BK channel agonists may affect matrix vesicle secretion and ameliorate vascular calcification via autophagy

Introduction

Vascular calcification (VC) is caused by mineral deposits of hydroxyapatite (1). VC is usually associated with cardiovascular diseases such as stroke, atherosclerosis, and heart disease, which further accelerate and worsen the disease (2). A meta-analysis of vascular diseases and risk factors revealed that individuals with VC were 2.94 times more likely to die from the disease than healthy individuals (3). Currently, no efficient therapy is available to prevent or reverse VC. Inhibition of vascular smooth muscle cell (VSMC) phenotypic transformation and calcium deposition are the two main strategies for reversing VC.

The potential mechanisms of VC include calcium and phosphorus metabolism disorders, osteogenic transformation, and VC regulator imbalance (4-6). High calcium disrupts the metabolism and function of VSMCs, resulting in calcification. High phosphorus promotes VC by inducing VSMC apoptosis and extracellular matrix remodeling (7). VSMCs transform into osteoblast-/chondrocytic-like cells, similar to those observed in bone formation, and secrete matrix vesicles (MVs) in response to physiological signals, which nucleate calcium phosphate crystals in the form of hydroxyapatite (8,9), eventually leading to the pathological deposition of calcium and phosphate in blood vessels. The upregulation of bone-related transcription factors [e.g., Runt-related transcription factor 2 (Runx2) and Osterix] in the VSMCs of calcified vessels increases the expression of markers of mineralization and bone or cartilage formation [bone morphogenetic protein-2 (BMP-2), Runx2, β-catenin, phosphorylated Smad1/5/8 (p-SMAD1/5/8), alkaline phosphatase (ALP), and osteocalcin (OCN)], leading to the transition to an osteoblast-like phenotype. However, the causes and mechanisms leading to the release of these particles are poorly understood (10,11).

A recent proteomic analysis of MVs released from rat valve interstitial cells revealed the expression of autophagic proteins, including lysosomal-associated membrane protein 1 (LAMP1), lysosomal-associated membrane protein 2 (LAMP2), and LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) (12). These findings suggest that MVs may overlap with the autophagy network, both during the autophagy vesicle formation phase, and during the release phase of VC. Recently, the emergence of a new field of secretory autophagy has led to the export of more complex cytoplasmic cargo, and the secretion of particulate substrates (13). In VC, various cellular compartments, such as endosomes, defective mitochondria, autophagic vesicles, and MVs, may interact. Calcium phosphate crystals or hydroxyapatite particles are internalized by the cell through endocytosis, forming endosomes that merge with autophagosomes, which are converted into autolysosomes. Hydroxyapatite in these autolysosomes can be broken down or directed toward MV-rich autolysosomes. These autolysosomes then fuse with the plasma membrane, facilitating the release of MVs into the extracellular matrix, where they contribute to the formation of hydroxyapatite. Additionally, mitochondria-derived vesicles, which can serve as a source of calcification, may be engulfed by autophagosomes or directly enter lysosomes. Within these compartments, they can be degraded or expelled from the cell through autolysosomal exocytosis (14,15).

Large-conductance calcium and voltage-activated potassium (BK) channels are key factors that regulate Ca²⁺ and the membrane potential in VSMCs, and play a role in controlling vascular tension and various pathophysiological conditions (16,17). BK is simultaneously affected by changes in several autocrine and paracrine factors, which in turn affect cardiovascular health, making BK the main target for treating cardiovascular diseases (18). Our previous studies revealed that BK channel agonists play important roles in bone metabolism and osteoblast differentiation, which is similar to the VC process (19,20). These results suggest that BK may be crucial to the VSMC calcification process. However, the role of BK in VC is still unclear. NS1619 is the only selective BK opener without any effect on other ion channels, such as ATP-sensitive potassium, voltage gated potassium, sodium, and calcium channels and safety in animal studies (21). Thus, we designed a study to clarify the regulatory effect of BK channel agonists on VC. Previous studies have shown that vitamin D3 induces calcification and mineralization in vivo (22,23). The mice were subcutaneously injected with Vitamin D to induce VC. We explored how BK channels inhibit the calcification process of primary VSMCs in vitro and in vivo. We present this article in accordance with the ARRIVE reporting checklist (available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-86/rc).

