Tammy Holm

A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2

We report heterozygous mutations in the genes encoding either type I or type II transforming growth factor β receptor in ten families with a newly described human phenotype that includes widespread perturbations in cardiovascular, craniofacial, neurocognitive and skeletal development. Despite evidence that receptors derived from selected mutated alleles cannot support TGFβ signal propagation, cells derived from individuals heterozygous with respect to these mutations did not show altered kinetics of the acute phase response to administered ligand. Furthermore, tissues derived from affected individuals showed increased expression of both collagen and connective tissue growth factor, as well as nuclear enrichment of phosphorylated Smad2, indicative of increased TGFβ signaling. These data definitively implicate perturbation of TGFβ signaling in many common human phenotypes, including craniosynostosis, cleft palate, arterial aneurysms, congenital heart disease and mental retardation, and suggest that comprehensive mechanistic insight will require consideration of both primary and compensatory events.

Angiotensin II type 1 receptor blockade attenuates TGF-β–induced failure of muscle regeneration in multiple myopathic states

Skeletal muscle has the ability to achieve rapid repair in response to injury or disease. Many individuals with Marfan syndrome (MFS), caused by a deficiency of extracellular fibrillin-1, exhibit myopathy and often are unable to increase muscle mass despite physical exercise. Evidence suggests that selected manifestations of MFS reflect excessive signaling by transforming growth factor (TGF)-β (refs. 2,3). TGF-β is a known inhibitor of terminal differentiation of cultured myoblasts; however, the functional contribution of TGF-β signaling to disease pathogenesis in various inherited myopathic states in vivo remains unknown. Here we show that increased TGF-β activity leads to failed muscle regeneration in fibrillin-1–deficient mice. Systemic antagonism of TGF-β through administration of TGF-β–neutralizing antibody or the angiotensin II type 1 receptor blocker losartan normalizes muscle architecture, repair and function in vivo. Moreover, we show TGF-β–induced failure of muscle regeneration and a similar therapeutic response in a dystrophin-deficient mouse model of Duchenne muscular dystrophy. NOTE: In the version of this article initially published, the same panels were inadvertently used to show negative pSmad2/3 and periostin staining in muscle of Fbn1C1039G/+ mice treated with TGF-β‐neutralizing antibody in both the steady-state (Fig. 1a, right column, second and third rows, respectively) and muscle-regeneration (Fig. 1b, right column, third and fourth rows, respectively) experiments. In reality, these images only relate to the steady-state experiment (Fig. 1a). The intended images for Figure 1b are provided (red, pSmad2/3 staining; green, periostin staining). As both sets of images show negative staining in neutralizing antibody–treated Fbn1C1039G/+ mice, this does not alter any observations or conclusions discussed in the manuscript. The error has been corrected in the HTML and PDF versions of the article.

Angiotensin II Type 2 Receptor Signaling Attenuates Aortic Aneurysm in Mice Through ERK Antagonism

Transforming growth factor–β promotes aortic aneurysm formation through activation of its “noncanonical” signaling pathway. Angiotensin II (AngII) mediates progression of aortic aneurysm, but the relative contribution of its type 1 (AT1) and type 2 (AT2) receptors remains unknown. We show that loss of AT2 expression accelerates the aberrant growth and rupture of the aorta in a mouse model of Marfan syndrome (MFS). The selective AT1 receptor blocker (ARB) losartan abrogated aneurysm progression in the mice; full protection required intact AT2 signaling. The angiotensin-converting enzyme inhibitor (ACEi) enalapril, which limits signaling through both receptors, was less effective. Both drugs attenuated canonical transforming growth factor–β (TGFβ) signaling in the aorta, but losartan uniquely inhibited TGFβ-mediated activation of extracellular signal–regulated kinase (ERK), by allowing continued signaling through AT2. These data highlight the protective nature of AT2 signaling and potentially inform the choice of therapies in MFS and related disorders.

Noncanonical TGFβ signaling contributes to aortic aneurysm progression in Marfan syndrome mice.

