Transforming Growth Factor-β
A Biomarker in Marfan Syndrome?
Marfan syndrome is an autosomal dominant disorder of connective tissue due to abnormal fibrillin-1 caused by mutations in the FBN1 gene on chromosome 15. Affecting 1 in ≈5000 individuals, Marfan syndrome has widespread features involving the cardiovascular system, eye, skeleton, lung, dura, and skin. After evaluation of individuals and families with Marfan syndrome over the years, it is remarkable how variable the phenotype and age of onset of various manifestations may be among affected individuals, even in the same family. Some individuals with Marfan syndrome require aortic root replacement early in childhood, whereas others may not require aortic surgery until late in life, if at all. This highlights the importance of thorough screening of all first-degree relatives of the Marfan patient, long-term follow-up, and consideration of mutation analysis for those with ambiguous features.
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The past 2 decades have witnessed remarkable scientific discovery and progress, providing hope to those affected by Marfan syndrome. In 1991, it was discovered that Marfan syndrome was due to a mutation in FBN1, the gene that encodes fibrillin-1.1 Hundreds of different mutations in FBN1 have subsequently been described, with most families having their own private mutation. Because fibrillin-1 is present in tissues affected by Marfan syndrome, it was considered that the underlying defect in fibrillin-1 led to a primary structural weakness in this connective tissue protein. Because of recent discovery, this concept has come into question.
Genetically engineered mouse models of Marfan syndrome, which recapitulate the phenotype observed in humans, have revolutionized understanding of basic pathophysiological mechanisms of this disease.2–4 Fibrillin-1 is a 350-kDa glycoprotein with multiple repeating, cysteine-rich, epidermal growth factor–like motifs, most of which are calcium binding. Fibrillin-1 also has motifs with homology to latent transforming growth factor-β–binding proteins. Fibrillin-1 proteins aggregate and associate to form microfibrils, which are important structural components of elastic and nonelastic tissue.
In addition to directing elastogenesis and providing structural support, fibrillin-1 also interacts with latent transforming growth factor-β–binding proteins and controls transforming growth factor-β (TGF-β) bioavailability.5 TGF-β is bound and kept inactive by the latent transforming growth factor-β–binding protein–fibrillin complex.5 Through the pioneering discoveries of Dr Hal Dietz and his colleagues, dysregulation of TGF-β activation and signaling has been demonstrated in the diseased tissues in mouse models of Marfan syndrome.2–4 The knowledge that abnormal TGF-β is present in these tissues has led to important breakthroughs in potential therapy. Blocking TGF-β, whether by neutralizing antibody or by the angiotensin II type I–receptor blocker (ARB) losartan, attenuated or prevented disease in genetically engineered mice with abnormal fibrillin-1. In landmark experiments, Neptune et al2 demonstrated that fibrillin-1 deficiency caused diminished sequestration of latent TGF-β in the extracellular matrix, which led to increased TGF-β signaling in abnormal lung tissue. When TGF-β neutralizing antibody was given to these mice, the abnormal alveolar septation was rescued.2 In a study of Marfan mice, Habashi et al4 observed that TGF-β neutralizing antibody or losartan prevented aortic root dilatation, elastic fiber degeneration, and Smad2 activation.
Angiotensin is important in TGF-β signaling, hence the study of drugs that block angiotensin in Marfan syndrome. Angiotensin stimulates TGF-β1 mRNA and protein expression and may, through its induction of thrombospondin, lead to TGF-β activation, which indicates that TGF-β acts downstream of angiotensin signaling.5 Angiotensin II and angiotensin type II receptor expression are increased in Marfan syndrome aortic tissue and have been associated with cystic medial degeneration.6 In cultured aortic cells from Marfan patients, angiotensin-converting enzyme (ACE) inhibition and angiotensin type II receptor antagonism significantly inhibited smooth muscle cell apoptosis.6 Angiotensin II type I–receptor blockade induces a decrease in TGF-β signaling, with a reduction in free TGF-β levels, tissue expression of TGF-β–responsive genes, and levels of mediators within the TGF-β signaling cascade.4,7
This basic work has translated to immediate clinical investigation. The Pediatric Heart Network of the National Heart, Lung, and Blood Institute trial of losartan versus atenolol in people with Marfan syndrome has enrolled 426 patients to date. This prospective randomized trial will examine the effects of angiotensin II type I blockade on aortic root growth.8 The results of this landmark clinical trial and ancillary studies are eagerly awaited.