Methods

Cell culture and treatment

Primary VSMCs were isolated from the rat or murine thoracic aorta by rinsing blood from the aorta with phosphate buffered saline (PBS), cutting it into small pieces (after removing the endothelium and adventitia), and plating it in a cell culture flask with Dulbecco’s modified Eagle’s medium (DMEM; Cat# 11995115; Gibco, Waltham, MA, USA) containing 20% fetal bovine serum (FBS; Cat# 10091148; Gibco) at 37 ℃ in an incubator containing 95% air and 5% CO₂ for 7–14 days. VSMCs that migrated from explants were collected and maintained in growth DMEM containing 10% FBS. VSMCs between passages 3 and 7 were used in the experiments. They were divided into the following five groups: (I) the control group; (II) the control + NS1619 group; (III) the calcify group; and (IV) the calcify + NS1619 group; (V) the calcify + NS1619 + 3-methyladenine (3-MA) group. To establish the calcification model of primary VSMCs, VSMCs were spread in a six-well plate at a density of 1×10⁵ per well and cultured until the number of cells reached 60–70%. Normal medium (complete medium containing 2% serum) or calcified medium [complete medium containing 2% serum, 10 mM β-glycerophosphate (β-GP, Sigma, St. Louis, MO, USA) and 3 mM Ca²⁺] was added, and the mixture was cultured for 7 days. Fresh medium was changed at the same time every other day, and the status was observed. The murine VSMC cell line MOVAS-1 was purchased from Sixin Biotechnology Company (ATCC^® CRL-2797TM, Shanghai, China). To investigate the effect of the BK channel on VSMC calcification, the BK channel agonist NS1619 (20 µM) (Sigma) was added to the calcified cells, and autophagy inhibitor 3-MA (10 mmol/L) (Selleck, Houston, USA) was added to the calcified cells with NS1619. After treatment for a certain time, the cells were collected for subsequent studies. Each experiment was performed at least three times, unless stated otherwise.

Animals

Twelve male C57BL/6 mice (20–25 g) were supplied by the Animal Center at the School of Pharmacy, Fudan University (Shanghai, China). All animal experiments were performed under a project license (No. 2017-03-YL-ZXM-01) granted by the Animal Experimentation Ethics Committee of Fudan University, in compliance with the Animal Management Rule of the Ministry of Health, China (documentation No. 55, 2001) as well as the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) guidelines for the care and use of animals. The mice were housed in individual cages at 25 ℃ and 25% humidity on a 12/12-hour light/dark cycle and fed a standard powder diet. The minimal possible number of animals was sacrificed, and all efforts were made to reduce their suffering. Without exception, all mice survived until the end of the experiment, and showed no signs of illness such as weight loss, increased temperature, fur changes, or trembling. A protocol was prepared before the study without registration.

Animal model of VC

To induce VC, the 12 C57BL/6 mice were intraperitoneally injected with 8×10³ IU of vitamin D (Sigma-Aldrich) at a dose of 10 µL/g once a day for three consecutive days (23). The vitamin mixture was prepared for use and kept at low temperature without light. Castor oil was used for dissolution. The volume ratio of injection to castor oil was 9:1. An injection of physiological saline alone was used as a negative control. The mice were divided into the following four groups: (I) the control group; (II) the vitamin D model group; (III) the vitamin D + 5 mg/kg NS1619 group; and (IV) the vitamin D + 10 mg/kg NS1619 group (19). Each group comprised three mice. The four groups of mice were weighed evenly and randomly selected. Randomization sequence was created using Stata 9.0 (StataCorp, College Station, TX, USA) statistical software and was stratified by center with a 1:1 allocation. Random allocation sequence was not concealed until interventions were assigned. NS1619 was injected intraperitoneally for five consecutive days. On Day 21, the mice were sacrificed under isoflurane anesthesia, and the tissues were collected in liquid nitrogen for further study.

Drug preparation, treatment and data collection were conducted blindly and independently by three investigators. All study coordinators and researchers were blinded throughout the entire study. The study was unblinded after all statistical analyses were completed.

BK plasmid transfection

We used a BK plasmid (Beyotime Biotechnology, Shanghai, China) to construct BK-overexpressing VSMCs. VSMCs were transfected with vector and BK plasmid (2.5 µg/well) for 6 hours using Lipofectamine™ 3000 Transfection Reagent. They were divided into the following four groups: (I) the control group; (II) the model group; (III) the vector group; and (IV) the BK group. Cell culture medium was then replaced with fresh medium incubated for 2 days. At the end of incubation, various treatments were applied.

