Genetic Variants in Smooth Muscle Contraction and Adhesion Genes Cause Thoracic Aortic Aneurysms and Dissections and Other Vascular Diseases

Chapter 97

Genetic Variants in Smooth Muscle Contraction and Adhesion Genes Cause Thoracic Aortic Aneurysms and Dissections and Other Vascular Diseases Dianna M. Milewicz and Callie S. Kwartler Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX

INTRODUCTION Smooth muscle cells (SMC) lack the characteristic crossstriations of cardiac and skeletal muscle, but contain contractile proteins organized in contractile units (1). Although smooth muscle cells line all hollow organs in the body, including the arteries, gastrointestinal tract, bladder, and uterus, genetic mutations that disrupt SMCspecific isoforms of contractile proteins, along with the mutations in the kinase that controls SMC contraction, lead primarily to vascular diseases. The major vascular disease resulting from these mutations is aortic aneurysm involving the ascending thoracic aorta immediately above the heart. These aneurysms predispose to a life-threatening complication, acute aortic dissections.

THORACIC AORTIC ANEURYSMS AND DISSECTIONS The major disease affecting the ascending thoracic aorta is aortic aneurysm, defined as a localized, permanent dilatation of an artery, and acute aortic dissection (Figure 97.1) (2). The natural history of thoracic aortic aneurysms located just above the heart is to asymptomatically enlarge over time until an acute tear in the intimal layer leads to an ascending aortic dissection (type A dissections based on the Stanford classification) or, rarely, an aortic rupture. Collectively, thoracic aortic aneurysms and their complications are designated as TAAD. With dissection, blood penetrates into the medial layer, separating the aortic layers and causing tearing through the adventitia (rupture) or other complications. Type A aortic dissections cause sudden death in up to 50% of individuals; survivors of the acute event have a 1% per hour

Muscle. DOI: http://dx.doi.org/10.1016/B978-0-12-381510-1.00097-1 © 2012 Elsevier Inc. All rights reserved.

death rate until they undergo emergent surgical repair. TAAD are a common cause of premature deaths, ranking as high as the 15th leading cause of death in the United States (3). Less-deadly aortic dissections can also originate in the descending thoracic aorta just distal to the branching of the subclavian artery (Stanford type B dissections) and are a further part of the TAAD disease spectrum. Although medical treatments can slow the enlargement of an aneurysm, the mainstay of treatment to prevent dissections and premature deaths is surgical repair of the thoracic aortic aneurysm before an aortic dissection occurs. This is typically recommended when the aneurysm reaches 5.0 5.5 cm in diameter; however, studies on patients presenting with acute type A dissections indicate that up to 60% present with aneurysms smaller than 5.5 cm (4). Studies to identify genetic causes of TAAD have determined that the specific gene can both identify individuals at risk for the disease and predict at what aortic diameter a dissection may occur, thereby optimizing the timing of aortic surgery. Risk factors for TAAD include poorly controlled hypertension and congenital cardiovascular abnormalities, such as a bicuspid aortic valve (BAV) and aortic coarctation. In addition, genetic predisposition plays a prominent role in the etiology of TAAD. Thoracic aortic disease is inherited in families in an autosomal dominant manner in the presence or absence of syndromic features. Marfan syndrome (MFS), caused by mutations in FBN1, is an example of a genetic syndrome in which essentially all affected individuals have TAAD, in addition to skeletal and ocular complications (see Chapter 71) (5). Studies using mice engineered with a heterozygous Fbn1 missense mutation known to cause MFS have suggested that defects in Fbn1 lead to excessive active transforming

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(A) Adventitia Elastic fibers

Intima (B)

Medial degeneration

FIGURE 97.1 Classification of thoracic aortic aneurysms and dissections. A thoracic ascending aortic aneurysm is a permanent, localized dilation of the ascending aorta immediately above the heart. An aortic dissection is a tear in the intimal layer of the aorta allowing blood to penetrate the medial layer and dissect along this layer. Dissections in the aorta are classified by their location: Stanford Type A dissections are dissections in which the intimal tear is in the ascending aorta, while Stanford Type B dissections are dissections in which the intimal tear is in the descending thoracic aorta just distal to the left subclavian artery.

