Calcific Aortic Valve Disease　― From Mechanism to Treatment ―

Abstract

Calcific aortic valve disease (CAVD) is one of the most prevalent heart valve diseases and is characterized by progressive stiffening and calcification of the aortic valve. For decades, CAVD has been treated with surgical intervention. In recent years, some progress has been made in understanding the pathogenesis of CAVD and the exploration of novel therapeutic strategies, leading to the identification of potential therapeutic targets and innovative treatment approaches. This review systematically outlines the pathophysiological advances in CAVD over the past 5 years, proposing a 3-stage model for disease progression: inflammatory, fibrotic, and calcification stages. In addition, recent clinical trials investigating pharmacological therapies, such as those targeting lipid metabolism, vitamin K pathways, and calcium-phosphorus balance, are summarized and discussed. These developments hold promise for improving patient outcomes and revolutionizing the management of CAVD.

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder characterized by progressive calcification and aortic valve narrowing. In 2017, it was estimated that CAVD affected 12.6 million individuals globally and accounted for 102,700 deaths.¹ Based on the 2023 Global Burden of Disease database, it is projected that the prevalence of CAVD will increase further with population aging,² which emphasizes the need for advances in our understanding of the pathogenesis of CAVD and the development of effective treatments.

At present, transcatheter aortic valve implantation^3–5 and surgical aortic valve replacement⁶ are the only effective interventions for the treatment of CAVD,⁷ and these procedures necessitate long-term, often lifelong, postoperative management.^8,9 Consequently, there is a need to develop pharmacological interventions for both the prevention and treatment of CAVD. Despite ongoing efforts, clinical trials of pharmacological therapies have yet to yield satisfactory outcomes,^7,10 and no effective drug-based treatments have been approved for clinical use.¹¹ These challenges underscore the clinical need for further research into the pathogenesis of CAVD to identify novel therapeutic targets.

Due to the technological limitations, previous drug trials focused on pathological abnormalities, such as hypertension, hyperlipidemia, and calcium deposition, as well as clinical symptoms such as aortic stenosis.^7,10 Reports on preventive treatment during the stage characterized by molecular or cellular abnormalities in the absence of clinical symptoms are relatively rare.^12,13 Advances in single-cell sequencing and omics technologies have enabled more comprehensive exploration of CAVD mechanisms, leading to the identification of early diagnostic markers and therapeutic targets.

Calcification of the aortic valve can be categorized into 3 distinct stages, namely inflammatory, fibrotic, and calcification stages.¹⁴ The inflammatory stage typically starts with inflammation triggered by endothelial mechanical stress injury^15–17 and lipid deposition,^18,19 which is identified through staining of inflammatory cells and quantification of inflammatory factors.²⁰ The fibrotic stage is regarded as the stage of propagation,¹⁴ and is characterized by intensified staining of collagen fibers and the initial appearance of α-smooth muscle actin (SMA)-positive valvular interstitial cells (VICs), a subset of activated myofibroblasts that can express contractile proteins and contribute to valve development.²¹ The calcification stage represents the terminal phase of the process and is characterized by the presence of stained mineralized nodules or upregulation of α-SMA-positive VICs.²² In addition, the gene and protein expression of VICs vary across the 3 stages,²³ and targeted treatment strategies addressing specific molecular targets in specific stages may be more effective in delaying disease progression.

Physiology of the Aortic Valve and Calcific Aortic Valve Pathology

The human aortic valve is a trilaminar structure composed of the fibrosa layer, facing the aorta, the spongiosa layer in the middle, and the ventricularis layer, facing the ventricle. The leaflet tissue is encapsulated by valve endothelial cells, whereas VICs are distributed throughout the tissue.²⁴ VICs constitute the major cellular component of the aortic valve and include at least 2 subtypes: α-SMA-positive and -negative cells. In adults, most α-SMA-negative VICs are quiescent fibroblasts (qVICs).²⁵ A 3-dimensional hydrogel culture experiment demonstrated differentiation of qVICs into activated (a) VICs, characterized by α-SMA expression. Notably, α-SMA expression decreases as aVICs differentiated into osteoblast-like VICs (oVICs).²⁶

