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. 2026 Mar 4;16:12006. doi: 10.1038/s41598-026-42767-7

Contactin-2 protects against aortic valve calcification via osteogenic differentiation inhibition

Zhongxing Zhou 1,2,#, Ruming Shen 1,2,#, Shuaijie Chen 1,2, Hailin Zhang 1,2, Longqing Chen 1,2, Xiuzhu Weng 1,2, Xiaoyan Lin 3, Dajun Chai 1,2,4,✉
PMCID: PMC13069110  PMID: 41781684

Abstract

Current pharmacological strategies for delaying the progression of calcific aortic valve disease (CAVD) remain inadequate. This study used Mendelian randomization (MR) analysis to identify a significant association between the plasma protein Contactin-2 (CNTN2) and CAVD. Transcriptomic and in vivo/in vitro experiments further validated these findings. An MR study was conducted to evaluate the exposure-outcome relationship between plasma proteins and CAVD. Transcriptomic profiling identified differentially expressed genes and also analyzed pathways related to the osteogenic differentiation phenotype of human primary aortic valve interstitial cells (hVICs), which further validated the MR analysis. Western blotting, Alizarin Red staining and immunohistochemistry were used to validate the MR result in hVICs and patients with CAVD. Furthermore, adenovirus-mediated CNTN2 overexpression in hVICs was performed to elucidate the role in the osteogenic phenotype. MR analysis demonstrated a significant causal association between CNTN2 and CAVD. Colocalization analysis indicated that CNTN2 shares genetic loci with CAVD. Gene Ontology (GO) analyses revealed that CNTN2 was downregulated in hVICs with an osteogenic phenotype. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that CNTN2 is enriched in pathways associated with the osteogenic phenotype. Immunoblotting further confirmed that CNTN2 protein expression was reduced in osteogenically induced hVICs and in clinical specimens obtained from patients with CAVD. Additionally, overexpression of CNTN2 significantly inhibited the osteogenic phenotype of hVICs. CNTN2 has a significant causal relationship with CAVD and contributes to protection during aortic valve calcification. These findings could provide new insights and therapeutic targets for the prevention and treatment of CAVD.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42767-7.

Keywords: Contactin-2, Calcific aortic valve disease, Valve interstitial cells, Osteogenic differentiation, Mendelian randomization

Subject terms: Cardiology, Diseases, Genetics, Medical research

Introduction

CAVD is a severe chronic condition that has experienced a steady rise in incidence over the past 30 years, a trend that is exacerbated by an aging population, thereby placing a substantial burden on public health systems in numerous countries globally1,2. As calcific aortic stenosis progresses, leading to obstruction of the left ventricular outflow tract, further impairing cardiac function and potentially leading to heart failure3,4. Several pharmacological treatments, such as statins, angiotensin-converting enzyme inhibitors, and calcification inhibitors, have been applied in patients with CAVD, but their efficacy is unsatisfactory5,6. Surgical interventions and transcatheter aortic valve replacement (TAVR) can improve patient symptoms; nevertheless, both procedures are invasive, costly, associated with significant risk, and entail various potential complications7–9. Thus, identification of effective intervention targets and therapeutic measures to prevent CAVD progression is urgently needed.

Emerging research is attempting to discover the genetic determinants of complex diseases to facilitate new intervention targets through genome-wide association studies (GWASs)10,11. MR is a genetic technique used to reveal the causal effects of predictable exposures on complex disease-related phenotypes or outcomes12,13. GWASs of plasma proteins have revealed genetic variations associated with these proteins, known as protein quantitative trait loci (pQTLs). Therefore, by selecting pQTLs as the exposure and CAVD as the outcome, we aimed to identify proteins that have a causal relationship with CAVD through MR analysis.

In this study, we first established a significant association between the plasma protein CNTN2 and CAVD utilizing MR analysis. Transcriptomic analysis indicated that the reduction in CNTN2 expression correlated with the osteogenic phenotype of hVICs. Additionally, CNTN2 exhibits a protective role in the osteogenic differentiation of hVICs. Furthermore, our findings revealed a reduced expression of CNTN2 in clinical specimens derived from patients with CAVD.