growth factor-β (TGF-β) being released from stores in the microfibrils (6,7). The identification of mutations in the TGF-β receptors type I and II (TGFBR1 and TGFBR2) as a cause of another syndrome predisposing to TAAD, Loeys Dietz syndrome (LDS), further implicate a role for altered TGF-β signaling in the pathogenesis of syndromic TAAD (8 10). Family aggregation studies indicate that up to onefifth of TAAD patients who lack features of a genetic syndrome have family histories of TAAD (11,12). TAAD in these families is typically inherited in an autosomal dominant manner, with decreased penetrance, particularly in women (familial disease is designated FTAAD) (13). FTAAD families demonstrate variable expression of TAAD, including varying age of disease onset, severity of presentation, and whether the aneurysm involves the aortic root or ascending aorta. Additionally, phenotypic variability between families is evident by the characterization of a subset of FTAAD families whose members experience aortic dissections with little to no enlargement of the ascending aorta, whereas other families present with large, stable aneurysms that are not prone to dissection. Clinical heterogeneity appears in other features inherited by subsets of families, which can include intracranial aneurysms (ICAs), bilateral iliac artery aneurysms, occlusive vascular diseases such as early onset strokes and coronary artery disease (defined as age of onset less than 55 years of age in men and 60 years in women), abdominal aortic aneurysms (AAAs), bicuspid aortic

FIGURE 97.2 Thoracic ascending aortic wall structure and pathology associated with thoracic aortic aneurysms and dissections. (A) The medial layer of the healthy aorta stained with Movat’s pentachrome stain. The wall is comprised of layers of elastic lamellae separated by layers of smooth muscle cells. In the vessel wall, the lamellae are arranged concentrically, and in large elastic arteries like the aorta, a single layer of smooth muscle cells separates each layer of elastin. (B) Aortic tissue from the surgical repair of an aortic aneurysm stained with Movat’s pentachrome stain. Medial degeneration is characteristic of the diseased aorta, and includes fragmentation of the elastic fibers, focal areas of smooth muscle cell loss, focal areas of smooth muscle cell hyperplasia, and accumulation of proteoglycans within the medial layer.

valve (BAV), and patent ductus arteriosus (PDA). Along with informing management of aortic disease, the specific gene causing FTAAD in a given family can determine the risk for vascular disease beyond TAAD and associated congenital heart defects, such as BAV and PDA. The aorta is comprised of three layers: a thin inner layer, the tunica intima; a thick middle layer, the tunica media; and a thin outer layer, the tunica adventitia. The tensile strength and elasticity of a normal aorta reside in the medial layer, which contains concentrically arranged elastic fibers and SMCs (Figure 97.2A). The SMCs are longitudinally oriented and dispersed among the circular elastic fibers. Contractile filaments composed of thick and thin filaments within the SMCs are linked up to elastin fibers through connections between focal adhesions on the cell surface and bundles of elastin-associated microfibrils in the matrix. Together, these form a continuous structure called the “elastin-contractile unit”, which provides the basis for uniform force generation (14). Humans have between 40 50 layers of elastin lamellae and SMCs in the ascending aorta. Aortic SMCs contract in response to pulsatile blood flow but, unlike the small muscular arteries, this contraction does not regulate blood flow and pulse pressure in the large diameter aorta. In fact, data indicate that the elasticity of the ascending aorta is due to the elastic fibers with little to no contribution from SMC contraction (15,16). The aortic pathology associated with TAAD is medial degeneration, previously termed “cystic medial

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degeneration”. Medial degeneration is characterized by loss and fragmentation of elastic fibers, accumulation of proteoglycans in the aortic media, and focal regions of the aortic media depleted of SMCs (Figure 97.2B). Although there are areas of SMC loss, there are also often adjacent areas of SMC hyperplasia. Debate persists as to whether SMC loss or hyperplasia is more important to the pathology, but more recent studies provide data to suggest that there is overall SMC hyperplasia in the aortic media with aneurysm progression (17). Inflammatory cells often accompany medial degeneration, but the role of inflammation in disease progression remains to be defined (18,19).