To date, there are limited clinical data available on angiotensin II blockade in patients with Marfan syndrome.9 In a small, randomized, double-blind, placebo-controlled trial of 17 adults with Marfan syndrome taking standard β-blocker therapy, patients treated with the ACE inhibitor perindopril for 24 weeks had a reduction in aortic root size compared with those given placebo.10 Plasma levels of active and latent TGF-β and matrix metalloproteinase-2 and -3 were collected at baseline and at 24 weeks. The authors concluded that perindopril reduced aortic size, possibly through attenuation of TGF-β signaling.10 However, there was a marked variability in the blood levels of TGF-β at baseline and in response to ACE inhibitor therapy.
In a nonrandomized, retrospective, uncontrolled study, Brooke et al7 reported the effects of angiotensin II blockade in 18 pediatric Marfan patients (14 months to 16 years of age) with aggressive aortic disease (average z-score of 7). Patients were treated for at least 1 year with losartan (n=17) or irbesartan (n=1) in addition to their β-blocker.7 Marfan patients treated with ARB therapy showed a dramatic stabilization of the aortic root size. Before ARB use, the rate of change in aortic root diameter was ≈3.5 mm per year. After initiation of ARB therapy, the aortic diameter only increased by 0.4 mm per year. Interestingly, there was also a decline in the rate of change of increase in body height after initiation of ARB therapy in this population.
Other pharmaceuticals may hold promise in Marfan syndrome. Doxycycline has been demonstrated to inhibit a broad spectrum of matrix metalloproteinases and suppress aneurysm formation, as well as to have favorable effects on elastic tissue and aortic wall architecture in animal models and in human abdominal aortic aneurysm.11 In a mouse model of Marfan syndrome, doxycycline was more effective than atenolol in preventing aortic aneurysm formation, preserving elastic fiber structure, and normalizing endothelial and vascular smooth muscle cell function.11 Notably, doxycycline suppressed the upregulation of TGF-β signaling and resulted in a significant reduction in matrix metalloproteinase activity.
The exact mechanisms of cellular events responsible for the pathogenesis of Marfan syndrome remain uncertain. In this issue of Circulation, Matt et al12 importantly advance the understanding of TGF-β and Marfan syndrome by examining circulating TGF-β levels in a mouse model of Marfan syndrome and in humans taking part in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). The authors set forth several hypotheses: If abnormal fibrillin-1 in Marfan mice leads to excessive activation and signaling of TGF-β, does this correspond to increased blood levels of TGF-β? Does treatment with losartan reduce circulating TGF-β levels in Marfan mice? Importantly, do circulating TGF-β levels correspond to aortic root size? Using the genetically engineered mouse line heterozygous for Fbn1 mutation C1039 (Fbn1C1039G/+), the authors compared serum TGF-β levels in Marfan mice treated with placebo, Marfan mice treated with losartan, and wild-type mice and then correlated TGF-β levels with aortic root size in these mice. The results are provocative. In mice, circulating TGF-β levels increased as the mice aged and were higher in Marfan mice than in wild-type mice. Losartan-treated Marfan mice had lower TGF-β concentrations than placebo-treated Marfan mice. Losartan not only prevented aortic disease in the Marfan mouse but also reduced TGF-β concentrations to a level that was identical to that of age-matched wild-type mice. Interestingly, there may be a dose-response to blocking TGF-β. In a small number of Marfan mice treated with double the dose of losartan, the TGF-β concentrations were further reduced.12
Does losartan treatment lead to lower TGF-β levels in people with Marfan syndrome, and does this have any effect on aortic root size? The authors examined TGF-β levels and clinical information in 207 patients with Marfan syndrome as part of the GenTAC registry.12 In this nonrandomized, uncontrolled cohort comparison, Marfan patients had higher circulating TGF-β levels than control subjects. The TGF-β concentrations were not age dependent. As one would expect, the majority of Marfan patients enrolled in GenTAC were being treated with medication, including β-blocker, losartan, or ACE inhibitor. Compared with the untreated Marfan patients, Marfan patients treated with β-blocker, losartan, or the combination all had lower TGF-β levels. There was a trend toward lower TGF-β levels in the few patients treated with the combination of β-blockers and ACE inhibitors; however, TGF-β concentrations remained significantly higher in the treated Marfan patients than in control subjects. There was no correlation between total TGF-β concentration and aortic root size in Marfan patients. Of interest, TGF-β levels were also lower in Marfan patients treated with β-blockers than in untreated Marfan patients.