Methods

Study design

Figure 1 illustrates the overall study design. First, we conducted two-sample MR analyses using predefined human blood plasma protein pQTLs and CAVD GWAS data to evaluate the causal associations between crucial proteins and CAVD while also performing sensitivity and colocalization analyses. Additionally, transcriptomic data further confirmed the osteogenic phenotype of hVICs induced in vitro, characterized by the downregulation of CNTN2 expression. Furthermore, the overexpression of CNTN2 in hVICs induced by osteogenic medium (OM) significantly mitigated their osteogenic differentiation phenotype. Ultimately, we validated the MR results using tissue samples from patients with CAVD. MR analysis followed the STROBE-MR guideline. All procedures involving patient samples and tissue handling were approved by the hospital’s ethics committee (MRCTA, ECFAH of FMU [2023] 449) and were carried out in accordance with the World Medical Association Declaration of Helsinki.

Fig. 1.

Fig. 1

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Diagram depicting the experimental protocol of the current study to identify crucial proteins related to CAVD. The scientific illustration was created in BioRender. Chen, S. (2024) https://BioRender.com/f19t533. pQTLs: protein quantitative trait loci; IVW: inverse variance weighted. CAVD: calcific aortic valve disease; CNTN2, Contactin 2. OM, osteogenic media. hVICs, human primary aortic valve interstitial cells.

Data sources of instrumental variables (IVs) and outcomes

The pQTL data for human blood plasma proteins from subjects with European ancestry originate from previously published literature14. The selection of IVs was conducted according to the following criteria: (1) SNPs were highly correlated with protein expression levels (P< 1 × 10⁻⁵); (2) there was no linkage disequilibrium (r² < 0.001); and (3) SNPs with weak instrumental strength were excluded (F statistic < 10)15. Ultimately, a total of 5,228 SNPs associated with 1,118 proteins were included as IVs in our MR analysis.

The GWAS data for CAVD were derived from the FinnGen consortium database with data from subjects with European ancestry, which includes 11,047 cases and 442,686 controls16. The diagnosis of CAVD within the FinnGen study adhered to the corresponding ICD-9 and ICD-10 standards. The IV data utilized for MR analysis, after the exposure and outcome data were merged, are presented in Supplementary Table S1.

MR analysis

For proteins associated with two or more SNPs (N = 1,061), the inverse variance weighted (IVW) method was employed to estimate the MR effects. Additionally, the weighted median method was utilized as a complementary validation measure. In contrast, for proteins associated with only one SNP (N= 57), the Wald ratio was used to determine the ratio of effect sizes on the outcomes17. We carried out two sample MR analyses by applying the R TwoSampleMR package (v0.5.7). To correct for multiple testing, the Bonferroni method was applied, yielding a significance threshold of P < 4.48 × 10⁻⁵ (calculated as 0.05/1,118). Proteins that demonstrated significant MR effects on CAVD were subsequently subjected to sensitivity analyses and colocalization analyses.

Sensitivity and colocalization analysis

Sensitivity analyses were conducted to assess the potential violations of MR assumptions18. The Cochran’s Q test was used to detect heterogeneity, with a P value of less than 0.05 indicating potential heterogeneity. The MR‒Egger intercept was employed to assess horizontal pleiotropy. Additionally, leave-one-out analyses were performed to examine whether the exclusion of any single SNP significantly impacted the overall results.

Colocalization analyses were conducted on significantly associated protein pQTLs using the “coloc” R package to determine whether the single variants associated with plasma proteins were causally associated with CAVD. Default prior probabilities were used, with P1 = P2 = 1e-4 and P12 = 1e-5. A posterior probability for H4 (PP.H4) greater than 80% was considered indicative of a colocalization relationship19..

Clinical samples

Human aortic valve specimens were obtained from the Department of Cardiothoracic Surgery at The First Affiliated Hospital of Fujian Medical University. Those with rheumatic valve disease and infective endocarditis were excluded. Patients with CAVD were established using echocardiography in conjunction with medical history, physical examination, and intraoperative observation under direct visualization. The main echocardiographic manifestations of calcified valves in CAVD patients are thickening, nodular plaques, or increased echogenicity of the aortic valves. Control valve tissue samples were collected from individuals without leaflet thickening or calcium deposition, primarily from patients with acute aortic coarctation who underwent Bentall surgery or from those with end-stage heart failure who underwent orthotopic heart transplantation. These individuals had no history of valvular heart disease; had normal aortic valve histology as diagnosed by ultrasound; and had thin, soft, noncalcified aortic valves. All enrolled patients provided informed consent. The collected valve tissue samples were promptly frozen at −80 °C. These sections were then used for Western blot and immunohistochemistry assays.

