Hereditary Influence in Thoracic Aortic Aneurysm and Dissection

Abstract

Thoracic aortic aneurysm (TAA) is a potentially life-threatening condition in that it places patients at risk for aortic dissection (AoD) or rupture. Nevertheless, our modern understanding of the pathogenesis of TAA is quite limited. A genetic predisposition to TAA has been established, and gene discovery in affected families has identified two major categories of gene alterations. The first involves mutations in genes encoding various components of the transforming growth factor beta (TGF-β) signaling cascade (FBN1, TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3 and SKI), and these conditions are known collectively as the TGF-β vasculopathies (TGFβVs). The second set of genes encodes components of the smooth muscle contractile apparatus (ACTA2, MYH11, MYLK, and PRKG1), a group termed the smooth muscle contraction vasculopathies (SMCVs). Mechanistic hypotheses based on these discoveries have shaped rational therapies, some of which are under clinical evaluation. This review will discuss published data on genes involved in TAA and attempt to explain divergent hypotheses of aneurysm etiology.

Introduction

Aortic aneurysm and dissection are associated with significant morbidity and mortality, accounting for more than 10,000 and contributing to more than 17,000 deaths annually in the United States¹. Numerous risk factors for aneurysms have been identified, although they differ depending on the segment of the aorta involved. Aneurysmal disease in humans has been shown to involve the strong influence of hereditary predisposition and genetic discovery has been progressing at an intensifying pace.

Genetic determinants are now understood to represent a major factor in determining aneurysmal risk for the individual patient and genetic testing is regular feature of clinical practice. In addition to clinical utility, genetic discovery has identified novel aspects of vascular biology that reveal cellular and tissue events contributing to aortic aneurysm, which may, in turn, offer new therapeutic targets. This review will attempt to integrate our current understanding of human genetic discoveries, cellular and animal modeling of disease, and implications for aneurysm biology with special focus on the emergence of thematic groups of genes implicated in aortic disease.

Epidemiology and Etiology

Initial clues in the etiologic understanding of aneurysm came from clinical observations of repetitive anatomic patterns of disease. While studying the incidence trends of aortic aneurysm in the population of Olmstead county Minnesota, researchers noted divergent incidence of thoracic aortic aneurysm and abdominal aortic aneurysm², which was a startling finding at the time as it had previously been assumed that all aortic aneurysms had a similar etiology. It is now well recognized that aortic root and ascending thoracic aortic aneurysms (ATAAs) are significantly different from abdominal aortic aneurysms (AAAs) in terms of risk factors, pathophysiology, and natural history, despite their common phenotypic manifestation. Abdominal aortic aneurysm is a disease driven primarily by atherosclerosis³. As a result, AAA shares many risk factors with coronary artery disease including cigarette smoking, hypertension, diabetes mellitus, and male gender.

In contrast, genetic influences play a more prominent, if not dominant, role in TAA expression, in that as many as 20% of classically affected individuals have a first- degree relative with a dilated thoracic aorta⁴. In keeping with this idea, the standardized incidence rates among sibling pairs for TAA is two-fold higher than that for AAA⁵. Mendelian pedigrees demonstrate autosomal dominant inheritance, suggesting large influences of single genes segregating with thoracic aortic disease^{6, 7}. Less well understood are the inherent differences between ATAAs and descending thoracic aortic aneurysms (DTAAs). Genetically-triggered thoracic aortic disease invariably involves the ascending aorta, whereas no Mendelian pedigree has yet been described with isolated DTAA segregation. This seems to imply that single gene disorders may uniquely associate with ATAAs. Indeed, clinical observations suggest that DTAAs are more often associated with atherosclerosis, age, and hypertension than are ATAAs, even in the absence of syndromic association⁸.

Syndromic versus Nonsyndromic Thoracic Aortic Aneurysms

When TAAs appear to be familial, clinicians tend to describe them as being either “syndromic” or “non-syndromic.” Specific syndromic aortic aneurysm conditions include Marfan syndrome (MFS;OMIM#154700), Loeys-Dietz syndrome (LDS;OMIM#609192), and vascular Ehlers-Danlos syndrome (vEDS; OMIM#103050), among others (Table 1). Classically, well-described external physical features associated with “connective tissue disorders” (CTDs) have included findings (and sometimes dysfunction) in the integumental, musculoskeletal, ocular, craniofacial, and cardiovascular systems. It should be understood that while some familial aortic conditions are correctly classified as CTDs, the presence of these external CTD features alone does not indicate cardiovascular risk. While associations with CTD and aortic disease were once exclusive, improved phenotyping has now described extracardiac phenotypes in some non-CTD aortic conditions, signs of which may be less familiar to cardiologists who encounter aortic disease patients. Perhaps unsurprisingly, investigation in such families have discovered deficits in genes with utility specific to the cardiovascular system, such as functions specific to the smooth muscle cells of the aortic media. When present, external phenotypic features may be difficult to detect without a high degree of suspicion; for example, livedo reticularis, iris flocculi, mydriasis, and peripheral vascular malformation have been described in such patients, in some cases with high penetrance^{9, 10} (Figure 1).

Table 1.

Syndromic and Nonsyndromic Aneurysm Conditions

Syndromic Aneurysm Conditions	Nonsyndromic Aneurysm Conditions
Marfan syndrome	Familial Thoracic Aortic Aneurysm and
Loeys-Dietz syndrome	Dissections (FTAAD)
Vascular Ehlers-Danlos syndrome	Familial Thoracic Aortic Aneurysm
Shprintzen-Goldberg syndrome	Bicuspid Aortic Valve with Aneurysm
Aneurysms-Osteoarthritis syndrome
Cutis laxa with aneurysm

Figure 1.

