NEXN protects against vascular calcification by promoting SERCA2 SUMOylation and stabilization

Guo, Wenjie; Guo, Wenjing; Chen, Boliang; Lin, Zexuan; Wen, Zhuohua; Ye, Jiamin; Feng, Wei; Feng, Xin; Yan, Jianyun; Yang, Pingzhen; Ouyang, Kunfu; Li, Yifei; Yang, Hanyan; Ou, Caiwen; Liu, Canzhao

doi:10.1038/s41467-025-63462-7

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Published: 29 August 2025

NEXN protects against vascular calcification by promoting SERCA2 SUMOylation and stabilization

Wenjie Guo^1,2^na1,
Wenjing Guo^1,2^na1,
Boliang Chen^1,2^na1,
Zexuan Lin^1,2,
Zhuohua Wen³,
Jiamin Ye^1,2,
Wei Feng⁴,
Xin Feng³,
Jianyun Yan^1,2,
Pingzhen Yang^1,2,
Kunfu Ouyang⁵,
Yifei Li ORCID: orcid.org/0000-0002-3096-4287⁶,
Hanyan Yang ORCID: orcid.org/0009-0003-4830-795X^1,2,
Caiwen Ou ORCID: orcid.org/0000-0002-9515-6125⁷ &
…
Canzhao Liu ORCID: orcid.org/0000-0002-9035-5723^1,2

Nature Communications volume 16, Article number: 8074 (2025) Cite this article

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Abstract

Vascular calcification, a key risk factor for cardiovascular diseases, is driven by the phenotypic transition of vascular smooth muscle cells from a contractile to an osteogenic phenotype. NEXN, a protein highly associated with heart function, has also been implicated as a potential susceptibility factor in the development of coronary artery disease, but its role in the progression of vascular calcification remains unclear. In this study, multi-transcriptomics analysis and various animal models of male mice were used to explore the cell-specific roles and molecular mechanisms of NEXN in vascular calcification. Here, we show that vascular smooth muscle cell-specific NEXN knockout exacerbates calcification, while NEXN overexpression alleviates it. Mechanistically, NEXN interacts with SERCA2, enhancing its SUMOylation, stability, and function, thereby protecting against calcification. These findings suggest potential therapeutic strategies by targeting NEXN-SERCA2 interactions or enhancing SERCA2 SUMOylation to prevent vascular calcification and its complications.

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Introduction

Vascular calcification, a pathological condition characterized by the ectopic deposition of calcium and phosphate crystals in the blood vessel wall, is recognized as a critical risk factor for cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD), diabetes, aging and atherosclerosis¹. Numerous pathological factors contribute to vascular calcification, encompassing hyperphosphatemia, increased oxidative stress, cell death, senescence, inflammation, and matrix remodeling^2,3. Accumulating studies have highlighted vascular calcification is not merely a passive consequence of aging or disease. Instead, it is an active and tightly regulated biological process that involves the reprogramming and phenotypic transition of vascular smooth muscle cells (VSMCs), which play a crucial role in vascular homeostasis^4,5. In the context of vascular calcification, VSMCs undergo a phenotypic switch from a contractile to an osteogenic phenotype, which is characterized by the upregulation of osteogenic markers such as RUNX2 and BMP2, and is accompanied by the secretion and deposition of calcifying matrix vesicles in the vessel wall. Understanding the mechanisms underlying VSMCs phenotypic switch and calcification is crucial for developing therapeutic strategies to prevent and treat vascular calcification and its associated cardiovascular complications.

Nexilin (NEXN) was isolated as an actin filament-binding protein in rat brain and fibroblasts and identified as a Z-disc protein abundant in striated muscles⁶. Our previous studies have revealed NEXN is a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules^7,8,9. In addition, it had been reported that NEXN suppresses the TLR4/NF-κB pathway, chemokine/cytokine expression in endothelial cells, and exerts a protective effect against atherosclerosis¹⁰. These findings suggest that NEXN plays a crucial role in maintaining the normal function of the cardiovascular system. More importantly, our preliminary data derived from the integration of multiple transcriptomic datasets, including bulk RNA-sequencing data from a rat vascular calcification model and single-cell RNA-sequencing data from atherosclerotic lesions in mice arteries, suggest that NEXN is a potentially important modulator in the phenotypic switch of VSMCs from contractile to osteogenic during vascular calcification. However, the functional role of NEXN in vascular calcification and the underlying mechanism remains unknown.

In this study, we observed a correlation between reduced NEXN expression and the progression of vascular calcification in human and mice. Knockdown of NEXN in VSMCs or VSMCs-specific knockout of NEXN in mice induced aggravation of vascular calcification. While overexpression of NEXN in VSMCs or AAV-mediated NEXN delivery in mice exhibited a protective effect against vascular calcification. To elucidate the mechanism of NEXN’s protective role, we generated Flag-Nexn transgenic mice, and Flag-pull down mass spectrometry data revealed SERCA2 as a key binding partner in VSMCs. Subsequent analysis demonstrated that NEXN maintains intracellular calcium homeostasis by stabilizing SERCA2 protein through a SUMOylation-dependent manner, thereby mitigating vascular calcification progression.

Results

Identification of NEXN as candidate gene associated with the phenotypic transformation of VSMCs in vascular calcification

To identify potential genes associated with the progress of vascular calcification, we performed differential expression analysis on a transcriptome dataset comparing a 5/6 nephrectomy-induced vascular calcification model in rats with a control group (GSE146638)¹¹. The differential expression analysis identified a total of 545 differentially expressed genes (DEGs), comprising 263 upregulated genes and 282 downregulated genes (Fig. 1a). Enrichment analysis of DEGs was shown in Supplementary Fig. 1a–d.

**Fig. 1: Identification of NEXN as a high-confidence gene associated with phenotypic transformation of VSMCs and vascular calcification.**

Osteogenic differentiation of VSMCs plays an important role in the process of vascular calcification. To further identify potential driver genes promoting osteogenic transformation of VSMCs, we analyzed scRNA-seq data from atherosclerotic vessels in mice (GSE131780), which was capable of VSMCs lineage tracing¹². VSMCs were categorized into modulated SMCs and normal SMCs (Fig. 1b). Using ossification-related markers (RUNX2, SPP1, SOX9, and ACAN) to label chondrocytes-like cells, we observed that a subset of cells within modulated VSMCs underwent osteogenic transformation (Fig. 1c). Differential analysis between these two cell groups revealed 893 DEGs.

