Circulating Transforming Growth Factor-β1 Levels in Asymptomatic Carotid Plaques
To the Editor:
It was with great interest we read the report by Cipollone et al1 on the role of transforming growth factor-β1 (TGF-β1) in the process of plaque stabilization. The authors demonstrate that TGF-β mRNA levels are increased up to 3-fold in asymptomatic as compared with symptomatic plaques, with a parallel increase in protein expression at immunocytochemistry and Western blot analyses.1 In addition, TGF-β1 expression was associated with a comparable increase in plaque procollagen and collagen content, thus providing a tangible mechanism of plaque stabilization.1 As Cipollone et al acknowledge in their discussion, one important issue would be to measure systemic levels of TGF-β1 to verify whether a correlation exist between the localized expression and circulating levels of this cytokine; this correlation would suggest a systemic process in these patients rather than a local phenomenon, and probably might add prognostic information to the management of patients with carotid plaques.
We have only recently completed reviewing the data collected in a 10-year prospective study of the incidence of major cardiovascular events in 42 patients with asymptomatic low-grade carotid stenosis. Patients were consecutively enrolled over a 1-year period from those presenting at the Department of Internal Medicine of the Palermo University Hospital for ultrasound evaluation (high-resolution B-mode ultrasonography using a 7.5-MHz duplex-type probe; Toshiba) of carotid atherosclerotic involvement because of the presence of at least 1 cardiovascular risk factor. Percent carotid lumen stenosis was graded as low-grade lumen stenosis because of plaque >15% but <50% (intima-media thickness >0.85 and <1.5 mm). Plasma TGF-β1 levels were determined by enzyme immunoassay (R&D Systems Inc, Minneapolis, Minn). Patients with asymptomatic low-grade carotid stenosis had markedly higher baseline levels of TGF-β1 (median, 3.7 pg/mL; interquartile range, 0.5 to 10.8 pg/mL) compared with control subjects in whom no lesion could be detected (mean, 0.5 pg/mL; interquartile range, 0.5 to 4.8 pg/mL; P<0.0001) independently of the presence of cardiovascular risk factors, which is in agreement with the finding of Cipollone et al.1
All patient were longitudinally followed-up for a median of 8.8 years. During this period, 14 (33%) patients with asymptomatic low-grade stenosis experienced a hard endpoint (nonfatal myocardial infarction, n=4; stroke, n=2; transient ischemic attack, n=4; intermittent claudication, n=2; percutaneous revascularization procedure, n=2). Surprisingly, these patients had significantly higher baseline values of TGF-β1 (median, 7.2 pg/mL, interquartile range, 0.8 to 10.8 versus median, 2.4 pg/mL, interquartile range, 0.5 to 10.6 pg/mL; P<0.02) than those who remained event-free during the follow-up. Nine of 19 (47%) patients with TGF-β1 levels >4.8 pg/mL (upper quartile of values observed in control subjects) experienced a hard endpoint compared with 5 of 23 (22%) patients with TGF-β1 levels <4.8 (log-rank test 1.9; P=0.06).
These results suggest that mechanism(s) other than local expression might be responsible for the increased TGF-β1 levels in the circulation. In this respect, we must consider that platelets are a major source of TGF-β1 in the circulation as they release and activate latent growth factor in response to activation.2 This has been clearly demonstrated in the setting of visceral obesity, a condition associated to increased incidence of cardiovascular events, in which we observed that TGF-β1 levels were independently related to prothrombin fragment F1+23 and correlate to the rate of urinary thromboxane metabolite excretion (Rho=0.37; P<0.05) (G. Davì, unpublished observation, 2003). It is therefore conceivable to hypothesize that the increased TGF-β1 levels found in plasma samples from patients with low-grade carotid stenosis might be caused by a procoagulant state. Specific trials should be designed to directly address the issue of platelet release of TGF-β1 in this clinical setting.
Supported by grants from Italian Ministry of Research (40% and Fondo per gli Investimenti Recerca di Base) to the “G. d’Annunzio” Foundation, Chieti.
We read with interest the letter from Ferroni et al about the circulating levels of transforming growth factor-β1 (TGF-β1) in patients with asymptomatic carotid plaques. In fact, in our recent study,1 we demonstrated that TGF-β1 generated locally within the atherosclerotic plaques is actively involved in the process of plaque stabilization in humans. However, an unresolved issue in this study was whether a correlation exists between the localized expression of TGF-β1 and the circulating levels of this cytokine in high-risk patients.
