Genetic and Developmental Contributors to Aortic Stenosis

Abstract

Aortic stenosis (AS) remains one of the most common forms of valve disease, with significant impact on patient survival. The disease is characterized by left ventricular (LV) outflow obstruction and encompasses a series of stenotic lesions starting from the left ventricular outflow tract to the descending aorta. Obstructions may be subvalvar, valvar or supravalvar, and can be present at birth (congenital) or acquired later in life. Bicuspid aortic valve, whereby the aortic valve forms with two instead of three cusps, is the most common cause of AS in younger patients due to primary anatomical narrowing of the valve. In addition, the secondary onset of premature calcification, likely induced by altered hemodynamics, further obstructs LV outflow in bicuspid aortic valve patients. In adults, degenerative AS involves progressive calcification of an anatomically normal, tricuspid aortic valve, and is attributed to life-long exposure to multifactoral risk factors and physiological wear-and-tear that negatively impacts valve structure-function relationships. AS continues to be the most frequent valvular disease that requires intervention, and aortic valve replacement is the standard treatment for patients with severe or symptomatic AS. While the positive impacts of surgical interventions are well documented, the financial burden, the potential need for repeated procedures, and operative risks are substantial. In addition, the clinical management of asymptomatic patients remains controversial. Therefore, there is a critical need to develop alternative approaches to prevent the progression of LV outflow obstruction, especially in valvar lesions. This review summarizes our current understandings of AS etiology; beginning with developmental origins of congenital valve disease, and leading into the multifactorial nature of AS in the adult population.

INTRODUCTION.

In the United States, aortic stenosis (AS) accounts for 3–6% of all congenital heart defects and is the second most prevalent valve-associated disease in the general population. AS is clinically defined as ‘left ventricular (LV) outflow obstruction’ and includes etiologies such as congenital malformations (particularly bicuspid and unicuspid aortic valve disease), calcification, and rheumatic valve disease.¹ AS is both sex and age-dependent, occurring more frequently in males,² affecting >2% of individuals over 60 years and up to 10% of patients over 80.^{3, 4} Known risk factors are typical of other cardiovascular diseases including hypertension, smoking, elevated low density lipoprotein (LDL) cholesterol and metabolic syndrome.⁴ While survival is high in asymptomatic patients, the mortality rate can be up to 50% within 2 years after the onset of symptoms, if left untreated.¹ Despite disease prevalence, effective treatment options for AS patients are limited. Pharmacological initiatives to treat AS have focused on targeting risk factors, particularly elevated lipoprotein levels; however outcomes of therapies such as statins remain unsubstantiated, with some reports demonstrating little beneficial effect on valve structure and function despite a significant drop in LDL-cholesterol levels.^5–12 For more severe AS patients, aortic valve replacement surgery or transcatheter approaches comprise contemporary standard levels of care. However, surgical interventions are accompanied by significant drawbacks, including the need for long-life anti-coagulation therapy in patients with a mechanical valve. There is also a high incidence of re-operation due to degeneration of implanted valves in adults, and somatic growth in pediatric-aged recipients whose hearts are too small to accommodate adult-size protheses. Ongoing clinical trials are currently focused on testing new treatments based on molecular targets in the signaling pathways identified to be associated with AS, largely calcification; however to date, no effective pharmacotherapy has been established to prevent or treat AS.³ As a result, AS remains a significant and growing healthcare burden and disease prevalence will rise as life expectancy increases. AS continues to be a financial drain with annual spending costing an estimated total of $10.2 billion in the US alone.^{13, 14} Therefore, the field is in need of new, and more effective drug remedies for the management of AS.

HEART VALVE ANATOMY AND PATHOLOGY.

Situated on the left side of the heart, the aortic valve serves to maintain unidirectional forward blood flow from the LV to the aorta and the systemic circulation. To do this, the aortic valve must maintain durability and strength in response to changes in mechanical stresses during the cardiac cycle; ensuring that the valve orifice is open during ventricular contraction and closed by the end of systole, with minimal regurgitation into the LV cavity.