Emerging evidence suggests that VSMCs senescence is a hallmark of calcified vessels, and the aging of VSMCs included the phenotypic transformation of VSMCs. Phenotypic transformation of VSMCs, characterized by the loss of the contractile phenotype, plays an important role in vascular calcification^13,14,15,16. To further narrow down the potential candidate genes modulating vascular calcification, we then introduced and reanalyzed a scRNA-seq data from aging vessels in mice (GSE164585)¹⁷. After cell clustering (Supplementary Fig. 2a), we performed Weighted Gene Co-expression Network Analysis (WGCNA) to identify modules of co-expressed genes and further calculated the correlation between these modules and various cell types (Supplementary Fig. 2b, c)^18,19. Our analysis revealed that the genes in the Turquoise module exhibited the highest positive correlation with VSMCs contractile phenotype. By screening the genes in the Turquoise module based on Gene Significance (GS) and Module Membership (MM) (GS > 0.6, MM > 0.8), we identified 64 hub genes which are positively associated with VSMCs contractile phenotype (Supplementary Fig. 2d). We intersected DEGs from these three datasets and identified 12 candidate genes (Fig. 1d). These 12 genes are likely to regulate VSMCs, leading to a loss of the contractile phenotype and simultaneous osteogenic differentiation, thereby promoting the occurrence of vascular calcification. NEXN is one of 12 candidate genes and is specifically expressed in VSMCs within the vessels (Supplementary Fig. 2e). NEXN has been reported to have a protective effect against atherosclerosis, while its single nucleotide polymorphisms (SNPs) are related to coronary artery disease, suggesting that NEXN has an important role in cardiovascular diseases.

To further validate the role of NEXN in regulating VSMCs, we detect the NEXN in modulated VSMCs, demonstrating that the NEXN expression was markedly diminished in modulated VSMCs that exhibited an osteoblast like phenotype, suggesting that the downregulation of NEXN promotes the osteogenic transformation of VSMCs (Fig. 1e). We then extracted VSMCs from the scRNA-seq data of aging vessels in mice (GSE164585) and performed pseudotime analysis (Supplementary Figs. 2f, 3a). Our analysis revealed that during vascular aging, the expression of NEXN exhibited a significant downward trend consistent with the VSMCs contractile phenotype markers MYH11 and ACTA2, indicating a positive correlation between NEXN and the maintenance of the VSMCs contractile phenotype (Supplementary Fig. 3b–d). Clinical evidence was used to further investigate whether NEXN expression is associated with vascular calcification in the clinical perspective, we leveraged large-scale artery-specific expression quantitative trait loci (eQTLs) for NEXN and genome-wide association study (GWAS) statistics for coronary artery calcification to explore potential relationships^20,21. Interestingly, our Mendelian Randomization (MR) analysis revealed a significant association between higher mRNA expression levels of NEXN in coronary artery tissue and a reduced genetic predisposition for coronary artery calcium (CAC) score in individuals of European ancestry (p = 0.034). Specifically, each increment of one transcript-per-million in NEXN expression in coronary artery tissue was linked to a 0.34 unit decrease in log (CAC + 1). These findings were further supported by a multi-ancestry GWAS meta-analysis of CAC, which confirmed a similar result (p = 0.005) (Fig. 1f). NEXN expression levels were examined across various organs, revealing high expression in the heart and aorta using a knockout-validated antibody (Fig. 1g). In addition, human aortic specimens from groups exhibiting calcification and those without calcification were collected based on the results of chest multi-detector computed tomography (MDCT). Immunofluorescence assays demonstrated a significant downregulation of NEXN expression in the calcified vessels while RUNX2 was upregulated (Fig. 1h). Collectively, these results strongly suggest NEXN is a high-confidence gene associated with vascular calcification and the NEXN deficiency may promote the transformation of VSMCs from contractile to osteogenic phenotype.

NEXN exhibits a significant reduction during the progression of vascular calcification

To further confirm the association of NEXN in vascular calcification, the expression of NEXN was detected both in vitro and in vivo model. Osteogenic differentiation model was induced using a calcified medium containing high phosphate (Pi) which was confirmed by increased calcium deposition and Alizarin red staining (Fig. 2a-c). Immunofluorescence assays revealed that NEXN was predominantly localized within the cytoplasm and its intensity was reduced in the calcified VSMCs (Fig. 2d). Osteogenic markers, including Collagen I, RUNX2, and BMP2, were significantly upregulated, while the NEXN protein levels were markedly repressed in calcified VSMCs (Fig. 2e, f). To validate the observed changes in NEXN levels in vivo, we utilized two mouse models. Firstly, we induced vascular calcification in mice by subcutaneous injection of vitamin D3 (VitD3). Compared to the control group, Alizarin red-positive areas constituted approximately 31.24% of the aorta (Fig. 2g, h). Aortic calcium deposition was also significantly increased in the calcified aorta ((Fig. 2i). Concomitantly, the upregulation of BMP2 confirmed the onset of arterial calcification, which was accompanied by a significant downregulation of NEXN (Fig. 2j, k). To investigate the dynamic changes in NEXN expression levels during vascular calcification, we established a CKD model in mice by performing 5/6 nephrectomy. Ultrasound images revealed a gradual decrease in vascular elasticity at different time points (Fig. 2l, m). As anticipated, the expression of NEXN gradually decreased during the progression of vascular calcification (Fig. 2n, o).

**Fig. 2: NEXN expression was downregulated during vascular calcification in vitro and in vivo.**

Knockdown of NEXN aggravates vascular calcification in vitro and in vivo

Next, we sought to determine whether NEXN suppression could aggravate vascular calcification. NEXN siRNA-mediated knockdown in VSMCs was validated (Supplementary Fig. 4a, b). To this end, VSMCs were transfected with NEXN siRNA with or without high-Pi stimulation. Knockdown of NEXN did not induce spontaneous calcification but significantly exacerbated high Pi-induced calcium deposition in VSMCs, as evidenced by Alizarin red staining (Fig. 3a). NEXN deficiency dramatically increased the expression of osteogenic genes Collagen I, RUNX2, and BMP2(Fig. 3b, c). Immunofluorescence assays further confirmed that knockdown of NEXN upregulated the expression of RUNX2 in calcified VSMCs (Fig. 3d).