Immunohistochemistry

Immunohistochemistry assays were conducted on 8 μm paraffin-embedded human aortic valve sections. For increased specificity of immunostaining, heat-induced antigen retrieval was performed on deparaffinized aortic valve sections. To prevent nonspecific binding of the antibody to the tissues, we blocked the sections with 5% goat serum for 1 h at room temperature. A primary antibody against CNTN2 (#67089-1-Ig, Proteintech, 1:500) was applied to the sections in a humidified chamber at 4 °C overnight. Subsequently, biotinylated secondary antibodies were applied, followed by the use of VECTASTAIN ABC reagent (#: 67089-1-Ig; Vector Laboratories).

Cell culture and osteogenic culture

Human primary aortic valve interstitial cells (hVICs) were purchased from Saibaikang Biotechnology Co., Ltd (HUM-iCell-c005, Shanghai, China), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal Bovine Serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. The cells were identified with immunofluorescence to ensure their phenotype (Supplementary, Figure S1) and then further passaged and cultured. Cultured cells were collected from the third to the sixth generations for research and experiments.

hVICs were plated in 6-well plates at a density of 2.0 × 104 cells/ml and cultured until 70–80% confluency was achieved. After 48 h, the medium was changed to either control media, composed of DMEM supplemented with 10% FBS and 1% P/S, or OM, composed of DMEM with 10% FBS, 1% P/S (Penicillin and Streptomycin), 10 nM dexamethasone, 10 mM β-glycerol phosphate, and 100 mM L-ascorbate phosphate. The medium was changed every 3 days for 3 weeks.

Immunofluorescence

For immunofluorescence, hVICs were plated in 12-well plates at a density of 1.0 × 104 cells/ml and cultured for 24 h. Gently wash the cells with 1 × phosphate-buffered saline (PBS), then add an appropriate 4% paraformaldehyde, and fix the cells at room temperature for 30 minutes. Treat the cells with a 0.5% Triton X-100 solution for 15 minutes., blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h, and incubated with the primary antibody overnight at 4°C. The sections were thoroughly washed with 1× PBS three times, followed by 1-h incubation with the appropriate fluorescent-labelled secondary antibodies at room temperature. The sections were thoroughly washed three times with 1× PBS three times, counterstained with 4’ 6-diamidino-2-phenylindole (DAPI) (#D1306, Invitrogen), and mounted with VECTASHIELD Anti-fade Mounting Medium (#H-1000-10, Vector Laboratories). Stained sections were visualized using a Zeiss 710 confocal microscope (Carl Zeiss). The primary antibodies used for immunofluorescence included: anti-CD31 (#3528, CST, 1:500), anti-α-SMA (#19245, CST, 1:500), anti-Vimentin (#5741, CST, 1:200). The secondary antibody used was goat anti-rabbit, Alexa Fluor 594 (#A-11037, Invitrogen, 1:500).

RNA sequencing (RNA-seq)

hVICs were cultured with CM or OM for 3 weeks, and the cells were promptly frozen at −80°C. The sequencing library preparation and RNA-seq were performed by using a HiSeq sequencing system (Illumina). HTSeq v0.6.0 was used to count the read numbers mapped to each gene. We applied the limma package (v3.58.1) to filter the differentially expressed genes under the following criteria: (i) |log2FC| > 1 and (ii) adjusted p-value using the Benjamini-Hochberg method for FDR correction < 0.05. Gene Ontology (GO) analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were implemented using the clusterProfiler package (v4.10.1)20. The results were visualized using the ggplot2 package (v3.5.1).