MUTATIONS IN GENES FOR SMC CONTRACTION PROTEINS CAUSE FAMILIAL THORACIC AORTIC DISEASE The clinical heterogeneity of FTAAD described above is due to underlying genetic heterogeneity, i.e., many genes can be altered to cause aortic disease to be inherited in families and the specific causative genetic alteration in a family leads to a particular disease presentation and associated features. Six FTAAD genes have been identified and are responsible for disease in 20% of families. Three of the genes are the genes responsible for MFS and LDS: FBN1, TGFBR1, and TGFBR2. In FTAAD families with mutations in these genes, the affected family members have no or minimal syndromic features of MFS or LDS and the onset of the thoracic aortic disease is later in life (9,20). The three additional genes that cause FTAAD, MYH11, ACTA2, and MYLK, all encode proteins critical for SMC contractile function and provide the focus of this chapter. Mutations in ACTA2, which encodes the SMC-specific α-actin (SM α-actin), a component of the contractile complex and the most abundant protein in vascular SMCs, are the most common cause of FTAAD identified to date (21,22). Thirty-three ACTA2 missense mutations, one in-frame deletion, and a splice site mutation deleting the second to last exon (exon 8) of the gene have been identified in TAAD patients (21,23 25). The penetrance of TAAD in family members heterozygous for ACTA2 mutations is low, with only approximately 50% of the mutation carriers experiencing aortic disease. In a subset of families, ACTA2 mutations segregate with a skin rash caused by dermal capillary and small artery occlusion referred to as livedo reticularis. Other features present in some families with ACTA2 mutations include iris flocculi, PDA, and BAV. ACTA2 mutations are heterozygous missense mutations predicted to produce a mutant α-actin monomer. These heterozygous mutations are located in all four

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P245H

I250L R258C/H

R212Q R185Q G160D

R39H P72Q W88R

R179H N117T R118Q Y135H T326N R292G

Y145C R149C

T353N

V154A

FIGURE 97.3 Distribution of identified ACTA2 mutations. The structure of the smooth muscle α-actin protein is represented; residues identified to harbor mutations are highlighted. Mutations represented in green (R258 and R39) are associated with a clinical presentation of thoracic aortic disease and early onset stroke (including Moyamoya disease). Mutations represented in red (R149, R118) are associated with a clinical presentation of thoracic aortic disease and early onset coronary artery disease. Mutations in yellow (W88 and G160) are only associated with thoracic aortic disease. The mutation in pink (R179) is associated with global smooth muscle cell dysfunction. For mutations shown in blue, insufficient data are available to characterize the clinical phenotype.

subdomains of actin and are predicted to produce structurally-altered actin monomers (Figure 97.3). Over 100 ACTA1 mutations, primarily missense mutation, have been identified to cause congenital myopathies (see Chapter 74). Similarly, ACTAC missense mutations cause either hypertrophic or dilated cardiomyopathies (see Chapter 33). As described in Chapter 33, characterization of ACTA1 mutations has provided genetic evidence for a dominant negative pathogenesis of these mutations. Preliminary assessment of the effect of ACTA2 missense mutations performed by visualizing all cellular polymerized actin in filaments with phalloidin, and SM α-actinspecific filaments using a specific monoclonal antibody (21). SMCs explanted from the aortas of unaffected individuals demonstrated abundant SM α-actin in stress fibers that extended across the cell. In contrast, SMCs explanted from individuals heterozygous for ACTA2 missense mutations had no SM α-actin-containing filaments extending across the cell. These observations suggest that missense mutations perturb SM α-actin incorporation into filaments or stability of assembled filaments in aortic SMCs, implicating a dominant negative pathogenesis of ACTA2 mutations, similar to the findings with ACTA1 mutations. ACTA2 missense mutations known to cause FTAAD have been engineered into yeast actin and the effect on actin function assessed (26). ACTA2 missense mutations N117T and R118Q were both introduced into yeast and led to reduced growth and abnormal mitochondrial