To further evaluate the role of NEXN in vascular calcification in vivo, we generated VSMCs-specific Nexn knockout mice (Nexn^ismKO) by breeding Nexn^flox/flox mice (Nexn^F/F) with Myh11-Cre/ERT2 mice (Myh11^Cre/ERT2) (Fig. 3e, Supplementary Fig. 5a, b). We then induced vascular calcification using two mouse models: an adenine and phosphate (AP) diet-induced chronic kidney disease (CKD) model (Fig. 3f) and a VitD3-induced model. Histopathological analysis of renal tissue morphology and serum blood urea nitrogen (BUN) levels indicated that the AP diet-induced CKD group exhibited marked renal dense granularity and notable atrophy, accompanied by a significant elevation in BUN concentrations, reflecting impaired renal function (Supplementary Fig. 6a, b). VSMCs-specific knockout of NEXN significantly aggravated the aortic calcium deposition induced by VitD3, as determined by Alizarin red staining (Supplementary Fig. 7a–c). NEXN deficiency enhanced the expression of osteogenic marker BMP2 compared to the control group (Supplementary Fig. 7d, e). Consistent with the results of the VitD3-induced calcification model, in the AP-induced model, the degree of vascular calcification in Nexn^ismKO mice was more severe, as evidenced by the increased calcification volume and surface through micro-computed tomography (micro-CT) scanning and three-dimensional CT reconstruction (Fig. 3g-i). Furthermore, we found that aortic stiffness, measured by pulse wave velocity (PWV), was significantly increased in Nexn^ismKO mice (Fig. 3j). The protein expression of RUNX2 and BMP2 were dramatically increased in arteries from VSMCs-specific knockout of Nexn mice (Fig. 3k, l). Collectively, these results suggest that deficiency of NEXN aggravates vascular calcification.

Overexpression of NEXN alleviates vascular calcification in vitro and in vivo

To evaluate whether NEXN overexpression could attenuate vascular calcification progression, we upregulated NEXN expression in VSMCs using adenovirus, with or without high-Pi stimulation. NEXN protein expression in VSMCs transfected with adenovirus was significantly upregulated compared to controls (Supplementary Fig. 8a). Alizarin red staining revealed that overexpression of NEXN significantly suppressed calcium deposition (Fig. 4a). Additionally, NEXN overexpression reversed the upregulation of osteogenic markers Collagen I, RUNX2 and BMP2 in calcified VSMCs induced by high Pi (Fig. 4b, c).

To further validate our findings in vivo, we generated adeno-associated virus serotype 9 specifically expressing murine Nexn in VSMCs driven by the Myh11 promoter (AAV-Nexn) (Fig. 4d). Mice injected with AAV-Nexn significantly decreased calcification in AP-induced model, as indicated by the reduced calcification volume and surface (Fig. 4e–h, Supplementary Fig. 9a, b). Accordingly, AAV-Nexn downregulated the expression of RUNX2 and BMP2 (Fig. 4i, j). Taken together, these data demonstrate that NEXN overexpression can inhibit the osteogenic phenotypic transformation and prevent the development of vascular calcification.

NEXN modulates vascular calcification through SERCA2

To explore the molecular mechanism by which NEXN modulates VSMCs calcification, we performed RNA-Seq in adenovirus mediated overexpression of NEXN group and vector group, both subjected to high-Pi stimulation. The differential expression analysis and the enrichment analysis of DEGs were applied (Fig. 5a–c, Supplementary Fig. 10a, b). The results showed that the Calcium signaling pathway and the endoplasmic reticulum lumen were affected. Moreover, GSEA revealed that, compared to Ad-Ctrl group, intracellular calcium ion homeostasis and vascular smooth muscle contraction were promoted in Ad-NEXN group (Fig. 5d, e). Additionally, we generated Flag-Nexn transgenic mice and performed NEXN interactome analysis (Fig. 5f). Anti-FLAG affinity immunoprecipitates and subsequent mass spectrometry were performed to identify potential binding partners of NEXN (Fig. 5g, h). Among these, SERCA2, a key calcium-regulating protein primarily located in the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR), draws our attention due to its role in maintaining intracellular calcium balance by pumping calcium ions from the cytoplasm back into the ER/SR. Dysregulation of calcium ion homeostasis can lead to increased cytosolic calcium levels, which in turn can activate various calcium-dependent signaling pathways that drive the phenotypic switch from contractile to osteogenic VSMCs^22,23,24,25. Additional analysis of the GSE146638 dataset, along with KEGG and GO enrichment analyses, revealed alterations in ER/SR function and calcium ion-related signaling pathways in vascular calcification. Therefore, we hypothesized that NEXN might regulate vascular calcification through SERCA2-dependent ER/SR function and subsequent intracellular calcium homeostasis. Co-immunoprecipitation experiments validated the interaction between NEXN and SERCA2 in VSMCs (Fig. 5i). Furthermore, immunofluorescence was used to investigate the co-localization of NEXN and SERCA2 in VSMCs, which revealed that NEXN and SERCA2 partially co-localized in the cytoplasm of VSMCs (Supplementary Fig. 11a). These findings demonstrated an intracellular interaction between NEXN and SERCA2 in VSMCs. We next investigated the effects of NEXN on SERCA2. Overexpression of NEXN significantly upregulated SERCA2 expression in calcified VSMCs induced by high-Pi stimulation (Fig. 6a, b). Accumulating evidence suggests that SERCA2 plays a pivotal role in vascular remodeling^26,27,28. However, its role in vascular calcification remains poorly understood. To validate the ameliorative effects of SERCA2 on VSMCs calcification, VSMCs were transfected with SERCA2 siRNA with or without high-Pi stimulation. Knockdown of SERCA2 did not induce spontaneous calcification but significantly exacerbated high Pi-induced calcium deposition i–n VSMCs, while promoting the upregulation of RUNX2 and BMP2 (Fig. 6c–e). Additionally, inhibition of SERCA2 by cyclopiazonic acid (CPA) eliminated the downregulation of Collagen I, RUNX2, and BMP2 in VSMCs overexpressing NEXN (Fig. 6f–h). While NEXN deficiency-aggravated VSMCs calcification was effectively abolished by additional treatment with the SERCA2 activator istaroxime (ISTA) (Fig. 6i-k), indicating that the inhibitory effect of NEXN on vascular calcification is mediated by SERCA2.

