TGFBR2 mutations alter smooth muscle cell ... - Semantic Scholar

Cardiovascular Research (2010) 88, 520–529 doi:10.1093/cvr/cvq230

TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections Sakiko Inamoto 1†‡, Callie S. Kwartler 1†, Andrea L. Lafont 1}, Yao Yun Liang 2,3, Van Tran Fadulu 1, Senthil Duraisamy 1, Marcia Willing 4, Anthony Estrera 1, Hazim Safi 1, Mark C. Hannibal 5, John Carey 6, John Wiktorowicz 7, Filemon K. Tan 1, Xin-Hua Feng 2,3, Hariyadarshi Pannu 1, and Dianna M. Milewicz 1* 1 Department of Internal Medicine and Cardiothoracic and Vascular Surgery, University of Texas Medical School at Houston, MSB 6.100, 6431, Fannin St, Houston, TX 77030, USA; 2Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA; 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; 4Department of Pediatrics, University of Iowa, Iowa City, IA, USA; 5Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA; 6Department of Pediatrics, University of Utah College of Medicine, Salt Lake City, UT, USA; and 7Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

Received 29 March 2010; revised 29 June 2010; accepted 6 July 2010; online publish-ahead-of-print 13 July 2010 Time for primary review: 8 days

Aims

Transforming growth factor-b (TGF-b) signaling is critical for the differentiation of smooth muscle cells (SMCs) into quiescent cells expressing a full repertoire of contractile proteins. Heterozygous mutations in TGF-b receptor type II (TGFBR2) disrupt TGF-b signaling and lead to genetic conditions that predispose to thoracic aortic aneurysms and dissections (TAADs). The aim of this study is to determine the molecular mechanism by which TGFBR2 mutations cause TAADs. ..................................................................................................................................................................................... Methods Using aortic SMCs explanted from patients with TGFBR2 mutations, we show decreased expression of SMC contracand results tile proteins compared with controls. Exposure to TGF-b1 fails to increase expression of contractile genes in mutant SMCs, whereas control cells further increase expression of these genes. Analysis of fixed and frozen aortas from patients with TGFBR2 mutations confirms decreased in vivo expression of contractile proteins relative to unaffected aortas. Fibroblasts explanted from patients with TGFBR2 mutations fail to transform into mature myofibroblasts with TGF-b1 stimulation as assessed by expression of contractile proteins. ..................................................................................................................................................................................... Conclusions These data support the conclusion that heterozygous TGFBR2 mutations lead to decreased expression of SMC contractile protein in both SMCs and myofibroblasts. The failure of TGFBR2-mutant SMCs to fully express SMC contractile proteins predicts defective contractile function in these cells and aligns with a hypothesis that defective SMC contractile function contributes to the pathogenesis of TAAD.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Thoracic aortic aneurysms and dissections † Smooth muscle cell differentiation † TGF-b † TGFBR2 mutations † Myofibroblast

1. Introduction The vascular smooth muscle cell (SMC) is a highly specialized cell whose principal function is contraction to regulate pulse pressure and blood flow. Smooth muscle cells found in the aorta and arteries

are quiescent, fully differentiated cells that harbour a unique repertoire of contractile proteins required for the cells’ function. Unlike terminally differentiated skeletal and cardiac muscle cells, SMCs retain phenotypic plasticity and can de-differentiate into proliferating and synthetic cells not expressing contractile proteins in response

†

These authors contributed equally to this work.

‡

Present address: 2-24-5-603 Akutagawacho, 569-1123 Takatsuki, Osaka, Japan.

}

Present address: Cell Cycle and Cancer Biology Research Program, MS 48, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, Oklahoma 73104, USA.

* Corresponding author. Tel: +1 713 500 6725; fax: +1 713 500 0693, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].

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TGFBR2 mutations alter smooth muscle cell phenotype