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Converter ATPase head

Coiled-coil tail

Arg712GIn

Δ R1241-L1264del

Smooth Muscle

FIGURE 97.4 Location of MYH11 mutations on a graphic representation of the β-myosin heavy chain.

Δ L1452-N1526del

Leu1264Pro

morphology. Additionally, both mutant actins exhibited altered thermostability and nucleotide exchange rates as well as abnormalities during polymerization, but exact results differed between the N117T and R118Q mutations. A large French family with TAAD associated with patent ductus arteriosus was used to map and identify mutations in MYH11 as a cause of FTAAD (27). MYH11 encodes the SMC-specific myosin heavy chain, a major component of the contractile unit in SMCs. Further studies determined that MYH11 mutations were responsible for 1% of FTAAD, and were specifically seen in families with TAAD associated with PDA (28). The spectrum of MYH11 mutations identified for the familial TAAD/PDA phenotype is limited to four mutations: a small deletion, a splice site mutation, and two missense mutations (Figure 97.4). The deletion and the splice site mutation cause removal of 24 and 71 amino acids respectively from the coiled-coil tail domain of the myosin heavy chain (27). Both of these mutations are likely to result in protein products that are either unstable or unable to assemble into polymerized filaments. A coiled-coil modeling tool predicts that both deletions would decrease the probability of coiled-coil formation, and wild-type and mutant rod domain constructs could not co-immunoprecipitate, suggesting altered interactions between the myosin monomers (27). These mutations likely result in a dominant negative effect on filament formation, similar to that observed with the ACTA2 mutations described above. A MYH11 missense mutation results in the substitution of a proline for a leucine at amino acid 1264, also in the coiled-coil domain. This substitution likely disrupts the formation of the coiled-coil as predicted by the in silico modeling analysis, and may affect filament assembly in a similar fashion to the deletion mutations. Only one motor domain mutation has been linked to aortic aneurysms: a missense mutation R712Q. This residue lies in the crucial SH1 helix, which links the enzymatic region of the molecule with the converter domain that functions as a lever and actively moves the rest of the head domain. An equivalent mutation in MYH9, the nonmuscle myosin heavy chain II, causes syndromic deafness (29,30). When engineered into a Dictyostelium myosin background, alteration of this arginine caused a decrease in the velocity of actin movement along the

myosin filament, but did not affect the enzymatic ATPase activity of the motor (29). These results suggest that the R712Q mutation likely disrupts force generation by the mutant myosin heavy chains. Heterozygous mutations in the gene for myosin light chain kinase (MYLK) have also been reported as a cause of FTAAD (31). Cell signaling events that increase intracellular [Ca21]i in SMCs, such as the opening of stretch-activated Ca21 channels, stimulate the Ca21/ calmodulin-dependent myosin light chain kinase (MLCK) (see Chapter 87). The kinase then phosphorylates a specific site on the N-terminus of the regulatory light chain (RLC) of myosin polymerized in thick filaments. RLC phosphorylation is sufficient to activate the myosin motor, and thereby affect cellular contraction. MLCK appears to be the only known kinase for this function and the only known physiological substrate for MLCK is myosin RLC; thus, it is a dedicated protein kinase (32 34). The aortic phenotype in FTAAD families with MYLK mutation is characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta (35). Mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta, though as with the human patients no dilation of the vessel was apparent (31). MYLK mutations lead to a loss of enzymatic function of MLCK: one mutation is a nonsense mutation, R1480X, that leads to a truncated protein lacking the kinase- and calmodulin-binding domains, while the other, S1759P, alters amino acids in the α-helix of the calmodulinbinding sequence. The latter alteration disrupts MLCK binding to calmodulin as shown by immunoprecipitation. An in vitro kinase assay also showed decreased enzymatic activity (decreased Vmax and increased Km) (31). The identification of mutations in the major structural proteins of the SMC contractile unit, along with mutations in the kinase controlling SMC contraction, as causes of FTAAD implicate disruption of the “elastin-contractile unit” as a factor that predisposes to TAAD (Figure 97.5). The major protein in microfibrils in fibrillin-1, and mutations in the gene for this protein, FBN1, also lead to a genetic predisposition to TAAD in patients with MFS and FTAAD as described above (36 38). Therefore, these data suggest that aortic SMCs may act as sensors for