Recent omics studies have revealed that the distribution and calcification potential of VICs with distinct markers exhibit spatial specificity. For example, glial fibrillary acidic protein (GFAP)-positive VICs are predominantly localized in the spongiosa layer;²³ CD44⁺ VICs, which have high calcification potential, are primarily distributed in the fibrosa layer;²⁷ and CD34⁺/platelet-derived growth factor receptor α-positive VICs, which have low calcification potential, are most abundant in the spongiosa layer, followed by the fibrosa layer, and are rarely observed in the proximal region of the cusp, which is prone to calcification.²⁸ Interestingly, the expression of specific receptors on VICs may contribute to the observed sex differences in CAVD,^29,30 with women having a lower incidence of CAVD than men. This sex disparity highlights the need for further investigations into the underlying mechanisms, including potential hormonal influences or differences in VIC behavior between the sexes.^31,32

Recent Studies Into Mechanisms Underlying CAVD

As noted above, recent studies have revealed that CAVD progresses through 3 interconnected pathological stages¹⁴ (inflammatory, fibrotic, calcification) driven by synergistic interactions between inflammatory signaling, mechanical stress, and metabolic dysregulation. Below, we synthesize these 3 mechanisms and therapeutic implications. The various signaling molecules involved in inflammation and their interactions during in the process of valve calcification are shown in the Figure.

Figure.

Three pathological stages in calcific aortic valve disease (CAVD) valvular interstitial cells (VICs): inflammatory stage, fibrotic stage and calcification stage. Open arrows indicate transformation and solid arrows indicate activation or initiation. The inflammatory, fibrotic, and calcification stages are sequentially linked, with cross-regulatory interactions and integrative signaling pathways driving disease progression. AHR, aryl hydrocarbon receptor; AMPK, AMP-activated protein kinase; ATF6, activating transcription factor 6; ATG13, autophagy related 13; BGN, biglycan; CSE, cystathionine γ-lyase; eLDL, enzyme-modified low-density lipoprotein; ERS, endoplasmic reticulum stress; Gln, glutamine; Glu, glutamate; hcy, homocysteine; Herpud1, homocysteine inducible ER protein with ubiquitin like domain 1; HuR, human antigen R; IFN, interferon; IL, interleukin; JAK3, Janus tyrosine kinase 3; MAPK, mitogen-activated protein kinase; MMP12, matrix metalloproteinase 12; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-κB; PERK, protein kinase R-like ER kinase; PIP4K2A, phosphatidylinositol-5-phosphate 4-kinase, type II, alpha; ROCK1, coiled-coil containing protein kinase 1; RUNX2, RUNX family transcription factor 2; STAT3, signal transducer and activator of transcription 3; TCA, tricarboxylic acid; TNF-α, tumor necrosis factor-α.

Inflammatory Stage: Molecular Triggering and Amplification of the Signaling Cascade

In the early inflammatory stage of CAVD, various endogenous molecules drive the inflammatory response by activating core pathways such as nuclear factor (NF)-κB. Hydrogen sulfide (H₂S) produced by cystathionine β-synthase and cystathionine γ-lyase in aortic endothelial cells significantly reduces interleukin (IL)-1β and tumor necrosis factor-α expression by inhibiting nuclear translocation of NF-κB, thereby alleviating valve inflammation.^33–35 However, under the stimulation of calcification, the accelerated metabolism of H₂S by mitochondria in human aortic valves leads to an imbalance in its compensatory synthesis, which ultimately exacerbates the transformation of inflammation to fibrosis. This finding provides a rationale for the development of mitochondrial-targeted H₂S donors as therapeutics.³⁶