Phenotypes of age-matched TGFβV and SMCV patients. (A, left) Aortic root aneurysm (3.5 cm, Z score = 6.2) in a patient with FBN1 mutation and MFS; (right) ascending aortic aneurysm (3.2 cm, Z score = 5.7) in a patient with ACTA2R179H mutation. (B, left) Normal pupillary width in MFS patient; (right) congenital mydriasis in patient with ACTA2 mutation. (C, left) Typical arachnodactyly seen with MFS versus (right) normal skeletal phenotype in ACTA2 mutation

In “nonsyndromic” TAAs, abnormalities are limited to the cardiovascular system. The majority of these conditions demonstrate autosomal dominant inheritance, but affected individuals do not exhibit external features of CTD or any other recurrent phenotype. Conditions that are usually considered “nonsyndromic” include Familial Thoracic Aneurysms and Dissections (FTAAD, which is also known as familial thoracic aortic aneurysm or FTAA)(OMIM#607086), as well as thoracic aortic aneurysm associated with bicuspid aortic valve (BAV). An extensive list of human genes associated with TAA is provided in Table 2.

Table 2.

Human Hereditary Aneurysm Conditions

Gene (Protein)	Human Aneurysmal Syndrome	OMIM Number	Inclusion on Available Clinical Panel Testing
Extracellular Matrix (ECM) Proteins
FBN1 (fibrillin-1)	Marfan syndrome11	#154700	+++
EFEMP2 (fibulin-4)	Cutis Laxa, Autosomal Recessive, Type IB12	#614437	+
ELN (elastin)	Cutis Laxa, Autosomal Dominant13	#123700	+
COL3A1 (Collagen 3 alpha-1)	Ehlers-Danlos syndrome, type 414	#130050	+++
COL4A1 (Collagen 4 alpha-1)	HANAC15	#611773	-
COL4A5 (Collagen 4 alpha-5)	X-linked Alport syndrome16	#301050	-
PLOD1 (lysyl hydroxylase 1)	Ehlers-Danlos syndrome, type 617	#225400	+
PLOD3 (lysyl hydroxylase 3)	Bone Fragility with Contractures, Arterial Rupture, and Deafness18	#612394	+
LOX (lysyl oxidase)	Thoracic aortic aneurysm and dissection19	Unassigned	-
MFAP5 (microfibrillar associated protein 5)	Familial Thoracic Aortic Aneurysm, AAT9 20	#616166	+
TGF-β Pathway
TGFBR1 (transforming growth factor, beta receptor 1)	Familial Thoracic Aortic Aneurysm, AAT5 /Loeys- Dietz syndrome 121, 22	#609192	+++
TGFBR2 (transforming growth factor, beta receptor 2)	Familial Thoracic Aortic Aneurysm, AAT3 /Loeys- Dietz syndrome 221, 22, 23	#610168	+++
TGFB2 (transforming growth factor beta 2)	Loeys-Dietz syndrome 324, 25	#614816	+++
TGFB3 (transforming growth factor beta 3)	Loeys-Dietz syndrome 526	#615582	+
SMAD2 (SMAD family member 2)	Aortic and Peripheral Arterial Aneurysm and Dissection27	Unassigned	-
SMAD3 (SMAD family member 3)	Aneurysms-Osteoathritis syndrome/ Loeys-Dietz syndrome 428	#613795	+++
SMAD4 (SMAD family member 4)	JP/HHT syndrome29	#175050	++
SKI (v-SKI sarcoma oncogene homolog)	Shprintzen-Goldberg syndrome30	#182212	+++
Cytoskeletal/ Smooth Muscle Contraction Apparatus Proteins
ACTA2 (α-smooth muscle actin)	Familial Thoracic Aortic Aneurysm, AAT6 31	#611788	+++
MYH11 (smooth muscle myosin)	Familial Thoracic Aortic Aneurysm, AAT4 32, 33	#132900	+++
FLNA (Filamin A)	Periventricular Nodular Heterotopia34	#300049	++
MYLK (Myosin Light Chain Kinase)	Familial Thoracic Aortic Aneurysm, AAT7 35	#613780	+++
PRKG1 (protein kinase, cGMP-dependent, type I)	Familial Thoracic Aortic Aneurysm, AAT8 36	#615436	++
Neural Crest Migration
NOTCH1 (notch1)	Bicuspid Aortic Valve with Aneurysm37	#109730	++
Unknown
SLC2A10 (Glucose transporter 10)	Arterial Tortuosity Syndrome38	#208050	+++
MAT2A (methionine adenosyltransferase II, alpha)	FTAAD39	Unassigned	+

Legend: Presence of gene in commercial and academic NGS Aortopathy Panel Tests: + = 0–25% of censored aortopathy panels, ++ = Inclusion in 34–66% of censored aortopathy panels, +++ = Inclusion in 67–100% of censored aortopathy panels (Table 1S); FTAAD = Familial Thoracic Aortic Aneurysm and Dissections; HANAC = Hereditary angiopathy with nephropathy, aneurysms, and muscle cramps., JP/HHT = Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome.