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FIGURE 97.5 Diagram of the “elastin-contractile unit”. Contractile proteins like α-actin and β-myosin are assembled into filaments that comprise the contractile unit of a smooth muscle cell. These units are then linked to integrin-containing focal adhesions at the cell surface, and the integrins in these adhesions form connections with microfibrils in the extracellular matrix. These fibers connect the cells to the large elastic fibers in the vessel wall. These “elastin-contractile units” connect all cells to the elastic fibers, allowing the aortic wall to contract coordinately.

biomechanical stress on the ascending aorta and that intact SMC “elastin-contractile units” serve as a critical component for this sensor function. The aortic SMCs respond to aberrant forces on this unit by activating cellular signaling pathways in an attempt to repair and remodel the wall to withstand these stressors. If SMCs continually respond to increased forces or stresses, resulting either from genetically-mediated disruption of the elastincontractile unit or from the increased pressures of poorly controlled hypertension, these cell signaling pathways may be continuously activated, leading to medial degeneration and TAAD. Analysis of human TAAD aortas and mouse models of thoracic aortic disease have implicated the following to be some of the signaling pathways leading to TAAD: proteoglycan accumulation, increased metalloproteinases (MMPs) activity, including MMP-2 (39,40), hyperplastic medial SMC cellular proliferation (17), increased TGF-β signaling (7,41,42), increased expression of insulin-like growth factor-1 (IGF-1), and evidence of increased angiotensin (AngII) signaling (28,43). Although many of the genes that predispose to TAAD directly encode proteins found in the “elastin-contractile unit,” how mutations in the TGF-β receptors could disrupt this unit is not immediately obvious. Heterozygous mutations in TGFBR1 and TGFBR2 cause both FTAAD and LDS. The causative mutations are missense mutations in the intracellular kinase domain of the receptor that are predicted, and for a few of the missense mutations

proven, to disrupt the kinase activity of the receptors (44,45). Therefore, these mutations are predicted to disrupt TGF-β-driven cellular signaling that is activated with ligand binding, and those same signaling pathways regulate the expression of contractile proteins by vascular SMCs (46). Explanted aortic SMCs from FTAAD patients with TGFBR2 missense mutations have intact canonical Smad signaling but decreased expression and protein levels of contractile proteins, including SM α-actin, β-myosin, and calponin, when compared with control SMCs. In contrast to control SMCs that have SM α-actin filaments extending across the cells, aortic SMCs from TGFBR2 mutant patients have no incorporation of SM α-actin into filaments, similar to the phenotype observed in SMCs with ACTA2 missense mutations. Analysis of proteins isolated from aortic tissue also showed decreased expression of contractile proteins in TGFBR2 patients compared with controls. Dermal fibroblasts from TGFBR2 patients similarly demonstrated defective transdifferentiation into myofibroblasts with TGF-β exposure when compared to control fibroblasts, a finding that may help explain the poor wound healing and atrophic scarring observed in these patients (45). These results suggest that heterozygous missense mutations in the TGF-β type II receptor intracellular kinase domain disrupt TGF-β signaling such that SMCs and fibroblasts fail to fully express contractile proteins that mark differentiation and transdifferentiation of these cells into contractile SMCs and