The intestinal metabolite indoxyl sulfate induces the NF-κB pathway by activating the aryl hydrocarbon receptor (AHR) to promote IL-6 secretion, as observed in experiments using human aortic valve tissue.³⁷ In parallel with this mechanism, recent evidence highlights the role of the RNA-binding protein Sam68 in amplifying inflammation-calcification cross-talk in CAVD. Sam68 drives osteogenic differentiation of VICs via the tumor necrosis factor-α/signal transducer and activator s of transcription 3 (STAT3)/autophagy axis: it binds to phosphorylated STAT3, upregulates IL-6 and osteogenic markers, and suppresses autophagy, thereby establishing a positive feedback loop. Notably, Sam68 may synergistically enhance the procalcific effects of inflammatory signaling by modulating the NF-κB pathway, a mechanism overlapping with indoxyl sulfate/AHR-mediated effects.³⁸ In addition, enzyme-modified low-density lipoprotein promotes the release of IL-33 and IL-6 by activating the proinflammatory protein p38 mitogen-activated protein kinase pathway, further amplifying inflammatory signals, as demonstrated in experiments using human aortic valve tissue.³⁹ These cascades not only maintain chronic inflammation but also set the stage for subsequent fibrosis.

Fibrotic Stage: Metabolic Reprogramming and Cell Phenotype Switching

As inflammation progresses, VICs undergo significant metabolic remodeling. Mechanical stimuli, such as ascending aortic coarctation, trigger calcium-dependent Yes-associated protein signaling by activating the mechanosensitive ion channel Piezo1, leading to the upregulation of glutaminase 1 expression in human aortic valve tissue. Glutaminase 1 catalyzes the decomposition of glutamine to glutamate, which is metabolized to produce acetyl-CoA via the tricarboxylic acid (TCA) cycle. This process promotes the transcription of RUNX family transcription factor 2 (RUNX2) through histone acetylation modification and enhances the expression of α-SMA and osteogenic differentiation markers such as osteopontin (OPN).⁴⁰ Notably, the VICs of CAVD patients show a significant “Warburg effect”, which favors anaerobic glycolysis even under oxygen-rich conditions. In human aortic valve tissue, the ras homolog family member A (Rho A)/coiled-coil containing protein kinase 1 (ROCK1) pathway can inhibit the phosphorylation of AMP-activated protein kinase (AMPK), reduce the degradation of RUNX2 protein, and promote the expression of OPN to accelerate the transition from fibrosis to calcification.⁴¹ Endoplasmic reticulum stress also plays a key role at this stage: hyperhomocysteinemia upregulates the homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) receptor through activation of the protein kinase R-like ER kinase/activating transcription factor pathway and induces autophagy-dependent fibrosis, as demonstrated in experiments using murine aortic valve tissue.⁴²

Calcification Stage: Terminal Differentiation and Microenvironment Remodeling

In the terminal calcification stage, multiple pathways converge on the regulation of osteogenic transcription factors such as RUNX2. Using a human aortic valve, Dhayni et al. demonstrated that IL-8 promotes inorganic phosphate-mediated calcium deposition by inhibiting OPN and upregulating matrix metalloproteinase-12 and elastin via the CXCR2/NF-κB axis.⁴³ Research has demonstrated that mechanical strain and endogenous ligands like biglycan activate Toll-like receptor 3 (TLR3) in human VICs, triggering a conserved calcification cascade through type I interferon (IFN) signaling. Post-translational modification of biglycan by xylosyltransferase 1 enables its interaction with TLR3, inducing phosphorylation of INF regulatory factor 3 and subsequent production of IFN-β. This activates Janus tyrosine kinase 1 (JAK1)/STAT3 signaling via the IFN-α/β receptor, promoting osteogenic differentiation of VICs through RUNX2 upregulation.⁴⁴ IL-22 drives valvular calcification in CAVD by binding IL-22 receptor, alpha 1 on human VICs, also activating JAK3/STAT3 signaling. This induces STAT3 phosphorylation, nuclear translocation, and upregulation of osteogenic markers, promoting calcium deposition, forming a cross-cell-type calcification regulatory network.⁴⁵

Epigenetic regulation is particularly highlighted at this stage: human antigen R protein activates the AKT/mammalian target of rapamycin/autophagy related 13 autophagy pathway by stabilizing phosphatidylinositol-5-phosphate 4-kinase, type II, alpha transcript, which ultimately promotes RUNX2 expression.⁴⁶ Loss of sirtuin 6, which can be caused by activation of AKT in human VICs, will reduce the deacetylation modification and upregulate expression of RUNX2, leading to irreversible progression of calcification.⁴⁷ These findings reveal the potential of epigenetic modifiers as therapeutic targets.