Marfan syndrome

The study of genetically triggered aortic disease has often been focused on Marfan syndrome, but the work has advanced the understanding and of TAA more broadly. Key accomplishments of MFS research include establishing the link between progressive aortic growth and aortic dissection (AoD)^{40, 41}, demonstrating the benefits of prophylactic aortic root surgery to prevent AoD⁴², and, most recently, introducing medical therapies directed at pathologic signaling events within the aortic wall^43–46. However, one must be careful not to be complacent in concluding that aortic dissection arises purely from progressive aneurysmal growth of the aorta. Indeed, sporadic type A and type B AoDs often occur at aortic dimensions not usually considered aneurysmal^{47, 48}.

MFS is a monogenic disorder caused by recurrent heterozygous mutations in the gene FBN1 encoding the protein fibrillin-1. Fibrillin-1 is a large extracellular matrix (ECM) protein that forms polymers called microfibrils, which closely associate with elastic fibers. Patients with MFS manifest abnormalities in multiple organs, especially the ocular, skeletal, and cardiovascular systems. Ectopia lentis (dislocation of the lens) is a highly specific manifestation of the Marfan syndrome. Typical skeletal manifestations include overgrowth of long bones, resulting in tall stature, arachnodactyly, pectus deformities of the chest, and characteristic facial features (Figure 1). The cardiovascular features of MFS were first described systematically by McKusick in Circulation in 1955⁴⁰, and in the intervening decades the cardiovascular phenotype of MFS has been more clearly defined. The primary cardiovascular abnormality is an aneurysm of the aortic root, which often extends into the proximal portion of the tubular ascending thoracic aorta to create pear-shaped aortic dilatation that is sometimes referred to as annuloaortic ectasia. Affected patients are at risk of AoD, and the risk increases with aortic diameter. However, medial degeneration is diffuse and all large and medium size arteries are at risk for dissection.

MFS is caused by a variety of genetic alterations in the FBN1 gene. While heterozygous missense mutations are most commonly observed, nonsense and whole gene deletions have been routinely described⁴⁹. Clinical genetic sequencing currently identifies ~90% of classically affected probands, raising the possibility of mutations outside of the coding region that affect the expression of fibrillin-1 such as intronic mutations that create abnormal splicing and premature termination^{50, 51}. Therefore, both human genetic observations and experiments in animal models have confirmed that loss-of-function of the FBN1 gene is the causal mechanism in MFS⁵².

At the protein level, loss of fibrillin-1 function was initially thought to affect the nature and composition of the ECM by affecting structural function. Early models of fibrillin-1 function emphasized the structural aspects of microfibrils and hypothesized that a lack of polymeric fibrillins reduced tissue strength and integrity, most manifest in elastin-rich tissues such as the aorta. In addition to their structural role, fibrillins also perform a regulatory role by influencing cell-signaling events; this regulatory action is dominated by the interaction between fibrillins and the class of cell signaling molecules known as the transforming growth factor-beta (TGF-β). TGF-β are developmental cytokines that control multiple aspects of cellular behavior including differentiation, motility, and proliferation. Multiple members of the TGF-β superfamily bind fibrillin, and therefore fibrillin deficiency indirectly influences ligand availability.

Fibrillin-1 binds to TGF-β ligands when they are complexed within the large latent complex (LLC) of TGF-β. On the basis of this knowledge and the human genetic evidence suggesting that loss of function of fibrillin-1 associates with pathology, it was hypothesized that decreased sequestration of TGF-β could result in an increase of bioavailable TGF-β, with a resultant increase in TGF-β dependent signaling. Consistent with this hypothesis, tissues from humans and animal models of Marfan syndrome demonstrate increased phosphorylation of smad2, a biochemical modification dependent on the kinase activity of the TGF-β receptor. Additionally, treatment of Fbn1^C1039G/+ mice (a murine model of MFS with aortic root aneurysms) with TGF-β neutralizing antibody significantly slowed growth and preserved histologic structure of the aorta, and was associated with a corresponding decrement in the level of phosphorylated smad2⁴³. Nevertheless, chronic antibody therapy in humans would be problematic, so other agents to antagonize TGF-β signaling were sought. Angiotensin converting enzyme inhibitors and angiotensin receptor blockers (ARBs) were known to co-antagonize TGF-β signaling, so investigators studied the effect of the ARB losartan in Fbn1^C1039G/+ mice⁴³.

They found that, just as with TGF-β neutralizing antibody, treatment with losartan normalized the rate of aortic growth and improved aortic wall structure. These findings raised the possibility that losartan may be effective in treating humans with MFS as well, prompting a number of clinical trials around the world, the results of which thus far have been mixed (discussed below)^{45, 46,53}.

Loeys-Dietz and Shprintzen-Goldberg syndrome

The association of TGF-β signaling with aortic aneurysm was affirmed through the surprising description of mutations in the genes TGFBR1 and TGFBR2 (encoding the two major human TGF-β receptor subunits) causing a human thoracic aortic aneurysm condition^{21, 22}. LDS demonstrates many overlapping phenotypes with MFS, including chest wall deformity, pes planus, highly arched palate, and, most significantly, aortic root aneurysms. But despite these overt similarities, several features are routinely described in LDS but not in Marfan syndrome, including bifid uvula, cervical arterial tortuosity, and hypertelorism; moreover, in more severe cases of LDS there may be cervical instability, cleft lip and palate, and craniosynostosis. And the arteriopathy in LDS is disseminated, with aneurysms and dissections occurring in peripheral arterial beds as well as the aorta. Conversely, ectopia lentis, a finding highly specific to dysfunction of the fibrillin-1 protein, is common in MFS but is not observed in LDS.