Transforming growth factor–β promotes aortic aneurysm formation through activation of its “noncanonical” signaling pathway. Transforming growth factor–β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal–regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase–1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Circulating Transforming Growth Factor-β in Marfan Syndrome

Background— Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-&bgr; (TGF-&bgr;). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-&bgr; activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-&bgr; might be mirrored in circulating TGF-&bgr; concentrations. Methods and Results— Serum obtained from MFS mutant mice (Fbn1C1039G/+) treated with losartan was analyzed for circulating TGF-&bgr;1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-&bgr;1 concentrations increased with age and were elevated in older untreated Fbn1C1039G/+ mice compared with wild-type mice (P=0.01; n=16; mean±SEM, 115±8 ng/mL versus n=17; mean±SEM, 92±4 ng/mL). Losartan-treated Fbn1C1039G/+ mice had lower total TGF-&bgr;1 concentrations compared with age-matched Fbn1C1039G/+ mice treated with placebo (P=0.01; n=18; 90±5 ng/mL), and circulating total TGF-&bgr;1 levels were indistinguishable from those of age-matched wild-type mice (P=0.8). Correlation was observed between circulating TGF-&bgr;1 levels and aortic root diameters in Fbn1C1039G/+ and wild-type mice (P=0.002). In humans, circulating total TGF-&bgr;1 concentrations were elevated in patients with MFS compared with control individuals (P<0.0001; n=53; 15±1.7 ng/mL versus n=74; 2.5±0.4 ng/mL). MFS patients treated with losartan (n=55) or &bgr;-blocker (n=80) showed significantly lower total TGF-&bgr;1 concentrations compared with untreated MFS patients (P≤0.05). Conclusions— Circulating TGF-&bgr;1 concentrations are elevated in MFS and decrease after administration of losartan, &bgr;-blocker therapy, or both and therefore might serve as a prognostic and therapeutic marker in MFS.

Altered TGFβ signaling and cardiovascular manifestations in patients with autosomal recessive cutis laxa type I caused by fibulin-4 deficiency

Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of 17 patients with cutis laxa revealed no FBLN4 mutations. In a second group of 22 patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the fibroblast culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming growth factor (TGF)β signaling. This was confirmed by pSmad2 immunoblotting of fibroblast cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFβ signaling in the pathogenesis of FBLN4 mutations in humans.

Murine Model of Surgically Induced Acute Aortic Dissection Type A

OBJECTIVES This study aimed at developing a murine model of surgically induced acute aortic dissection type A for investigation of the formation and progression of acute aortic dissection and to test whether this system could be used for biomarker discovery. METHODS Adult fibrillin-1 deficient, Fbn1(C1039G/+) mice and wild-type mice were anesthetized, ventilated, and the ascending aorta exposed via hemisternotomy. We hypothesized that acute aortic dissection could be induced either by injecting autologous blood into the aortic wall or by injury to the wall with aortic clamping. Echocardiography was done preoperatively, and serum samples were collected before and 30 minutes after the operation and analyzed by enzyme-linked immunosorbent assay. RESULTS Echocardiography revealed larger aortic root diameters in Fbn1(C1039G/+) compared with wild-type mice (P = .001). Histologic examination showed that aortic clamp injury but not injection of blood leads to large intimal tears, disruption of aortic wall structures, and localized dissection of the aortic media in Fbn1(C1039G/+) mice. Acute aortic dissection developed in 4 of 5 Fbn1(C1039G/+) mice versus 0 of 5 wild-type mice after aortic clamping (P < .01). Elastin staining showed higher elastic fiber fragmentation and disarray in Fbn1(C1039G/+) compared with wild-type mice. Enzyme-linked immunosorbent assay analysis revealed elevated circulating transforming growth factor beta1 concentrations after induction of acute aortic dissection in Fbn1(C1039G/+) mice (P = .02, 150 +/- 61 ng/mL vs 456 +/- 97 ng/mL), but not in wild-type or sham-operated mice. CONCLUSIONS Aortic clamp injury can induce AAD in Fbn1(C1039G/+), but not in wild-type mice. This murine model of surgically induced acute aortic dissection is highly reproducible and nonlethal in the short term. Using this system, we revealed that circulating transforming growth factor beta1 is a promising biomarker for acute aortic dissection.