The aortic valve is an avascular tricuspid structure connected to the aortic root via the fibrous annulus.¹⁵ Each of the three leaflets, referred to as cusps, are <1mm thick and named according to their location relative to the coronary artery ostia: the right coronary (RC), left coronary (LC) and noncoronary (NC) cusps.¹⁶ Each cusp is composed of an organized connective tissue system comprising a highly ordered network of extracellular matrix (ECM) components, which, in addition to specialized populations of cell lineages, are crucial for supporting biomechanical demand as the cusps open and close >100,000 times each day.¹⁵

Within each cusp, the ECM is stratified into 3 layers that each provide unique biomechanical properties. In the aortic position, the fibrosa is localized to the aortic side of the cusp away from blood flow, and is highly abundant in aligned collagen fibers that provide tensile strength to this high load-bearing layer. On the opposite surface of the cusp adjacent to blood flow is the ventricularis layer; enriched with elastin fibers that allow the valve configuration to return to the closed state once external forces have been released at the end of systole.¹⁶ Sandwiched between the fibrosa and ventricularis layers is the spongiosa, predominantly comprised of proteoglycans and glycosaminoglycans that aid both valve resistance to cyclic compression, and provide lubrication to the adjacent layers as they deform during each cardiac cycle.¹⁷ In addition to these layer-specific ECM components, the aortic valve cusps contain other minor but essential ECM components, including fibronectin and lamin.¹⁸ While this organization has been described for the aortic valve cusps, conserved patterning relative to blood flow is observed in the other three valves (mitral, tricuspid, pulmonary), as well as across species.¹⁹ Unlike the mitral and tricuspid valves, the aortic and pulmonary valves lack externally supporting chordae tendineae, but instead, possess tensile-providing ECM components within the high-load bearing region of the valve cusps (fibrosa) which including proteins such as Tenascin and Type 3 Collagen.^{19, 20}

The cellular composition of the aortic valve, like all cardiac valves, is predominantly comprised of two populations: valve interstitial cells (VICs), and valve endothelial cells (VECs). The VIC population has been described as dynamic and heterogenous,²¹ and their primary role is to secrete ECM during valve formation to establish the trilaminar structure, and then serve to maintain this structure throughout adulthood.^21,19 In healthy adult valves, VICs are described as ‘quiescent’ and share phenotypic characteristics with resting fibroblasts, including expression of vimentin, and an absence of smooth muscle α-actin.²² However, even in homeostatic valves, a small population (<5%) of VICs express molecular markers consistent with myofibroblast- and smooth muscle cell-like phenotypes and are therefore referred to as ‘activated’.^23–27 The physiological need for activated VICs (aVIC) in healthy valves is not clear, but based on studies in other fibroblast cell types, it has been suggested that these cells may mediate physiological remodeling or turnover of the valve ECM in response to ‘wear-and-tear’ throughout life in order to maintain structure-function relationships. Additional insights into the role of aVICs have also been gained from studies of diseased valves, which demonstrate an association between an abnormal increase in the number of smooth muscle α-actin-positive VICs,²² and pathological changes in the composition and organization of the valve ECM, subsequently compromising biomechanical function.^23–29 The mechanisms underlying the transition of a quiescent VIC to an activated state in health and disease is thought to be influenced by the biomechanical environment (increased stretch, stiffness, shear stress),^{28, 30–33} however the contribution of activated VICs to disease pathogenesis remains unknown.

In addition to VICs within the core of the valve cusp, a single layer of VECs forms a protective endothelium over the surface of the cusp. As a barrier, the endothelium prevents infiltration of disease-causing risk factors and inflammatory cells from the circulation. In addition, VECs have been shown to be mechanosensitive; in response to hemodynamic cues, VECs initiate signaling pathways that relay to the VICs to prevent activation, thus mediating adaptive changes in ECM homeostasis.^30–38 In healthy aortic valves, VECs experience a range of physiological hemodynamic forces including wall shear stress that are influenced by cyclic flow patterns. Further, biomechanical studies have utilized in vitro flow systems to demonstrate changes in VEC molecular profiles in response to mechanical and physiological hemodynamic conditions that are likely critical for mediating VEC and VIC function and maintaining ECM homeostasis.^{30, 37, 39} Together, these collective studies highlight the complexity of the normal valvlar environment in the beating heart, and shed light on the the balance of finely tuned biological processes that are needed to establish and maintain healthy valve structure-function relationships.

CONGENITAL VALVE MALFORMATIONS AND AS.

AS present at birth is referred to as congenital (see Figure 1), and accounts for approximately 3–5% of all congenital heart diseases. LV outflow tract obstruction may arise from a variety of causes, including muscular obstruction below the valve, obstruction at the valve itself, or aortic narrowing immediately above the valve, termed subvalvar, valvar and supravalvar AS, respectively. Patent ductus arteriosus, aortic coarctation, and ventricular septal defects are commonly associated congenital heart defects, found in 15–20% of AS patients,² in addition to isolated bicuspid aortic valve (BAV).⁴⁰ In the newborn, symptomatic presentation of diminished systemic cardiac output due to LV outflow tract obstruction is deemed “critical AS”, and requires life-saving urgent or emergent intervention.² While the presentation of AS or BAV in older children and adolescents can be associated with minimal symptoms, there is nevertheless a risk of sudden death in individuals with moderate to severe obstruction.⁴¹

Figure 1. Representation of congenital AS is largely influenced by genetics and pathogenesis is initiated in the embryo and present at birth.