There is a much wider phenotypic variation in the extra-cardiovascular manifestations of LDS than in MFS. The variation is so great, in fact, that non-syndromic multigenerational families have been prospectively diagnosed with FTAAD without connective tissue features and discovered to have mutations in TGFBR1 and TGFBR2⁵⁴. Such observations have led to controversy regarding the diagnostic nomenclature of patients with mutations in the TGF-β signaling pathway^{55, 56}.

Mutations causing vascular disease in TGFBR1 and TGFBR2 have been exclusively missense and commonly, although not exclusively, occur in the kinase domain of the receptors. Whole gene deletions or nonsense mutations that would be predicted to cause nonsense-mediated decay have not been identified. In fact, genomic deletions in TGFBR1 do not cause vascular disease at all, and are instead associated with Ferguson-Smith disease, a disorder of self-healing squamous epitheliomas⁵⁷. Such genetic observations have led to predictions that missense mutations work through either a dominant negative or complex gain of function mechanism. Experiments in cell culture have documented a loss of function for signaling competency for many mutations; however, TGF-β signaling, as assayed by phosphorylation of smad2 protein, is paradoxically increased in tissues from patients with LDS.

Both human genetic discoveries and experiments in animal models of LDS have reaffirmed these paradoxical observations. Mutations in SMAD3 were identified to cause a syndrome of TAA and systemic findings including widely spaced eyes, bifid uvula, and early onset osteoarthritis called Aneurysms-Osteoarthritis syndrome⁵⁸. Mutations in the gene TGFB2 (encoding isoform 2 of TGF-β) were described as causal in a human TAA condition sharing significant phenotypic overlap with LDS^{24, 25}. And, more recently, mutations in the closely related TGFB3 have been shown to extend the spectrum and cause syndromic TAA²⁶. Patients shared features of aortic root aneurysm, arterial tortuosity, congenital talipes equinovarus, and pectus excavatum. Proteins encoded by TGFB2, TGFB3, and SMAD3 are unequivocal positive regulators of canonical TGF-β signaling, and reported mutations suggest loss of function of the cognate protein. Furthermore, mice harboring deletion mutations in either Tgfb2 or Smad3 demonstrate an independent aneurysm phenotype, and an exacerbation of the aneurysm is observed in Fbn1^C1039G/+ mice when combined with haploinsufficiency for the co-smad Smad4⁵⁹. These data suggest that loss of canonical TGF-β signaling potency correlates with TAA.

Further work on receptor mutations causing LDS has reinforced these findings. An allelic series of mutations introduced into mice demonstrated that germline deletion of TGFBR1 or TGFBR2 is unable to produce vascular phenotypes, while introduction of a heterozygous mutation in the native locus mimicking a human LDS mutation induced aortic root aneurysm, arterial tortuosity, and extracardiac phenotypes⁶⁰. Vascular disease also developed in animals in which a mutant receptor was ectopically over- expressed. These observations superficially support a dominant negative mechanism; however, when signaling potency was directly studied in cells isolated from mutant embryos, there was a 50% decrement in TGF-β signaling potential upon activation with ligand. Interestingly, haploinsufficient cells showed no decrement in signaling potential except at supra-physiological doses of ligand, perhaps accounting for the lack of phenotype from deletion mutations⁶¹. This result is consistent with studies demonstrating that individual TGF-β receptor subunit pairs signal independently within the tetrameric TGF-β receptor complex, obviating a classical dominant negative mechanism of action. These data indicate loss of canonical TGF-β signaling, by either haploinsufficient expression of SMAD3 or TGFB2 or by missense mutations in TGFBR1 or TGFBR2 with resultant decrement in signaling potential, can cause TAA and systemic signs consistent with LDS (Figure 2A).

Figure 2.

Cellular Phenotypes in Genetically-Triggered TAA (A.) Gene products affected by genetic perturbation in TGFβVs delineate the major components of the canonical TGFβ signaling cascade (Leftward Cell). Activation of the TGF-β receptor by the ligand TGF-β2 or TGF-β3 causes receptor-mediated phosphorylation of the proteins SMAD2 and SMAD3. Phospho-SMAD2/3 bind the co-SMAD, SMAD4, and translocate to the nucleus where they bind DNA and direct transcriptional events. The activity of the SMAD proteins can be inhibited by the protein, SKI. SCMV genes affect multiple proteins involved in smooth muscle cell contraction (Rightward Cell). Myosin light chain kinase (MLCK) phosphorylates myosin light chain (MLC) and phospho-MLC allows interaction of smooth muscle myosin (smMHC) with actin isoforms, such as alpha-smooth muscle actin (α-SMC). The type 1, cGMP-dependent protein kinase (PKG-1) inhibits the activity of myosin light chain phosphatase (MLCP) that negatively regulates MLC phosphorylation. (B.) Healthy VSMCs (Leftward Cell) demonstrate stable association with the extracellular matrix through matrix binding receptors, a highly developed cytoskeletal network, and restrained biosynthetic capacity. Convergent phenotypes of VSMCs in TGFβVs and SMCVs (Rightward Cell) include loss of matrix adhesion structures with disorganized and delocalized actin cytoskeleton, hypertrophy of endoplasmic reticulum and activation of matrix degrading enzymes. Multiple signaling pathways are activated in aneurysm including TGFβ receptor signaling, angiotensin II receptor signaling receptor (AT2R), insulin-like growth factor receptor (IGF-1R) signaling, and platelet-derived growth factor receptor (PDGFR) signaling. The resultant expression and elaboration of matrix degrading enzymes with degradation of extracellular matrix components compromise the structural integrity of the aortic media, resulting in the end phenotype of aneurysm.

