Contribution of Oxidative Stress (OS) in Calcific Aortic Valve Disease (CAVD): From Pathophysiology to Therapeutic Targets

Calcific aortic valve disease (CAVD) is a major cause of cardiovascular mortality and morbidity, with increased prevalence and incidence. The underlying mechanisms behind CAVD are complex, and are mainly illustrated by inflammation, mechanical stress (which induces prolonged aortic valve endothelial dysfunction), increased oxidative stress (OS) (which trigger fibrosis), and calcification of valve leaflets. To date, besides aortic valve replacement, there are no specific pharmacological treatments for CAVD. In this review, we describe the mechanisms behind aortic valvular disease, the involvement of OS as a fundamental element in disease progression with predilection in AS, and its two most frequent etiologies (calcific aortic valve disease and bicuspid aortic valve); moreover, we highlight the potential of OS as a future therapeutic target.


Introduction
Valvular heart disease (VHD) is currently a major contributor to the loss of physical functioning, quality of life, and longevity [1]. Between 1990 and 2017, the global burden of VHDs showed a wide range of variations; over this time, period rheumatic heart disease, considered the leading cause of VHD in developing countries, substantially decreased. In this matter, The Euro Heart Survey (EHS) noted a predominance of functional and degenerative valvular diseases in high-income countries, where degenerative aortic valve disease and degenerative mitral valve disease represented 70% and 46% of VHD cases [1][2][3][4]. Additionally, the elderly population (≥60 years) is expected to grow from 962.3 million in 2017 to 2080.5 million in 2050, which will lead to a substantial increase in the incidence of non-rheumatic VHD (NRVHD), and its most frequent subtype-non-rheumatic calcific aortic valve disease (NRCAVD) [1,2].
Aortic stenosis (AS) is the most common type of valvular heart disease [5]; its prevalence varies between 2% and 7% in patients over 65 years old and it is estimated that by the year 2030, 4.5 million patients will be diagnosed with this VHD [6]. The most common etiology of aortic stenosis is calcific disease; however, the most common congenital valve defect causing AS is the bicuspid aortic valve (BAV) [7]. Calcific aortic valve disease (CAVD) is no longer considered a simple passive calcium depositing process with consequent fibrosis and valve leaflets thickening, it is also an active cellular process that causes endothelial dysfunction throughout oxidative stress (OS) and inflammatory activation [8,9]. BAV is characterized by the abnormal cusp development where two of the three cusps fuse into a single cusp resulting in abnormal leaflet architecture; it has a prevalence of 0.5-2% in the general population and is three times more prevalent in males than in females [10,11]. One of the most frequent complications of this congenital malformation is BAV-associated aortopathy (BAVAA), which predisposes patients to the risk of developing thoracic aortic aneurysm, aortic dissection in the ascending aorta, and is linked to a faster aortic valve degeneration [10,12]. In both aortic diseases, valvular interstitial cells initiate and maintain mineralization, calcification, and fibrosis; all of these processes are supported by mechanical and biochemical pathways that include lipid deposition, inflammation, and OS [13,14].
In this review, we describe the mechanisms behind aortic valvular disease and the involvement of oxidative stress as a key element in disease progression, with predilection in AS and its two most frequent etiologies (calcific aortic valve disease and BAV); moreover, we highlight the potential of OS as a future therapeutic target.