Structural malformations of the aortic valve as a result of developmental defects are a common cause of congenital AS in the pediatric population. Healthy aortic valves form with three cusps (tricuspid) but congenital aortic valve malformations include those that form with one (unicuspid), two (bicuspid) or four (quadricuspid) valve cusps. Bicuspid aortic valve is the most common and presents as fusion between the right-left cusps (R/L), right-noncoronary cusps (R/NC) and left-noncoronary cusps (L/N). As a result of these structural abnormalities, the hemodynamic environment around the bicuspid valve is disturbed and dependent on the fusion pattern. The tricuspid valve features a centrally-aligned flow jet through the valve orifice at physiologic velocity as well as symmetrical vortex formation in the leaflet sinuses. The smaller valve orifice and impaired mobility of the fused leaflet in both BAV R/L and BAV R/NC result in a higher velocity flow jet that is skewed toward the nonfused leaflet and impinging on the wall of the ascending aorta (right posterior wall in R/NC and right anterior wall in R/L). This leads to increased wall shear stress at these locations as well as the formation of asymmetrical vortices in the leaflet sinuses and abnormal helical flow patterns (not shown) downstream in the aorta. Secondary effects of BAV, likely mediated by disturbed hemodynamics, include aortopathies (not shown) in addition to calcification which further exacerbates left-sided outflow obstruction. AAo, Ascending Aorta; L, left; R, right; NC, non-coronary; BAV, bicuspid aortic valve

BAV is the most common aortic valve malformation, while unicuspid and quadricuspid aortic valves are rarer anatomic variants (Figure 1). The unicuspid aortic valve is an infrequent congenital malformation, noted in only 0.019% in patients,⁴² but most frequently observed in critical AS. It is morphologically characterized by a single thickened cusp that causes significant outflow obstruction² but can also present as aortic regurgitation, or as a mixed lesion with both stenosis and regurgitation.^{40, 43}

Bicuspid aortic valve (BAV) is the most common congenital malformation with an estimated prevalence of 1–3% and a male predominance of 3:1.⁴⁴ BAV may be asymptomatic in early years, but is the most common cause of AS in patients under the age of 70.¹ In broad terms, the structural defect at birth is characterized by the presence of two, rather than three cusps, functional cusps. While this often leads to a smaller valve orifice (Figure 1) and variable obstruction to the systemic circulation, in some cases of BAV the more significant functional defect is aortic valve regurgitation (AR).^{2, 44} In addition to valve dysfunction, BAV may exert secondary detrimental effects on myocardial growth leading to heart failure in ~16.9% of patients.⁴⁵ This is in addition to the premature onset of calcification observed in over 50% of BAV patients, resulting in less compliant valve tissue that further exacerbates the severity of AS, as will be discussed in detail below.⁴⁶

The identifying morphological feature of BAV is abnormal valvulogenesis, leading to 2 unequal cusps and a central raphe. Based on the number of raphes, there can be 3 forms - type 0 (no raphe present); type 1 (1 raphe present) or type 2 (2 raphe present). Type 1 is the most common with a frequency of 90% of all BAV cases and prognostically most likely to exhibit AS in the young adult population. However type 2 has also been associated with complications including AS, AR, infective endocarditis and myocardial dysfunction in the younger age group, although these phenomena are less common.^{47, 48} On the basis of raphe position relative to coronary artery origins, types 1 and 2 are classified as R/L (right-left fusion), R/NC (right-noncoronary fusion) and L/NC (left-noncoronary fusion). R/L is the most common BAV morphology (accounting for 80%), followed by R/NC (17%), and then L/NC (~2%).⁴⁸

Aortic Valve Development.

Going back to cardiac embryology, the field has defined the complex mechanisms underlying heart valve initiation, beginning with the formation of endocardial cushion swellings in the outflow tract and atrioventricular regions. This process, occurring between E9.5-E11.5 in the mouse, requires a subset of endocardial cells overlying the cushions to undergo endothelial-to-mesenchymal transformation (endMT); giving rise to a population of highly proliferative and migratory, mesenchyme valve precursor cells (reviewed⁴⁹). endMT is a tightly regulated process and restricted to endothelial cells only within the valvlar regions of the developing heart. Many complex molecular networks between differential cell types have been reported to be required for endMT, and include initiation of the process by local Transforming Growth Factor-β (Tgfβ),^50–53 and Bone Morphogenetic Protein (BMP)^{51, 54–61} signaling pathways emanating from the adjacent myocardium to overlying endothelial cells, as well as several T-box transcription factors, Wnt and Notch in valvlar cell types.^{52, 62–70}

