherosclerosis. In the present study, we have used in situ hybridisation to examine the gene expression of JE, the murine homologue of MCP-1, and its receptor, CCR2, during the development of atherosclerotic lesions in the ApoE knockout mouse. Interestingly, the earliest expression of JE detected during lesion development was found to be localised in mesenchymal cells in the adventitia and not in the intima. Macrophages were subsequently found to accumulate in these affected regions of the adventitia and these cells were found to express high levels of JE. At this stage, early macrophage-rich lesions with high expression of JE were also seen in the intima, but expression of mRNA for the receptor for JE (CCR2) was only found on adventitial macrophages and not in the intima. This sequence of events suggests that adventitial inflammation may be an important early event in lesion development and responsible for the subsequent accumulation of macrophages in the intima possibly by recruitment from the adventitia as well as via the vessel lumen.

Induced expression of adipophilin mRNA in human macrophages stimulated with oxidized low-density lipoprotein and in atherosclerotic lesions.

Oxidized low-density lipoprotein (OxLDL) plays a critical role in foam cell formation and atherosclerogenesis. A cDNA encoding adipophilin was identified in cultured human macrophages stimulated with OxLDL using mRNA differential display. Adipophilin is a 50 kDa protein known to be a specific marker for adipocyte cell differentiation and lipid accumulation in a variety of cells. The time-dependent induction of adipophilin mRNA in macrophages was specific to OxLDL but not native LDL, and not to various cytokines and serum. In human atherosclerotic lesions, adipophilin mRNA expression was localized in a subset of lipid-rich macrophages. These data suggest that adipophilin-expressing macrophages may represent foam cells and this gene expression is likely to be associated with the lipid accumulation in foam cells of the atherosclerotic lesions.

Chemokines and atherosclerosis.

Chemokines or chemotactic cytokines represent an expanding family of structurally related small molecular weight proteins, recognised as being responsible for leukocyte trafficking and activation. Soon after the discovery of this class of cytokines, about a decade ago, monocyte chemoattractant protein-1 (MCP-1) was found to be highly expressed in human atherosclerotic lesions and postulated to be central in monocyte recruitment into the arterial wall and developing lesions. In this review, we will discuss our present knowledge about MCP-1 and its receptor CCR2 and their role in atherogenesis. Although less well established, other chemokines such as RANTES, MIP-1alpha and MIP-1beta have also been implicated in atherosclerotic lesion formation as are a number of more recently discovered chemokines like MCP-4, ELC and PARC. The role of these chemokines in the progression of atherosclerosis will be discussed as well as the emerging role of IL-8, mostly know for its effects on neutrophils. Particular attention will be given not only to the involvement of chemokines in the inflammatory recruitment of monocytes/macrophages, but also to their role in the related local immune responses and vascular remodelling which occur during the formation of unstable atherosclerotic plaques.

Expression and cellular localization of the CC chemokines PARC and ELC in human atherosclerotic plaques.

Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.

Detection and cellular localization of heparin-binding epidermal growth factor-like growth factor mRNA and protein in human atherosclerotic tissue.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family which binds to and activates the epidermal growth factor (EGF) receptor. HB-EGF mRNA is expressed by monocytes and vascular smooth muscle cells (VSMC) in culture, and has been shown to be a potent VSMC mitogen in vitro. The aim of this study was to screen normal and human atherosclerotic arteries and SMC cultured from these arteries for expression of HB-EGF, and to determine its cellular localization in human lesions. Using the highly sensitive technique of reverse transcription polymerase chain reaction (RT-PCR), we screened biopsies taken from normal human vessel walls and atherosclerotic tissue, for expression of HB-EGF mRNA. Northern blotting and RT-PCR were employed to determine levels of HB-EGF gene expression in SMC, cultured from normal and atherosclerotic arteries. Cellular localization of mRNA and protein, within human atherosclerotic plaques, was assessed using in situ hybridization with 35S labelled riboprobes, and immunohistochemistry with polyclonal antibodies specific for human HB-EGF. HB-EGF mRNA was found to be expressed in human atherosclerotic lesions and in VSMC cultured from these lesions. Expression of HB-EGF could not be detected in quiescent aortic VSMC using Northern blotting, but was highly up-regulated in these cells after treatment with basic fibroblast growth factor (bFGF) for 24 h. Although HB-EGF mRNA was detected in all vascular tissue examined using RT-PCR, in situ hybridization and immunohistochemistry revealed expression of HB-EGF in small portions of diseased arteries only. Immunohistochemistry showed strong staining for macrophages in all areas of HB-EGF expression. No association of HB-EGF with SMC was observed in any of the specimens examined. In conclusion, HB-EGF, a potent mitogen for VSMC, is expressed by macrophages in human.

