Perturbations of mechanotransduction and aneurysm formation in heritable aortopathies  Richmond W. Jeremy, Elizabeth Robertson, Yaxin Lu, Brett D. Hambly  International Journal of Cardiology  Volume 169, Issue 1, Pages 7-16 (October 2013) DOI: 10.1016/j.ijcard.2013.08.056 Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Cartoon showing the major elements of protein structure and relationships in the aortic media, including extracellular matrix and vascular smooth muscle cell (VSMC) linkages. Contractile VSMC phenotype is shown in brown and synthetic phenotype is shown tan coloured. Major structural proteins include elastin and collagen types III and VI. Collagen type I is a major adventitial protein. Microfibrillar proteins include fibrillin I, fibulin V and Emilin. Proteoglycans include versican, decorin and perlecan. Note the bifunctional location of microfibrils as investing elastin lamellae and linking VSMC to endothelium. The beads on the fibrillin microfibrils are associated with fibulins and microfibril associated glycoproteins. International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Panel A: Relationship between ascending aortic diameter and aortic impedance, modelled for different degrees of aortic stiffness index (S.I. normal range 3.6±1.3 (ref)) at constant mean central aortic pressure of 75mmHg. Normal resting ascending aortic impedance is reported as 0.065±0.019 and is shown as the shaded region. Normal range of aortic root diameter for age 20years and BSA 1.8m2 is shown by the vertical dashed lines. Note the match between aortic diameter and impedance when stiffness index is normal. Panel B: Relationship between aortic stiffness and impedance at different aortic diameters. Data are shown for a mean arterial pressure of 75mmHg. The shaded regions indicate previously reported values of aortic stiffness index for normal (N) and Marfan populations. As aortic diameter increases, the effect of increases in arterial stiffness upon impedance is lessened. International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Illustration of VSMC mechanotransduction signal loops in normal and disease states. In normal aortic wall (left), contraction of VSMC is matched to tensile stress on the aortic extracellular matrix (ECM), predominantly elastin (ELN). Linkage of wall tension and VSMC contractility is mediated by integrins and intracellular linkers, such as talin or vinculin. When cycling time of the VSMC actin–myosin complex (T clutch) is normal, intracellular growth signaling is suppressed. Matched deformation of the VSMC and ECM also limits release of TGFβ from the fibrillin–LTBP1 complex. If the VSMC actin–myosin interaction is impaired by mutations (upper right), then T clutch is prolonged, leading to activation of intracellular growth signaling, including increased integrin expression, TGFβ synthesis and release of TGFβ by membrane bound MMP activity. In contrast, the VSMC actin–myosin interaction may be normal, but abnormal ECM mechanical properties may result in slippage of the VSMC-ECM linkage (lower right). As a result, T clutch will increase and intracellular growth signaling will be stimulated. However, in addition, there may also be direct release of TGFβ from the fibrillin/LTBP1 complex due to mechanical stress upon the complex and/or impaired fibrillin binding of the LTBP1/TGFβ. International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Illustration of the multiple signaling pathways for TGFβ in the VSMC, including identification of known proteins affected by gene mutations in TAAD. Key signaling variants are identified by callout boxes (I–VII) and mutations are shown by red markers (A–E). In the normal state (Box I) TGFβ is sequestered with LTBP, in complex with MAGP-1 and Fibulin-4, associated with the fibrillin microfibrils and is released in response to mechanical stimulus e.g. stretch. Binding of TGFβ to a functional receptor (TGFβRI/TGFβRII heterodimer) (Box II) results in phosphorylation of Smad2 and Smad3, which complex with Smad4, before nuclear translocation and activation of transcription factors (canonical signaling), with resultant synthesis of pro-fibrotic growth factors. Alternate Smad-independent (non-canonical) signaling can also occur via the MAPK pathway, resulting in activation of transcription factors controlling both pro-fibrotic growth factors and also degradative factors, such as matrix metalloproteinases. TGFβ can also activate an alternate heterodimer receptor (TGFβRII/ALK1) (Box III) resulting in activation of a second Smad cascade (Smad 1/5/8) which then undergoes nuclear translocation and activation of transcription factors. Unlike the paths downstream of the TGFβRI/TGFβRII receptor, activation of these transcription factors predominantly results in synthesis of enzymes and growth factors associated with degradation of the extracellular matrix, collagen and elastin. Mutations in genes encoding fibrillin-1, fibulin-4 or MAGP-1 (A) may interfere with normal sequestration of TGFβ in the microfibrils, leading to overstimulation of TGFβ receptors (Box IV). Mutations in fibrillin-1 which impair its mechano-transductive capability (B) may result in increased TGFβ signaling (Box V) and overstimulation of both TGFβRI/TGFβRII and TGFβRII/ALK1 receptors. Mutations in the TGFβRII receptor unit may impair TGFβ binding (C) whilst mutations in the TGFβRI unit (D) may impede phosphorylation and subsequent Smad activation, resulting in non-functional receptors (Box VI) and fostering promiscuous signaling via TGFβRII/ALK1 (Box VII). Mutations within the canonical Smad pathway (E) may also result in increased non-canonical and alternate pathway signaling. CTGF=connective tissue growth factor; GAG=glycosaminoglycan; Il-6=interleukin 6; LTBP=latent TGFβ1 binding protein; MAGP-1=microfibril associated glycoprotein 1; MAPK=mitogen-activated protein kinase; MMP=matrix metalloproteinase; P=phosphorus; TGFβ=transforming growth factor β1; TF=transcription factors; TIMP=tissue inhibitor of metalloproteinases. International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Cartoon illustrating the multiple pathways of endothelial–VSMC signaling, which contribute to regulation of VSMC phenotype and synthetic activity. Parallel endothelial–VSMC pathways promoting either a contractile or synthetic VSMC phenotype exist, with activation of either pathway in response to the cellular environment. Endothelial release of NO supports a synthetic VSMC phenotype, whilst endothelial secretion of perlecan inhibits TGFβ signaling to the contractile phenotype and promotes TGFβ signaling to the synthetic phenotype. In contrast, signaling via the NOTCH pathway supports a contractile VSMC phenotype. CTGF=connective tissue growth factor; PDGF=platelet derived growth factor; MAGP=microfibril associated glycoprotein; LTBP=latent TGFβ binding protein; eNOS=endothelial nitric oxide synthase; MAPK=mitogen-activated protein kinase; MMP=matrix metalloproteinase; IP3=inositol triphosphate; TIMP=tissue inhibitor of metalloproteinases. International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Second and third generation interactome map for fibrillin showing major gene products associated with fibrillin expression. The multifunctional pattern of interactions is illustrated by interactions associated with TGFβ signaling (red); vascular smooth muscle cell contractility (green); extracellular matrix structure (orange); and growth factor signaling (blue). (Figure drawn from data in NCBI interactome). International Journal of Cardiology 2013 169, 7-16DOI: (10.1016/j.ijcard.2013.08.056) Copyright © 2013 Elsevier Ireland Ltd Terms and Conditions