Sporadic de novo mutations in the gene SKI encoding a TGF-β repressor were found to cause Shprintzen-Goldberg syndrome (SGS; OMIM#182212), which is associated with TAA^{30, 62}. In contrast to LDS, aneurysmal disease in SGS seems not to be as aggressive and dissections have not been reported. Also experiments in SGS patients’ cells show clear upregulation of TGF-β mediated signaling events, in contrast to LDS cells, which generally show partial loss of signaling potency at the cellular level^{30, 60}. The results of these cellular experiments would largely be predicted by the known function of SKI as a potent inhibitor of SMAD protein function. SKI binds directly to the MH2 domains of SMAD2 and SMAD3, displacing the transcriptional activator p300 and recruiting mSin3A and HDACs^{63, 64}. SKI-SMAD complexes are still competent to bind SMAD binding DNA elements, thereby converting the local chromatin environment to a repressive state and inhibiting TGF-β mediated transcription. Described mutations in SKI are clustered in the N-terminal SMAD2/3 binding domain and the Dachshund-homology domain (DHD) responsible for binding SMADs and other cofactors, predicting loss of function for the SKI protein in agreement with cellular experiments^{30, 62}.

Analysis of TGF-β signaling in vascular tissues has not yet been published in SGS models or patient samples. It therefore remains unclear how mutations that superficially produce opposite biochemical effects on the same pathway produce human syndromic conditions with such a high degree of phenotypic overlap. These data argue for the involvement of counterregulatory or compensatory signaling events, a contention that has been emphasized in pathogenic models of TGF-β vasculopathies (TGFβVs).

Smooth Muscle Contraction Vasculopathies

In contrast to syndromic forms of TAA, some families exhibit autosomal dominant inheritance of aneurysmal disease with few outward physical manifestations. Syndromic aortic conditions tend to be caused by genes with wide expression patterns in multiple organ systems. In contrast, gene defects causing FTAAD represent defects in proteins with functionality specific to the aorta, and in particular to vascular smooth muscle cells. The first reported gene defect in the SMCV gene family was MYH11, encoding a smooth muscle specific myosin isoform^{32, 33}. Smooth muscle myosin heavy chain (smMHC) is expressed in definitive vascular smooth muscle cells, as well as uterine and enteric smooth muscle. Reported patients with smMHC mutations exhibit typical ascending thoracic aortic aneurysm with a high penetrance of patent ductus arteriosis. Penetrance is incomplete but individuals carrying the abnormal MYH11 alleles demonstrate evidence of increased arterial stiffness, a hallmark of aneurysmal tissue⁶⁵.

The next gene identified as causal in FTAAD was ACTA2, the locus encoding the smooth muscle specific isoform of actin (alpha smooth muscle actin, α-SMA). α-SMA is a protein well described component of smooth muscle cells, but it is also expressed widely in cells during inflammation and is a known transcriptional target of TGF-β signaling. Patients with mutations in ACTA2 exhibit a diverse vasculopathy characterized primarily by ATAA. Other cardiovascular abnormalities include cerebral aneurysm, myocardial infarction, and a neurovascular malformation resembling moyamoya disease⁶⁶. In ACTA2-associated vasculopathy there appear to be distinct allele-specific differences in disease severity ⁶⁷. In particular, missense mutations at arginine 179 produce a severe syndrome associated with multiple congenital anomalies, early onset aortic aneurysm and dissection, and congenital mydriasis^{9, 10} (Figure 1). Severe aortic disease is not, however, limited to the R179 mutation, as childhood AoD has also been described in mutations at different positions⁶⁸. So far described mutations in ACTA2 have been missense in nature and vascular disease has not been associated with large gene deletions or nonsense mutations that would invoke simple haploinsufficiency as a mechanism. The Acta2-/- knockout mouse is viable with a normal lifespan, and explanted Acta2-/- vascular smooth muscle cells are hyperproliferative⁶⁹. The human mutational repertoire in ACTA2 is more consistent with a dominant negative effect (individual missense mutations spread throughout the protein) however pathogenetic clarity will likely require more advanced gene modeling experiments in small animal models to formally exclude a gain of function mechanism for these alleles.

Subsequent reports have reinforced the importance of the actin-myosin interaction in genetically trigged TAA. Putative loss-of-function mutations in MYLK were reported to cause TAA in a large family with autosomal dominant inheritance³⁵. MYLK encodes myosin light chain kinase (MLCK), a positive regulator of the actin-myosin interaction, and mutations predicted loss of function of this regulator. Conversely, a recurrent mutation in PRKG1 (c.530G>A, p.Arg177Gln) was recently described to cause TAA³⁶. The mutation appears to disinhibit activity of the type I cGMP-dependent protein kinase (PKG-1). PKG-1 inhibits myosin light chain phosphatase, thereby functioning as a negative regulator of actin-myosin interaction. Consequently, increased PRK-1 activity results in decreased phosphorylation of myosin light chain and inhibition of actin-myosin interaction. In summary, genetic perturbations that tend to decrease actin-myosin interaction—either though decreased function of the actin-myosin pair (ACTA2 or MYH11) or their regulators (MYLK or PRKG1)—within vascular smooth muscle cells tend to cause TAA (Figure 2A).