Western blot

Human valve tissues and cultured hVICs were sonicated in 1x RIPA lysis buffer (#WB3100, NCM) containing protease inhibitors (#P001, NCM) and phosphatase inhibitors (#P001, NCM). After centrifugation, the lysates were processed to collect the supernatants as whole-cell proteins. Bicinchoninic Acid Assay (BCA) assay was performed to determine protein concentrations. Total protein was separated by SDS‒PAGE and then transferred to a PVDF (Millipore) membrane. The primary antibodies used were as follows: anti-RUNX2 (#12556T, CST, 1:1500) rabbit mAb, anti-ALP (#30971S, CST, 1:1500) rabbit mAb, anti-CNTN2 (#67089-1-Ig, Proteintech, 1:1000) mouse mAb, and anti-GAPDH (#2118, CST, 1:2000) rabbit mAb. Anti-rabbit IgG, HRP-linked antibody (#7074, CST, 1:4000) and anti-mouse IgG, HRP-linked antibody (#7076, CST, 1:4000) were used to visualize the binding of primary antibodies in combination with the enhanced chemiluminescent (ECL) substrate (#RPN2109, GE). Quantitative analysis of the protein bands was performed with ImageJ software. All original Western blot images are available in the Supplementary Materials.

Alizarin Red S staining of cultured cells

Following treatment, hVICs were fixed in 4% PFA for 20 min at room temperature and then rinsed with 1× PBS and distilled water. The fixed cells were then stained with 2% Alizarin Red stain (Sigma‒Aldrich) for 15 min. This solution was prepared by dissolving 2 g of Alizarin Red (Sigma-Aldrich, USA) in 100 ml of distilled water to ensure thorough mixing. The pH was subsequently adjusted to 4.2 with 1 mM NaOH. Quantitative analysis of relative calcium deposits was performed using ImageJ software.

Adenovirus-mediated overexpression of CNTN2

Recombinant adenovirus vectors expressing the CNTN2 gene were designed and purchased from GeneChem Co., Ltd. (Shanghai, China). hVICs were seeded at a density of 2.0 × 104 cells/well in 6-well plates. After reaching 70–80% confluence, the cells were incubated with adenovirus for 48 h at an MOI of 100 and then used for subsequent experiments. Immunofluorescence confirmed the transfection efficacy. Western blotting was used to verify the differential expression of CNTN2.

Statistical analysis

The means and standard errors (SEMs) of continuous data are presented, following a normal distribution test to guide the choice of statistical test. One-or-two-way analysis of variance (ANOVA) with a post hoc Bonferroni/Dunn test was used for multiple-group comparisons, and Student’s t test was used for two-group comparisons. The in vitro and clinical data statistical analyses were performed using GraphPad Prism version 8.3.0 (GraphPad Software, Inc.). P values < 0.05 were considered significant.

Results

MR analysis

Supplementary Table S2 details the findings from the two-sample MR analysis conducted on 1,118 human blood plasma proteins and CAVD. By applying a Bonferroni-corrected significance threshold, we identified a causal link between CNTN2 and CAVD (Fig. 2), with an IVW OR of 0.89 ([95% confidence interval (CI): 0.85 to 0.94]; P = 1.04e-05; Fig. 3). Cochran’s Q test and the MR‒Egger intercept further suggested that the association between CNTN2 and CAVD was free of heterogeneity and pleiotropy, with P values for the Q test and MR‒Egger of 0.695 and 0.366, respectively. The leave-one-out cross-validation analysis demonstrated that after excluding rs4951168, rs11108976, and rs35130600, the odds ratio (OR) values for the association between CNTN2 and CAVD were 0.95 [95% CI, 0.87 to 1.04], 0.89 [95% CI, 0.83 to 0.95], and 0.88 [95% CI, 0.85 to 0.92], respectively (Supplementary Figure S2). Moreover, colocalization analysis revealed strong colocalization effects between CNTN2 and CAVD, with a PP.H4 of 99.39% (Table 1).

Fig. 2.

Fig. 2

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Manhattan plot illustrating the MR results. P values were calculated using the IVW method for proteins instrumented by two or more single SNPs and the Wald ratio method for proteins associated with a single SNP. The dotted line represents the significance threshold for MR estimates (P = 4.49 × 10-⁵). Red data points highlight target proteins that exhibit statistically significant associations. MR, mendelian randomization; SNP, single-nucleotide polymorphism.

Fig. 3.