to vascular injury or environmental cues.1 TGF-b induces the differentiation of SMCs both in development and with phenotypic switching.2 Mouse models deficient in TGF-b1, or its receptors (TGFBR1, TGFBR2) or signaling molecules (SMAD5), are all associated with defective vascular maturation or loss of SMC differentiation, supporting a role of TGF-b in vascular development and SMC differentiation.1 Identification of heterozygous missense mutations in the transforming growth factor-b type 2 receptor gene (TGFBR2) as a cause of inherited predisposition to thoracic aortic aneurysms and dissections (TAADs) provides evidence that TGF-b signaling is involved in the pathogenesis of the disease.3,4 Heterozygous mutations in both TGFBR2 and TGFBR1 have been identified in patients with Loeys-Dietz syndrome (LDS), a syndrome with TAAD and associated arterial, skeletal, and craniofacial abnormalities, and patients with an inherited predisposition to TAAD in the absence of syndromic features.4 – 6 These missense mutations alter amino acids in the intracellular kinase domain of these receptors, a domain critical for TGFBR-induced signaling after TGF-b binding. Disease-causing TGFBR2 missense mutations have been shown to reduce receptor signaling activity in response to TGF-b binding.3 However, limited data on aortic tissue from LDS patients show evidence of paradoxically increased TGF-b signaling as assessed by enrichment of nuclear phosphorylated Smad2 in SMCs and increased expression of collagen and connective tissue growth factor in the aortic media.5,6 More recently, identification of mutations in the SMC-specific isoforms of a-actin and b-myosin heavy chain as causes of inherited predisposition to TAAD has suggested a role for proper SMC contraction in preserving aortic structure and preventing TAAD.7 – 9 Here we report that SMCs harbouring heterozygous TGFBR2 mutations have decreased global expression of contractile proteins when compared with control SMCs. These data suggest that TGFBR2 mutations disrupt SMCs from transforming into fully functional contractile cells.

2. Methods 2.1 Tissue samples and cell cultures Ascending aortic tissue above the sinuses of Valsalva was obtained from unrelated individuals with the following diseases and TGFBR2 mutations: familial TAAD, R460C (n ¼ 2) and R460H (n ¼ 1) and LDS, R528H (n ¼ 1) (detailed methods in the, Supplementary material online, Table S1).4,6 Control aortic tissue was obtained from the International Institute for the Advancement of Medicine from individuals who died of non-vascular disease and age- and gender-matched to the patients as closely as possible (n ¼ 6). Tissue was harvested and cells explanted as previously described.6,10 Human skin fibroblasts were derived from the following TGFBR2 mutation patients: R460C (n ¼ 6), C461Y (n ¼ 1), and R528H (n ¼ 1), and age-/gender-matched controls (n ¼ 9) and explanted as previously described.11 All SMCs and fibroblasts were used at passages 2 or 3. The investigation conforms with the principles outlined in the Declaration of Helsinki. This investigation is approved by the Committee for the Protection of Human Subjects at UTHSC-Houston (approval #054505). For detailed explant and culture protocols, see Supplementary material online, Methods.

2.2 Luciferase assays TGFBR2 negative DR-26 cells were transfected with the p3TP-Lux reporter plasmid3 along with various TGFBR2 receptor constructs (vector, WT receptor, dominant negative receptor DNIIR, R460H mutation receptor, R460C mutation receptor). After transfection, cells were incubated in

DMEM for a minimum of 24 h before being treated with or without 10 ng/mL of TGF-b. Cells were harvested 8 h after stimulation and luciferase activity was assayed.

2.3 Analysis of TGF-b signaling pathways in TGFBR2-mutant SMCs SMCs were grown to confluence and then seeded at 70 cells/mm2 in a 100-mm dish, and allowed to attach to the dishes for 24 h. Cells were cultured in serum-free SMC medium for 24 h prior to TGF-b stimulation. After 24 h of serum starvation, cells were collected as a base line (day 0). The remaining dishes of cells were stimulated with 10 ng/mL recombinant human TGF-b1 (R&D Systems) up to 72 h. Cells were harvested and scraped from the dish in RIPA buffer (see Supplementary material online) and gently shaken for 1 h at 48C.

2.4 Cell proliferation assays Cells were seeded into 96-well plates at a density of 622 cells/mm2 for 24 h prior to serum-starvation. After 24 h of culture in serum-free medium (either SMC medium for SMCs or DMEM for fibroblasts), the untreated plates were fixed and the remaining plates were treated with 10 ng/uL TGF-b1 for 72 h. All plates were analysed using the BrdU ELISA kit from Millipore (catalog #2750). The kit was used according to the manufacturer’s specifications.

2.5 Analysis of SMC contractile protein expression Confluent SMCs were seeded at 70 cells/mm2 in a 100-mm dish. Twentyfour hours later, the cells were washed with PBS, and cultured in serumfree SMC medium. After 24 h serum starvation, total RNA was isolated from SMCs and measured by two-step quantitative RT-PCR. These reactions were performed in triplicate. All values were adjusted to corresponding GAPDH expression levels.

2.6 SDS-PAGE and western blotting analysis The protein lysates were run on SDS-PAGE, transferred to the PVDF membrane, and probed with antibodies (see Supplementary material online). The experiments were done in triplicate with the same results; the figures in the manuscript are representative of the results.