Bicuspid Aortic Valve/ Thoracic Aortic Aneurysm

Bicuspid aortic valve (BAV) is the most common developmental malformation of the heart, affecting between 0.5–1% of the general population. In about 40–50% of those with BAV there is an associated dilatation of the ascending thoracic aorta (or aortic root). Therefore BAV-associated TAA (BAV/TAA) is likely the most common type of aneurysm affecting humans. Familial predisposition in BAV/TAA is a well-established; indeed, it is so common that screening of first-degree relatives is recommended in routine clinical practice. Some forms of monogenic TAA have a high predisposition for BAV such as LDS, ELN-related cutis laxa, and TAA associated with LOX mutations amongst others, however these conditions likely only represent a very small fraction of human BAV disease^{22, 70}. The most common forms of familial BAV appear to occur in an autosomal dominant pattern but with incomplete penetrance. Often cosegregating in families with BAV are other forms of left heart obstruction, including coarctation of the aorta, mitral stenosis, and, in severe cases, hypoplastic left heart syndrome. Although they have been associated with one large pedigree segregating with bicuspid aortic valve, coarctation of the aorta, and tetralogy of Fallot³⁷, mutations in NOTCH1 are thought to be rare amongst the general population of BAV patients⁷¹. Bicuspid aortic valve with, or without, aortic dilatation is also a common feature of Turner’s syndrome, which is caused by monosomy of the X chromosome⁷².

Available data suggests that typical BAV/TAA is genetically complex. Although pedigrees with apparent autosomal inheritance with incomplete penetrance are common, causative single loci have proved elusive. The inability to identify simple Mendelian loci with whole exome approaches suggests that many pedigrees may be due to polygenic influence rather than simply incomplete penetrance, which has been the commonly cited explanation⁷³. Unraveling the complex genetic architecture of BAV/TAA will likely require large-scale collections combined with deep sequencing approaches⁷⁴.

Pathogenic Models of Genetically-Triggered Aortic Disease

Research in experimental aneurysm has repetitively revealed overactivity of the TGF-β pathway in TAA^{43, 75, 76}. Furthermore, the additional evidence of human mutations in genes encoding effectors of canonical TGF-β signaling have lead to the hypothesis that aberrant TGF-β signaling drives aneurysm progression⁴³. Although postnatal observations of increased TGF-β signaling are robust, genetic perturbations tend to result in loss of TGF-β signaling potency, illustrating the complexity of the signaling perturbation. How then do these mutations induce increased TGF-β signaling? Loss-of- function mutations have been proposed to cause disease through upregulation of counterregulatory pathways that may directly drive aneurysm^{24, 77}. An example of is upregulation of the MAPK pathway noted in Fbn1^C1039G/+ mice⁵⁹, with inhibition of this noncanonical pathway retarding aneurysm progression. It should be noted that human aortic samples available for examination often represent late-stage disease and it remains possible that at an earlier stage of development low TGF-β signaling precedes upregulation. Other pathogenic models posit dysfunction of the smooth muscle contractile apparatus as the fundamental anomaly in TAA, based on multiple loss-of- function mutations discovered in components and regulators of smooth muscle contraction⁷⁸. This hypothesis emphasizes the importance of the actin-myosin unit in aortic homeostasis. Failed contractile structure leads to focal adhesion complex and other cell surface receptor rearrangement. As a result, the VSMC fails to sense and attach properly to the surrounding ECM. These VSMCs undergo phenotypic change and secrete matrix-degrading enzymes with nonproductive remodeling of the aortic media. Importantly, this model naturally incorporates hypertension, which is thought to be a clinical risk factor for AoD⁷⁸.

While these models have clear and compelling supporting evidence from the genetic literature and from experimental aneurysm studies, it is difficult to reconcile the two. Is increased TGF-β signaling a final common pathway for genetic perturbations causing aneurysm? Dysregulation of TGFβ is believed to induce VSMC phenotypic change and the secretion matrix-degrading enzymes such as MMPs, and would therefore represent a candidate final common pathway. Increased phosphorylation of the TGF-β effector SMAD2 has been demonstrated in tissue from patients with SMCVs⁷⁹ leading to the concept that increased TGF-β signaling could be a commonality across genetic perturbations causing aneurysm. However, dependence of aneurysm pathology on TGF-β has yet to be demonstrated in any SMCV disease model, leading to continued questions of correlation and causality. Alternatively, there is a lack of evidence that increased TGF-β signaling, which has been repeatedly observed in patient samples, drives decreased contraction of smooth muscle cells. In contrast TGF-β is a signal associated with transcriptional activation of contractile gene expression through SMAD3 and myocardin⁸⁰. Although increased contractile protein expression has recently been described in aneurysm samples taken from patients with MFS⁸¹, the exact opposite has been observed in patients with mutations in TGFBR2⁸² and sporadic TAA⁸³. These conflicting observations may indicate that relative “upregulation” or “downregulation” is less important than disruption, per se, of proper contractile protein homeostasis. This would also be consistent with observations that both loss-of-function and duplications in the contractile gene MYH11 are associated with TAA^33,84. These observations would be are consistent with disruption of cellular structures that are highly dependent on a regular stoichiometry. The polymeric actinomyosin cytoskeleton would certainly qualify in this respect. (discussed below).