While endMT is critical for generating the pool of mesenchyme valve precurosrs within the endocardial cushion of the outflow tract,^{19, 71} there is additional contribution from other cell lineages, including the cardiac neural crest and secondary heart field.^72–74 The importance, or relevance of the lineage diveristy has not been extensively examined, however, cell fate studies have significantly improved our understanding of lineage-specific anatomy of the valve. Following migration from the aortic sac, neural crest cells occupy the distal outflow tract cushion. This structure later gives rise to the aorticopulmonary septum, and divides the distal outflow tract into the aorta and pulmonary artery by E12.5.⁷⁵ In contrast, the proximal outflow tract cushions largely contain endMT-derived mesenchyme cells.^{71, 76, 77} During the process of cushion development, the neural crest- and endothelial-derived mesenchyme cells meet at the distal-proximal boundary, where the two cushions fuse, and separate the promoximal outflow tract into two ventricular outlets. Subsequently, the free (unfused) cushions undergo extensive ECM remodeling, and morphological ‘sculpt’, and elongate to become the coronary cusps of the aortic valve.^{61, 78, 79} Together these cell lineage studies conclude that the mature L/C and R/C coronary cusps predominantly originate from endothelial-derived (endMT) mesenchyme precursors with less contribution from neural crest cells,^{20, 71, 80} while the N/C cusp contains cardiomyocytes from the secondary heart field lineage.^81–84 Despite a wealth of knowledge regarding the developmental mechanisms of aortic valve development, the field has yet to fully delineate which aspect(s) of the valvulogenesis process is disturbed in giving rise to congenital structural malformations. A seminal question surrounding the developmental origins of BAV asks if the malformation is the result of abnormal separation or alternatively, irregular fusion of the cusps. Further, how, and why does this defect occur? In consideration of what is known about aortic valvulogenesis, it is suggested that inefficient cushion formation as a result of inadequate endMT, neural crest cell migration and SHF-derived cardiomyocyte differentiation, is a significant contributor. In addition, incomplete formation of three defined cusps may be the result of cushion displacement, poor cushion fusion, or even compromised sculpting and remodeling of the mature cushions, post-endMT.⁸⁵ To date there is no clear answer, but insights have been gained from human genetic sequencing studies in parallel with mouse models of congenital aortic valve malformations (as summarized in Table 1).⁸⁶

Table 1.

Mouse models of bicuspid aortic valve disease.

Gene	Mouse model	Known human mutations	BAV Penetrance	BAV subtype	Cushion defect?	Aortic aneurysm?	Other cardiac defects?	Reference
Exoc5	Nfatc1Cre+; Exoc5f/−	none	45%	80% R/NC & 20% L/N	yes	none	dysmorphic AoV, VSD, vascular hemorrhages	88
Gata5	Gata5−/−	yes70, 104, 105, 155	25%	R/NC	none	none	Mild LV hypertrophy	106
Gata6	Gata6+/−	yes70, 107, 156	56% males & 27% females	R/L	none	none	none	107
Hoxa1	Hoxa1−/−		24%	_	none	none	VSD, TOF, IAAB	157
Matr3	Matr3Gt-ex13	yes158	15%	_	none	none	PDA, AoC, IAA, HAA, VSD, DORV	158
Nkx2.5	Nkx2.5HDneo	yes109	1.4%	_	none	aneurysm of the septum primum	ASD	110
Nos3	Nos3−/−	none	~42%159	R/NC160	yes160	none	none	155, 159, 160
			27%160
			30%155
Notch1	Notch1+/−	yes89–91, 156, 161	~64%	_	none	none	AI	31
	Nos3−/−
Krox20	Krox20+/−;Nos3+/−	none	7.9%	Krox20lacz/lacz R/NC or L/N	none	none	none	162
	Krox20 lacz/lacz		27%
	Tie2cre/Krox20fl/fl		10%
	Wnt1cre/Krox20fl/fl		10.5%
Adamts5	Adamts5−/−;Smad2+/−	yes156	41.2%	_	none	none	Ascending aortic defects	163
RBPJ	Rbpjflox;Nkx2.5Cre	none	54%	75% R/NC & 25% R/L	none	none	VSD, DORV	97
Npr2	Npr+/− & Npr+/−;Ldlr−/−	none	9.4%	R/NC	none	Aneurysmal changes in ascending aorta	LV dysfunction, and Ascending aortic dilatations	34
Jag1	Jag1flox;Nkx2.5Cre	none	47%	R/NC or R/L	none	none	VSD, enlarged valve cusps	98
Brg1	Nfatc1Cre;Brg1 fl/fl	none	34.6%	66.6% L/NC & 33.3% R/NC	none	none	none	164
Alk2	Gata5cre+;Alk KOFX	none	78%	_	none	none	VSD	165