2.7 Expression array analysis Total RNA was isolated using the TRIzol reagent. The quality of RNA was monitored on an Agilent RNA Bioanalyzer. Total RNA was amplified, labeled and hybridized on Illumina Sentrix HumanRef-8 Expression BeadChips (v. 1). Signal intensity, quality data were extracted and normalized using the Illumina BeadStudio Gene Expression Module. For exploratory analysis of differentially expressed genes between TGFBR2-mutant SMCs and control SMCs (referent), we selected genes with a differential expression score ≥ + 13. These data were then analysed through the use of Ingenuity Pathways Analysis (Ingenuity Systemsw, www.ingenuity.com).

2.8 Differential protein estimation by two-dimensional gel electrophoresis The cells were harvested in lysis buffer (see Supplementary material online). The protein extracts were analysed in triplicate by 2DE as described previously.12 For the detailed protocol, see Supplementary material online, Methods.

2.9 Protein extraction from aortic tissue sample Protein extraction was done with frozen aortic tissue of TGFBR2 R460C (n ¼ 1) and controls (n ¼ 2). After dissecting of the aortic media and

522 weighing the tissue (100 mg/each), the samples were minced into small pieces and put into 1 mL of protein extraction buffer. Each sample was homogenized with tissue homogenizer on ice. After centrifugation of homogenate, the supernatant was used for SDS-PAGE and immunoblot analysis.

2.10 Immunohistochemistry of contractile proteins in aortic tissue Fixed and embedded aortic samples were de-paraffinized and re-hydrated followed by heat-induced epitope retrieval in citrate buffer. Slides were stained with antibody, which was revealed by an alkaline phosphatase substrate solution, and counterstained with haematoxylin.

2.11 Immunofluorescence of SMCs After reaching confluence, cells were seeded onto coverslips in 6-well plates with the density of 13.15 cells/mm2 for 24 h prior to serum starvation. After 24 h of culture in serum-free SMC medium, cells were stimulated with 10 ng/mL TGF-b1 for 72 h. Immunofluorescence was carried out as described previously.7

2.12 Stable transfection of human TGFBR2 in mouse embryonic mesenchymal cells 10T1/2 CMV expression plasmids carrying human wild-type TGFBR2 or R460C or R460H mutant, together with a puromycine-containing vector pBabe, were transfected into mouse embryonic mesenchymal cells 10T1/2 (under passage 13) with the calcium phosphate method. Individual clones were isolated and expanded after 2 weeks of selection with puromycine (2 mg/mL). Control cells and stably transfected 10T1/2 mouse embryonic mesenchymal cells were serum starved overnight in D-MEM plus 0.2% FBS, then treated with TGF-b1 (2 ng/mL) for the time periods indicated. Cells were harvested in Laemmli sample buffer (BIO-RAD) for direct western blot analysis

2.13 TGF-b1 induced fibroblast to myofibroblast transformation An established protocol was used to transform quiescent dermal fibroblasts into myofibroblasts expressing SMC contractile proteins with TGF-b1 stimulation.13 – 15 Cells were cultured in complete DMEM. Fibroblasts were kept confluent for 5 days and then plated into 60 mm dishes at 300 000 cells/dish and allowed to attach for 24 h. Cells were then treated 24 h in serum-free DMEM. RNA and protein were harvested 24 h later (base line, day 0) as described above. The remaining cells were cultured in serum-free DMEM with 10 ng/mL TGF-b1 and harvested at various times up to 72 h.

2.14 Statistical analysis Data are expressed as mean + s.e.m. Statistical differences between TGFBR2 mutation cells and controls were analysed by a Student’s t-test. Differences were considered statically significant at values of P , 0.05.

3. Results 3.1 TGF-b signaling pathways in TGFBR2-mutant cells The effect of missense TGFBR2 mutations, R460C and R460H, on propagating downstream TGF-b signaling was initially assessed by transfecting wild-type and mutant receptor constructs into TGFBR2negative DR-26 cells and assaying signaling using a p3TP-Lux reporter system (Figure 1A). The p3TP system has the luciferase reporter gene under the control of the PAI-1 promoter with three TPA responsive