Extracellular Matrix Genes and the Relationship to Matrix-Independent Gene Groups

Conduit arteries such as the aorta have one primary function: to accept the output of ventricular systole and carry blood to target organs. The engineering requirements of this function primarily involve components of the ECM; collagens (primarily I and III), elastin, and microfibrils necessary to provide structure and strength. Pathologic observations of aortic tissue have consistently noted striking abnormalities of the ECM of the aortic media with elastin fiber fragmentation and VSMC disarray^{6, 7}. It therefore comes as no surprise that defects in members of the ECM itself cause aneurysm when dysfunctional. In humans, examples include genetic variation in genes encoding ECM proteins, such as fibrillins (FBN1)¹¹, collagens (COL3A1)¹⁴, elastin (ELN)⁷⁰, and matrix-stabilizing enzymes such as fibulin-4 (EFEMP2)¹² and lysyl oxidase (LOX)¹⁹, a copper-containing oxidase responsible for cross linking of collagens and elastin. Recently, defects in the ECM component microfibrillar-associated protein 5, encoded by MFAP5, have been shown to cause TAA²⁰. EFEMP2 and LOX mutations in particular illuminate the importance of elastogenesis to TAA pathogenesis. Fibulin-4 is a critical factor required for the recruitment of lysyl oxidase to tropoelastin (the building block of amorphous elastin fibers) in vivo⁸⁵, and inactivation of either fibulin-4 or lysyl oxidase causes aneurysm in both humans and mice^{12, 19, 86, 87}.

While a model of TAA induced by improper developmental elastogenesis is experimentally validated^86,87, how do we account mechanistically for alteration in cell autonomous gene products such as members of the TGF-β signaling cascade or members of the smooth muscle contraction apparatus? Available data have widely implicated failure of ECM homeostasis (with elastin destruction) through upregulation of matrix degrading enzymes and/or decreased activity of inhibitor proteins^{88, 89}. Upregulation and altered activation of matrix metalloproteinases (MMPs) and other matrix degrading enzymes are commonly observed in TAA, specifically MMP-2 and MMP-9^{88, 90}. MMP upregulation has been associated with upregulation of various signaling pathways observed to be dysregulated in aneurysmal VSMCs including insulin-like growth factor-1 receptor⁹¹, TGF-β receptor⁹², platelet-derived growth factor⁹³, and angiotensin II type 1 receptor signaling⁹² (Figure 2B). There are many connections between angiotensin II signaling and TGF-β signaling. Angiotensin II signaling is known to upregulate autocrine production of TGF-β signaling in cell culture⁹⁴ and both TGF-β and angiotensin II signaling can directly activate SMAD proteins⁹⁵, and activate MMPs. In addition to degradation of ECM, MMPs can release and activate latent matrix associated signaling molecules, notably including TGF-β⁸⁹. ECM destruction by MMPs can lead to aortic medial weakening and aneurysm owing to the limited ability of the mature VSMCs to generate new elastin fibers⁹⁶. Enzymatic activation in genetically triggered human TAA is mediated by VSMCs (rather than inflammatory cells) and linked to changes in cellular phenotypes. Ascending aortic aneurysms demonstrate phenotypic modulation of VSMCs to a “synthetic” phenotype involving loss of stress fibers, endoplasmic reticular hypertrophy, and secretion of matrix degrading enzymes⁸³ (Figure 2B).

Loss of stress fibers and deficiency of the filamentous actin cytoskeleton is a particularly notable aspect of TAA pathology (Figure 3). Interestingly, several classes of cell autonomous TAA gene defects have close associations with the actin cytoskeleton. Actin reorganization is a hallmark of migratory mesenchymal cells induced by canonical TGF-β signaling, and underdeveloped stress fibers result when the canonical TGF-β pathway is inhibited⁹⁷. Mutations resulting in disruption of the VSMC contractile apparatus more directly influence cytoskeletal dynamics. Fibroblasts from patients with ACTA2 mutations demonstrate underdevelopment of stress fibers and fail to fully express contractile proteins when stimulated with TGF-β⁹⁸. Perturbation of actin dynamics through mutation in the gene FLNA, encoding filamin A, directly implicates the filamentous cytoskeleton in TAA pathogenesis. Women with X-linked periventricular nodular heterotopia (PVNH) associated with mutations in FLNA have a risk of TAA caused by filamin A deficiency³⁴. Filamin A is a broadly expressed integrator of cell signaling events (RhoA and SMADs) and mechanical forces with the actin cytoskeleton, necessary for orthogonal branching of actin as well as filament linkage to multiple extracellular receptors^{98, 99}. Cells deficient in filamin A show defective TGF-β signaling, as assessed by SMAD2 activation, providing a link between TGF-β signaling and proper actin assembly¹⁰⁰. Genetic evidence from patients with cervical artery dissection, a phenotype seen in genetically triggered aortopathy, further supports the association of vascular fragility with cytoskeletal dynamics. Sporadic cervical artery dissection has recently been linked to common variation at the PHACTR1 locus¹⁰¹. PHACTR1 binds G- actin through its four RPEL domains and directs assembly of stress fibers and cellular motility¹⁰².

Figure 3.

Loss of Cytoskeletal Structure in Thoracic Aortic Aneurysm Tissue. TAA stained with Verhoeff-Van Gieson (VVG) stain demonstrates familiar elastin fiber paucity and fragmentation (Upper panels). Staining of aortic tissue for filamentous actin (F-Actin) shows a nearly complete loss of intracellular organized cytoskeletal architecture in TAA (Lower panels).