Immunopathogenesis behind Aortic Disease
Aortic valve disease is a growing health condition that has a considerable impact on quality of life. The lack of effective strategies to prevent aortic valve degeneration and progression into calcific aortic valve stenosis (CAVS), besides the surgical option, reinforces the necessity of comprehending the immunopathogenic mechanism in order to develop targeted therapies [15,16].
Genetic susceptibility, endothelial shear stress, chronic inflammation, oxidative stress, lipid deposition, and valve calcification are all contributors to the progression of fibrocalcific remodeling of the aortic valve. The presence of overlapping disease development risk variables and a link between the severity of CAVD and coronary calcification suggest a shared disease pathway, at least in the early stages between CAVD and atherosclerosis [8,17]. As seen in atherosclerosis, endothelial dysfunction secondary to mechanical stress is the initiating event in CAVD. Damaging the structure of the endothelium enables lipids, primarily low-density lipoproteins (LDL) and lipoprotein (a) (Lp(a)) to penetrate the aortic leaflets, and can also trigger the migration of inflammatory cells within [17,18]. Plaque-like lesions consisting of LDL, Lp(a), and inflammatory cells form in the subendothelial region contributing to the production of reactive oxygen species (ROS) that promotes the oxidation of LDL and vascular dysfunction [17,19,20]. The persistent inflammatory process enables valvular interstitial cells (VICs) to attain an osteogenic phenotype resulting in the production of microcalcifications and leaflet mineralization [7,11]. The end stages of CAVD and atherosclerosis differ in the main cell types involved. Whilst in atherosclerosis, smooth muscle cells are known to be the major effectors in chronic inflammation, in CAVD, the pathophysiological process is enhanced by the addition of fibroblasts [17,21,22].
Given the various treatments that have been proven to delay the progression of atherosclerosis, none are effective at preventing aortic stenosis [7]. By stimulating inflammation through a deficiency of antioxidant protection (superoxide dismutase, heme oxygenase-1, glutathione peroxidases) or excessive formation of ROS, OS draws attention as one of the key mechanisms in the early stages of the disease [23,24]. Standard cardiovascular medications evaluated in clinical trials failed to slow disease progression or reduce negative outcomes. More research into alternative therapeutic options that target oxidative stress in VHD is needed [25].

Mechanical Stress and Endothelial Dysfunction
Endothelial dysfunction is recognized as a primary phase in the genesis of vascular disease and one of the most promising domains for the development of new therapeutic targets [26]. Mechanical stress, lipid infiltration, OS, inflammation, and the fibro-calcific response are all examples of interrelated events that trigger endothelial dysfunction, translated as the loss of its barrier properties [18,27]. The repeated opening and closing of aortic valvular cusps during the cardiac cycle is a continuous source of mechanical stress, which contributes directly to compromising the structural stability of the endothelial layer [5,18,28].
The aortic side of the valve is usually the area of choice for the above-mentioned pathological modifications, especially the noncoronary leaflet [28]. During systole, the blood is forcibly ejected past the ventricle leaving the aortic surface of the valve exposed during the diastole to a higher velocity gradient blood flow and oscillatory shear stress [29,30]. The bicuspid aortic valve also depicts the key role of hemodynamic stress through the high prevalence, accelerated initiation, and progression of CAVD [27,28], mainly at the site of the fused BAV leaflet [11]. In order to properly maintain endothelial homeostasis on both valvular and vascular endothelial cells (VECs), laminar blood flow is a mandatory condition. Fulfilling this requirement, the endothelial relaxing factors (mainly the nitric oxide signaling pathway) are stimulated while the expression of adhesion molecules (vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin), and vasoconstrictors (endothelin-1, angiotensin II, thromboxane A2) are suppressed [27,31]. The most common local laminar flow disturbances are inflicted by atherosclerosis, arterial stiffening, and remodeling [32], arterial hypertension, or valvular disorders [27,31]. Valvular endothelial cell changes relate to shear stress by aligning perpendicular to the flow direction opposed to vascular endothelial cells, which align parallel to the flow [29,30].
Ontological and transcriptional profiles suggest that valve endothelial cells (VECs) have the particularity to display side-specific heterogeneity being innate suppressors of calcification with antioxidative and anti-inflammatory phenotypes [30,[33][34][35]. VECs have the ability to not only detect alterations in sheer stress but also modulate these signals by regulating valvular interstitial cell (VIC) functions, preventing them from differentiating into osteoblast-like cells [11]. The process is very similar to the endothelial to mesenchymal transition (EndMT) seen throughout valve development and formation [21,36].
Apart from the classic wear and tear pathogeny, biomechanical factors are now recognized as driving forces in the development of CAVD [37,38]. For example, genes, such as the KLF2/PLPP3 axis, inhibitor of DNA binding-1 (ID1), heme oxygenase 1 (HMOX1), and nitric oxide synthase 3 (NOS3) may play hemodynamic-related roles in the development of CAVD by regulating other procalcification genes, such as nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB) [39], by promoting endothelial repair [40] and by protecting against OS and atherosclerosis [37].
The association between hemodynamics and endothelial oxidative stress in the genesis of CAVD is not yet completely defined and lacks sufficient research [37].