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elements.3 Our results indicate that these mutations impair TGF-b signaling, as predicted by their location in the kinase domain of the receptor. Next, SMCs explanted from the ascending aorta and fibroblasts explanted from the skin were obtained from patients harbouring these TGFBR2 missense mutations, along with mutations R528H and C461Y, and used to assess the effect of receptor mutations on cell signaling pathways and gene expression (Table S1, Supplementary material online). Early and late Smad2 phosphorylation in response to recombinant TGF-b1 were preserved in both aortic SMCs and dermal fibroblasts and was indistinguishable from the response in control cells explanted from healthy individuals (Figure 1B and Supplementary material online, Figure S1). These data support previously published data that TGFBR2 mutations do not disrupt canonical signaling pathways as assessed by Smad2 phosphorylation.5 TGF-b1 also activates non-canonical signaling pathways through the TGFBR1 and TGFBR2 complex, including the p38MAPK and Akt signaling pathways.16 Activation of p38MAPK and Akt pathways was assessed in TGFBR2-mutant SMCs. Across multiple repeats of the experiments, the phosphorylation of both p38MAPK and Akt in response to TGF-b was lower in the TGFBR2 R258H SMCs than in controls (Figure 1C and D). Activation of p38MAPK was biphasic in the control SMCs as previously reported, with a peak at 10 min and then another increase at 2 h with continued activation.17 In contrast, p38MAPK activation was completely suppressed in the TGFBR2-mutant SMCs. Additionally, Akt phosphorylation was both delayed and diminished in mutant compared with control cells. Akt is a known regulator of cellular proliferation in both SMCs and fibroblasts and TGF-b signaling drives Akt-activated proliferation in fibroblasts.18 We assayed proliferation in mutant and matched control cells to determine if the diminished Akt activation decreased proliferation in response to TGF-b1. Both SMCs and fibroblasts with TGFBR2 mutations had decreased proliferation at baseline as well as with TGF-b1 treatment when compared with matched control cells (Figure 1E and F).

3.2 TGFBR2 mutations alter expression of SMC contractile proteins Activation of the p38MAPK and Akt signaling pathways in SMCs is involved in the TGF-b induced expression of SMC-specific contractile proteins.17,19 Therefore, SMC contractile gene expression was analysed in patient and control SMCs using quantitative PCR (Q-PCR). The expression levels of a variety of contractile proteins, including a-actin (ACTA2), calponin (CNN1), caldesmon (CALD1), smoothelin (SMTN), a and b tropomyosin (TPM1 and TPM2), b-myosin heavy chain (MYH11) and SMC g-actin (ACTG2) in TGFBR2 R460C SMCs (n ¼ 2) were significantly lower than in SMCs explanted from age -matched controls (n ¼ 2) (Figure 2A). The expression of these SMC contractile proteins in TGFBR2 R528H SMCs and R460H SMCs were analysed separately and were also decreased compared with matched control SMCs (Figure 2B and C). In contrast, the expression of the cytoskeleton proteins b-actin (ACTB) and g-actin (ACTG1) was not altered in the mutant SMCs compared with controls. Immunoblot analysis of TGFBR2 R528H SMCs compared with control confirmed a corresponding decrease in protein levels of SMC a-actin, b-myosin, and calponin (Figure 2D). Interestingly, Q-PCR also revealed increased expression of S100A4 in the TGFBR2-mutant SMCs. S100A4 expression in SMCs has been reported to be correlated with


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Figure 1 Assessment of TGF-b signaling in cells from individuals with TGFBR2 mutations. (A) In vitro analysis shows impaired activity of receptors harbouring TGFBR2 mutations. TGFBR2 constructs were co-transfected with a TGF-b responsive luciferase reporter plasmid (p3TP-Lux) into TGFBR2-deficient DR-26 cells, standardized to a b-galactosidase expression plasmid. Wild -type TGFBR2 transfected cells showed an increase in the luciferase activity in response to TGF-b stimulation (black bar) when compared with basal activity in the absence of TGF-b (white bars), while those transfected with the vector alone, a dominant negative TGFBR2 lacking the entire cytoplasmic domain (DNIIR), a missense mutation previously reported in an MFS patient (R537C), or the two missense mutations found in familial TAAD patients (R460C and R460H) did not show an increase in luciferase activity, suggesting that these mutations impair TGFBR2 function. Error bars represent + standard deviation. (B) Assessment of Smad2 phosphorylation in aortic SMCs from an affected patient and age-matched control after TGF-b1 for various times up to 48 h (figure represents similar data collected from one of the three patients’ SMC cell strains assessed). (C) Western blot analysis of p38MAPK and phosphorylatedp38MAPK protein in SMCs on stimulation with TGF-b shows reduced phosphorylation of p38 in TGFBR2-mutant SMCs (R528H) compared with control SMCs. (D) Western blot analysis of Akt and phosphorylated- Akt protein in SMCs on stimulation with TGF-b shows reduced phosphorylation of Akt in TGFBR2-mutant SMCs (R528H) compared with control SMCs. (E) SMCs with TGFBR2 mutations proliferate significantly less rapidly than control SMCs at baseline and after 72 h of stimulation with TGF-b. *P , 0.0001 (F) Fibroblasts with TGFBR2 mutations proliferate significantly less rapidly than control fibroblasts at baseline and after 72 h of TGF-b stimulation. *P ¼ 0.015, **P ¼ 0.00015.