(LV, left ventricular; OFT, outflow tract; VSD, ventricular septal defect; TOF, tetralogy of Fallot; IAAB, Interrupted Aortic Arch type B; AoC, Aortic Coarctation; VSD, Ventricular Septal Defect; DORV, Double Outlet Right Ventricle; AI, Aortic Insufficiency).

The Etiology of BAV.

Studies have shown that the heritability of BAV is as high as 89%, indicating that it is genetically determined in most cases.⁸⁶ Despite the widely acknowledged notion that BAV is heritable, the inheritance of this valve malformation cannot be explained by a single gene model alone. Indeed, emerging data from GWAS studies suggests the involvement of many genes or a ‘multi-hit’ approach, with divergent inheritance patterns and possible environmental interactions.^{87, 88}

Studies of familial BAV provided initial insights into the genetic etiology when mutations in NOTCH1 were shown to segregate in families with autosomal-dominant valve disease,⁸⁹ and inherited from unaffected family members, with affected individuals showing left ventricle outflow tract defects including BAV.⁹⁰ Four unique NOTCH1 variants were further identified with both BAV and thoracic aortic aneurysms. Of these, 2 novel missense mutations (A1343V, P1390T) were identified in patients with BAV and aortic aneurysm, suggesting synergistic regulatory mechanisms.⁹¹ Another study from systematic mutation-analysis based on DNA-sequencing of all coding exons and neighboring splice consensus sequences of NOTCH1 revealed 2 sequence variants (p.T596M and p.P1797H) located in highly conserved regions of the NOTCH1 protein in a BAV patient population.⁹² The exact mechanism(s) underlying how mutations in NOTCH1 cause BAV have been explored in mouse models targeted divergent components of the Notch signaling pathway. Notch1 homozygous mice are embryonically lethal due to vascular endothelial abnormalities prior to cushion formation, and therefore insights into the requirement for aortic valve formation are limited.⁹³ However, Notch1 heterozygotes are viable, and develop aortic valve calcification but largely in the absence of BAV,^94–96 suggesting that Notch1 reduced function alone, is not sufficient to cause BAV, at least in mice, but rather, can underlie calcification of the tricuspid aortic valve later in life.

While Notch1 knockdown in mice does not cause aortic valve development abnormalities or BAV, cell-specific deletion or inactivation of other mediators of the Notch signaling pathway in mice do. Conditional inactivation of RBPJ, a major transcriptional effector of Notch1 signaling in both the endocardial and myocardial progenitor cells using an Nkx2.5cre driver results in BAV at a penetrance of ~54%, with no associated cushion defects.⁹⁷ In addition, mice lacking endocardial Jag1 (Jag1^flox;Nkx2.5cre) (a Notch ligand) display enlarged valve cusps, septal defects, and 47% have BAV, characterized by R/NC or R/L subtypes. Interestingly, early stages of endocardial cushion formation are grossly unaffected in Jag1^flox;Nkx2.5cre, however the bicuspid phenotype is associated with increased proliferation of mesenchyme precursor cells during post-endMT stages related to remodeling and sculpting of the immature cusps.⁹⁸ Conversely, endocardial deletion of the E3 ubiquitin ligase that ubiquitinates and promotes endocytosis of Notch ligands (Mib1), leads to poorly cellularized cushions, but there is no development of BAV.⁹⁸ Nonetheless, murine models targeting other Notch ligands have described BAV in the absence of endMT defects, suggesting that in relation to this signaling pathway, congenital left-sided valve disease is likely the result of compromised processes that occur after endMT, potentially during the cusp remodeling process.