Lipid Deposition and Oxidative Stress in SA and BAV
Nowadays, western dietary patterns (consisting of a high intake of saturated fatty acids, processed foods, and low intakes of unsaturated fatty acids, dietary fiber, and micronutrients) combined with sedentary lifestyles and increased life expectancies have, as a consequence, contributed to the increase in noncommunicable diseases (NCDs), such as cardiovascular diseases, diabetes, and cancers [41,42]. Vascular OS and inflammation are clearly distinguished mechanisms underlying endothelial dysfunction, which lowers the bioavailability of the key vasodilator molecule nitric oxide (NO) and promotes inflammatory cell recruitment, lipid peroxidation, and aortic valve remodeling [32,43].
Although oxidative stress and atherosclerosis are prominent traits in CAVD, statins used as successful monotherapies in atherosclerosis have not been proven effective in CAVD treatment amidst the significant evidence that LDL contributes to disease progression [6,18,20,54]. The depositions of oxidized LDL and LP(a) molecules in the subendothelial layer sustain chronic inflammation at the expense of proinflammatory cytokines and fibrocalcific remodeling by promoting osteogenic differentiation of VICs (via toll-like receptors 2 and 4) and the upregulation of bone morphogenetic protein 2 (BMP-2) (via extracellular signal-regulated kinase (ERK)1/2 and NF-κB) [7,14,21,27,55].
At the ROS imbalance, besides eNOS uncoupling in BAV aortopathy, the ineffective antioxidant protection is demonstrated by the inadequate SOD activity in the ascending aorta [11,[63][64][65], whose expression is also dependent on the valve morphotypes [10,12,66,67]. Alongside NOS, NOXs are also part of the ROS milieu seen in CAVD. NOX2 and NOX4 are the main members of the NOX assembly (NOX1 to 5 and DUOX1 and 2) in the vascular system that are required for LDL oxidation, as shown in experimental mice studies [68]. However, the exact role of every NOX in CAVD is not very well defined and further studies are needed. The significance of NOX2 in CAVD has been illustrated in hypercholesterolemia-induced mice who received western-type diets [69], in a rabbit model fed cholesterol-enriched chow and vitamin D [70], and in human aortic tissue [44,71]. From the standpoint of mitoROS, oxidative stress generated through this pathway is observed in the VIC initiation of calcific degeneration [72].
In conjunction with the direct mechanism that NOX exerts, it is also intricated in the crosstalk between OS and inflammation through the cytokine release, controlling the nod-like receptor protein 3 (NLRP3) inflammasome [25,[73][74][75][76]. Additionally, ROS assists various inflammation pathways via the redox modulation of inflammation mediators (damage-associated molecular patterns (DAMP)s, high mobility group box 1 (HMGB1), and S100 proteins), transcription factors (Nrf2, NF-κB, HIF-1α, and AP-1), and by the formation of redox-dependent protein complexes (Nrf2-Keap1) [25,49]. Semicarbazidesensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1) has been identified as an additional injurious component of ROS in CAVD and has been linked to disease activity in several research studies [16,44,77]. ROS components cannot be viewed as separate effectors because they are part of an intricate crosstalk process supporting self-propagation and amplification of oxidative stress [25,49].