The report by Matt et al12 is important because it furthers the link between TGF-β and Marfan syndrome pathogenesis. In a homogeneous population of genetically engineered mice, significant correlation exists between circulating TGF-β levels and aortic disease, and ARB therapy, by blocking TGF-β signaling, lowers circulating TGF-β levels and prevents aortic aneurysm formation. The lack of correlation between TGF-β levels in the human population with Marfan syndrome and aortic disease in this study may have several explanations. The human study was uncontrolled and nonrandomized. Marfan patients with multiple genotypes and degrees of disease progression were included. The Marfan patients were treated with medications based on their physician preferences, with no standardization of drugs or doses. Many patients in GenTAC had received no medical treatment for many years. Additionally, TGF-β levels were taken at 1 point in time, which provided only a snapshot of the TGF-β concentration in the course of their disease.
Differences in mutation type can affect the Marfan phenotype, including age of onset and severity of disease.13 Genetic or environmental modifiers, as well as individual susceptibility to matrix degradation or intrafamilial variation in FBN1 expression, have also been suggested as potential mechanisms for the marked clinical heterogeneity in Marfan syndrome.13 Patients of various FBN1 genotypes may have different blood levels of TGF-β concentration, depending on the specific mutation, severity of disease, and point of time in the course of their disease.
Pharmacogenetic bases of drug responsiveness may also play a role in efficacy of therapy in disease states. Marfan syndrome is no exception. Pharmacogenetics may explain differences in response to β-blockers and drugs that block the angiotensin system, and this is being investigated in an ancillary study of the Pediatric Heart Network trial.8 Polymorphisms in CYP2C9 are associated with reductions in transformation of losartan to its active metabolite. An open-label study of 291 patients with FBN1 gene mutation–proven Marfan syndrome and aortic root dilatation randomized to losartan, nebivolol, or both has been initiated.14 The primary end point for this study is aortic root growth rate. Importantly, secondary end points include comparison of TGF-β levels, pharmacokinetics of the drugs by age and dosages, quantitative assessment of the mutated gene expression, and pharmacogenetics of drug responses by evaluation of variants of CYP269 (for losartan) and CYP2D6 (for nebivolol) genes.
Whether measurement of circulating levels of TGF-β will prove useful in patients with Marfan syndrome is unknown. It would be a tremendous advantage to have a biomarker in Marfan syndrome to predict disease activity or severity, progression, and response to specific types of pharmacotherapy. Assessment of circulating TGF-β levels in the ongoing Pediatric Heart Network trial in Marfan syndrome would be an important addition to this trial and could be correlated with aortic growth and pharmacogenomics studies. Data from prospective investigation should provide insight into the role of circulating TGF-β and its manipulation with regard to these issues and many others in Marfan syndrome.
Dr Braverman is a member of the Professional Advisory Board of the National Marfan Foundation and a member of the Vascular Advisory Panel of the Ehlers-Danlos National Foundation.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Nagshima H, Sakomure Y, Aoka Y, Uto K, Kameyama Ki, Ogawa M, Aomi S, Koyanagi H, Ishizuka N, Naruse M, Kawana M, Kasanuki H. An angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis in cystic medial degeneration associated with Marfan’s syndrome. Circulation. 2001; 104: 1282–1287.
Chung AWY, Yand HHC, Radomski MW, van Breemen C. Long-term doxycycline is more effective than atenolol to prevent thoracic aortic aneurysm in Marfan syndrome through the inhibition of matrix metalloproteinase-2 and -9. Circ Res. 2008; 102: e73–e85.
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