Fig. 3

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MR results for the expression levels of the CNTN2 pQTL in relation to calcific aortic valve stenosis. CAVD, calcific aortic valve disease; pQTL, protein quantitative trait loci; SNP, single-nucleotide polymorphism; CI, confidence interval; OR, odds ratio.

Table 1.

Colocalization analysis results of CNTN2 pQTL gene expression in CAVD.

Trait a: protein Trait b outcome N tested-SNPs PP.H0.abf PP.H1.abf PP.H2.abf PP.H3.abf PP.H4.abf Top SNP
CNTN2 CAVD 155 0% 0.40% 0% 0.22% 99.39% rs3851294
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CNTN2, Contactin 2; pQTLs, protein quantitative trait loci; PP.H4, posterior probability of colocalization; CAVD, calcific aortic valve disease; SNPs, Single Nucleotide Polymorphism; GWAS, genome‒wide association study.

Transcriptomic analysis of in vitro-induced hVICs with osteogenic differentiation

VICs and valvular endothelial cells (VECs) are primarily involved in the progression of CAVD4. The osteogenic differentiation of VICs is closely associated with microhemorrhage in calcified valves and abnormal production of the extracellular matrix21. Inhibition of osteogenic phenotype differentiation in VICs can alleviate calcification of aortic valve leaflets and reduce valve damage22. Therefore, we induced hVICs with osteogenic differentiation medium in vitro and performed transcriptomic analysis. The volcano plot revealed 408 significantly upregulated genes in the OM group compared with those in the control group, whereas 517 genes, including CNTN2, were significantly downregulated (Fig. 4A). GO biological processenrichment analysis indicated that the differentially expressed genes were concentrated in pathways associated with cell‒cell adhesion, ossification, vasculature development, positive regulation of cell differentiation, and extracellular matrix organization (Fig. 4B). KEGG enrichment analysis revealed that the differentially expressed genes were enriched in cytokine‒cytokine receptor interactions, focal adhesion, ECM‒receptor interactions, cell adhesion molecules and other pathways (Fig. 4C). Heatmaps revealed that multiple cell adhesion molecules, including CNTN2, were significantly downregulated in the OM group compared with the control group (Fig. 4D). To further validate the results of the transcriptomic analysis, we observed calcification nodules in hVICs induced with osteogenic differentiation medium for 3 weeks. Additionally, OM increased the expression of ALP and RUNX2 in hVICs, whereas the protein expression of CNTN2 was decreased in osteogenically differentiated hVICs (Fig. 4E, Supplemental Figure S3A). In summary, CNTN2 may play a crucial role in the osteogenic differentiation of hVICs.

Fig. 4.

Fig. 4

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CNTN2 is downregulated in osteogenically induced hVICs, which is associated with osteogenic differentiation. (A) Volcano plot showing the upregulated and downregulated genes in osteogenically induced hVICs (CNTN2: logFC=−4.286, adjusted p-value < 0.001). (B) GO analysis reveals differentially expressed genes enriched in biological processes. (C) KEGG analysis of differentially expressed genes enriched in pathways. The KEGG imagery used in this figure was obtained from the KEGG database, and we acknowledge the permission provided by the Kanehisa laboratory. This figure is cited in accordance with KEGG citation guidelines (www.kegg.jp/kegg/kegg1.html)20. (D) Heatmap showing that the expression of CNTN2 is significantly downregulated in hVICs induced by OM. (E) Alizarin Red staining demonstrated the osteogenic differentiation of hVICs and relative calcium deposits induced by OM, n = 6. Western blotting revealed the expression levels of CNTN2, ALP, and RUNX2 in the control (con) and OM groups, n = 6. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes, CNTN2, Contactin 2; OM, osteogenic media; hVICs, human primary aortic valve interstitial cells; ALP, alkaline phosphatase; RUNX2 runt-related transcription factor 2.