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Figure 2 Decreased contractile protein expression and protein levels in patients’ explanted aortic cells and tissue. (A – C) Q-PCR analysis of mRNA isolated from serum-starved SMCs from patients with TGFBR2 mutations (black bars) and matched controls (white bars). The affected patients’ SMCs consistently have reduced expression of the SMC contractile complex genes, no change in expression of cytoskeletal genes (ACTB, ACTG1) and increased expression of S100A4. Gene expression level was standardized to GAPDH. *P , 0.01, **P , 0.001. (D) Immunoblot analysis of SMC lysates from patients’ SMCs versus matched control showed reduced SMC contractile protein levels in patients’ SMCs compared with control. (E) Immunoblot analysis of proteins isolated from frozen aortic tissue from a patient showed reduced SMC contractile protein levels compared with control tissue. (F to G) Immunohistochemistry staining of aortic tissue from patients shows decreased intensity of staining of the SMC contractile proteins calponin (F) and b-myosin (G) compared with controls. Patient images are shown below their age-matched control images. Magnification is 200x, and scale bars represent 100 mm.


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decreased expression of contractile proteins and increased proliferation.20 The expression of SMC contractile proteins was also assessed in SMCs in vivo using ascending aortic tissue from patients and controls. Immunoblot analysis of aortic proteins harvested from the medial layer of a TGFBR2 R460C patient confirmed decreased protein levels of calponin and SMC b-myosin heavy chain compared with controls in vivo, although a-actin expression was similar (Figure 2E). Immunostaining of the aortic media of three patients harbouring TGFBR2 mutations showed decreased signal intensity for calponin and SMC b-myosin heavy chain compared with matched control aortas, providing additional evidence that decreased expression of calponin and SMC-specific b-myosin also occurs in vivo in the aortic media (Figure 2F and G). To further confirm decreased expression of contractile proteins, gene expression array analysis was used to compare differences between SMCs from two unrelated patients with TGFBR2 R460C mutations and three unaffected control SMCs (Supplementary material online, Table S3). A two-dimensional gel electrophoresis (2DGE) was also performed in triplicate with fluorescence detection using TGFBR2 R528H SMCs and control SMCs (Supplementary material online, Table S4). Both gene and protein changes were analysed within the context of canonical pathways in the Ingenuity Knowledge base. Based on these analyses, the actin/cytoskeletal signaling pathway was the most significantly altered cellular pathway (P ¼ 0.001 for array, P ¼ 1.03 × 1023 for 2DGE). Taken together, these results all confirm a disruption of the smooth muscle contractile protein levels in TGFBR2-mutant SMCs.

fibres that span the cell body. Analysis of co-localization of red and green fluorescence confirmed significantly decreased co-localization in patients’ SMCs compared with control cells of the stress fibres and either a-actin (P ¼ 0.007) or calponin (P ¼ 0.003) (Supplementary material online, Table S5).

3.3 TGFBR2-mutant SMCs fail to increase contractile gene expression with TGF-b exposure

3.5 TGFBR2 mutations disrupt fibroblast transformation into myofibroblasts

We sought to determine whether the TGFBR2-mutant cells could increase expression of contractile genes with exposure to TGF-b1. After exposure to TGF-b1, control SMCs demonstrated significantly increased expression of a-actin, calponin, and b-myosin at 72 h (Figue 3A). In contrast, TGFBR2 R528H SMCs showed no significant increase in expression of these genes 72 h after TGF-b1 exposure. Interestingly, S100A4 expression was decreased significantly in both the control and TGFBR2 R528H SMCs with TGF-b1 treatment. Further, expression levels of SMC contractile genes were not increased in either TGFBR2 R460C or R460H mutant SMCs following TGF-b1 exposure, whereas matched controls demonstrated increased expression of these genes (Figure 3B and C, respectively). To determine if TGFBR2-mutant SMCs could assemble contractile filaments, immunofluorescence of a-actin and calponin was performed on SMCs 72 h after exposure to TGF-b1. Alpha-actin and calponin are both incorporated into actin filaments of the contractile unit in vascular SMCs, while b-actin is found in the actin filaments of the cytoskeleton.21,22 Antibodies specific for either a-actin or calponin were used (green); in addition, all cellular polymerized actin in filaments was visualized with phalloidin (red). Confocal images of control SMCs after TGF-b stimulation showed organized stress fibres spanning the cell body containing a-actin and calponin (Figure 3D and E, respectively, and Supplementary material online, Figure 2A and B, respectively). In all four of the TGFBR2-mutant SMCs, actin stress fibre formation appeared normal but immunostaining for a-actin and calponin was diminished in intensity and these proteins were not incorporated into the stress

