Atherosclerosis goes to the wall atherosclerosis research For many years concentrated on the role of circulating lipids, with less attention being paid to the cellular abnormalities of lesions in the arterial wall. A conferencei held in Colorado, USA, in January illustrated how the balance has been redressed, largely because atherosclerosis has benefited from the heat of research into cellular communication by cytokines, growth factors, and adhesion molecules. That research, in turn, depended heavily on the power of molecular
biological techniques. This trend owes much to Russell Ross, whose work in Seattle has indicated how platelet-derived growth factor (PDGF) causes arterial smooth muscle cells to proliferate. Such proliferation contributes to the arterial wall thickening in atherosclerosis that leads to ischaemic disease.2 The potency of growth factors in causing proliferation can be shown by transfecting cells of an artery wall in vivo with growth factor genes, as has been achieved by Nabel et al. These researchers found that PDGF-B, acidic fibroblast growth factor, and transforming growth factor beta were all active. Macrophages may well be the most important cells in atherosclerosis. Derived from blood monocytes, they are prominent in all stages of the disease; they are not present in normal arterial wall. Although best known for phagocytosis, macrophages are also secretory cells and can produce PDGF.Two new macrophage-derived cytokines were discussed in Colorado. Heparin-binding epidermal growth factor, unlike the original epidermal growth factor, is a potent stimulant of smooth muscle cell growths and therefore may promote thickening of the arterial wall, like PDGF. Monocyte chemoattractant protein,66 a member of an extensive group of low molecular weight cytokinesis synthesised by endothelial cells and macrophages8 and attracts blood monocytes to the site of its production. Fogelman’s group6 found that if human aortic endothelial cells were co-cultured with smooth muscle cells and exposed to low-density lipoprotein (LDL) there was mild oxidation of the lipoprotein, and monocytes migrated into the subendothelial space. Migration is due to production of monocyte chemoattractant protein and can be
high-density lipoprotein (HDL)
antioxidants. These inhibitory properties may help to explain the epidemiological evidence for a protective action of both HDL9 and antioxidants1O against atherosclerosis. Monocytes also gain entry to tissues via expression of adhesion molecules on the endothelial cells of inflamed vessels." These molecules are induced by cytokines from other cells, especially by interleukin-1I (IL-1) and tumour necrosis factor from macrophages. Monocytes bind to intercellular adhesion molecule-1I (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelia, via their beta-2 integrin and VLA-4 receptors, respectively. VCAM-1 has been found in rabbit atherosclerotic lesions;13,14 in human beings, by contrast, ICAM-1 is increased in the endothelium and in other cells of atherosclerotic plaques but little VCAM-1 can be detected.15,16 Analysis of the promoter regions for the genes of these molecules may aid understanding of their control 17 and perhaps of these species differences. The findings suggest that, once a plaque has become established, its growth may be self-promoting, further entry of monocytes being encouraged by positive feedback. This theory could explain the characteristic growth of atherosclerosis in localised plaques.16 How do macrophage processes and lipids interrelate? Oxidation of LDL allows uptake of lipids by macrophages because they bear the scavenger receptor that binds oxidised LDL. In this way macrophages are converted into the large foamy cells found in plaques. Oxidised LDL has been detected in atherosclerotic plaques, and macrophages themselves are capable of oxidising extracellular LDL
extensively.7 In view of the secretory activity of macrophages, it is important to determine the nature of the inciting stimulus. The scavenger receptor, when occupied, may be able to stimulate macrophages, but there are other stimulatory factors such as macrophage colony stimulating factor present in the lesions.18 In addition to cytokine secretion, atherosclerosis macrophages are activated to produce c-fos and apo-lipoprotein E, but it is not yet clear to what extent inflammatory cytokines such as IL-1and tumour necrosis factor are induced. 1920 Since endothelial cells in vitro can synthesise adhesion molecules in response to scavenger receptor ligands/1 thrombin, 22 and activated platelets/3 other mechanisms might stimulate expression of these molecules. What about thrombosis? The effects of varying the concentrations of plasminogen activator inhibitor-1 were studied in transgenic mice which had been transfected with the gene coupled to a promoter that enhanced its expression.24 The development of spontaneous thrombosis in the mice signifies the importance of this molecule in coagulation. Studies such as these give us some idea of the molecular machinery that creates an atherosclerotic plaque. We now know that the building blocks are the same as those in a conventional inflammatory
constructed in different ways and the mechanism is driven by lipids in a manner that we are now starting to understand. response, but
Keystone symposia 1992; 16A: 1-58.
2. Ross R. The
molecular and cellular
pathogenesis of atherosclerosis:
biology. J Cell Biochem
update. N Engl J Med
1986; 314: 488-500. 3. Nabel GJ, Yang Z, Derynck R, Haudenschild C, Maciag T, Nabel EG.