CNTN2 is downregulated in calcified aortic valves and mitigated osteogenic differentiation of hVICs

To further confirm the important role of CNTN2 in the process of aortic valve calcification, we overexpressed CNTN2 in hVICs using adenoviral vectors (Supplementary Table S3). Fluorescence initially confirmed successful cell transfection, and Western blotting confirmed the overexpression of CNTN2 (Fig. 5A). After hVICs were induced with OM for 3 weeks, Alizarin Red staining confirmed the formation of calcified nodules, and the overexpression of CNTN2 reduced the formation of these nodules (Fig. 5B). Furthermore, the expression of RUNX2 and ALP was significantly increased in the OM-induced group, and the overexpression of CNTN2 substantially reduced the expression levels of these two proteins (Fig. 5B, Supplemental Figure S3B). Therefore, CNTN2 can alleviate the osteogenic phenotype of hVICs induced by OM, thereby reducing calcification. Moreover, the protein expression level of CNTN2 in patients with CAVD was significantly lower than that in patients with no aortic valve calcification (Fig. 5C and D). These findings further confirmed that CNTN2 is involved in the progression of CAVD and indicated that CNTN2 may have a protective effect on valve leaflet calcification.

Fig. 5.

Fig. 5

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CNTN2 is downregulated in calcified aortic valves and mitigates the osteogenic differentiation of hVICs. (A) Immunofluorescence image showing the transfection of CNTN2 in hVICs, and immunoblotting image showing the expression level of CNTN2 in both the CNTN2-OE and CNTN2-OE-NC groups. (B) Alizarin Red S staining and immunoblotting analysis of the osteogenic differentiation markers ALP and RUNX2 in hVICs from the control, OM, CNTN2-OE + OM and CNTN2-OE-NC + OM groups, n = 6. (C) Immunoblotting and quantification of the protein levels of CNTN2 in patients with and without CAVD, n = 6. (D) Typical immunohistochemical images showing the expression level of CNTN2 in the two groups of patients. CNTN2, Contactin 2; hVICs, human primary aortic valve interstitial cells; OM, osteogenic media; ALP, alkaline phosphatase; RUNX2 runt-related transcription factor 2; OE, overexpression; OE-NC, overexpression negative control; CAVD, calcific aortic valve disease.

Discussion

Plasma proteomic research has revealed new therapeutic targets for a multitude of cardiovascular diseases23,24. This study employed two-sample MR analysis and showed that CNTN2, from 1118 plasma proteins, is significantly associated with CAVD. Transcriptomic analysis revealed that CNTN2 expression is downregulated in hVICs with an osteogenic phenotype, and we further confirmed the result in human aortic valve calcification tissue. In vitro, the overexpression of CNTN2 in hVICs significantly mitigated the osteogenic differentiation phenotype. In summary, we report for the first time that CNTN2 inhibits hVICs osteogenic differentiation and may play an active protective role in CAVD.

Current treatments for CAVD including, reducing LDL and Lp(a) to slow the burden of atherosclerosis25,26. Reductions in calcium‒phosphate precipitation and anti-inflammatory effects have shown limited effectiveness and are controversial27–29. The identification of biomarkers and potential therapeutic targets to prevent CAVD progression is a major challenge. Inhibition of sorting protein (SORT1) can suppress osteogenic differentiation of VICs and mitigate the calcification process of damaged valves30. Activation of a mechanosensitive nonselective ion channel (Piezo1) or lumican (LUM) promotes osteogenic phenotypes of hVICs and aortic valve calcification in mice31,32. Concurrently, Mendelian whole-genome research has revealed that the key mitochondrial gene aldehyde dehydrogenase 9 family member A1 (ALDH9A1) has a protective role in CAVD33. However, intervention strategies for these molecular targets still have poor accessibility. Plasma proteomic analysis directly targets soluble proteins in the circulation—these proteins naturally exhibit inherent “accessibility”34.The plasma proteome-derived MR analysis can further identify causal relationships between exposures and outcomes, thereby providing new intervention targets for diseases12,13. Through MR analysis, our team discovered that CNTN2 has a significant positive effect on CAVD. Furthermore, by integrating our in vitro transcriptomic data, we confirmed that CNTN2 plays an active protective role in the calcification process of osteogenic differentiation. Thus, targeting CNTN2 may become an effective therapeutic strategy for CAVD.