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myofibroblasts, respectively. Animal models of these mutations, along with further cellular studies, are needed to determine if the cellular defects described in TGFBR2 mutant cells also disrupt contractile properties of SMCs. Finally, there is also genetic evidence that disruption of the connection between α-actin filaments and cell surface integrin receptors also predisposes to thoracic aortic disease. Filamin A is a large, multi-domain, homodimeric actin-binding protein that interacts with the actin cytoskeleton and integrin receptors, thereby regulating various aspects of cell shape, motility, and function. Mutations in filamin A (FLNA) result in X-linked inheritance of a brain malformation known as periventricular heterotopia (47). The disorder occurs mostly in females and affected women have an increased number of miscarriages of male fetuses, suggesting that hemizygous males die perinatally. In addition, FLNA mutations also cause aortic dissections in women with periventricular heterotopias (48). The identification of thoracic aortic disease in women with FLNA mutations further highlights a potential role for filamin A in the “elastin-contractile unit”.

GENETIC VARIANTS CONTRIBUTING TO SPORADIC THORACIC AORTIC DISEASE DISRUPT SMOOTH MUSCLE CELL CONTRACTION AND ADHESION The majority of patients with TAAD do not report a family history of the condition: their disease is classified as sporadic TAAD (STAAD) and accounts for approximately 80% of thoracic aortic disease (11,49). Identification of patients at risk for TAAD via a genetic strategy could potentially prevent sudden deaths due to acute aortic dissections. Initial studies have begun to identify the genetic variants that predispose to disease in STAAD patients and have highlighted a role for genomic copy number variants (CNVs) in predisposing individuals to STAAD. CNVs are large regions of the genome that are either deleted or duplicated in the population. They can encompass multiple genes or involve regions that contain no genes, leading to decreased or increased gene dosage. They may also disrupt the structure of genes at the boundaries of the affected region. CNVs have been shown to confer increased risk for common multifactorial diseases such as autism and schizophrenia, as well as congenital cardiovascular disorders such as tetralogy of Fallot (50 52). The findings in these studies support a genetic model wherein any of a large number of individually rare copy number mutations contribute to disease causation or predisposition (53). This model is supported by the observation that CNVs for neuropsychiatric conditions are enriched for genes involved in neuronal function and activity (54,55).

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Through the analysis of over 750 STAAD patients, the CNV burden in these patients was found to be significantly increased when compared with a control population (56,57). Furthermore, ontology, expression profiling, and network analysis showed that genes within and disrupted by the CNVs in patients with STAAD regulate SMC focal adhesions and contractility, and many of the CNV-involved genes encode proteins that interact with SM α-actin and β-myosin. Therefore, the CNVs in patients with STAAD disrupt the ability of the SMC to adhere and contract, once again implicating the “elastincontractile unit” in the maintenance of the integrity of the ascending aorta. It is notable that a recurrent CNV involving duplications of chromosome 16p13.1 was more commonly found in STAAD patients than controls (p value 5 1.4 3 1028, Odds Ratio 10.7 [Confidence Interval 5.1 21.1]) (58). Nine genes are in the chromosome 16p13.1 duplicated region, including MYH11. Since MYH11 mutations cause FTAAD, increased gene dosage of MYH11 may be responsible for the increased risk for thoracic aortic disease associated with this duplication. Increased MYH11 gene expression was found in aortic tissues from TAAD patients with the 16p13.1 duplications when compared either with unaffected aortas or aortas from patients without the 16p13.1 duplication. Studies in C. elegans have shown that a precise ratio of β-myosin to its cellular chaperone, UNC45, is required for proper folding of myosin and its assembly into thick filaments, and an imbalance in this ratio causes the degradation of myosin heavy chain protein and dysfunction of the contractile complex (59). Further studies are needed to determine if overexpression of MYH11 in aortic SMCs similarly leads to an imbalance of β-myosin to its chaperone, leading to degradation of β-myosin and dysfunction of the SMC contractile unit.

