Transforming Growth Factor-β

• atherosclerosis
• inflammation
• fibrosis
• transforming growth factor-β
• unstable plaque

Atherosclerosis is mainly considered to be a chronic inflammatory disease.^1,2 The importance of inflammatory mediators in the initiation and progression of atherosclerosis is reflected by the composition of the atherosclerotic lesion and by many intervention studies in mouse models of atherosclerosis. Activated macrophages and T-lymphocytes are already observed in fatty streak lesions,³ and the contribution of inflammatory cells and mediators increases when the atherosclerotic lesion progresses.⁴ Furthermore, intervention studies in atherosclerotic mouse models that inhibit major (anti)inflammatory mediators such as CD40L,^5–9 gm-CSF,^10,11 MCP1,¹² IFNγ,¹³ and IL-10^14,15 have a profound effect on lesion initiation, progression, and plaque composition.

So far, IL-10 is the only antiinflammatory cytokine that has been reported to be protective in atherosclerosis.^15,16 In the present issue of Circulation Research, the study by Mallat et al¹⁷ showed that inhibition of the antiinflammatory cytokine transforming growth factor (TGF)-β resulted in an acceleration of atherosclerosis. Moreover, atherosclerotic lesions exhibited an increased inflammatory cell content and a decrease in collagen content, which are features of plaque instability. The data of Mallat et al indicate that TGF-β plays a protective role in the initiation of atherosclerosis and may be an important factor for the maintenance of plaque stability.

The feature that inhibition of inflammatory mediators or stimulation of antiinflammatory mediators modulate atherosclerotic plaque stability has already been shown in several studies. For example, interventions in atherosclerotic mouse models with major inflammatory regulators such as CD40L,^5,6,8,9 IFNγ,¹³ and IL10¹⁴ are able to modulate features of plaque stability, such as the amount of inflammation and fibrosis. IFNγ −/−/ApoE −/− mice show a decreased inflammatory cell content and an increased collagen-content,¹³ whereas inhibition of the antiinflammatory cytokine IL-10 showed the reverse.¹⁴

The inflammatory mediator with the most profound effect on plaque stability is CD40L. Besides a profound decrease in plaque area, deficiency of a functional CD40L gene resulted in a less lipid-containing, collagen- and smooth muscle cell–rich plaque phenotype, with a reduced macrophage and T-lymphocyte content in their advanced atherosclerotic plaques.^5,7 In follow-up studies, pharmacological interruption of CD40L-CD40 signaling (anti-CD40L antibody) induced a quite similar phenotype.^6,7,9 This phenotype could even be established when the antibody treatment was delayed until advanced plaques had developed.

Further dissection of the pathways involved in the development of the stable plaque phenotype revealed an increased immunoreactivity of TGF-β.⁶ These data suggest a key role for TGF-β in the development of a stable plaque phenotype during CD40L inhibition. Moreover, with the data of Mallat et al,¹⁷ a key role for TGF-β in the regulation of plaque stability in general can been proposed.

The first evidence for an important role for TGF-β in vascular biology has been shown in studies in the balloon-injured rat carotid artery, a model for neointima formation. TGF-β levels had increased during the first day after the procedure.¹⁸ Furthermore, overexpression or inhibition of TGF-β influenced the extent of neointima formation, extracellular matrix deposition, or smooth muscle cell proliferation.^19,20

Although the effects of TGF-β on neointimal formation have been extensively investigated, data regarding the effects of TGF-β on primary atherosclerosis are still limited and, moreover, solely descriptive. The localization of the different isoforms of TGF-β and its receptors in the atherosclerotic plaque are well described. TGF-β1 and -β3 are present in all stages of atherosclerosis and are predominantly expressed by the macrophage and the smooth muscle cell.^21,22 On the other hand, TGF-βRI and -βRII are abundantly present in fatty streaks, whereas only low, patchy expression is observed in advanced atherosclerotic lesions.²³ Interestingly, mutations in the TGF-βRII that disable proper signaling in atherosclerotic lesions have also been reported by some,²¹ but not all,²⁴ indicating that absence of TGF-β contributes to disease progression.²¹