CNTN2 is a contact protein that plays a crucial role in the initiation of axonal elongation, axonal guidance, and membrane potential stabilization35,36. We report that CNTN2, a plasma protein, has a causal relationship with CAVD, and its expression is reduced in human aortic valve calcification tissue, confirming the correlation between CNTN2 and aortic valve calcification. VICs with osteogenic phenotypes play an important role in the progression of aortic valve calcification, and previous studies have shown that reversing the osteogenic differentiation of VICs can alleviate aortic valve calcification and stenosis31,32. In vitro transcriptomic analysis revealed that the expression of CNTN2 was downregulated in hVICs with an osteogenic phenotype induced by OM and that the overexpression of CNTN2 significantly reduced the osteogenic phenotype of VICs. These findings suggest that CNTN2 may play a protective role during the progression of aortic valve calcification by inhibiting the osteogenic differentiation of VICs. As delivery technologies continue to mature, we propose that nanoparticle-based delivery systems loaded with CNTN2 protein could be a promising strategy for targeting aortic valves and preventing the progression of aortic valve calcification.

The limitations of this study are that the clinical samples were derived from patients with severe aortic valve calcification and impaired cardiac function, and the potential protective role of CNTN2 in the early stages of aortic calcification is the subject of our forthcoming research. Due to inherent limitations in tissue acquisition, we only included six clinical samples, and this limitation should be considered when interpreting our findings. The GWAS data for our MR was from individuals of European ancestry, which might limit the generalizability of the findings. Age and gender play crucial roles in the pathogenesis of CAVD1,37. Our study did not perform separate analyses for different genders or age groups, which suggests that we should further conduct clinical cohort studies to investigate the expression differences of CNTN2 across populations of different ages and genders.

Conclusion

Through MR studies and transcriptomic analysis, we established a causal association between CNTN2 and CAVD, indicating a potential protective role of CNTN2 in the progression of CAVD. This finding may provide new targets for intervention and treatment strategies for CAVD.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (39.3KB, docx)
Supplementary Material 2 (1.1MB, xlsx)
Supplementary Material 3 (385.4KB, pdf)
Supplementary Material 4 (959.9KB, pdf)

Acknowledgements

We thank Liangliang Yan and HanFan Qiu for providing human aortic valve tissues and clinical patient baseline data.

Abbreviations

CAVD

Calcific aortic valve disease

CNTN2

Contactin-2

GWAS

Genome‒wide association study

hVICs

Human primary aortic valve interstitial cells

GO

Gene ontology

KEGG

Kyoto encyclopedia of genes and genomes

TAVR

Transcatheter aortic valve replacement

BNP

B-type natriuretic peptide

PQTLs

Protein quantitative trait loci

IVW

Inverse variance weighted

ATX

Autotaxin

ALDH9A1

Aldehyde dehydrogenase 9 family member A1

PCSK9

Proprotein convertase subtilisin/kexin type 9

Author contributions

Z.X.Z., R.M.S and S.J.C: Conceptualization. Writing-Reviewing and Editing. L.Q.C., H.L.Z: Conceptualization, Methodology, Software. X.Z.W., X.Y.L: Supervision, Formal analysis, Visualization. Z.X.Z., D.J.C: Data curation, Writing-Original draft preparation. D.J.C: Funding acquisition.

Funding

The Fujian Provincial Health and Family Planning Commission (grant number 2021ZQNZD005) and the Joint Funds for the Innovation of Science and Technology of Fujian Province (grant number 2023Y9107).

Data availability

Raw data for MR analysis can be found in FinnGen https://www.finngen.fi/en), and GWAS Catalog (https://www.ebi.ac.uk/gwas/publications/28240269, https://www.ebi.ac.uk/gwas/publications/38494474). The population data can be obtained from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All procedures involving patient samples and tissue handling were approved by the First Affiliated Hospital of Fujian Medical University ethics committee (MRCTA, ECFAH of FMU [2023] 449).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zhongxing Zhou and Ruming Shen contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (39.3KB, docx)
Supplementary Material 2 (1.1MB, xlsx)
Supplementary Material 3 (385.4KB, pdf)
Supplementary Material 4 (959.9KB, pdf)

Data Availability Statement

Raw data for MR analysis can be found in FinnGen https://www.finngen.fi/en), and GWAS Catalog (https://www.ebi.ac.uk/gwas/publications/28240269, https://www.ebi.ac.uk/gwas/publications/38494474). The population data can be obtained from the corresponding author upon reasonable request.

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