ACTA2 MUTATIONS CAUSE OCCLUSIVE VASCULAR DISEASES IN ADDITION TO FTAAD Analysis of families with inherited heterozygous mutations ACTA2 determined that these mutations predispose not only to TAAD but also to occlusive vascular diseases, including early onset coronary artery disease (CAD), early onset strokes, and Moyamoya disease (23). The investigation into occlusive vascular diseases in FTAAD patients with ACTA2 mutations was initiated when it was observed that, as described above, in some families with ACTA2 mutations, mutation carriers had livedo reticularis (21). Interestingly, the rash was present in mutation carriers whether or not they had aortic disease. Subsequent pathologic studies of the vasa vasorum in the outer layers of the aorta in ACTA2 mutation patients identified

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occlusion or stenosis of these arteries due to thickening of the medial layer. Further linkage analysis and association studies from 20 families with ACTA2 mutations began to uncover a concurrent predisposition among mutation carriers for occlusive vascular diseases, including greaterthan-expected incidences of early-onset ischemic stroke and CAD. Additionally, a recurrent mutation in TAAD families, ACTA2 R258C, was associated with an unusual number of cases of primary Moyamoya disease (23). Moyamoya disease is a rare cerebrovascular syndrome often leading to ischemic stroke at a young age. Diagnostic features on angiography of Moyamoya disease include bilateral occlusion or stenosis of the terminal internal carotid artery and the formation of collateral vessel networks at the base of the brain, the so-called “Moyamoya vessels”. These occlusive lesions occur in young and middle-aged mutation carriers despite minimal risk factors for vascular disease such as hyperlipidemia, smoking, or diabetes. These data demonstrated that diffuse vascular diseases, resulting from either occluded or dilated arteries, can be caused by a mutation in a single gene. Analysis of the mutated sites along the SM α-actin sequence suggested that different vascular diseases were associated with specific ACTA2 missense mutations (21). ACTA2 mutations R118Q and R149C are significantly associated with CAD and are less frequently associated with strokes. In contrast, mutations that alter R258 are primarily associated with strokes, including Moyamoya disease, and not with CAD. The thickened medial layer of the vasa vasorum in the arteries of ACTA2 patients appeared to be composed of increased numbers of SMCs, suggesting that excessive SMC proliferation might be the mechanism of occlusive lesion formation in these patients. Occlusive diseases due to unchecked SMC hyperplasia were further suggested by the observation that atherosclerotic coronary artery lesions in ACTA2 mutation carriers were SMC-rich and lipid-poor (60). Interestingly, the occlusive lesions in the distal internal carotids of Moyamoya disease patients have also been described as SMC-rich and lipid-poor (61). Given the evidence of hyperplasia of SMCs in vascular lesions in ACTA2 mutation patients, it was not surprising that SMCs and myofibroblasts from ACTA2 mutation carriers proliferate more rapidly in vitro than matched control cells (21). Based on these data, it is hypothesized that ACTA2 mutations lead to a “gain of function” in SMC, specifically an excessive hyperplasia in response to vascular injuries, in addition to the “loss of” contractile function leading to the thoracic aortic disease. Extensive evidence has linked polymerization of G actin monomers into F actin filaments to differentiation and proliferation of SMCs through serum response factor (SRF), an axis that may be responsible for the increased proliferation of SMCs harboring ACTA2 mutations