3.4 Expression of mutant TGFBR2 in mouse mesenchymal cell line To validate that missense mutations in the TGFBR2 kinase domain disrupt the differentiation of precursor cells into SMCs, TGFBR2 R460C and R460H mutant constructs were stably expressed in 10T1/2 cells, a mouse embryonic mesenchymal cell line that differentiates into an SMC phenotype expressing contractile proteins with TGF-b1 stimulation.17 Expression of TGFBR2 wild-type, R460C, and R460H receptors was confirmed through an antibody directed against the myc tag at the carboxyl-terminus of the receptor protein (Figure 4A). Introduction of an exogenous wild-type receptor into the cells lead to more pronounced and sustained phosphorylation of Smad2/3 after TGF-b1 stimulation (Figure 4A). In contrast, expression of the exogenous mutant TGFBR2 R460C and R460H decreased the TGF-b-induced phosphorylation of Smad2/3, along with reducing the TGF-b-induced activation of Akt and p38 (Figure 4A and C ). As a consequence, expression of SMC contractile proteins induced by TGF-b, specifically a-actin and SM-22a, was reduced in cells expressing either of the mutant receptors but not in cells expressing wild-type receptor (Figure 4B).

The studies described above are limited by access to low passage number SMCs explanted from the ascending aorta of patients. In contrast, explanted dermal fibroblasts can be readily obtained. Additionally, the translucent skin and atrophic scars observed in some patients with TGFBR2 mutations suggest an in vivo defect in myofibroblast formation.6 The differentiation of fibroblasts into myofibroblasts can be achieved with exposure of quiescent fibroblasts to TGF-b1 and is defined by the induction of expression of SMC contractile genes.13 We sought to determine if fibroblasts explanted from patients heterozygous for TGFBR2 mutations poorly differentiated into myofibroblasts with TGF-b1 exposure. Since myofibroblast differentiation can vary in fibroblast cell strains, combined data from six passage-matched dermal fibroblast cell lines explanted from patients with TGFBR2 R460C mutation were compared with passage-, age-, and gender-matched control fibroblasts. There was no significant difference in the expression levels of ACTA2, CNN1, and MYH11 after serum deprivation between the six TGFBR2 R460C fibroblast cell strains and six matched control fibroblast strains (Figure 5A, 0 time). Although expression of SMC contractile proteins was increased to a similar extent in both TGFBR2 R460C and control fibroblasts 24 h after exposure to TGF-b1, the expression was significantly lower in the mutant SMCs at 48 h, and even more dramatically lower at 72 h. Fibroblasts explanted from patients with TGFBR2 mutations C461Y and R528H showed similar results when compared with matched control fibroblast strains (Figure 5B and C ). These data demonstrate that TGFBR2-mutant fibroblasts poorly transform into myofibroblasts

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Figure 3 TGFBR2-mutant SMCs fail to demonstrate significantly increased expression of contractile proteins and fail to assemble contractile fibres with exposure to TGF-b. (A– C) Q-PCR analysis of mRNA isolated from TGFBR2 R528H (A), R460C (B) and R460H SMCs (C) harvested 72 h after exposure to TGF-b fails to show increased expression of contractile protein genes ACTA2, CNN1 and MYH11 but showed significantly decreased expression of S100A4 (*P , 0.05). Control SMCs demonstrate significant increases in the expression of these same genes (*P , 0.05). For this experiment, each sample’s gene expression was normalized to its own 0 h timepoint to assess the changes that occurred with TGF-b stimulation (except S100A4). (D to E) Immunofluorescence analysis of a-actin (D) and calponin (E) and stress fibres in cultured SMCs from patients and controls. In the control cells, there is extensive co-localization of either a-actin (D, green) or calponin (E, green) and polymerized actin (stained red with phalloidin). The stress fibres containing a-actin tend to cross the cell body, whereas the calponin tends to be incorporated into stress fibres near the nucleus of the cell, as previously reported.22 In contrast, the intensity of a-actin and calponin stain is diminished in all four of the patients’ SMCs (shown below their age-matched control) with minimal staining at the periphery of the cells. SMC nuclei were counterstained with DAPI (blue). Scale bars 40 mm.

with TGF-b stimulation and are consistent with the observation that TGFBR2 mutations lead to poorly differentiated SMCs.