**Fig. 5: Identifying protein binding partners of NEXN in vascular calcification.**

**Fig. 6: NEXN regulates vascular calcification through SERCA2 mediated ER/SR function.**

Considering the importance of SERCA2 in ER/SR function, we next examine the morphological changes of the ER in VSMCs using transmission electron microscopy (TEM). Under calcifying conditions, a marked swelling of the ER, indicative of ER dysfunction, was observed. In contrast, NEXN overexpression significantly improved ER morphology, restoring its normal slender shape, suggesting an effective recovery of ER function (Supplementary Fig. 12a). In addition, NEXN overexpression effectively increased the Ca²⁺ transient amplitude, which could be facilitated by restoring SERCA2 pump activity (Supplementary Fig. 12b). SERCA2 deficiency could lead to ER dysfunction which significantly contributes to the development of ER stress, a process known to promote vascular calcification. To further clarify the regulatory role of SERCA2 in ER stress in vascular calcification, SERCA2 was downregulated in VSMCs with or without high-Pi stimulation. The results showed that knockdown of SERCA2 significantly upregulated the ATF4 and CHOP expression, demonstrating that SERCA2 deficiency exacerbates ER stress and further aggravates vascular calcification (Supplementary Fig. 13a, b). Moreover, we found that high-Pi stimulation significantly increased ATF4 expression, while overexpression of NEXN notably decreased ATF4 levels (Supplementary Fig. 14a, b). To further validate this finding, we induced vascular calcification in Nexn^ismKO mice and simultaneously administered the ER stress inhibitor 4-Phenylbutyric acid (4PBA). The treatment with 4PBA efficiently inhibited the ER stress and alleviated vascular calcification in Nexn^ismKO mice as evidenced by the reduction in calcification area and downregulation of BMP2 level (Supplementary Fig. 14c–f). These results indicate the exacerbation of vascular calcification due to NEXN deficiency relies on the ER stress. Collectively, these findings suggest NEXN alleviates ER stress by improving the function of SERCA2, thereby inhibiting vascular calcification.

The 299-671 fragment of NEXN is indispensable for inhibiting vascular calcification

To further delineate the functional fragment of NEXN in vascular calcification, we constructed several truncated mutants (D1: aa 1-465, D2: aa 1-671 (full length), D3: aa 168-671, D4: aa 299-671, D5: aa 466-671) (Fig. 7a). GFP-tagged NEXN mutants and the FLAG-tagged SERCA2 plasmid were co-transfected into HEK293T cells. Co-Immunoprecipitation experiments revealed SERCA2 could be specifically immunoprecipitated by full-length NEXN, D3, and D4 using an anti-GFP antibody. These results suggest amino acids 299-671 of NEXN, which contains one ABD fragment and an Ig fragment, is necessary for mediating the binding of NEXN and SERCA2 (Fig. 7b). To examine whether this functional fragment could exert the same inhibitory effect on calcification as the full length of NEXN, we transfected VSMCs with adenovirus overexpressing NEXN 299-671 aa, with or without high-phosphate stimulation. Overexpression of NEXN 299-671 aa effectively alleviated high-Pi-induced calcification, as demonstrated by Alizarin red staining (Fig. 7c), as well as the protein levels of Collagen I, RUNX2 and BMP2 (Fig. 7d, e). These results indicate that the NEXN 299-671 aa, acting as a functional region, exerts protective effects on VSMCs calcification.

**Fig. 7: NEXN 299-671 aa acts as a functional region in protecting VSMCs from calcification.**

NEXN enhances SERCA2 expression by promoting its SUMOylation

To determine the mechanism by which NEXN upregulates SERCA2 expression during vascular calcification, we assessed SERCA2 mRNA following NEXN intervention. The data show NEXN overexpression had no effect on SERCA2 mRNA levels in VSMCs, suggesting that NEXN may regulate SERCA2 at the post-translational level (Fig. 8a). It has been reported that SERCA2 could be SUMOylated, resulting in an increase in the molecular weight of the protein. SUMO1-dependent SUMOylation has been shown to be essential for maintaining the activity and stability of SERCA2^29,30. As shown in Fig. 6a, a clear band could be observed above SERCA2 in the Ad-NEXN group, suggesting that NEXN overexpression may promote the SUMOylation of SERCA2. To further confirm that NEXN promotes SERCA2 SUMOylation, VSMCs overexpressing NEXN were treated with the SUMOylation inhibitor ML792. The results showed that the higher molecular weight band above endogenous SERCA2, which was upregulated by NEXN overexpression, was inhibited by ML792 treatment (Fig. 8b, c)³¹. Conversely, in NEXN knockdown VSMCs, the SERCA2 specific SUMOylation agonist N106 (N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine) restored the reduced band intensity, supporting that NEXN enhances SERCA2 SUMOylation (Fig. 8d, e)³⁰. To further investigate the effect of NEXN on SERCA2 SUMOylation, full-length and the 299-671 aa of NEXN were transfected into HEK293T cells along with FLAG-SUMO1 and SERCA2, or into VSMCs along with FLAG-SUMO1. Co-Immunoprecipitation experiments revealed that the presence of full-length NEXN and the 299-671 fragment significantly increased SERCA2 SUMOylation (Fig. 8f, g, Supplementary Fig. 15a, b). These findings indicate that NEXN elevates the SERCA2 SUMOylation, a process primarily mediated by the 299-671 fragment of NEXN.

**Fig. 8: NEXN promotes SERCA2 SUMOylation to inhibit vascular calcification.**

To verify whether NEXN deficiency aggravates vascular calcification through reduced SERCA2 SUMOylation in vivo, N106, a SERCA2 specific SERCA2 SUMOylation activator was used to treat mice. N106 treatment significantly alleviated vascular calcification in VSMCs-specific Nexn knockout mice as evidenced by the reduction in calcification volume and surface (Fig. 8h-k). Additionally, the protein levels of BMP2 were downregulated (Fig. 8l, m). These pharmacological intervention experiments strongly indicate the exacerbation of vascular calcification due to NEXN deficiency is dependent on the reduction of SERCA2 SUMOylation. To clarify whether the regulation of ER stress by NEXN depends on its effect on SERCA2 SUMOylation, VSMCs overexpressing NEXN were treated with ML792 and the results indicated that NEXN overexpression effectively inhibited ER stress, but this inhibition was blocked by ML792 (Supplementary Fig. 16a, b). Additionally, N106 effectively reversed the exacerbation of ER stress caused by NEXN knockdown (Supplementary Fig. 16c, d). These findings collectively demonstrate that the inhibitory effect of NEXN on ER stress depends on its ability to promote SERCA2 SUMOylation.

Discussion

Vascular calcification is a complex and multifactorial process influenced by various biochemical, mechanical, and genetic factors. It is a serious complication of chronic kidney disease, diabetes, and atherosclerosis and is associated with increased cardiovascular morbidity and mortality. Here, through integrative analysis of transcriptomic datasets, including vascular tissue transcriptomes from a chronic kidney disease rat model (GSE146638), scRNA-seq transcriptomic datasets from mouse aortic root atherosclerotic plaques (GSE131780), and scRNA-seq transcriptomic datasets from mouse aortas during aging (GSE164585), we identified NEXN as a potential modulator implicated in vascular calcification. In the current study, we demonstrate that NEXN plays a crucial role in suppressing vascular calcification by inhibition of ER stress.

