Modulation of growth control mechanisms critical to atherogenesis

The principal lesion characteristic of atherosclerosis is the plaque. This lesion mainly consists of smooth muscle cells, connective matrix and large amounts of extracellular lipids. Smooth muscle cell hyperplasia is an integral event in atherosclerotic plaque formation and the resultant occlusion of blood vessels. These abnormal cell proliferations are primarly caused by defects in the autocrine and/or paracrine growth regulation. A novel mechanism critical to atherogenesis introduced in the present study is gap junctional mediated growth control. The atherogen-induced effects on gap junctional communication between human smooth muscle cells are described in chapters 2,3 and 4. The role of autocrine growth stimulation in atherogenesis is shown by the demonstration of the presence of activated transforming genes in atherosclerotic lesions (chapter 5) and the cell transforming potential of (atherogenic) low density lipoproteins (LDL) as shown in chapter 6. Chapter 5 also demonstrated that several transforming genes may be present in human plaque DNA, one of them being PDGF-A (platelet-derived growth factor chain A). The expression of the latter gene can be modulated by oxidized LDL (chapter 7).The present investigation provides additional evidence for the hypothesis that autocrine production of growth modulating factors may contribute to the characteristic abnormal smooth muscle growth in atherogenesis. We demonstrated that lipoproteins with atherogenic potential, such as LDL, in contrast to the non-atherogenic HDL, are able to transform fibroblasts in vitro. Genetical alteration of intimal cells present in atherosclerotic lesions was demonstrated using DNA-mediated transfection techniques. It could be demonstrated that human aortic plaque DNA contains active transforming genes, as PDGF-A. This is in agreement with studies by Penn et al. (1986) and Ahmed et al. (1990), showing transforming genes in human coronary artery plaques. Even DNA of cultured human atherosclerotic plaque cells appeared to have cell transforming capacities (Parkes et al., 1991). Only Yew et al. (1989) could not demonstrate a transforming potential of human plaque DNA. The variable results obtained sofar might indicate that different mechanisms are involved. It should be stressed in this respect that in all these studies pooled plaque samples were used and it should be realized that the transforming potential is not necessarily present in each plaque. The observed transforming potential of plaque DNA supports very well the monoclonal hypothesis of Benditt and Benditt (1973). They observed that atherosclerotic lesions start as singular focal masses containing monoclonality and suggested that these masses were in general similar to benign tumors as observed in other tissues. However, it has been argued that apparent monoclonality could also arise because of strong selection pressure favoring a subpopulation of cells, although findings of transforming genes, such as PDGF-A, present in atherosclerotic lesions and the transforming potential of atherogens indicate the opposite. Additional arguments in favor of the monoclonal hypothesis are that tumor initiators like chemical mutagens and promutagens (Albert et al., 1975; Bond et al., 1981), radiation (Gold, 1961) and oncogenic viruses (Minick et al., 1979) can induce atherosclerotic lesions in laboratory animals. Hence, molecular alterations underlying the proliferation of smooth muscle cells could show resemblance to the molecular events, which are critical in the development of cancer.The current view on atherosclerosis is based on the "response to injury" hypothesis, implying a paracrine stimulation of smooth muscle growth as a result of injury of endothelium (Ross, et al., 1976; Ross, 1981). Most of the studies of experimental atherogenesis are implemented in the framework with this hypothetical model. Support for this hypothesis is based on the following observations. Atherosclerotic lesions can be induced in experimental animals by endothelial denudation and factors derived from platelets can induce smooth muscle growth in vitro. Injury to arterial endothelium by mechanical, chemical, toxic, viral, or immunological agents caused endothelial denudation, and was followed by platelet adhesion and aggregation, with consequent release of platelet-derived growth factors (PDGF), in turn leading to migration into and proliferation of smooth muscle cell in the intima and secretion of connective tissue components. Over the years, the hypothesis has been modified (Ross, 1990). First of all, it became apparent that actual denudation was not a consistent early feature of atherosclerosis (Davies et al., 1976). Secondly, platelet adherence is neither necessary nor sufficient to cause the lesions (Schwartz and Reidy, 1987). Presently, the view is: a. the endothelium can respond to a variety of stimuli, by subtle changes in function (endothelial dysfunction) and/or by the induction of new endothelium properties; thus non- denuding injury may be important in initiating the lesions of atherosclerosis and b. platelets are not the sole initiators of smooth muscle proliferation, since growth promoting and growth inhibitory factors secreted by other cell types, including macrophages and endothelial cells, may modulate smooth muscle growth.However, not all observations made can be explained