4. Discussion In arteries, SMCs are quiescent, contractile cells expressing a full repertoire of contractile proteins.1 Here we report that aortic SMCs explanted from patients with heterozygous TGFBR2 kinase domain mutations have decreased expression of SMC contractile

proteins when compared with control SMCs. Furthermore, TGFBR2-mutant SMCs fail to increase expression of these contractile proteins with exposure to TGF-b1. Assessment of calponin and b-myosin protein levels in the aortic media of TGFBR2 patients suggests decreased in vivo expression of these proteins compared with control aortas. Finally, 10T1/2 cells stably transfected with TGFBR2 kinase domain mutations also fail to express contractile proteins with exposure to TGF-b1 when compared with 10T1/2 cells transfected with wild-type TGFBR2. Taken together, we hypothesize


Figure 4 Disruption of TGF-b signaling by over-expressed TGFBR2 mutants R460C and R460H in 10T1/2 cells. Western blot analysis shows decreased phosphorylation levels of Smad2/3 (A) and Akt and p38 (C) in cells transfected with mutant receptors. Contractile protein expression (B) was also decreased in cells transfected with mutant compared with wild-type receptors.

that heterozygous TGFBR2 mutations disrupt downstream TGF-b1 cellular signaling such that vascular SMCs fail to express a full repertoire of contractile proteins necessary for contractile function. These studies have a number of limitations, including the following: (1) SMCs from only four patients with TGFBR2 mutations were available for study; (2) the SMCs were explanted from diseased tissue and may have adopted an altered phenotype secondary to the disease process; (3) primary SMC cultures cannot be manipulated for mechanistic studies. We addressed the first two limitations, in part, by confirming decreased SMC contractile gene expression with TGF-b1 stimulation in both 10T1/2 cells stably transfected with TGFBR2 mutations and fibroblasts harbouring heterozygous TGFBR2 mutations. In addition, decreased contractile protein expression was observed in the aortic SMCs in tissues from TGFBR2-mutant patients when compared with the control tissue. We attempted to overcome the third limitation by stably transfecting 10T1/2 cells with mutant TGFBR2 receptors. This over-expression model with high levels of mutant receptors showed decreased Smad signaling, making this model less physiologically relevant than the patients’ primary cells, where we and others have found no defect in Smad signaling. Therefore, the 10T1/2 cell model did not fully recapitulate the findings in the patients’ cells, most likely due to difficulty in obtaining a 1:1 ratio of mutant to wild-type receptors.

527 The lack of expression of contractile proteins by TGFBR2-mutant SMCs is not surprising given what is known about aortic SMC differentiation into a quiescent contractile cell. The SMCs explanted from the patients and controls were from the ascending aorta and are therefore neural crest in origin.23 – 25 The differentiation of a neural crest cell line, Monc1 cells, into SMCs can be achieved in culture with exposure to TGF-b1.25 Specific inactivation of TGF-b signaling in neural crest stem cells through loss of the TGFBR2 alleles in neural crest cells results in cardiovascular defects, but also results in a lack of SMC a-actin expression in neural crest derived cells, suggesting that TGFBR2 deficient cells do not differentiate into SMCs.26 Similarly, TGF-b1 signaling has been demonstrated to be important for SMC development from mesenchymal cells.2 Although our studies focused on SMCs explanted from the ascending aorta, the clinical disease in patients with TGFBR2 mutations includes aneurysms and dissections of other arteries and arterial tortuousity. Based on the diffuse nature of the vascular disease associated with TGFBR2 mutations and our data that TGFBR2-mutant fibroblasts also poorly transform into myofibroblasts, mesenchymal-derived SMCs may also have disrupted TGF-b driven expression of SMC contractile proteins. Indeed, our data showed defective differentiation of the mesenchymal-derived SMC precursor 10T1/2 cells when transfected with mutant TGFBR2. Similar to the TGFBR2-mutant SMCs, fibulin-4 (Fbln4) deficiency in the mouse model, both germline and SMC-specific Fbln4 deficiency, leads to the SMCs in the aortic walls exhibiting reduced expression of SMC-specific contractile genes, including Acta2, Myh11, and Cnn1.27 This failure of SMCs to express a full repertoire of contractile proteins is associated with development of aneurysms primarily in the ascending thoracic aorta. In vitro Fbln4-deficient SMCs exhibit an immature SMC phenotype with a marked reduction of b-myosin heavy chain levels. These studies concluded that Fbln4 played a role in aortic SMCs maturation into contractile SMCs. Similarly, it is known the TGF-b signaling is important for differentiation of SMCs into mature, contractile cells,28 and our findings suggest that the TGFBR2 mutations disrupt this TGF-b-driven differentiation. We and others have failed to demonstrate a defect in the canonical TGF-b1 signaling pathways as assessed by Smad2 phosphorylation in SMCs and fibroblasts explanted from patients with heterozygous TGFBR2 mutations.5 Fibroblast to myofibroblast transformation with exposure to TGF-b1 is initially mediated through adhesionindependent Smad signaling, which increases the expression of contractile protein genes (i.e. ACTA2, MYH11 and CNN1), along with genes encoding integrins and extracellular matrix proteins. TGFBR2-mutant fibroblasts increase expression of contractile protein genes similar to control fibroblasts the first 24 h after TGF-b1 exposure, suggesting that this phase of fibroblast to myofibroblast transformation, dependent on canonical Smad signaling, is intact.29 After 24 h, adhesion-dependent signaling through integrin receptors and focal adhesion kinase, leading to activation of Akt, is required for continued transformation to myofibroblasts.29 Our data indicate that the Smad signaling after TGF-b1 exposure in TGFBR2-mutant fibroblasts remains intact; however these cells are unable to complete the fibroblast to myofibroblast transformation, suggesting a possible defect in adhesion-dependent signaling and perhaps indicating a previously undefined role for non-Smad pathways in controlling this differentiation. In contrast to what is known for myofibroblasts, accumulating evidence in the literature indicates TGF-b activation of non-canonical