Intracellular calcium homeostasis is a tightly controlled process that plays a pivotal role in almost every aspect of cell function. The delicate balance between Ca²⁺ channels and pumps ensure precise control of cytosolic Ca²⁺ levels, enabling cells to respond appropriately to various stimuli and maintain overall health. Disruptions in intracellular calcium homeostasis can lead to cell dysfunction and disease³². Notably, a deficiency in the ER/SR calcium sensor STIM1 has been shown to disrupt intracellular calcium homeostasis, leading to the promotion of vascular calcification and stiffness in diabetes. This suggests the critical role of ER/SR-mediated calcium signaling in vascular calcification. In this study, differential expression analysis of our transcriptomic data revealed enrichment of several signaling pathways, including collagen-extracellular matrix organization, ER lumen, PI3K-Akt signaling, and calcium signaling (Fig. 5b, c, Supplementary Fig. 1a, b). These findings suggest that NEXN may regulate VSMCs osteogenic differentiation through a complex signaling network. Notably, our previous work demonstrated that NEXN is predominantly localized at plasma membrane-ER/SR contact sites in cardiomyocytes, where it modulates excitation-contraction coupling by regulating ER/SR calcium homeostasis. Based on these observations, we focused our mechanistic investigations on ER/SR functional regulation and its involvement in calcium signaling.

The SERCA2 pump is a membrane protein residing within the ER/SR Ca²⁺ store, acting as the primary regulator of cellular calcium homeostasis. Its fundamental role involves actively transporting calcium ions from the cytosol into the ER/SR lumen. Accumulated evidence indicates that SERCA2 plays a central role in both normal cardiac function and pathological conditions^33,34,35. Recent research has extended our understanding of SERCA2’s function beyond contractile regulation. Studies have shown that SERCA2-mediated calcium homeostasis also plays a significant role in glucose homeostasis and pancreatic β-cell function^36,37. Through Flag-IP mass spectrometry analysis of mouse aorta, we identified SERCA2 as a binding partner of NEXN, and the results of immunoprecipitation and immunofluorescence indicated that NEXN interacts with SERCA2 in VSMCs. Then, we utilized pharmacological and molecular interventions to demonstrate that SERCA2 deficiency could exacerbate ER stress which is known to aggravate vascular calcification, and SERCA2 plays a crucial role in inhibiting the phenotypic transition of VSMCs towards an osteogenic lineage. In addition, the results showed that the protective effect of NEXN on vascular calcification depends on SERCA2 expression. SUMOylation is a crucial post-translational modification essential for maintaining the stability and activity of SERCA2 in cardiomyocytes^29,30. Interestingly, we found that the interaction between NEXN and SERCA2 is capable of promoting SERCA2 SUMOylation. N106, a specific agonist for SERCA2 SUMOylation, could significantly ameliorate vascular calcification in Nexn^ismKO mice treated with VitD3. Moreover, enhancing SERCA2 SUMOylation by N106 mitigated the pro-calcifying effects of NEXN knockdown by reducing ATF4 and CHOP levels. These findings demonstrate that the protective effects of NEXN against vascular calcification via inhibition of ER stress are dependent on the SERCA2 SUMOylation.

NEXN, initially identified as an actin filament-binding protein, is characterized by two actin-binding fragments (ABDs) separated by approximately 130 amino acids (aa) and a C-terminal immunoglobulin like fragment. Interposed between these ABDs is a coiled-coil region, conferring F-actin cross-linking activity to NEXN. We identified that the NEXN 299-671 aa fragment lacking the first ABD is necessary for SERCA2 binding and SUMOylation. Functional studies validated that the NEXN 299-671 aa fragment could suppress the phenotypic switch of VSMCs toward the osteogenic lineage. These findings indicate that actin cross-linking activity is not essential for osteogenic differentiation of VSMCs. Further in-depth studies are needed to elucidate the precise mechanism by which NEXN modulates SERCA2 SUMOylation in future research.

NEXN has previously been implicated in various cardiovascular diseases, with gene mutations in NEXN associated with hypertrophic and dilated cardiomyopathy^38,39,40. Furthermore, SNPs near NEXN were found to associate with coronary artery disease⁴¹. Studies on atherosclerosis have shown that NEXN can suppress its development by regulating the TLR4/NF-κB pathway, and NEXN deficiency promotes the occurrence of atherosclerosis and increases the vulnerability of atherosclerosis plaque¹⁰. The findings from the present study expand and refine our understanding of biological functions of NEXN in cardiovascular disease by revealing its important role and underlying mechanism in regulating vascular calcification. However, the upstream mechanisms regulating NEXN expression during vascular calcification remain unclear. Previous studies have shown that NEXN can be modulated by myocardin-related transcription factors (MRTFs), which play a key role in the phenotypic switching of VSMCs⁴². Additionally, research has demonstrated that the long noncoding RNA (lncRNA) NEXN-AS1 regulates NEXN expression by interacting with the chromatin remodeler BAZ1A and the 5’ flanking region of the NEXN gene¹⁰. Whether the downregulation of NEXN during vascular calcification is mediated by MRTFs or epigenetic mechanisms involving NEXN-AS1/BAZ1A-mediated chromatin remodeling requires further investigation.

In summary, we discovered that NEXN confers a strongly protective effect against vascular calcification. Our results identified SERCA2 as a critical binding partner of NEXN and mediated its effects on vascular calcification. Pharmacological targeting of SERCA2 could significantly alleviate vascular calcification in NEXN deficiency mice. These findings suggest that targeting the NEXN-SERCA2 interaction may offer a potential therapeutic strategy for vascular calcification.

Methods

Detailed description of the studies included and methods used is provided in the article and Data Supplement. The origins of antibodies are outlined in Supplementary Table 1, while the reagents are specified in Supplementary Table 2. The siRNA sequences are displayed in Supplementary Table 3. The primers used for qRT-PCR analysis are displayed in Supplementary Table 4. The primers used for mice genotyping are listed in Supplementary Table 5.