Mutations and variants in several members of the GATA gene family have been associated with the development of BAV and CAVD in humans particularly those in GATA4, GATA5, and GATA6.^{70, 99} The burden of rare variants in GATA4 are significantly enriched in early onset complications of BAV, primarily with concomitant thoracic aortic anuerysms.¹⁰⁰ Disruption of GATA4 by CRISPR-Cas9 in induced pluripotent stem cells has demonstrated dysfunctional transition of endothelial to mesenchymal transformation; a critical step for early valve formation and endocardial cushion development.¹⁰¹ GATA4 null mice die embryonically (E8.5) prior to valve formation,¹⁰² however mice heterozygous for a human mutation in GATA4 (p.Gly294Ser) develop AS, abnormal outflow tract cushion development and endMT defects.¹⁰³

Loss-of-function mutations in GATA5 are associated with accelerated predisposition to BAV in human patients.¹⁰⁴ Rare, non-synonymous mutations in the transcription activation domains of GATA5 are also crucial in the development of BAV aortopathy.¹⁰⁵ Two such rare nonsynonymous GATA5 variants were identified in BAV patients in the presence of coarctation of the aorta.¹⁰⁴ In mice, Gata5 deletion causes R/NC subtype BAV at a penetrance of 25%¹⁰⁶. Despite this phenotype, neither endocardial cell proliferation nor cushion formation is altered in these mice, however dysregulation of several components of the Notch pathway were reported and thought to be a contributing factor.¹⁰⁶

While Gata5^+/− mice do not develop overt valve disease, Gata6 heterozygous mice develop a highly penetrant R/L BAV subtype (56% male, 27% female mice), associated with abnormal embryonic valve remodeling during mid-stages.¹⁰⁷ Cell-specific loss of Gata6 within the Isl1-lineage (secondary heart field), but not Tie2- (endothelial) or Wnt1- (neural crest) lineages, recapitulated the phenotype of GATA6^+/− mice suggesting the involvement of secondary heart field-derived myocytes in the development of BAV.

Nkx2.5 is a synergistic transactivational partner to GATA5 and is critical for the normal cardiovascular development and valvulogenesis.¹⁰⁸ A study published in 2014 with a cohort of 142 BAV patients and 200 controls, described a novel heterozygous Nkx2.5 mutation, with no transcriptional activity (p.K192X) preventing transcriptional synergy between Nkx2–5 and GATA5 leading to valve disease.¹⁰⁹ Despite this loss-of-function mutation enhancing susceptibility to BAV in humans, in mice Nkx2–5 heterozygosity leads to stenotic BAV at a very low penetrance (1.4%).¹¹⁰ Together, these studies have increased our understanding of the genetic causes of BAV.

Together, these studies highlight key regulators of aortic valve development, then when mutated in humans and mice, increase susceptibility to the development of BAV. In addition to these candidates, other syndromic disorders have been associated with the development of BAV including connective tissue disorders that interface BAV with aortic aneurysm and dissection.¹¹¹ Continued efforts in the areas of integrating genetic, clinical and biological studies, including those by the International BAV Consortium will provide additional insights into the complex etiology of this prevalent disease.¹¹²

BAV Hemodynamics Leading to Secondary Calcification, AS and Aortic Dilation.

Young adults (>35 years old) with BAV can experience early onset cusp calcification and develop severe AS, that interestingly, is more rapid and severe for the R/NC configuration. In general, calcification of the valve is characterized by the presence of calcium-rich calcific nodules on the fibrosa surface of cusp, which as expected stiffens the biomechanical compliance leading to outflow obstruction and AS. Studies of human patients and mouse models of calcific aortic valve disease have shown that calcification is a chronic and active process, rather than degenerative and passive as previously perceived. The process presents significant similarities to bone mineralization with overlap of several molecular markers and phenotypes that suggest osteoblast-like changes in VICs (as reviewed^{113, 114}). This involves expression of the osteoblast markers Runx2 and Osterix, as well as other key transcriptional regulators of osteoblast development,^{115, 116} which in turn, promotes the secretion of proteins highly expressed in mineralized tissue, including Osteopontin and Osteocalcin.²²

Although, the mechanisms of accelerated calcification in BAV remains largely unexplored, there is no doubt that the physical dysmorphogenesis of the aortic valve anatomy gives rise to disturbed hemodynamics, which in turn is reciprocated back onto the already abnormal valve structure. A normally functioning BAV exhibits an asymmetrical orifice with increased flow angle patterns in the ascending aorta, associated with regional increase in wall shear stress.⁸² Irrespective of the BAV fusion type, the most prevalent flow abnormality due to BAV is increased right-handed helical flow in the ascending aorta;¹¹⁷ although specific BAV subtypes lead to different patterns of dilation of the ascending aorta.¹¹⁸ For example, R/NC fusion results in flow streaming towards the right-posterior or posterior aortic valve compared to the R/L subtype which was associated with flow jets directed towards the right anterior aortic wall.¹¹⁸ The R/NC subtype also exhibits more severe flow abnormality including a higher proportion of left-handed flow, associated with larger ascending aortic diameters compared to R/L. This abnormal hemodynamic environment has been proposed as a driving factor in the acceleration of early onset of calcific aortic valve disease, but the number of direct studies supporting this hypothesis are fewer than those for aortopathies. Interestingly, the onset of calcification and AS is most rapid with R/N fusion, and a plausible but inadequately tested theory suggests that differences in the flow patterns as described above may account for these differences.^{113, 119–122}