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S. Inamoto et al.

Figure 5 Defective transformation of quiescent fibroblasts to myofibroblasts with TGF-b in patient and control fibroblasts. (A) Q-PCR analysis of mRNA expression of SMC contractile protein genes in fibroblasts various times after exposure to TGF-b1 showed reduced expression in TGFBR2-mutant cells (R460C, n ¼ 6) compared with matched controls (n ¼ 6). (B to C) Similarly expression analysis of mRNA or SMC contractile protein genes in fibroblasts explanted from two unrelated patients with TGFBR2 mutations (C461Y, n ¼ 1) in panel B, and R528H, (n ¼ 1) in panel (C) also showed reduced expression of these genes.

pathways mediates SMC differentiation, including activation of MAP and JNK kinases, P13 kinase and RhoA.17,19,30 TGF-b-driven transformation of Monc1 cells into SMCs has found that RhoA signaling controls the expression of SMC contractile proteins mediated through myocardin/SRF-dependent transcription. The RhoA downstream target, PKN, interacts with p38 MAPK to regulate SMC contractile protein promoter activity.19 Additional analyses suggest that p38MAPK can also be activated directly by TGF-b associated kinase 1 (TAK1), which is itself directly activated by the TGFBR2.16,31 TAK1 has been implicated as a major effector of TGF-beta induced vascular differentiation in an in vivo model.32 In 10T1/2 cells, the

differentiation from mesenchymal cells to SMC occurs with TGF-b signaling through both p38MAPK and PI3 kinase/Akt signaling.17 In our studies, we found that both TGFBR2-mutant SMCs and 10T1/2 cells expressing the mutant TGFBR2 demonstrated a defect in both p38MAPK signaling and Akt signaling with TGF-b exposure when compared with controls. The mechanism by which heterozygous TGFBR2 mutations have little effect on Smad signaling but disrupt non-Smad signaling in SMCs is not known. One-fifth of patients with TAAD have inherited a predisposition to the disease, most commonly due to a single autosomal gene mutation inherited in a dominant manner. Mapping and identification of genes


causing TAAD have established significant genetic heterogeneity for the disease.8 Recent studies have identified mutations in the SMC-specific isoform of a-actin (ACTA2) as the major gene for familial TAAD, responsible for 14% of the disease in families with inherited disease.7 In addition, mutations in the SMC specific isoform of MYH11 are responsible for the infrequent association of inherited TAAD and patent ductus arteriosus.9,33 Identification of mutations in the SMC-specific isoform of these contractile proteins has emphasized the role of SMC contraction in preventing TAAD and maintaining structural integrity of the ascending aorta. Thus, our data indicating that TGFBR2-mutant SMCs fail to fully express contractile proteins when compared with control SMCs further supports a hypothesis that disruption of SMC contractile function is an underlying defect leading to TAADs.

Supplementary material Supplementary material is available at Cardiovascular Research online.

Acknowledgements The authors are grateful to the patients and other individuals who donated a skin sample and/or an aorta for these studies and to Dr Joan Massague´ for the mink lung epithelial DR-26 cell line and 3TP-lux plasmid. Control aortas were obtained through the International Institute for the Advancement of Medicine (www.iiam.org). Conflict of interest: none declared

Funding This work was supported by the National Institutes of Health [grant numbers RO1 HL62594 to D.M.M., P50HL083794-01 to D.M.M., R01 CA108454 to X.H.F. and UL1 RR024148 to CTSA]. D.M.M. is a Doris Duke Distinguished Clinical Scientist. X.-H. F. is a Leukemia and Lymphoma Scholar.

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