Animals

All experiments were approved by the Institutional Animal Care Committee at Zhujiang Hospital, Southern Medical University, China (Approval number: LAEC-2023-162) and performed in accordance with the regulations of the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China. Nexn floxed (Nexn^F/F) mice were provided by Professor Ju Chen from UCSD, and Myh11^Cre/ERT2 mice were purchased from The Jackson Laboratory. Nexn^F/F mice were crossed with Myh11^Cre/ERT2 mice to generate Nexn^F/F; Myh11^Cre/ERT2 mice. The inducible VSMCs-specific Nexn knockout (Nexn^ismKO) was induced by intraperitoneal injection of tamoxifen in Nexn^F/F; Myh11^Cre/ERT2 mice for 5 days at a dose of 40 mg/kg body weight. As the Myh11Cre/ERT2 transgene was randomly inserted in the Y chromosome, only males were used in this study. In experiments, the 8-week old male mice were randomly allocated to each group. All mice were housed in rooms maintained at a temperature of 22–24 °C, 45–65% relative humidity, with free access to food and water. An appropriate light-dark cycle, typically 12 h of light and 12 h of darkness, was implemented to mimic the mice’s natural circadian rhythm, all under Specific Pathogen-Free conditions. Animals were weighed daily and systematically assessed for health status throughout the study. Predefined humane endpoints for CO₂ euthanasia included significant weight loss, severe distress, impaired mobility, or other signs of suffering, ensuring animal welfare was prioritized. All experimental animals in this study were anesthetized by intraperitoneal injection of 250 mg/kg tribromoethanol before surgery or sacrifice to reduce the pain.

Human samples

All procedures involving human samples were conducted ethically in accordance with the principles of the Declaration of Helsinki. Approval for the study was granted by the medical ethics committee of Zhujiang Hospital, Southern Medical University, China (Approval number: 2023-KY-209). Written informed consent was obtained from all participants prior to their involvement in the study. Human coronary arteries samples were obtained from non-CKD patients and CKD patients, respectively. Non-CKD patients had normal kidney function, while CKD patients were all diagnosed with Stage V chronic kidney disease and required long-term dialysis treatment. The clinical data regarding kidney function and diagnosis of patients are provided in Supplementary Table 6.

Cell culture and transfection

Human primary vascular smooth muscle cells (VSMCs) were purchased from iCell Bioscience Inc (HUM-iCell-c010) and cultured in a primary smooth muscle cell low serum culture system (PriMed-iCell-004) according to the vendor’s protocol. The tissue origin of VSMCs is human aorta. Passages 2-8 of the cultured VSMCs were used for the experiments. HEK293T cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained in a humidified incubator (5% CO₂, 37 °C). VSMCs were cultured with differently treatments for 7 days, such as 10 μM cyclopiazonic acid (CPA, HY-N6771, MedChemExpress), 200 nM istaroxime (ISTA, HY-15718, MedChemExpress), 10 μM ML792 (HY-108702, MedChemExpress), and 5 μM N106 (HY-110273, MedChemExpress). To induce calcification, VSMCs were treated with calcified medium containing 3.0 mM sodium phosphate buffer (Sigma, 94046-100ML-F) for 7 days, with fresh medium changes every two days.

Small interfering RNA (siRNA) oligonucleotides targeting human NEXN (stB0014693), SERCA2 (stB0005645), and a scrambled siRNA were designed and synthesized by RiboBio. Human VSMCs were transfected with siRNAs using Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000015) and Opti-MEM™ I Reduced Serum Medium (Gibco, 31985070), according to the manufacturer’s protocol. The cultured VSMCs were infected with 20 MOI adenovirus encoding GFP-NEXN (Ad-NEXN) for 5 h, and then the cells were washed with phosphate-buffered saline (PBS) and incubated in fresh culture medium before experiment. Separately, HEK293T cells were transfected with plasmids using the same Lipofectamine 3000 Transfection Reagent and Opti-MEM™ I Reduced Serum Medium, according to the manufacturer’s protocol. Cells were harvested at the indicated time points.

Mendelian randomization analysis

Two-sample Mendelian Randomization (MR) was conducted to investigate the potential association between NEXN and coronary artery calcification (CAC) measured by Agatston Score. We obtained human coronary artery tissue-specific expression quantitative trait loci (eQTL) data for NEXN from GTEx v8 and selected single nucleotide polymorphisms (SNPs) associated with NEXN at a significance level of p < 1 × 10⁻⁵. The largest genome-wide association study (GWAS) on coronary artery calcification was used as the outcome dataset^20,21. To ensure independence of the selected SNPs, we pruned those in linkage disequilibrium (R² < 0.01 in the 1000 Genomes European population) with a clumping window of 5000 kb. Consequently, only a single variant remained, which allowed us to quantify its MR effect using Wald ratio tests through the TwoSampleMR (0.5.5) in R.

Immunofluorescence assay

Human arteries were rinsed with PBS and fixed overnight in 4% PFA. They were then dehydrated in 10% and 20% sucrose solutions for 30 min each, followed by overnight incubation in 30% sucrose. The dehydrated tissues were embedded in OCT tissue and cut into 6 μm thick cryosections. VSMCs were fixed with 4% PFA for 15 min. The cryosections from human arteries and the VSMCs were washed three times for 5 min with PBS and then incubated with PBS containing 5% BSA, 5% donkey serum, and 0.1% Triton for 1 hour. They were then incubated with primary antibodies overnight and secondary antibodies for 1 hour. Nuclei were stained with DAPI (Vector Laboratories, H-1200-10). Immunofluorescence images were acquired using a Leica DMi8 confocal microscope.

Protein extraction and Western blot

Total protein was extracted using RIPA lysis buffer. Lysates were collected and centrifuged by centrifugation at 13,523 × g for 15 min at 4 °C. Protein content was quantified using the BCA Protein Assay Kit (Thermo Scientific, 23227). The protein was electrophoresed in SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked in TBST containing 5% skimmed milk for 1 h at room temperature. They were then incubated with primary antibodies at 4 °C overnight, followed by secondary antibodies for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence (ECL) detection system (Millipore, WBKLS0100).

Quantitative real time PCR (qRT-PCR) analysis

Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen, 15596026). The extracted RNA was then reverse transcribed to cDNA using PrimeScript™ RT Master Mix (Takara, RR036A). qRT-PCR was performed according to the manufacturer’s protocol.

Characterization of calcified nodules by Alizarin Red Staining

Alizarin red staining was used to visualize calcified nodules on VSMCs. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 minutes. They were then washed three times with deionized water and incubated with 2% Alizarin red in deionized water at room temperature for 30 minutes. Following incubation, the Alizarin red solution was removed, and the cells were washed with deionized water to remove excess dye before observation. To quantify the extent of calcification, the Alizarin red dye was incubated with 10% formic acid for 5 minutes, and the optical density at 405 nm was measured using a microplate reader.

Plasmids DNA constructs

The overexpression plasmids were constructed by PCR amplification of the corresponding coding sequences (CDS) and subsequent cloning into vectors (restriction enzyme site: HindIII and BamHI) using the In-Fusion Snap Assembly Master Mix (Clontech, 638948). The truncated plasmids were constructed by inverse PCR using pairs of nonoverlapping primers and Hi-T4 DNA Ligase (NEB, M2622S).