As discussed, VECs are mechanosensitive and biomechanical studies have utilized in vitro flow systems to determine molecular changes following exposure to physiological hemodynamic conditions. Interestingly, high throughput screens have shown that eNOS and Tgfβ1, among others, are increased in response to physiological laminar shear stress,^{30, 37, 39} and thought to maintain quiescent VIC phenotypes and ECM organization.^{28, 30–33} Therefore, it can be appreciated that in healthy valves, endothelially-mediated paracrine factors serve to protect against valve disease, including calcification. In fact, reduced function of eNOS or Tgfβ1 in VECs promotes early onset calcification in mice.^{31, 32} In contrast to tricuspid aortic valves, the wall shear stress indices are disturbed and skewed in BAV, leading to turbulent blood flow, and thus increasing endothelial permeability and putative dysfunction. In turn, this may negatively impact the ability of VECs to maintain normal paracrine signaling, thereby causing disruption of the anti-calcific eNOS and Tgfβ1 pathways leading to osteogenic-like changes.^{119, 121–125} However, the complete relevance of endothelial dysfunction and its impact on BAV has not been explored, and requires further investigation.¹²⁶

Studies of human bicuspid valves excised at end-stage have reported molecular changes over tricuspid aortic valve controls. These include several miRNAs (miR-26a, miR-30b and miR-195) that were shown to be mechanosensitive and modulate expression of calcification-related genes.¹²⁷ Additional high throughput screening studies have also highlighted differential miRNA profiles which may be reflective of the perturbed shear forces absorbed by the BAV.^128–131 Importantly, these studies are retrospective and largely investigate end stage calcification and thus minimally informative with respect to the mechanisms of disease onset and progression.

More frequently encountered than congenital AS, acquired AS is defined as narrowing of the aortic valve opening in the absence of underlying congenital malformation and occurs much later in life (<75 years).¹³² Several diseases can lead to acquired AS, the most common cause in developing countries being rheumatic heart disease.¹³³. In contrast, acquired AS in developed countries is associated with chronic progressive tissue remodeling and age-related calcific degeneration.¹³⁴ Indeed, degenerative aortic valve disease and AS is present in more than 25% of those over age 65 years.¹³⁵ Accelerated calcification further exacerbates AS by age 75 years,¹³⁶ associated with a two year mortality of >50% in elderly patients.¹³⁷ Interestingly, recent studies have reported aortic valve calcification in mouse models of accelerated aging as a result of telomerase shortening¹³⁸ or deletion of the putative age-suppressing gene Klotho.¹³⁹

In humans, age-related calcification is further associated with additional cardiovascular risk factors such as smoking, hyperglycemia, elevated LDL cholesterol, hypertension, metabolic syndrome and chronic kidney disease (CKD) (Figure 2).⁴ However, in the absence of the bicuspid anatomy and disturbed hemodynamics acting affecting VECs, the contribution of these modifiable cardiovascular risk factors in promoting osteogenic changes in VICs remains an active area of research. Studies in humans have correlated degenerative calcification in familial cases of hypercholesterolemia associated with high LDL-cholesterol due to mutations in the LDL receptor (LDLr),¹⁴⁰ as well as patients with increased serum cholesterol independent of any known genetic defect.¹⁴¹ Similarly, aged hypercholesterolemic Ldlr^−/−;ApoB^100/100 mice fed a Western (high fat) diet progressively develop calcification of the aortic valve, with functional impairment similar to humans.¹⁴² Additional studies have associated hyperlipidemia in Ldlr^−/− mice fed on the Western diet with activation of the osteogenic gene-regulatory program in affected valves.¹⁴³ Similar observations have been reported in wild type mice fed high fat, but low cholesterol diets.¹⁶ Because genetic and/or multiple risk factors are needed to induce significant phenotypes, these pre-clinical studies highlight the multifactorial nature of degenerative calcification in AS. Accordingly, designing therapeutic programs may not be as simple as targeting a single risk factor; rather, combinatory and personalized approaches are likely needed. This is evident by the unsubstantiated outcomes of clinical trials utilizing “lipid lowering” drugs to treat AS patients, in which attenuated hyperlipidemia did not conclusively result in beneficial effects on valve function.¹⁴⁴ Similar findings were also reported in “Reversa mice” (Ldlr^−/−/Apob^100/100/Mttp^fl/fl/Mx1Cre^+/+) that develop hyperlipidemia, valve degeneration and AS. Following Cre-mediated inactivation of the Mttp gene resulted in lower lipid levels, but valve dysfunction remained despite improvements in cusp structure.¹⁴⁵ It may prove challenging or even impossible for a degenerative valve to ‘recover’ from chronic insults of excess risk factor exposure.