by the "response to injury" hypothesis, including the findings that (intimal) smooth muscle cells themselves can secrete and can respond to growth modulating factors and the cell transforming potential of plaque DNA as demonstrated above, thus implying an involvement of autocrine growth stimuli. A good candidate for autocrine growth factor stimulation of smooth muscle cells is PDGF-A. High transcript levels of this transforming gene were detected in intimal cells of human atherosclerotic lesions (Libby et al., 1988; Wilcox et al., 1988). The present study showed that PDGF-A was identified as one of the transforming genes present in human plaque DNA and the expression of this gene can be induced by LDL (oxidized). HDL, a protective lipoprotein to atherosclerosis, did not modulate PDGF-A transcript levels in smooth muscle cells. While both oxidized LDL as well as PDGF-A transcripts are detected in atherosclerotic lesions this molecular mechanism could play a role in atherogenesis.Furthermore, the present study provides strong evidence that disturbance of intercellular communication is another mechanism, which is probably important in the etiology of atherosclerosis. It is shown that compounds with atherogenic potential inhibit communication between human smooth muscle cells. The potency to modulate cell-cell communication correlates well with their atherogenic potential. For example, low density lipoproteins (LDL) in oxidized form and cholesterol oxidation products inhibited cell-cell communication. A non-atherogenic lipoprotein as high density lipoprotein (HDL) did not influence gap junctional communication between human smooth muscle cells. Otherwise, growth factors like PDGF and EGF can modulate cell-to-cell communication (Maldonado et al., 1988; Madhukar et al., 1989). This would imply that both paracrine and autocrine growth factor production are involved in atherogenesis, in which their influence on the gap junctional mediated growth control mechanism is a vital step in atherogenesis. At this stage, disruption of intercellular communication results in an uninhibited multiplication of these cells leading to the formation of (monoclonal) atherosclerotic lesions.The main conclusion of the present study is that the three phenomena 1) production of growth modulating factors by vascular cells, including endothelium, monocytes, platelets and smooth muscle cells 2) DNA modification (e.g. PDGF-A) of smooth muscle cells 3) loss of intercellular communication together are important factors in the process of atherosclerosis. The two main theories on atherosclerosis, the "monoclonal" and the "response to injury" theory, are compatible in many respects and can be fit into one unifying hypothesis as described in Fig. 8.1The figure shows that one of the earliest events in atherosclerosis is an increased adhesion of monocytes to what appears to be intact arterial endothelium, a phenomenon which can be well demonstrated using scanning electron microscopy (Faggiotto et al., 1984). In this process lipid factors like hypercholesteraemia and other factors as IL-1 or TNF (which introduces adhesion molecules. on endothelial cells) are important triggers. As a consequence of monocyte emigration, there is focally increased permeability to LDL and macromolecules (Territo et al., 1984; Gerrity et al., 1979). An altered endothelial function (dysfunction) causing increased permeability may also be induced by risk factors such as hypertension, hyperlipidaemia, smoking, immunological factors, stress and diabetes mellitus (Reidy, 1985; Sieffert et al., 1981; Gordon et al., 1981, Munro and Cotran, 1988). In this way the agents present in the bloodstream (such as LDL) accumulate and are modified in the arterial wall and thereafter exert their effects locally on vascular cells.The next important step could be DNA modification(s) of smooth muscle cells as shown by the presence of transforming genes, as PDGF-A, in lesion DNA and the effects of the atherogenic LDL on cell transformation. This defect of an autocrine growth regulation is followed by another vital growth control mechanism, the gap junctional communication. Communication between human smooth muscle cells has been modified by several atherogens, like LDL (oxidized) and oxysterols. In case of impairement of both growth regulation mechanisms clonal growth may occur.The processes described on the left side of the scheme are consistent with the paracrine proliferation mechanism of "the response to injury" theory of Ross (1990). The first trigger in this process is a dysfunction of the endothelium. This process introduces the adhesion and aggregation of platelets and/or monocytes to the endothelium, which results in a release of growth modulating factors as platelet-derived growth factor. At this stage, paracrine growth stimulating factors do not yet cause abnormal smooth muscle growth, because autonomous growth of cells still is controlled by gap junctional communication with surrounding cells. Only when growth factors (and atherogens) inhibit gap junctional communication abnormal smooth muscle cell proliferation occurs. Thus, inhibition of gap junctional intercellular communication could play a predominant role in the onset of atherogenesis.A better understanding of these growth modulating processess will improve the possibility to study the possible atherogenic properties of chemicals within the framework of toxicological research.