Analysis of vessel function by direct
gene transfer in vivo. J Cell Biochem 1992; 16A: A005. 4. Ross R, Masuda J, Raines EW, et al. Localisation of PDGF-B protein in all stages of atheroscerlosis. Science 1990; 248: 1009-12. 5. Klagsbrun M, Marikovsky M, Abraham J, Thompson S, Damm D, Higashiyama S. Heparin-binding EGF-like growth factor, structural and biological properties. J Cell Biochem 1992; 16A: A013. 6. Schall TJ. Biology of the RANTES/SIS cytokine family. Cytokine 1991; 3: 165-83. 7. Navab M, Imes SS, Hough GP, et al. Monocyte transmigration induced by modification of low density lipoprotein in co-cultures of human aortic wall cell is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Cell Biochem 1992; 16A: A008. 8. Takeya M, Yoshimura T, Leonard EJ, Takahaski K. The role of the monocyte chemoattractant protein-1 (MCP-1) in atherosclerosis: immunohistochemical detection in human atherosclerotic lesions by an anti-MCP-1 monoclonal antibody. J Cell Biochem 1992; 16A: A322. 9. Gordon DJ, Rifkind BN. High density lipoprotein: the clinical implications of recent studies. N Engl J Med 1989; 321: 1311-16. 10. Riemersma RA, Wood DA, MacIntyre CCA, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentrations of vitamins A, C, and E and carotene. Lancet 1991; 337: 1-5. 11. Pober JS. Cytokine activation of vascular endothelium: physiology and pathology. Am J Pathol 1988; 133: 426-33. 12. Harlan J, Carlos T, Kovach N, et al. Mononuclear leukocyte binding to endothelium. J Cell Biochem 1992; 16A: A006. 13. Cybulsky MI, Gimbrone MA. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 1991; 251: 788-91. 14. Cybulsky MI, Kume N, Collins T, Gimbrone MA. Expression of vascular cell adhesion molecule-1 by endothelial cells during atherogenesis. J Cell Biochem 1992; 16A: A002. 15. Poston RN, Johnson-Tidey RR, Coucher JR, Gall NP. Expression of the adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in normal and atherosclerotic human arteries. J Cell Biochem 1992; 16A: A218. 16. Poston RN, Haskard DO, Coucher JR, Gall NP, Johnson-Tidey RR. Expression of intercellular adhesion molecule-1 in atherosclerotic
plaques. Am J Pathol 1992; 140: 665-73. T, Williams AJ, Neish AS, et al. Transcriptional control of the ELAM-1 and VCAM-1 genes. J Cell Biochem 1992; 16A: A004. Clinton SK, Schaub RG, Kufe DW, Libby P. Macrophage-colony stimulating factor (M-CSF) gene expression in vascular cells and in experimental and human atherosclerosis. J Cell Biochem 1992; 16A:
17. Collins 18.
Libby P, Clinton SK. Possible roles for cytokines in atherogenesis. J Cell Biochem 1992; 16A: A003. 20. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Tumor necrosis factor expression in human vascular smooth muscle cells detected by in situ hybridisation. Am J Pathol 1990; 137: 503-09. 21. Palkama T, Mattila P, Majuri M-L, Hurme M, Renkoven R. Endothelial adhesion molecules involved in monocyte extravasation are regulated via scavenger receptor. J Cell Biochem 1992; 16A: CA315. 22. Bender JR, Sadeghi MMM, Mills LK, Pardi R, Watson C. Endothelial cell adhesion molecule introduction by &agr;-thrombin. J Cell Biochem 19.
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Hawrylowicz CM, Howells G, Feldmann M. Platelet derived IL-1 modulates endothelial function. J Cell Biochem 1992; 16A: CA106. 24. Loskutoff DJ, Keeton M, Sawdey M, Eguchi Y, Schneiderman J. Regulation of PAI-1 gene expression m the vascular wall. J Cell 23.
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Peripheral stem cells made to work Most drugs used in the treatment of cancer kill dividing normal cells in other tissues. This drawback is especially troublesome (and often dose-limiting) in the bone marrow, and may lead to life-threatening neutropenia, thrombocytopenia, and anaemia. One
way to overcome such difficulties is to collect and cryopreserve the patient’s own marrow before giving
further intensive chemotherapy at potentially curative doses. The bone marrow contains stem cells with unlimited self-renewal capacity; these very primitive cells can generate progenitor cells, which amplify and mature to form the cells that are released in the peripheral blood. Production of mature blood cells is mediated by various endogenous haemopoietic growth factors. After further treatment of the patient with potentially marrow ablative chemotherapy, the cryopreserved bone marrow is reinfused. The haemopoietic cells migrate to the bone marrow, reconstitute haemopoiesis, and lead to regeneration of peripheral blood neutrophils in 3 weeks and of platelets by 4-6 weeks. Despite intensive support with antibiotics and blood products during the panycytopenic phase, infection and bleeding are still important causes of mortality and morbidity in patients undergoing autologous bone marrow transplantation. In this issue (p 640) Sheridan et al describe how the period of dangerous neutropenia and especially thrombocytopenia can be shortened if bone marrow cells are supplemented with peripheral blood cells collected before transplantation and mobilised by haemopoietic growth factor treatment.
Peripheral blood normally contains very few Treatment with haemopoietic progenitors. recombinant haemopoietic growth factors (granulocyte colony stimulating factor [G-CSF], filgrastim; granulocyte-macrophage colony factor stimulating [GM-CSF], ecogramostim) only modestly increases cell division in normal marrow, mainly affecting late progenitor cells,12 but promotes circulation of abundant peripheral blood stem cells (PBSC) as well as of committed progenitor cells that produce erythrocytes, neutrophils, and platelets.3PBSC can also increase during marrow recovery after treatment with cytotoxic agents such as cyclophosphamide.4 Numbers of PBSC are greatly enhanced if cyclophosphamide treatment is followed by an infusion of haemopoietic growth factor.5 The mechanisms underlying stem cell release from bone marrow are unknown but presumably involve disruption of the controls that normally impede the release of progenitors. What is the quality of these PBSC and are they as effective at haemopoietic reconstitution as marrow stem cells? In animal studies PBSC (recruited under the influence of G-CSF) transplanted into syngeneic irradiated recipients produce long-term lymphomyelopoietic regeneration, as confirmed by the presence of genetic markers in mature cells in blood.6In this case, PBSC seem to be as good as marrow stem cells. Human PBSC mobilised by G-CSF have been tested in an in-vitro system in which they are seeded onto preformed previously irradiated allogeneic stroma. In the two-stage long-