TGF-beta in Atherosclerosis
Our first indirect evidence in support of the role of TGF-beta in atherosclerosis came from animal model studies. David Mosedale in the group demonstrated that the level of smooth muscle differentiation at different sites through the vasculature was closely correlated with the amount of active TGF-beta present (J. Cell Sci. 111:2977). Furthermore, agents that modulated TGF-beta levels (whether pharmacologic or genetic) resulted in changes in smooth muscle differentiation. We concluded that TGF-beta dynamically regulates smooth muscle differentiation in vivo. In a second series of studies in mice, we demonstrated that at least one mechanism that could be responsible for lowering TGF-beta activity at sites of atherogenesis. Extending the observations made by Rifkin’s lab and by ourselves, showing that the atherogenic lipoprotein Lp(a) could inhibit TGF-beta activation in vitro (Science 260:1655), we went on to demonstrate a similar inhibition of TGF-beta activation in the blood vessel wall in vivo (Nature 370:460).
More recently, we have identified a range of other mechanisms that could plausibly lower TGF-beta activity thereby promoting atherogenesis. For example, the protease inhibitor PAI-1, which is elevated during inflammatory reactions, can inhibit TGF-beta activation (Atherosclerosis 140:45). High levels of LDL- and VLDL cholesterol can also reduce TGF-beta activity both directly, through sequestration of the hydrophobic TGF-beta protein (J. Lipid Res. 38:2344), and indirectly through stimulated PAI-1 production.
Studies in mouse models of atherosclerosis have been consistent with the protective cytokine hypothesis; that low levels of TGF-beta do cause atherosclerosis-like lesions to develop, and that agents which elevate TGF-beta in the vasculature can reduce or prevent such lesions from forming. The first direct evidence to support the protective cytokine hypothesis came from our studies of TGF-beta knockout mice. Although the homozygous null mice die shortly after birth from a massive inflammatory response in many different organ systems, the heterozygous null mice survive into adulthood. When these mice, which have reduced levels of TGF-beta protein in their blood vessel walls, are exposed to dietary lipid challenge they develop atherosclerosis-like lesions to a much greater extent than do their wild-type littermate controls (J. Cell Sci. 113:2355). These studies demonstrate clearly that reduced levels of the protective TGF-beta signal renders the blood vessel wall more susceptible to atherogenic changes. Our original observations have now been confirmed by at least two other independent research groups (Circ. Res. 89:930, ATVB 22:975).
Less direct supporting evidence came from our extensive studies with the anticancer drug Tamoxifen. Our interest in this drug, originally identified for its anti-estrogenic properties, stemmed from its ability to stimulate TGF-beta production both in cancer cell lines and in VSMCs in culture (Biochem. J. 294:109). Based on these properties, we predicted that Tamoxifen would inhibit atherosclerosis in mice (Nature Med. 2:381). We have shown that this is correct, and that treatment with Tamoxifen completely blocks atherogenesis in a range of mouse lines, suggesting (though not conclusively proving) that elevation of TGF-beta signaling can protect against atherogenesis irrespective of the underlying molecular cause of the disease (Nature Med 1:1067 and Circulation 95:1542). These studies in mice have been replicated by others in monkeys, and early studies in man suggest that Tamoxifen may have similar beneficial properties.
Recent studies on the role of TGF-beta in atherosclerosis have focused on the role of TGF-beta in T cell function, in particular the role of TGF-beta in natural regulatory T cell activity (J. Clin. Invest. 112:1342, Nat. Med. 12:178). The anti-atherogenic effects of macrophages that have ingested apoptotic cells has also recently been a topic of prolific research activity (J. Immunol. 173:6366, Circulation 115:2168). Taken together, these recent studies suggest that deficiency in apoptotic cell clearance, leading to altered macrophage/dendritic cell function, results in decreased regulatory T cell function and accelerated atherosclerosis. TGF-beta is implicated in this process because TGF-beta1 is produced by macrophages in response to phagocytosis of apoptotic cells (J. Clin. Invest. 101:890) and is a known regulator of natural regulatory T cell activity.
As discussed in a recent review (Cardiovasc. Res. 74:213), it is clearly very difficult to carry out definitive experiments in man to prove a causal link between low TGF-beta signaling and increased risk of atherosclerosis. However, we and others, have looked for evidence for such a correlative association. Our first approach was to measure the levels of circulating TGF-beta in individuals with angiographically-proven atherosclerosis and compare them with healthy individuals. Consistent with other workers, we found that total level of circulating TGF-beta to be slightly, but significantly, higher among individuals with atherosclerosis. However, using an assay we had developed to measure active TGF-beta (that portion of the TGF-beta which is capable of binding to the type II TGF-beta receptor) (Clin. Chim. Acta 235:11), we found evidence for a striking reduction in TGF-beta activity (Nature Med. 1:74). Clearly such a reduction is consistent with the protective cytokine hypothesis in humans, but needs to be interpreted with care. Firstly, there are considerable technical difficulties associated with measuring TGF-beta activity in human biological fluids which have yet to be resolved (Biochem. J. 343:125 and Cyt. Growth Factor Rev. 11:133), and secondly it is entirely unclear whether the circulating levels of TGF-beta are likely to be associated with specific changes in the blood vessel wall.
Nevertheless, encouraged by these findings we pursued a parallel line of investigation looking at the possibility that genetic polymorphisms in the tgfb1 gene might be associated with altered TGF-beta production in vivo, and hence be associated with atherosclerosis. We and others identified a range of polymorphisms in the TGFb1 gene, and we went on to show that circulating levels of TGF-beta were under genetic control (Hum. Mol. Gen. 8:93). Whether these polymorphisms are associated with the presence of atherosclerosis remains unclear: two large studies suggested that polymorphisms associated with low TGF-beta production were associated with increased risk of atherosclerosis (consistent with the protective cytokine hypothesis) but our own large study found no evidence at all for such an association in the UK population (Clin. Sci 95:659). Further studies are required to clarify this link.
Stimulated by our attempts to ascertain whether altered TGF-beta production or activation might be involved in human atherogenesis, we have performed a number of studies examining the factors which regulate TGF-beta production by various cell types in culture, as well as examined the mechanism by which TGF-beta is activated. Despite the considerable interest in this problem for over a decade, there remains no universally accepted molecular mechanism for TGF-beta activation in vivo (Biochem. J. 350:291). This may be because TGF-beta activation occurs by a wide range of distinct processes, or because the dominant mechanism has yet to be uncovered.
One interesting study examined the release of TGF-beta from human platelets. Platelets are a major store of latent TGF-beta, and during platelet activation and degranulation, much of this TGF-beta is released. We demonstrated that platelets actually contain two distinct pools of TGF-beta: one pool, characterised by the presence of a large binding protein called LTBP, is released rapidly on degranulation, while the other pool which has no similar binding protein is bound up by the activated platelets and only released much more slowly (Nature Med. 1:932). This biphasic model for TGF-beta release from human platelets has considerable implications for the pathophysiology of wound healing and scar formation.
In summary, evidence has accumulated in support of the protective cytokine hypothesis, which states that TGF-beta, through its role in maintaining tissue homeostasis, is protective against atherosclerosis. However, more work is needed to establish if TGF-beta is dysregulated in atherosclerosis and whether modulation of TGF-beta activity represents a good therapeutic target for atherosclerosis.
Vascular lipid uptake in tgfb1+/- mice. Representative sections from the aortic sinus of wild-type mice (a,c) and tgfb1+/- mice (b,d) on a lipid-enriched diet, stained for neutral lipid with oil red O and counterstained with fast green. (Journal of Cell Science 113, 2355).