Quantitative polymerase chain reaction (qPCR)

Previous research has revealed that BK channel agonists play important roles in bone metabolism and osteoblastic transformation (20). To verify the correlation between the BK channel and VC, we first performed a comparative genomics analysis. The genomic sample data (GSE89130) were sourced from the Gene Expression Omnibus database.

Total RNA was extracted from the cells, and gene expression was assessed by qPCR as described by Wang et al. (24). Briefly, total RNA was extracted from the VSMCs using RNAiso Plus reagent (# 9109, TaKaRa, Tokyo, Japan) and reverse transcribed into cDNA using PrimeScript RT Master Mix (#RR036A, TaKaRa). qPCR was performed using 2 µL of the cDNA mixed with SYBR® Premix Ex Taq™ solution (#RR0420A, TaKaRa). Each reaction was performed in triplicate and analyzed on a BIO-RAD CFX Connect Real-time PCR system and GAPDH was used for normalization. The specific primers are as follows:
Runx2, 5'-CAAGCACAAGTGATTGGCCGAACT-3' (forward) and 5'-CTCAACCACGAAGCCTGCAATTT-3' (reverse);
BK, 5'-AGGAATGCATCTTGGCGTCACTC-3' (forward) and 5'-CCTCGAAGTGCATTCTCCTCAGC-3' (reverse);
Alpha-smooth muscle actin (α-SMA), 5'-GTCCCAGACATCAGGGAGTAA-3' (forward) and 5'-TCGGATACTTCAGCGTCAGGA -3' (reverse);
GAPDH, 5'-GGCACAGTCAAGGCTGAGAATG-3' (forward) and 5'-ATGGTGGTGAAGACGCCAGTA-3' (reverse).

Von Kossa staining

Von Kossa staining was used to analyze aortic calcification. A 1-cm segment of the aorta was fixed with 10% formalin (pH 7.4, 0.1 M) at room temperature for 24 hours. Aortic tissue was dehydrated with an ethanol gradient, embedded in paraffin, treated with 5% silver nitrate for 30 min at room temperature, exposed to ultraviolet light for 1.5–2 hours, and then washed three times with deionized water.

MV extraction and nanoparticle tracking analysis (NTA)

An exoEasy Maxi spin column was used to extract the MVs. The cell culture supernatant was collected, and the supernatant was filtered to remove particles larger than 0.8 µm. After mixing the buffer XBP with the same volume of the sample thoroughly, it was placed on the exoEasy spin column and centrifuged at 500 ×g for 1 min, after which the flow through was discarded. The mixture was poured into XWP buffer and centrifuged at 5,000 ×g for 5 min. XE buffer was added, and the mixture was incubated for 1 min. After centrifugation at 500 ×g for 5 min, the eluate was collected to obtain the MVs. An NTA was used to detect the size and concentration of the MVs. The Brownian motion of each particle was tracked and analyzed, and the hydrodynamic diameter and concentration of the nanoparticle were calculated using the Stokes-Einstein equation (25).

Western blot (WB) analysis

Immunoblotting was used to assess the protein content. Total protein was extracted from VSMCs using RIPA lysis buffer (Keygen Biotech, Shanghai, China). Each sample (20 µg) was electrophoresed on sodium dodecyl-sulfate-polyacrylamide gels. The prestained protein molecular weight markers (size range 10–180 kDa, Sigma, Poole, UK) were run on every gel in the first and last lane. The separated proteins were transferred onto polyvinylidene difluoride membranes (Cat# IPV00010; Millipore, USA). After blocking with 5% skim milk [prepared in 1× Tris-buffered saline Tween (TBST)] for 1 hour at room temperature, the membranes were incubated with primary antibodies against BK channels (1:1,000) (Alomone Laboratories Ltd, Jerusalem, Israel), Runx2 (1:1,000) (Santa Cruz, TX, USA), α-SMA (1:1,000) (Sigma), CD63 (1:1,000) (Abcam), CD9 (1:1,000) (Abcam, Cambridge, UK), p62 (1:1,000) (Abcam), LC3 (1:1,000) (Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000) (Santa Cruz) overnight at 4 ℃. After washing three times with 1× TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit immunoglobin G, 1:10,000) against the primary antibodies for more than 1.5 hours at room temperature. After washing again three times with 1× TBST, the protein content was analyzed by Image Lab (ImageLab, Haifa, Israel) and normalized to the GAPDH level. All the experiments were repeated at least three times.

Statistical analysis

All the data were analyzed using GraphPad Prism 8.4 software San Diego, CA, USA). The results are presented as the mean ± standard deviation. Differences among groups were analyzed by the Kruskal-Wallis non-parametric test. P<0.05 (*) and P<0.01 (**) were considered statistically significant.