Starting from this point, Ferroni et al provide 2 important observations in their study. The first is that patients with proved asymptomatic carotid stenosis had markedly higher baseline levels of TGF-β1 compared with control subjects, in whom no lesion could be detected independently of the presence of cardiovascular risk factors, thus suggesting that systemic TGF-β1 may be associated with the development of atherosclerotic damage. The second observation is that patients with high circulating TGF-β1 levels may experience a higher incidence of hard end point compared with patients with low TGF-β1 levels, thus suggesting a role for systemic TGF-β1 also in the evolution of atherosclerotic plaques developing complications.
Some limitations exist in this study. The first is that the absence of a control group of patients with symptomatic plaques does not permit a complete interpretation of the role of circulating TGF-β1 in the process of atherothrombosis. The second is that the absence of a second measurement of TGF-β1 after the follow-up period does not permit us to know the modifications of circulating TGF-β1 levels during the progression of vessel damage.
Nevertheless, some aspects of the methodology adopted in this study are noteworthy. In particular, the long-term period of observation (8.8 years) permitted to the authors, despite the limited number of studied patients, the observation of a fair number of hard end points and their correlation with the circulating level of TGF-β1. Thus, using this approach, authors observed that TGF-β1 level at enrollment was positively associated with higher incidence of cardiovascular events at 8.8 years of follow-up, thus suggesting the circulating TGF-β1 may predict the risk of future cardiovascular complications in high-risk patients.
Therefore, the evidence rising from the Ferroni study together with our own seems to resolve the question as to whether a correlation exists between the localized expression of TGF-β1 within the plaques and the circulating levels of this cytokine. In fact, TGF-β1 generated from tissue macrophages and smooth muscle cells is an active player involved in the process of plaque stabilization by modulation of the turnover of extracellular matrix. In contrast, circulating TGF-β1 appears to be simply a marker of risk rather than an active mediator. In fact, the results from Ferroni et al on the platelet origin of this mediator, despite being based only on correlative data and indirect observations and therefore fairly speculative, nevertheless identified TGF-β1 as a potential marker of platelet reactivity in humans.
In this light, unfortunately Ferroni et al did not provide any information about the pharmacological treatment of patients, particularly with respect to the antiplatelet therapy. In fact, the recent observations of aspirin-insensitive platelet activity in patients with acute vascular events,2,3,4 probably a consequence of the expression of the aspirin-insensitive cyclooxygenase 2 in young platelets,5,6 seem to suggest that a subgroup of platelets with higher (aspirin-insensitive) reactivity may exist. Whether they are also responsible for the generation of systemic TGF-β1 remains unknown. Thus, further studies using different antiplatelet strategies as a pharmacological tool will be necessary to confirm the role of platelets in the systemic generation of TGF-β1 and to answer the question as to whether TGF-β1 could be the (for a long time searched) marker of the abnormal platelet reactivity responsible for important clinical conditions, such as the so called “aspirin-resistance phenomenon.”7
Cipollone F, Fazia M, Mincione G, Iezzi A, Pini B, Cuccurullo C, Ucchino S, Spigonardo F, Di Nisio M, Cuccurullo F, Mezzetti A, Porreca E. Increased expression of transforming growth factor-beta1 as a stabilizing factor in human atherosclerotic plaques. Stroke. 2004; 35: 2253–2257.
Cipollone F, Patrignani P, Greco A, Panara MR, Padovano R, Cuccurullo F, Patrono C, Rebuzzi AG, Liuzzo G, Quaranta G, Maseri A. Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina. Circulation. 1997; 96: 1109–1116.
Cipollone F, Ciabattoni G, Patrignani P, Pasquale M, Di Gregorio D, Bucciarelli T, Davi G, Cuccurullo F, Patrono C. Oxidant stress and aspirin-insensitive thromboxane biosynthesis in severe unstable angina. Circulation. 2000; 102: 1007–1013.
Cipollone F, Ganci A, Greco A, Panara MR, Pasquale M, Di Gregorio D, Porreca E, Mezzetti A, Cuccurullo F, Patrignani P. Modulation of aspirin-insensitive eicosanoid biosynthesis by 6-methylprednisolone in unstable angina. Circulation. 2003; 107: 55–61.
Rocca B, Secchiero P, Ciabattoni G, Ranelletti FO, Catani L, Guidotti L, Melloni E, Maggiano N, Zauli G, Patrono C. Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets. Proc Natl Acad Sci U S A. 2002; 99: 7634–7639.
Cipollone F, Rocca B, Patrono C. Cyclooxygenase-2 expression and inhibition in atherothrombosis. Arterioscler Thromb Vasc Biol. 2004; 24: 246–255.