Translational Direction of Treatment Strategies

Current mechanistic studies have indicated multidimensional intervention pathways: (1) targeting the upstream regulatory points of NF-κB (e.g., H₂S replacement therapy or AHR antagonist) during the inflammatory phase;^34,37 (2) regulating metabolic remodeling during fibrosis (e.g., ROCK1 inhibitors or AMPK activators);⁴¹ and (3) intervention in terminal differentiation during calcification (e.g., STAT3 inhibitors or epigenetic editing tools).^38,47 Of particular note, mitochondrial-targeted H₂S donors have shown stage-specific efficacy in animal models, suggesting the importance of precise timing of drug delivery.³⁶ Future studies need to integrate single-cell sequencing and dynamic imaging technology to establish a multistage treatment evaluation system.

Exploration of Pharmacological Therapy

Currently, there are no approved pharmacological therapies for CAVD; however, research on its underlying mechanisms and potential therapeutic strategies remains ongoing.

Vitamin K

Vitamin K plays a critical role in carboxylating and activating vitamin K-dependent Gla-rich protein,⁴⁸ inhibiting aortic valve calcification through the upregulation of α-SMA and downregulation of OPN expression.⁴⁹ Warfarin, an anticoagulant that inhibits vitamin K-dependent carboxylation of γ-glutamine proteins,⁵⁰ has been shown to exacerbate aortic valve calcification in both in vitro and in vivo models of human VICs.⁵¹ Despite this, warfarin remains the preferred anticoagulant for many patients with valvular atrial fibrillation. Because the use of warfarin may exacerbate valve calcification and worsen valve lesions, it may increase the risk of atrial fibrillation and embolism in patients. However, this potential adverse effect has not received sufficient attention in clinical practice. Notably, vitamin K1 supplementation in CAVD patients receiving vitamin K antagonists (VKAs) for anticoagulation has been shown to slow the progression of aortic calcification.⁵² Research by Sønderskov et al. supports this mechanism, demonstrating that the use of VKAs, but not non-vitamin K antagonist oral anticoagulants (NOACs), is associated with increased aortic valve calcification scores.⁵³ This finding underscores that CAVD progression is linked to vitamin K antagonism rather than anticoagulation itself. However, supplementation with vitamin K2 and vitamin D in patients with severe CAVD has not been shown to effectively delay disease progression.⁵⁴ Omarjee suggested that this negative outcome may be attributed to the lack of guarantee that all the MK-7 (a form of vitamin K2) used in that study was in the biologically active cis configuration.⁵⁵ Hariri et al. further noted that factors such as the timing of the intervention, dosage, and patient adherence to supplementation protocols may have contributed to the observed negative results.⁵⁶ Consequently, further clinical trials investigating the use of vitamin K2 in the treatment of aortic valve calcification are needed.

Biochemical reactions in metabolism play a pivotal role in the development of aortic calcification. Consequently, pharmacological targeting of these biochemical pathways holds significant potential as a therapeutic intervention. Beyond vitamin K supplementation to counteract warfarin-induced calcification, other conventional drugs, which were originally developed for non-calcification indications but subsequently found to inhibit calcification pathways, represent promising candidates for further investigation.