Results

BK negatively regulates the VC process

The messenger RNA (mRNA) profile of the sample was compared with that of the normal control. The comparative genomics analysis revealed that VC was associated with the expression of BK. During VC, BK gene expression is significantly decreased (Figure 1A,1B).

Figure 1 Calcification downregulates BK channels in vivo and in vitro. (A) Hierarchical clustering heatmap showing decreased expression of BK gene in vascular calcification. (B) In the calcify group, the expression levels of the BK channel gene and the α-SMA and Runx2 genes changed (n=5). (C) Vitamin D-induced VC model in mice. VK staining of the mouse thoracic aorta (n=3) was used to detect the degree of remission of calcification caused by NS1619. (D,E) The expression of the BK channel and Runx2 was detected using WB to verify the effect of NS1619 on the primary VSMC calcification model (n=3). The data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. α-SMA, alpha-smooth muscle actin; VC, vascular calcification; VK, Von Kossa; VSMC, vascular smooth muscle cell; WB, Western blot.

Vascular staining of NS1619 significantly reversed VC of the mice (Figure 1C). Von Kossa staining of the thoracic aorta revealed that NS1619 had the most significant effect on relieving VC in the high-dose group. The WB results revealed that the Runx2 protein level increased in the calcification group, and NS1619 treatment reversed the change in the Runx2 protein (Figure 1D,1E). In summary, the BK protein level was significantly reduced in the calcified blood vessels, and the BK agonist NS1619 improved the calcification process.

BK agonist NS1619 inhibits MV secretion to relieve VC

MVs play central roles in the process of calcification; thus, we tested whether NS1619 regulates calcification by affecting the formation of MVs. The WB results revealed that the expression of the MV markers CD63 and CD9 was significantly increased in the calcification group, while the NS1619 treatment downregulated the expression of CD63 and CD9 (Figure 2A,2B). The increased expression of CD63 and CD9 suggested that the secretion of MVs may be increased.

Figure 2 NS1619 relieves calcification by affecting the secretion of MVs. (A,B) The expression of CD63 and CD9 was detected by WB blot to verify the effect of NS1619 on the secretion of MVs in the calcification model. (C) NTA was used to detect the specific particle size and corresponding concentration of MVs in each group, and the results were fitted with a curve diagram. The data are expressed as the mean ± standard deviation (n=3). *, P<0.05; **, P<0.01. MV, matrix vesicle; NTA, nanoparticle tracking analysis; WB, Western blot.

The NTA results revealed that the concentration of MV particles increased in the calcification group, while the mean size of MV particles decreased. Conversely, the concentration of MV particles significantly decreased and became greater following NS1619 treatment (Figure 2C). These results suggest that NS1619 inhibits the calcification of primary VSMCs and inhibits the secretion of MVs during calcification. We investigated the effect of BK channels on VC via the BK channel opener NS1619, taking advantage of its ability to increase BK channel activity. However, as Figure 2 shows, NS1619 increased the concentration of the BK protein. This phenomenon may be due to NS1619 stimulating cell proliferation and migration (26).

BK channels regulate MV secretion to affect VC

We verified that inhibiting MV release to improve VC is related to the BK channel through BK channel openers. Next, we used BK plasmids to confirm the role of the BK channel in altering MV secretion to improve VC. The overexpression of BK significantly reduced the upregulation of Runx2, and reversed the downregulation of α-SMA (Figure 3A,3B). Similarly, the expression of BK affected the secretion of MVs. Compared with calcification alone, BK overexpression significantly reversed the increase in CD63 and CD9 expression (Figure 3C,3D). Similarly, BK overexpression inhibited the secretion of MVs via calcification (Figure 3E). These results indicate that BK channels can inhibit the calcification of primary VSMCs, and can inhibit the formation and secretion of MVs.

Figure 3 BK channels regulate MV secretion to affect VC. (A,B) WB was used to detect the expression of the BK channel, Runx2, and α-SMA in the calcification model of the smooth muscle cell line MOVAS to verify the effect of the BK plasmid on calcification. (C,D) WB was used to detect the expression of CD63 and CD9 in the calcification model of the smooth muscle cell line MOVAS to verify the effect of the BK plasmid on the secretion of MVs. (E) NTA was used to detect the specific particle size and corresponding concentration of MVs in each group, and the results were fitted with a curve diagram. The data are expressed as the mean ± standard deviation (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. α-SMA, alpha-smooth muscle actin; MOVAS, mouse vascular smooth muscle cell line; MV, matrix vesicle; NTA, nanoparticle tracking analysis; VC, vascular calcification; WB, Western blot.