Quantification of VSMCs and aorta calcification (calcium assay)

The calcium content of VSMCs and aortas was measured using a commercial Ca²⁺ assay kit (Leagene). The protein lysates were collected and centrifuged by centrifugation at 845 × g for 15 min at 4 °C. After mixing the lysates with the working solution, it was incubated at room temperature for 10 min, and the absorbance was measured at 575 nm using a microplate reader⁴³.

Immunoprecipitation

For immunoprecipitation, cells were lysed with IP lysis buffer supplemented with 1% protease inhibitor. 1 mg of the lysates was incubated with either anti-GFP antibody/anti-FLAG antibody or control IgG along with 25 μL Protein A/G Magnetic Beads overnight at 4 °C. The immunocomplexes were washed three times with wash buffer and then resuspended in 2 × SDS-PAGE loading buffer. The resulting samples were analyzed by western blot analysis.

HEK293T cells were transfected with plasmids. The HEK293T cells were lysed and incubated with anti-GFP magnetic beads or anti-FLAG magnetic beads. The beads were washed and eluted with 2 × SDS-PAGE sample loading buffer. The samples were analyzed by western blot analysis.

Measurement of intracellular free calcium concentration (Ca²⁺)

Human VSMCs were seeded onto 25 mm diameter circular glass coverslips, washed with PBS, and incubated with Fura 2-AM for 30 min at 37 °C. Ca²⁺ measurement was determined from the ratio of F340/F380 at room temperature^44,45.

RNA sequencing

Total RNA was extracted from mouse arteries using Trizol reagent (Invitrogen, 15596026) following the manufacturer’s instructions. The quality of the RNA was evaluated, and qualified samples were used to construct libraries and perform sequencing. RNA library sequencing was performed on the Illumina NovaSeq 6000 platform by Novogene (Beijing, China). Differential expression analysis of RNA-seq data was performed using DESeq2 (1.36.0) with the following parameters: absolute fold change (FC) ≥ 1 and adjusted p-value < 0.05⁴⁶. Gene set enrichment analysis (GSEA) was performed using the clusterProfiler (4.10.1) package, with an adjusted p-value < 0.05 considered statistically significant⁴⁷.

Analysis of RNA-seq and scRNA-seq data

The RNA sequencing data are available and can be downloaded from the Gene Expression Omnibus (GEO) under the accession number GSE146638¹¹. GSE146638 provides bulk RNA sequencing transcriptomic data from rat aorta samples, comparing a vascular calcification model induced by 5/6 nephrectomy to normal controls. Differential expression analysis of RNA-seq data was performed using the Limma (3.52.4) package with the following parameters: absolute fold change (FC) ≥ 1 and adjusted p-value < 0.05. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed with the clusterProfiler package, with an adjusted p-value < 0.05 considered statistically significant. The scRNA sequencing data are available and can be downloaded from the Gene Expression Omnibus (GEO) under the accession number GSE131780 and GSE164585. GSE131780 offers scRNA sequencing data of VSMCs isolated from mouse aortas of atherosclerotic versus normal mice¹². GSE164585 contains scRNA sequencing data detailing mouse aortas across different age groups¹⁷. The Seurat package (4.4.0) was used for quality control, data normalization, and dimensional reduction. Cells with a mitochondrial ratio >10% and <500 genes were filtered out. The “NormalizeData” function of Seurat was used for normalization. Highly variable genes were identified using the “FindVariableFeatures” function with modified parameters (nfeatures: 2000). The “FindIntegrationAnchors” and “IntegrateData” functions were used for sample integration. Heterogeneities were regressed out using the “ScaleData” function, and then dimensional reduction analysis was conducted using the “RunTSNE” function. “FindNeighbors” and “FindClusters” were used for cell clustering, and then marker genes for each cluster were determined using “FindAllMarkers”⁴⁸. Cell types were assigned to each cluster. To construct the gene co-expression network, the R package WGCNA (1.73) was used. Genes with coherent expression profiles were grouped into modules using average linkage hierarchical clustering, with the topological overlap measure used as the dissimilarity metric. The modular gene centrality, defined as the sum of within-cluster connectivity measures, was used to rank modular genes for hubness within each gene module^18,19. Pseudotime analysis was then performed on the identified VSMCs using the Monocle 2 (2.34.0) R package according to the manual. Cells from 4-week old mice were set as the beginning point for trajectory analysis. Cell trajectory analysis, differentiation fate-related gene analysis, branched expression analysis modeling (BEAM), and clustering were sequentially performed to identify differentiation fate-related genes and potential differentiation trajectories. Statistical models were built to characterize the data using the “estimateSizeFactors”, “estimateDispersions”, and “dispersionTable” functions with default parameters. Subsequently, the ‘DDRtree’ method was employed to reduce dimensionality and identify branching points across differential cell fates. Finally, differentiation fate-related genes identified by DEG analysis were utilized to distinguish cells in different states, illustrating how differential gene expression prompted the divergence of cell fates toward distinct directions⁴⁹.

Generation of the 3×Flag-Nexn Mouse Line Via CRISPR-Cas9

The 3×Flag-Nexn mouse line was generated using CRISPR-Cas9 technology⁵⁰. Cas9 protein, crRNA, tracrRNA, and a single-stranded oligodeoxynucleotide (ssODN) repair template were diluted and mixed in IDTE buffer. The mixture was microinjected into the pronuclei of C57BL/6 N zygotes. The sequences used in this study were as follows:

crRNA: 5’-uuuucuaguggaugacuacuGUUUUAGAGCUAUGCUGUUUUG-3’
ssODN template, including the 3×Flag tag sequence (indicated by lowercase letters): 5’-ATAGGAAAAAAAATCATCCTTCTCCTTCTTCTTTTCTAGTGGATGACTACgactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagTAGGCTTCCCTCTGTCCTTGGGACTCTCTCTCTCGCTGCATCTCTGTGGA-3’

Following microinjection, the zygotes were transferred into the oviductal papillae of pseudopregnant ICR host female mice. After birth, genomic DNA was extracted from the pups, and the insertion of the 3×Flag sequence at the Nexn locus was confirmed using genomic PCR and direct sequencing. The homozygous 3×Flag-Nexn knock-in mice and their littermate controls were used in Fig. 5.