In vivo studies investigating the effects of TGF-β on atherosclerosis are sparse. ApoE −/− mice that were treated with tamoxifen (an antiestrogen) exhibited increased levels of TGF-β, which was associated with a decrease in initial plaque area.²⁵ In addition, TGF-β1 +/− mice that were treated with high cholesterol diet showed increased endothelial activation and lipid retention compared with TGF-β1 +/+ mice.²⁶ The first in vivo correlation between plaque rupture and TGF-β was reported by Grainger et al,²⁷ who showed that humans suffering from unstable angina have decreased plasma levels of TGF-β. However, the study by Mallat et al is the first in vivo intervention study that describes the effects of TGF-β in plaque progression and phenotype in primary atherosclerosis.

Mallat et al treated ApoE −/− mice for a long period (9 weeks) with a neutralizing antibody that inhibits TGF-β1, -β2, and -β3. As may be expected from such an approach, they did observe systemic effects.¹⁷ The authors correctly mention in the article that some of these systemic effects of anti–TGF-β treatment might have confounded their results. Indeed, besides the effects of TGF-β inhibition on inflammation and fibrosis in atherosclerotic lesions, treatment also induced inflammatory changes in the heart and resulted in a 3-fold increase in CD3-positive cells in the adventitia, suggesting a systemic vasculitis. Therefore, the question raises whether the systemic effects are due to local inhibition of TGF-β or a generalized immunosuppression. Mallat et al were able to find decreased levels of phospho-Smad2, indicating that the effects were mediated by TGF-β¹⁷; however, measurement of systemic inflammatory parameters in serum, such as CRP, TNFα, IL-6, etc, and whole body autopsy would have been more appropriate to exclude systemic inflammation.

As has been reported, vasculitis, as well as other systemic inflammatory diseases, are able to accelerate and aggravate atherosclerosis. Patients suffering from Takayasu arteritis have an increased incidence of advanced atherosclerotic lesions compared to their age matched controls.²⁸ This is also true for patients suffering from systemic lupus erythematosus (SLE). In both the carotid and coronary arteries of patients with SLE, the extent of atherosclerosis is 30% to 50% more than in age matched controls. ^29,30 In a large prospective population study (Bruneck Study³¹), it was shown that even common infections, such as respiratory infections, urinary tract infections, dental infections, or other infections, amplify the risk of atherosclerosis. Atherosclerotic risk was highest among subjects with chronic infections. Furthermore, the association between chlamydia pneumonia infection and atherosclerosis is also thought to result from systemic inflammation rather than from direct infection of the atherosclerotic plaque.³²

Using a different approach, we found similar results as reported by Mallat et al.¹⁷ In a recent study, we treated ApoE −/− mice with a murine TGF-βRII fusion protein (TGF-βRII:Fc), which acts as a competitive inhibitor of TGF-β signaling (data will be presented at the Scientific Sessions of the AHA, November 2001). As Mallat et al have shown,¹⁷ we also observed a profound increase in inflammatory cells and mediators in initial and advanced atherosclerotic plaques after TGF-β inhibition, whereas the amount of fibrosis had decreased. Moreover, in advanced atherosclerotic plaques, the increase in inflammation and decrease in fibrosis in plaques were associated with a significant increase in the frequencies of recent and older intraplaque bleedings, fibrin deposition, iron deposition, and small plaque ruptures with disruption of the endothelial coverage after TGF-βRII:Fc treatment.

These data provide in vivo evidence that inhibition of TGF-β signaling induces characteristics of plaque instability in mouse atherosclerotic plaques. The data indicate that TGF-β plays an important role as immunomodulator and in extracellular matrix biology in atherosclerotic lesions. Thus, activation of TGF-β–signaling may provide a therapeutic target in atherosclerosis. It might not prevent the initiation of atherosclerosis, but it may prevent the transition into an unstable plaque phenotype due to its immunosuppressive and profibrotic effects.