Inflammation Mechanism
Apart from being present from the onset of CAVD, inflammation orchestrates all subsequent events that lead to aortic valve remodeling [78]. In summary, as part of the initial innate immune response, activated VICs from prior exposure to mechanical stress and cytotoxic oxLDL prompt the recruitment and infiltration of macrophages, T cells, and mast cells via toll-like receptors 2, 4 (TLRs-2,4) and the NF-kB pathway [6,14,15,17,21,37].
As a peculiarity in BAV, enhanced densities of macrophages [11] that produce IL-12 and Th17 activation have been observed, as well as changes in the proteomics of canonical pathways characterized by significant upregulation of α-1-antitrypsin (AAT), α-1antichymotrypsin (ACT), and α-1-acid glycoprotein 2 (AGP) [64]. In addition to macrophages and inflammatory cells, a cluster of CD4+ and CD8+/CD28− T cells are present in early valve lesions adjacent to calcific loci [82,84].
As far as the adaptive immune response in aortic stenosis is concerned, this remains a topic with considerable potential for further research, especially with regard to intracellular pathogens [17] or epitopes [82].
Degradation and fibrosis of the extracellular matrix serve as the foundations for the two subsequent calcification processes that follow. Dystrophic calcification is enabled by the precipitation of calcium and phosphates in potent calcification nuclei, such as degraded collagen and elastin fibers in the absence of the active participation of osteoblasts [77]. In the definitive stages of CAVD evolution, dystrophic calcification is completed by biomineralization. The process is governed by VICs differentiated into osteoblasts under the transcription factor RUNX2, acquiring the expressions of ALP, BMP-2, BMP-4, osteopontin (SPP1), osteocalcin (BGLAP), Sry-related HMG box-9 (SOX9), and bone sialoprotein [6,34,53]. Under hypercholesterolemia and oxidative stress, endothelial valvular cells secrete Wnt3 that binds to receptors LRP5 and Frizzled on the VIC surface [34,58,82], further inducing the RUNX2 expression via the Wnt pathway [7]. The Notch signaling pathway, consisting of four receptors (Notch1/2/3/4), ligands, Jagged-1/2, and delta-like proteins (DLL1/3/4), is a physiological neutralizer of the Wnt pathway, upholding indirect regulatory effects on RUNX2 [20], being in close conjunction with NF-κB and TLR4 activation in calcific areas of aortic leaflets [60,82].
In summary, the interplay of oxidative stress, endothelial dysfunction, and inflammation alter valvular homeostasis expressed as a damaged and hyper-vascularized extracellular matrix containing VICs differentiated into active myofibroblasts ( Figure 1). The anarchic organizational pattern of collagen fibers promotes calcium and phosphate deposition, and VIC osteogenic differentiation, conclusively leading to valve ossification, and ending the complex active chain reaction of biomineralization [27].

Therapeutic Targets of Oxidative Stress
CAVD is extremely common and a rapidly advancing disease with no effective drug therapies at present. Surgical aortic valve replacement (SAVR) is the last resort in severe cases;the window of opportunity being limited for elderly frail patients as they represent the majority of concerned patients and, thus, it is not the most desirable option [4,7,92]. In the last decades, transcatheter aortic valve replacement (TAVR) gained popularity as a more tolerable procedure with promising outcomes [48,[93][94][95].

Therapeutic Targets of Oxidative Stress
CAVD is extremely common and a rapidly advancing disease with no effective drug therapies at present. Surgical aortic valve replacement (SAVR) is the last resort in severe cases;the window of opportunity being limited for elderly frail patients as they represent the majority of concerned patients and, thus, it is not the most desirable option [4,7,92]. In the last decades, transcatheter aortic valve replacement (TAVR) gained popularity as a more tolerable procedure with promising outcomes [48,[93][94][95].
Several therapies that were attempted, including statins, antihypertensives, and medications that target the phospho-calcic pathways, were unable to arrest the naturally occurring progression of CAVD [14,20,96]. As the research into the pathology of calcific aortic stenosis progressed and revealed the multifactorial processes and cellular mechanisms, the demand for targeted therapeutics effectively preventing or delaying the progression of CAVD has emerged [17,20,[97][98][99].
Regarding oxidative stress as a unifying mechanism involved in the initiation and propagation phases of aortic stenosis, counteracting it may represent a major future in targeted therapy. Thus, Table 1 provides an overview of potential strategies for reducing the interplay of oxidative stress in CAVD.