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(reviewed in Chapter 95). Actin polymerization leads to the translocation of myocardin-related transcription factors (MRTF; myocardin, MRTF1 and MRTF2) to the nucleus and co-activation with SRF for the transcription of genes encoding SMC-restricted contractile proteins, which marks the differentiation of these cells (62 67). Dedifferentiation of SMCs is associated with the nuclear export of MRTF-A/B and downregulation of contractile protein expression, allowing for the binding of SRF to ternary complex factors (TCFs, members of the Ets family of transcription factors) (68,69). The TCF SRF complex activates a subset of SRF-regulated growth responsive genes, leading to cellular proliferation. Why ACTA2 mutations lead to dilation of the aorta but occlusion of smaller arteries may be related to one or more of the following: (i) the differences between large elastic arteries and smaller muscular arteries, including the different developmental origins of SMCs in the ascending aorta versus other arteries and the organization of elastin fibers in relation to SMCs (70,71); (ii) differences in force experienced by these two types of arteries may activate different pathways (23); or (iii) differential response of SMCs to the underlying mutation. This differential SMC response may result from two roles for α-actin in SMCs: force generation and mechanotransduction (linking mechanical stresses to transcription) (62 64). The differential response of elastic versus muscular arteries to ACTA2 mutations is in part suggested by the observation that occlusive lesions in the distal internal carotid arteries in Moyamoya disease patients stereotypically form in the region where the distal carotid artery transitions from an elastic to a muscular artery (72).

SYNDROME OF GLOBAL SMOOTH MUSCLE DYSFUNCTION DUE TO A DE NOVO ACTA2 MUTATION ACTA2 mutations have also been found in a rare syndrome that is characterized by global SMC dysfunction (25). Recurrent mutations in ACTA2 altering R179H have been identified de novo in seven children with this syndrome. Children heterozygous for this ACTA2 missense mutation are diagnosed with TAAD and have cerebrovascular lesions diagnostic or similar to Moyamoya disease under the age of 20 years. Interestingly, these patients also form fusiform aneurysms of the carotid artery proximal to the MMD occlusive lesions in the distal carotids. Therefore, these children have earlier onset of the vascular diseases found in FTAAD patients with ACTA2 mutations. Additional phenotypic features in these children indicate that the R179H mutation disrupts more than just the vascular SMCs and leads to global SMC contractile failure. The children have fixed and dilated pupils

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(congenital mydriasis), which is most likely due to loss of function of the SMCs responsible for contraction of the pupil. Affected children have hypotonic bladders diagnosed shortly after birth and a subset of patients also have hypoperistalsis of the gut. Another complication observed in these children is primary pulmonary hypertension, which occurs due to occlusion or stenosis of pulmonary arteries due to SMC hyperplasia. The severity and onset of clinical complications at a young age, along with the identification of this mutation de novo in patients, indicates that this is the most clinically severe ACTA2 mutation identified to date. Similar to what is observed with ACTA2 mutations leading to TAAD and other occlusive vascular diseases, there is a significant phenotype to genotype correlation with the ACTA2 R179H mutation.

CONCLUSION Identification of genes that when mutated predispose to thoracic aortic disease has uncovered a previously unconsidered mechanism for aortic disease pathogenesis: disruption of the smooth muscle cell “elastin-contractile unit”. Disease-causing mutations have been identified in the genes encoding the smooth muscle-specific isoforms of α-actin and β-myosin, as well as the kinase governing the contractile function of myosin, myosin light chain kinase. SMCs harboring TGFBR2 mutations failed to express and assemble contractile proteins, suggesting that a loss of contractile function may underlie aortic disease in these patients as well. Analysis of CNVs in STAAD patients also points to defects in SMC contractility and adhesion as predisposing to disease. Taken together, the results of all these inquiries suggest that an intact “elastin-contractile unit” in the medial layer of the aortic wall is crucial to maintaining vessel integrity and preventing aneurysm formation. Furthermore, the identification of both occlusive vascular diseases and aneurysms in ACTA2 mutation carriers signifies a shift in the paradigm for vascular disease, i.e., one gene can cause diverse and diffuse vascular disease. Given that vascular SMCs line the arteries throughout the body, it is perhaps not surprising that SMC-specific gene mutations can cause such a diffuse vasculopathy. Further studies are needed to confirm that SMC hyperplasia in response to ACTA2 missense mutations is responsible for the occlusive diseases in these patients and to identify the link between the mutant α-actin and the SMC proliferative response.

REFERENCES 1. Small JV. Contractile units in vertebrate smooth muscle cells. Nature 1974;249:324 7.

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