Animals models of vascular calcification

The cholecalciferol (vitamin D3) overload-induced calcification model was conducted. Mice were treated with subcutaneous injections of VitD3 or vehicle for 3 consecutive days, and the aortas were collected 4 days later. Mice were fed with a standard chow diet for the entire procedure⁵¹.

The 5/6 nephrectomy-induced chronic renal failure vascular calcification model was conducted with a two-step procedure. Mice were anesthetized, and the left kidney was separated. The upper and lower poles of the left kidney were then ligated and excised with scissors. One week later, mice were subjected to a total right nephrectomy to establish 5/6 nephrectomy. One week later, the CKD mice were fed with a high phosphate (1.8%) diet for two months⁵².

For the adenine and phosphate (AP) diet-induced chronic kidney disease (CKD) model, mice were fed a specialized diet containing 0.2% adenine and 1.2% phosphorus (Dyets, D201223). The control group received a standard chow diet⁵³.

The mice treated with N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)- 1,3,4-oxadiazol-2-amine (N106, HY110273, MedChemExpress) were received with N106 orally 10 mg/kg/day. The mice treated with 4-Phenylbutyric acid (4PBA, HY-A0281, MedChemExpress) were received with 4PBA at 20 mg/kg/day intraperitoneally.

Adeno-associated virus (AAV) injection

The AAV9-Myh11-Nexn (AAV-Nexn) and AAV9-Myh11-vector (AAV-Ctrl) constructs were obtained from Genechem. The viruses were injected into mice at a titre of 5 × 10¹¹ viral genomes/mL, and subsequent experiments were performed 10 days post-injection.

Measurement of aortic pulse wave velocity (PWV) by echocardiography

Mice were anesthetized with inhaled isoflurane and underwent transthoracic echocardiography using a Vevo 2100 system. Ascending and descending aortic peak velocities were measured from the pulse wave (PW) Doppler-mode aortic arch view. Aortic arch PWV was calculated as PWV (m/s) = aortic arch distance (D1)/transit time (T2 − T1). T1 was measured from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform in the ascending aorta. T2 was measured from the onset of the QRS complex to the onset of the descending aortic Doppler waveform as distally as possible in the descending aorta. D1 was measured between the two sample volume positions along the central axis of the aortic arch.

Micro-CT

Mice were anesthetized with inhaled isoflurane and underwent whole-body computed tomography (CT) scans on a micro-CT scanning bed. The CT images were analyzed using Mimics Medical 21.0 software, which enabled three-dimensional reconstruction. Surface area and volume of calcification were calculated from the reconstructed images.

Statistics analysis

All data were analyzed using GraphPad Prism 8.0 software. Values are presented as mean ± standard deviation of the mean (SD). Statistical significance between two groups was analyzed by two-sided and unpaired t-test. Ordinary one-way ANOVA and Turkey’s post hoc test were used for comparisons among more than two groups. A p-value < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the main text, Supplementary Information, and Source data. The RNA sequencing data GSE146638¹¹ [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146638], the single-cell RNA sequencing (scRNA-seq) data GSE131780¹² [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131780] and GSE164585¹⁷ [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164585] are available and can be downloaded from the Gene Expression Omnibus (GEO). The datasets including GSE146638, GSE131780, and GSE164585 were re-analyzed from previous publication. The RNA-seq data generated in this study have been deposited in GEO under the accession codes GSE297578. The raw data of the mass spectrometry have been deposited in the Figshare under https://doi.org/10.6084/m9.figshare.29648861 [https://doi.org/10.6084/m9.figshare.29648861.v1]. Source data are provided with this paper.

Code availability

Code is available and deposited in Zenodo under https://doi.org/10.5281/zenodo.15856149, (https://zenodo.org/records/15856149).

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Acknowledgements

We would like to express our gratitude to Ju Chen from the University of California, San Diego for generously supplying the Nexn^F/F mice. This work was supported by grants from the National Natural Science Foundation of China (No. 82300469, Hanyan Yang; No. 82170231 and No. 82370410, Canzhao Liu; No. 82172103 and No. 32371428, Caiwen Ou), and the Guangdong Basic and Applied Basic Research Foundation (No. 2023B1515020103, Canzhao Liu).

Author information

These authors contributed equally: Wenjie Guo, Wenjing Guo, Boliang Chen.

Authors and Affiliations

Department of Cardiology, Laboratory of Heart Center, Heart Center, Center for Translational Medicine Research, Zhujiang Hospital, Southern Medical University, Guangzhou, China
Wenjie Guo, Wenjing Guo, Boliang Chen, Zexuan Lin, Jiamin Ye, Jianyun Yan, Pingzhen Yang, Hanyan Yang & Canzhao Liu
Guangdong Provincial Key Laboratory of Cardiac Function and Microcirculation, Guangdong Provincial Biomedical Engineering Technology Research Center for Cardiovascular Disease, Guangzhou, China
Wenjie Guo, Wenjing Guo, Boliang Chen, Zexuan Lin, Jiamin Ye, Jianyun Yan, Pingzhen Yang, Hanyan Yang & Canzhao Liu
Neurosurgery Center, Department of Cerebrovascular Surgery, Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Zhujiang Hospital, Southern Medical University, Guangzhou, China
Zhuohua Wen & Xin Feng
Department of Medicine, University of California San Diego, La Jolla, CA, USA
Wei Feng
Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
Kunfu Ouyang
Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital Sichuan University Chengdu, Sichuan, China
Yifei Li
The Tenth Affiliated Hospital of Southern Medical University (Dongguan People’s Hospital), Southern Medical University, Dongguan, China
Caiwen Ou

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Wenjie Guo
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Wenjing Guo
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Boliang Chen
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Zexuan Lin
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Hanyan Yang
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Caiwen Ou
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Canzhao Liu
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Contributions

W. Jie. Guo, W.Jing. Guo, B. Chen and Z. Lin performed the experiments. W.Jie. Guo, Z. Wen, and J. Ye analyzed the results. W. Feng, X. Feng, J. Yan, P. Yang, K. Ouyang, and Y. Li contributed to the experimental design. C. Liu, W. Jie. Guo, H. Yang, and C. Ou conceived the idea, designed experiments, and co-wrote the manuscript. The manuscript was read and approved by all authors.

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Correspondence to Hanyan Yang, Caiwen Ou or Canzhao Liu.

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Guo, W., Guo, W., Chen, B. et al. NEXN protects against vascular calcification by promoting SERCA2 SUMOylation and stabilization. Nat Commun 16, 8074 (2025). https://doi.org/10.1038/s41467-025-63462-7

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Received: 26 February 2025
Accepted: 19 August 2025
Published: 29 August 2025
DOI: https://doi.org/10.1038/s41467-025-63462-7