Lipid-Lowering and Antihypertensive Therapy

Rosuvastatin administration in asymptomatic patients with moderate to severe aortic stenosis has been shown to effectively inhibit disease progression.⁵⁷ However, that study only assessed the degree of stenosis using ultrasound and did not evaluate calcification.⁵⁷

The antisense oligonucleotide drug ISIS-APO(a)_Rx targets and inhibits the synthesis of lipoprotein(a), a known risk factor for aortic valve calcification. A randomized, double-blind, placebo-controlled study demonstrated the efficacy of ISIS-APO(a)_Rx in reducing lipoprotein(a) levels, a significant risk factor for CAVD,⁵⁸ thereby providing a promising direction for further exploration of pharmacological treatment.⁵⁹

An original investigation in 2005 reported a positive effect of angiotensin-converting enzyme inhibitors (ACEi) in inhibiting aortic valve calcification,⁶⁰ although the study did not identify a specific ACEi responsible for this effect. In addition, as a retrospective study, it could not establish a causal relationship between ACEi use and the progression of CAVD.⁶⁰

Vitamin D and Calcium-Phosphorus Metabolism

The inhibition of aortic valve calcification in patients with CAVD could theoretically be achieved by reducing serum calcium concentrations, such as by promoting osteogenesis and other methods of redistributing calcium within the body. However, these approaches have not yet been validated in practice. In addition, the use of non-toxic, metabolically active exosomes to bind calcium ions in the bloodstream is a promising direction for pharmacological treatment.

One study in mice demonstrated that dietary vitamin D deficiency leads to aortic valve calcification, which can be reversed by subsequent adequate vitamin D supplementation.⁶¹ Similarly, aortic valve calcification has been observed in patients with vitamin D deficiency due to underlying diseases.^62,63 Kassis et al. stratified patients into 3 groups: those receiving vitamin D alone, those receiving calcium with or without vitamin D, and those receiving neither of these treatments.⁶⁴ In that study, no significant difference in the progression of aortic stenosis was observed among the 3 groups.⁶⁴ This intriguing phenomenon may be explained by the fact that elevated serum phosphate levels, but not calcium or vitamin D, contribute to the increased risk of aortic stenosis.⁶⁵

Topical administration of the zoledronic acid, an osteoporosis drug, has been shown to inhibit aortic valve calcification in rabbits.⁶⁶ However, in a randomized double-blind trial in humans, neither denosumab nor alendronate, 2 other osteoporosis drugs, delayed calcification-induced aortic stenosis.⁶⁷ Myo-inositol hexaphosphate, also known as phytic acid or phytate, has been formulated into an intravenous preparation (SNF472) for enhanced absorption and has been shown to inhibit of calcium phosphate crystal formation in serum.⁶⁸ SNF472 also effectively suppresses calcification of VICs in vitro.⁶⁹ Furthermore, SNF472 has been shown to delay aortic valve calcification in hemodialysis patients in vivo.⁷⁰

Conclusions

CAVD progression involves 3 stages (inflammatory, fibrotic, calcification) driven by NF-κB signaling, metabolic reprogramming, and epigenetic dysregulation. Although preclinical studies highlight stage-specific targets, clinical trials on vitamin K, lipid-lowering, and calcium regulators remain inconclusive. Future research must prioritize precision therapies, leveraging multiomics and artificial intelligence to bridge mechanistic insights and clinical translation.

Sources of Funding

This study was supported by the National Science Foundation of China (No. 82422007, 82241205, 82170487), Beijing Natural Science Foundation (JQ24039), Undergraduate Scientific Research Innovation Project of Capital Medical University and Beijing Anzhen Hospital Major Science and Technology Innovation Fund (No. KCZD202203, KCQY202201).

Disclosures

The authors have no conflicts of interest to declare.

Author Contributions

Y.C.: conceptualization, data curation, formal analysis, visualization, writing-original draft; S.J.: conceptualization, data curation, formal analysis, writing-original draft; J.Z. and J.H.: conceptualization, data curation, formal analysis; H.Z.: study concept or design, data analysis or interpretation, administrative support; provision of study materials or patients; W.J.: study concept or design, data analysis or interpretation, administrative support.

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