Modulation of gap junctional intercellular communication between human smooth muscle cells by leukocyte-derived growth factors and cytokines in relation to atherogenesis

In this thesis, the effect of leukocyte-derived growth factors and cytokines on GJIC between SMC was investigated. GJIC is regarded as an important mechanism in the control of cell growth, cell differentiation and tissue homeostasis. Disturbance of SMC growth control is regarded to be a key event in the pathogenesis of atherosclerosis in which growth factors and cytokines are thought to play a central role. In the present study, cultured human SMC were incubated with (human) recombinant growth factors and cytokines. TNFα, IFN-γ, PDGF, bFGF and IL-6 were chosen as representatives of several classes of growth modulating factors. These growth factors and cytokines are known to be products of macrophages and/or T lymphocytes and have been detected in human atherosclerotic lesions. After an incubation period, GJIC between SMC was measured. In addition, human SMC were co-cultured with J774A.1 murine macrophages or human monocyte-macrophages in the Transwell-COL cell culture system, to account for the complexity of macrophage secretion patterns. After removal of the macrophages, GJIC between the co-cultured SMC was determined.The experiments described in <strong>chapter 2 and 3</strong> clearly demonstrate that all factors tested reduced GJIC between SMC with ~20 - 50%, except for bFGF which strongly increased GJIC. Furthermore, these experiments revealed that effects of growth factors and cytokines on GJIC are not univocal and thus cannot be generalized. PDGF, IL-6 and bFGF caused transient effects on GJIC, whereas in experiments with TNFαor IFN-γ, a persistent inhibition of GJlC was obtained.The most remarkable result of the study described in <strong>chapter 4</strong> was that upon combining TNFα and IFN-γ, GJIC between SMC strongly reduced (up to 86%) in an additive or synergistic manner. Upon long term incubation with the combination of TNFα and IFN-γ, some SMC did not communicate with neighbouring cells at all. This may result in an escape from growth control mechanisms, which, in turn, may lead to disturbance of SMC proliferation, a key event in atherosclerosis. In incubations with other combinations of growth factors and cytokines, (antagonistic) interactive effects on GJIC were observed.The present investigation provided evidence that reactive oxygen species may play a role in cytokine-induced inhibition of GJIC between SMC. Experiments described in <strong>chapter 2</strong> revealed that pretreatment of SMC with antioxidants like ascorbic acid, α-tocopherol or GSH prevented the inhibition of GJIC upon exposure of SMC to TNFα. Studies with SOD <strong>(chapter 4)</strong><TT></TT>demonstrated that the superoxide radical may be involved in GJIC reduction by TNFα, since incubation with SOD, even hours after the addition of TNFα, restored GJIC to control values. Furthermore, SOD partly restored IFN-γeffects on GJIC in the short - but not in the long term. When SMC were incubated with TNFα and IFN-γsimultaneously for 24 h, high levels of SOD could not even partly counteract the strong inhibition of GJIC caused by these cytokines. Thus, other, superoxide-unrelated mechanisms may affect GJIC more predominantly in long term incubations with the combination of TNFα, and IFN-γ, One such mechanism may be represented by the reduced Cx43 staining which was observed in immunofluorescence studies on SMC cultures incubated with these cytokines <strong>(chapter 4)</strong> ,<TT></TT>which may be an indication for the reduced presence of functional gap junction channels.PDGF-AA, PDGF-BB, IL-6, IFN-γ, TNFαand bFGF all stimulated SMC proliferation in our cell culture system, as individual factors as well as in combinations <strong>(chapter 3 and 4).</strong> Upon comparing these cell proliferation results with GJIC data, a complex relationship between modulation of GJIC, cell proliferation and the process of atherosclerosis is suggested.Experiments described in <strong>chapter 5</strong> demonstrated that macrophages cultured on pore membrane inserts modulate GJIC between SMC co-cultured in Transwell-COL cell culture chambers. Since these results were obtained in an indirect co-culture system which prevents direct cell-cell contact, it was hypothesized that soluble factors, released by macrophages, may be involved in the modulation of GJIC between SMC. At this moment, one can only speculate about the nature of the factors involved in this macrophage-dependent modulation of GJIC. The results clearly indicate that the source and activation state of macrophages were of importance in these co-culture experiments. Therefore, further research should be aimed at studying the effect of different types of macrophages on GJIC between co-cultured SMC. Heterogeneity in atheroma macrophages exists; the most noticeable difference being the presence of 'normal' macrophages and the presence of macrophage-derived foam cells, which are likely to differ in endocytic and secretory repertoire. Furthermore, macrophages should be exposed to different (patho-)physiological agents with relevance to the process of atherosclerosis, in order to study their effects on GJIC between SMC in even more detail.The present study provides a good starting point for further research aimed at the understanding of mechanisms by which enviromnental contaminants or drugs might interfere with atherogenesis. It is already known that widespread food chain and cigarette smoke contaminants like for example benzo[a]pyrene, polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo- <em>p</em> -dioxin may affect the pathogenesis of atherosclerosis in several ways, for instance by damaging SMC DNA, disrupting endothelial barrier function, or by modulation of plasma cholesterol and lipoprotein levels). Furthermore, chemicals like components in cigarette smoke condensate may modulate GJIC between SMC. Considering that growth factors and cytokines like TNFα, and IFN-γ, may have marked effects on GJIC, one may assume that enviromnental contaminants and drugs capable of affecting the expression of growth factors and cytokines or their receptors may interfere with GJIC in an indirect manner. In the case of atherogenesis for instance, chemicals may stimulate growth factor- and cytokine production by SMC and/or macrophages which, in turn, may influence homologous GJIC between SMC. In addition, exogenous chemicals may influence heterologous GJIC between macrophages and SMC as well; either directly, or indirectly via the induction of growth factor and cytokine expression by these cells. As a consequence, macrophage-derived reactive substances will have more - or just less- impact on SMC functioning.Relatively short exposures to enviromnental contaminants or drugs in individuals in which plaques have already passed some critical phases in the atherosclerotic process might enhance the severity of the lesions. Further research along this line may also lead to the identification of nutritional or chemical factors that may have beneficial (protectiveregressive) effects on the development of atherosclerotic lesions.Modulation of GJIC by growth factors and cytokines may affect a response-to-injury. On the other hand, modulation of GJIC may also play a role in the monoclonal expansion of cells. Therefore, the response-to-injury hypothesis and the monoclonal theory may be compatible is some respects, as was previously suggested by Zwijsen.The results of the present study may also be applicable to other pathophysiological phenomena, in which growth factors and cytokines may play a prominent role in the onset or progression of the disease. Proliferative diseases like pulmonary fibrosis, glomerulosclerosis and liver cirrhosis share some pathobiologic mechanisms with atherosclerosis, including leukocyte infiltration, mesenchymal cell proliferation and enhanced matrix synthesis. Leukocyte-derived growth factors and cytokines may modulate GJIC between the mesenchymal cells concerned, which in turn may result in abnormal cell proliferation. It is known that certain chemicals may contribute to the development of these diseases. The mechanisms by which these chemicals act may be linked to the processes studied and discussed in this thesis.Overall, the information presented in this thesis concerning the possible role of growth factors and cytokines in the pathophysiology of atherosclerosis provides a useful instrument to study possible modulatory effects of chemicals on the process of atherosclerosis via the mechanisms mentioned above.