How could disturbance in actin assembly mediate aneurysmal phenotypes? Interestingly, regulation of filamentous actin assembly is intimately linked to both cellular morphology and MMP expression, cellular phenotypes displayed within TAA tissue. For instance, depolymerization of actin cytoskeleton with cytochalasin D, but not the microtubulin destabilizing toxin nocodazole, has been shown to directly induce the activation of MMPs, including MMP2^{103, 104}. In this way, TAA cells share features in common with cancer cells that have achieved anchorage independent growth. In fact, M2 melanoma cells deficient for filamin A exhibit constitutive secretion of active MMP9, an activity that is downregulated upon stable reintroduction of filamin A expression¹⁰⁵.

Identifying a unifying pathogenetic mechanism for VSMCs under genotoxic stress has been difficult, despite the known transcriptional interplay between TGF-β signaling and VSMC contractile gene expression. Direct experimentation will be required to interpret which of the functions of the actin cytoskeleton (organelle trafficking, mechanosensing, cellular signaling, and the maintenance of cellular junctions) is affected by human mutations causing aneurysm. Recent hypotheses have emphasized the role of the cytoskeleton in the process of mechanosensing^{106, 107}, although many functions of the cytoskeleton are difficult to separate experimentally. Whether effects are mediated through direct alteration of actin dynamics (FLNA or PHACTR1), altered mechanosensing (ACTA2, MYH11)⁷⁸, or as a downstream target of disordered TGF-β signaling (TGFBR1, TGFBR2, SMAD3)⁶, disruption of the filamentous cytoskeleton is an emerging theme of genetic discovery in aneurysm.

Implications for Therapy

Just as we’ve come, over the years, to recognize that the thoracic aorta behaves differently from the abdominal aorta and, further still, that the ascending thoracic aorta behaves differently from the descending thoracic aorta, so too have we come to appreciate that TAAs of different etiologies can behave quite differently. Indeed, because BAV-associated TAAs are genetically mediated and demonstrate medial degeneration histologically, for many years experts had thought that such aneurysms were especially vulnerable to AoD, just as in MFS. However, a recent study found that in patients presenting with an acute AoD, the mean aortic diameter among those with an underlying BAV was actually significantly larger (i.e., not smaller) than among those with tricuspid aortic valves (66±15 mm vs. 56±11 mm, respectively; p=0.0001)¹⁰⁸, refuting that the notion that a genetic underpinning predicts more virulent disease. Conversely, many patients with MFS suffer type B AoD at aortic diameters that are quite normal.

Even more provocative is recent evidence that among those with MFS, the underlying gene mutation may significantly impact outcome. In a registry of patients with Marfan syndrome and FBN1 mutations, patients with haploinsufficient mutations had a 2.5-fold increased risk for cardiovascular death and a 1.6-fold increased risk for any aortic complication compared with patients with a dominant-negative mutation¹⁰⁹. And while the clinical trials of losartan to slow aortic root growth in MFS have been by and large disappointing, a related study by the same investigators discovered that therapy with losartan significantly reduced aortic the rate of aortic growth in haploinsufficient MFS patients but not in dominant-negative patients¹¹⁰.

Collectively these findings highlight both the challenges and opportunities that face us in determining optimal treatment strategies for TAAs. We simply can no longer assume that all aneurysms, even when they involve the same aortic segment, will behave similarly. The underlying etiology and even the underlying genetic mutation appear to impact both risk and response to therapy. Therefore, efforts to better define individual patients’ specific genetic defects may inform future research efforts and define the groups in which specific therapies may be most efficacious.

Future Directions

There are many questions to address in the field of hereditary aneurysm, the answers to which may inform therapeutic approaches. What are the processes by which cells within the aortic media undergo phenotypic change in response to mutations in VSMC contractile or the canonical TGF-β signaling pathways? Cellular phenotypic changes are typically accompanied by large-scale epigenetic chromatin remodeling and in fact; extensive epigenetic changes have been described at the SMAD2 promoter in cells from TAA^{111, 112}. Modulation of these and similar pathways may useful to improve aortic performance.

In addition to epigenetic investigation, next-generation sequencing approaches have much to offer in the analysis of the genetics of aortic disease. Although great progress has been made in the identification of rare Mendelian forms of aortic disease, very little is known about more common genetic variation conferring susceptibility to TAA or to AoD. Sporadic TAA patients tend to be older and have more comorbidities than patients with Mendelian forms of TAA and AoD^{113, 114}, suggesting etiologic diversity. Considering the rarity of identifiable genetic causality in described TAA and AoD cohorts, it is clear that the large majority (80–90%) of patients with these diseases have poorly understood genetic predisposition. A better understanding of the genetic landscape of TAA using these approaches will no doubt identify new targets for therapeutic intervention.

A single GWAS study has been performed on nonsyndromic individuals with TAA, which identified a susceptibility locus at 15q21.1 overlapping the gene FBN1¹¹⁵; indeed, the redundant identification of genes involved in both monogenic and sporadic forms of the same disease has become a theme in the investigation of cardiovascular disorders¹¹⁶. While this important study cemented the association of fibrillin-1 with forms of nonsyndromic TAA, only this one association reached genome wide significance, likely as a consequence of limited power. Research groups studying myocardial infarction, hyperlipidemia, atrial fibrillation, stroke, and other cardiovascular conditions have made more significant progress through the establishment of large-scale genetic collections. Considering the progress made by the combination of both common and rare human variation in these conditions, it would seem worthwhile to expand future efforts aortic research more aggressively into large-scale populations.