Figure 2.

Acquired AS is most prevalent in the aging population and largely influenced by long-life exposure to several known (and unknown) risk factors as indicated. Degenerative calcification of the otherwise anatomically normal, tricuspid aortic valve is a major contributor to acquired AS as a result of mineralization of the cusp tissue leading to reduced compliance, increased stiffness leading to restricted movement of the cusp.

Multiple clinical and population-based studies have demonstrated that patients with CKD harbor a significant risk of cardiovascular complications including calcification of the vasculature and the aortic valve, the latter leading to AS. Notably, calcific abnormalities are present in 28–38% of CKD patients, and severe AS is observed in 6–13% of patients undergoing hemodialysis.¹⁴⁶ The mechanistic underpinnings of how CKD induces cardiovascular calcification is largely unknown, although correlations between high serum phosphate levels, and calcification indices have been reported.^{53, 147–149} Furthermore, mice with increased serum phosphate exhibit calcification of the aortic valve and aorta.¹³⁹ Several elegant studies have delineated the mechanisms underlying vascular calcification (reviewed¹⁵⁰), however, similar studies in VICs are lacking and less is known about phosphate-driven heart valve calcification relative to CKD.

CONCLUSIONS.

AS represents an increasing health care burden, but there is no effective pharmacological therapy to halt progression towards intervention. Despite a deeper understanding of the risk factors that contribute to congenital and acquired AS, effective medical therapies remain elusive. Most attractive are therapeutic interventions focused on preventing the onset of premature calcification during young adult life, but we lack full understanding of the multifactorial and interactive contributors to disease pathophysiology. The association of BAV with altered hemodynamics is a starting point. Moving forward we can begin to think about how, via mechanosensitive pathways elicited by VECs, the disturbed flow patterns convert VICs to osteoblast-like phenotypes. These signaling axes and cell types may ultimately serve as a platform for new treatment options.

Pharmacological therapies to halt the progression of acquired AS remain imperfect. Indeed, studies suggest that addressing modifiable risk factors following the diagnosis of acquired AS may not prove beneficial. Early detection of subtle valvular changes may allow successful medical intervention prior to irreversible dysfunction, and may be advanced by identification of circulating biomarkers predictive of nascent AS. The field is moving towards using high resolution imaging (e.g., MRI and 4D flow) to characterize patient-specific flow and WSS patterns to predict the onset of secondary complications. The latter has been well studied in adult BAV patients relative to aortopathy development,¹⁵¹ and the field is beginning to extend this approach to calcification of anatomically normal aortic valves in the elderly.¹⁵² Studies of calcific aortic valve disease in the pediatric BAV population are still lacking.

In closing, AS is a complex, progressive and lifelong condition affecting both children and adults. Calcific aortic valve degeneration in pediatric BAV patients represents an investigational target with the potential for lifelong benefits if effective preventive measures may be applied early in life. For adults with AS, the introduction of transcatheter aortic valve implantation has reduced the number of procedure-related complications and long-term mortality. However, the prevalence of BAV is predicted to double over the next four decades,¹⁵³ even as five year survival rates for medically managed patients is estimated to fall.¹⁵⁴ Oversimplified and generalized treatment strategies to treat individual patients have generally not been effective, which is not surprising considering the complex genetic and environmental factors contributing to AS. Genome-scale molecular information integrated with clinical phenotyping, cellular processes and bioengineering are needed to advance the field. In doing this, we can take a precision medicine approach to improve medical decisions, practices and interventions for individual patients based on their risk of disease and predicted response.

Non-Standard Abbreviations.

AoC

Aortic Coarctation

AI

Aortic Insufficiency

aVIC

Activated Valve Interstitial Cell

AS

Aortic Stenosis

BAV

Bicuspid Aortic Valve

CKD

Chronic Kidney Disease

DORV

Double Outlet Right Ventricle

IAAB

Interrupted Aortic Arch type B

LC

Left Coronary

L/NC

Left/Noncoronary Fusion

LV

Left Ventricular

NC

Noncoronary

OFT

Outflow tract

RC

Right Coronary

R/L

Right/Left Fusion

R/NC

Right/Noncoronary Fusion

TOF

Tetralogy of Fallot

VEC

Valve Endothelial Cell

VIC

Valve interstitial Cell

VSD

Ventricular septal defect