Targeting Lipid Oxidation
Since atherosclerosis and CAVD share similar risk factors and pathways in the initial pathological stages, targeting LDL to further prevent oxLDL formation was the first subject of interest in regard to medical therapies for aortic stenosis.
Proprotein-converting enzyme subtilisin/kexin type 9 (PCSK9) inhibitors can simultaneously decrease the level of LDL-C via LDL-R internalization and Lp(a) synthesis [115,116]. The FOURIER (Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk) (NCT01764633) [115,117] and the GLAGOV (Global Assessment of Plaque Regression with a PCSK9 Antibody as Measured by Intravascular Ultrasound) (NCT01813422) studies [118] reinforced the important roles of PCSK9 inhibitors in CAVD treatment.
Niacin (nicotinic acid) also raises awareness by reducing Lp(a), LDL-C, and triglycerides plasma levels and by increasing HDL-C [119]. There are currently only two trials evaluating niacin (the Early Aortic Valve Lipoprotein (a) Lowering (EAVaLL; NCT02109614)) and PCSK9 inhibitor (South Korean clinical trial (NCT03051360)), supporting the necessity of additional studies on this matter [6,14,34,108].
Presently, there is a paucity of data from RCTs to endorse the notion that lowering Lp(a) levels alone can improve the CAVD outcome without taking into account other lipid risk factors. New therapies consisting of antisense oligonucleotide against apolipoprotein(a), which consistently lowers plasma Lp(a) levels, such as IONIS-APO(a)-Rx, IONIS-APO(a)-LRx [120], and hepatocyte-directed antisense oligonucleotide AKCEA-APO(a)-LRx (TQJ230) have recently emerged [106]. Additionally, E06, a natural antibody that prevents macrophages from absorbing oxLDL, demonstrated a 49% reduction in the AV gradient and a lower overall calcium load in E06 mice [121].

Antioxidants from Natural Compounds to Targeted Experimental Therapies
A general trait observed in calcified aortic valves is the general reduction of the major antioxidant systems and excessive ROS accumulation; hence, mechanisms approaching these imbalances were sought as novel therapeutic strategies [44,124,125]. Most natural antioxidants are obtained from herbal sources, as will be described in the following paragraphs.
Improved endothelial dysfunction and anti-proliferative impacts on smooth muscle cells were exemplified by ellagic acid, naturally present in nuts and red berries [142,143], and gallic acid that further decreased the expressions of the calcification markers RUNX2, BMP2, osteocalcin, and MSX2 [87,144]. Anticalcific and anti-inflammatory properties by interfering with the PI3K-AKT, ERK1/2, and NF-κB/NLRP3 inflammasome pathways are also displayed by caffeic acid phenethyl ester (CAPE), found in honeybee propolis and conifer barks [145,146].

Vitamins
Inhibitory effects on lipid oxidation, cardiovascular inflammation, vascular calcification, and the improvement of vascular endothelial dysfunction are depicted by vitamins C and E (α-tocopherol), which have been extensively researched in cardiovascular preventive medicine [19,163,164]. Several observational studies have revealed an unfavorable connection between the nutrient intake of antioxidants and cardiovascular morbidity and mortality, despite the supporting idea that antioxidant vitamins help reduce oxidative stress and vascular illnesses [165].
The cardiovascular morbidity and mortality rates in the placebo and antioxidant groups were found to be identical in meta-analyses of RCTs on the benefits of vitamins B, C, E, and S, or b-carotene [166,167]. The unsatisfactory results can be explained by a number of factors, such as the lack of standard dosages and the follow-up periods of insufficient lengths, but further research is needed on the so-called antioxidant paradox in therapeutics [45].
Recently, the pineal hormone melatonin showed antioxidant effects in atherosclerotic lesions excising NLRP3 inflammasome activation [49,177]. In patients with underlying pathologies that additionally amplify ROS production, such as rheumatic valvular heart disease, or those undergoing cardiopulmonary bypass or diabetes, L-carnitine and glycine represent promising potential in targeting NF-κB, Nrf2 [178], and the RAGE-NOX-NF-κB signaling pathway [75]. Studies have also been conducted on ascending aortic aneurysms and patients with Marfan syndrome. Thus far, Hibiscus sabdariffa Linne (HSL), which is rich in polyphenols, anthocyanin, flavonoids, and L-ascorbic acid [19], and the inhibitor of the kappa B kinase epsilon (IKKε) inhibitor, amlexanox [179], are potential antioxidants meriting further research.