IGF-I increases bFGF-induced mitogenesis and upregulates FGFR-1 in rabbit vascular smooth muscle cells.

Insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) have both been implicated in the abnormal proliferation of vascular smooth muscle cells (VSMC) that occurs after injury to the arterial wall in vivo. We have investigated the effects of these growth factors on proliferation of rabbit aortic smooth muscle cells (RASMC) in vitro. IGF-I, in contrast to bFGF, is a weak mitogen for RASMC. However, when IGF-I (10 ng/ml) was added in combination with bFGF for 24 h, the effect of the two growth factors on DNA synthesis was synergistic at all concentrations tested (P > 0.001 compared with summed values of bFGF alone plus IGF-I alone), and this synergy was also observed at the level of RASMC proliferation (P < 0.001). Time-course experiments indicated that although bFGF was able to stimulate DNA synthesis after 16 h, activity peaked at 24 h, and a synergistic response with IGF-I was not observed before 24 h. Northern blot analysis demonstrated that IGF-I (10 ng/ml) could selectively upregulate fibroblast growth factor receptor-1 (FGFR-1) mRNA 4.0 +/- 0.24-fold (P < 0.001) without a significant effect on FGFR-2, and this induction in FGFR-1 mRNA occurs in a time- and dose-dependent manner. In addition, IGF-I increases FGFR-1 protein levels in RASMC 2.7 +/- 0.12-fold (P < 0.01), as demonstrated by Western blotting, and this upregulation occurs before the peak in DNA synthesis. These results suggest that IGF-I may be capable of increasing the responsiveness of VSMC to bFGF through modulation of FGFR-1.

Inhibition of insulin production by cyproheptadine in RINm5F rat insulinoma cells.

The clonal insulin producing cell line RINm5F was evaluated as a model for the action of cyproheptadine (CPH)-like diabetogenic compounds in the rat pancreas. Treatment with 10 microM CPH and selected structural analogs under culture conditions produced a progressive loss of cellular insulin which reached 30% of control within 24 hours. Comparison of the activities of the analogs 4-diphenylmethylpiperidine (4-DPMP) and 2-diphenylmethylpiperidine (2-DPMP) to produce cellular insulin depletion showed that 4-DPMP was as active as CPH but 2-DPMP had no activity at the highest concentration employed (10 microM). The CPH metabolite desmethyl CPH-epoxide was five times more active than the parent compound in producing loss of insulin in RINm5F cells. These results are consistent with previously published results of CPH actions in vivo. An inhibition of insulin biosynthesis with no loss of preproinsulin mRNA occurred in RINm5F cells treated with CPH or DMCPH-epoxide. This suggests that an effect on transcription may not be the primary action by which CPH and its analogs inhibit insulin synthesis in vivo.

Enzyme induction in recovering dauer larvae of the nematode Caenorhabditis elegans in response to increasing concentrations of food source in the recovery medium.

Exposure of recovering dauer larvae of Caenorhabditis elegans to increasing concentrations of Escherichia coli in the recovery medium produced dramatic increases in the enzymes of intermediary metabolism. There was no significant difference between the rates of development of recovering dauer larvae grown on different concentrations of E. coli. When the activity of several key enzymes was assayed after 12, 22 and 32 hours of recovery in 0.5% w/v E. coli it was found that the activities recorded never reached levels observed at 12 hours for larvae grown on the optimum concentration of E. coli. These results imply that enzymes of intermediary metabolism in the nematode C. elegans are capable of being induced in response to changes in nutrient intake, as previously described for mammals and microorganisms.