Targeting mitoROS and SOD
One of the most important systems involved in maintaining redox balance is SOD, which is downregulated in aortic stenosis [24]. In an attempt to promote SOD upregulation, polyethylene glycol-SOD (peg-SOD) and other SOD-mimetics consisting of cerium oxide nanoparticle (CNPs) rods and sphere-shapes [23] were utilized [180,181]. With the application of several aortic VICs, controversial outcomes were observed in terms of the calcification process [70], raising the question as to whether this therapeutic method depends on the dosage or a specific CAVD model. A redox-active MnSOD mimetic, manganese compound MnTnBuOE-2-PyP5 (MnBuOE) reduced aortic valve remodeling by targeting collagen buildup [182].

NO Bioavailability and NOX Inhibition
Increasing NO bioavailability was proposed as an alternative to reduce oxidative stress relying on the concomitant Notch1 regulation to also target osteogenic VICs differentiation [183]. From the aforementioned potential therapies, statins illustrate pleiotropic NO increased levels but their failure in aortic stenosis therapy mandates the use of alternative approaches [34].
In the hope of finding a more targeted approach, NOX-specific inhibitors were explored. Apocynin and celastrol from the antioxidant category demonstrated their potential as NOX2-selective inhibitors in addition to other non-specific antioxidant activities described in previous paragraphs. Apocynin significantly prevents the phosphorylation of the NOX2 cytosolic component [172], and celastrol demonstrates a general favorable effect in terms of calcium deposition and AV fibrotic remodeling by blocking the NOX2-driven GSK3β/β-catenin pathway [70,191].
Antidiabetic therapies, such as sitagliptin, showed pleiotropic effects in NOX by additionally inhibiting the cross-talk between RAGE/NF-κB with implications in calcific remodeling [21,89,122]. By reducing TNF-α, Il-6, iNOS, and NOX2 expression, the GLP-1 analog liraglutide also reduced vascular dysfunction, aortic inflammation, and oxidative stress in mice with polymicrobial sepsis [192]. Given that numerous other anti-inflammatory properties of pioglitazone were documented, such as improving TNF and IL-6 levels and cusp mobility, it is unclear as to the specific role it plays in NOX2 inhibition [69,193]. Triazolopyrimidines, such as VAS2870 and VAS3947 [194][195][196], and pyrazolopyridine derivatives, such as GKT136901/137831 [197], were developed under the idea of creating substances that specifically block NOX enzymes, although more research is needed to determine their safety and precise effects in CAVD [45,56,63]. As seen, efforts are being made in finding new therapeutic approaches via OS in CAVD, with potential for future implementation as additional therapy to current approaches (Tables 1-3). GLAGOV [118] Evolocumab 420 mg every month -Added statin treatment in angiographic coronary artery disease decreased atheroma volume.

Conclusions
Aortic stenosis is a significant worldwide medical issue and a disabling disease that diminishes a patient's life quality. Even though the pathogenesis of aortic stenosis cannot be explained by a single mechanism, current data reveal the deleterious effects of increased OS activity in the complex heterogeneity of active cellular and molecular mechanisms that support the progression of CAVD and endothelial dysfunction through various pathways. Nonetheless, research continues to address new molecules that, in addition to playing roles in cardiovascular pathophysiology, may also represent novel therapeutic targets. Due in part to the fact that the multifactorial, self-replicating cellular mechanisms causing CAVD have already been established by the time a patient presents with the disease, proposed strategies to inhibit oxidative stress, either systemically or locally in CAVD, have so far delivered unclear results.
Furthermore, the spectrum of early paraclinical diagnosis might be expanded; the risk stratification and long-term prognosis could be improved through the development of new protein assays and technologies that speed up and increase the detectability of aortic stenosis. Currently, there are few in vivo/in vitro and cohort observational studies that could determine whether there is a relationship between the degree of valvular oxidative stress, the rate of disease progression, and its relationship to clinical severity. Therefore, more research studies with adequate follow-up periods are essential for translating the OS concept into clinical practice therapies.