BK agonist NS1619 inhibits the secretion of MVs by promoting autophagy

Based on the possible novel mechanistic avenues related to secretory autophagy mentioned above, we then examined the correlation between the BK channel and autophagy. In both the control and calcification groups, after NS1619 treatment, the ratio of LC3II/I increased significantly, and the content of p62, a selective autophagy adaptor, decreased, suggesting the activation of autophagy (Figure 4A,4B). We hypothesized that the mechanism by which BK channels alleviate VC is that agonists inhibit the secretion of MVs by promoting autophagy (16). 3-MA is a generally recognized autophagy inhibitor. The WB results revealed that 3-MA treatment inhibited the significant downregulation of CD63 and CD9 expression induced by NS1619 (Figure 5A,5B). Moreover, the NTA results also revealed that the 3-MA treatment reversed the decrease in MV concentration caused by the NS1619 treatment (Figure 5C). In summary, in the case of calcification, the BK channel agonist NS1619 can inhibit the secretion of MVs by activating autophagy, thereby alleviating calcification.

Figure 4 NS1619 activates autophagy to ameliorate calcification. (A,B) Western blot was used to detect the expression of LC3II/I and p62, which verified the activation of autophagy by NS1619 via calcification. The data are expressed as the mean ± standard deviation (n=3). *, P<0.05; **, P<0.01.

Figure 5 NS1619 inhibits the secretion of MVs by activating autophagy. (A,B) WB was used to detect the expression of CD9 and CD63 to verify the effect of the autophagy inhibitor 3-MA on the activation of autophagy by NS1619. (C) NTA was used to detect the effect of the autophagy inhibitor 3-MA on the inhibition of MV secretion by NS1619. The data are expressed as the mean ± standard deviation (n=3). (D) BK channels affect matrix vesicle secretion and ameliorate vascular calcification via autophagy. Smooth muscle cells deposit calcium and phosphate, secreting MVs that further promote the deposition of calcium and phosphate. BK channels enhance the autophagy of matrix vesicles, leading to a decrease in secretion and subsequently alleviating calcification. *, P<0.05; **, P<0.01. 3-MA, 3-methyladenine; MV, matrix vesicle; NTA, nanoparticle tracking analysis; WB, Western blot.

Discussion

Our studies showed that BK channels play an important role in regulating VC, and BK channel agonists inhibit the calcification of VSMCs in vivo and in vitro. Our results also confirmed that BK channel agonist treatment and BK overexpression significantly inhibit the production and secretion of MVs, which is achieved by activating autophagy. The decreased secretion of MVs eventually alleviated the calcification of VSMCs (Figure 5D).

One of the main mechanisms of VC is the secretion of MVs caused by abnormal calcium and phosphorus metabolism, which form the initial crystal nucleus for the formation of calcified crystals (4,27). For a long time, research on the mechanism has been slow. High calcium levels are thought to cause an increase in intracellular calcium, leading to changes in the composition of MVs, such as changes in phospholipid components, which increase the release of MVs (28,29). Moreover, changes in phospholipid components play a more significant role than high phosphorus and high calcium contents in promoting MV release (10). Our study used the concentration and particle size of MVs, as well as the degree of calcification, as indicators. Our results revealed a close correlation between the two.

In recent years, several research groups have reported that the essence of MVs is Exos, which may be derived from intracellular multi-vesicle bodies rather than materials derived from cell membranes (30,31). MVs express the marker proteins of Exos (e.g., CD9, CD63, and Hsp70), which suggests that MVs play a bridging role in the communication between cells and the extracellular matrix (32). In addition, some factors related to exocytosis, such as tubulin and sortilin, also regulate the secretion of MVs from calcified VSMCs (33,34). Studies have shown that BK has a regulatory effect on both the classic exocytosis pathway and non-classical lysosomal exocytosis, and BK-mediated abnormal exocytosis is one of the pathogeneses of lysosomal storage diseases (35,36). In addition, Exos represent another form of non-classical exocytosis. There is no evidence as to whether the BK channel is regulated. In view of the potential connection between BK and Exos, and between Exos and MVs, we speculate that the BK channel may exert a regulatory effect on the production and secretion of MVs, and further affect VC. In this study, we discovered for the first time that a BK channel agonist can alleviate VC by affecting the secretion of MVs.

Our previous study showed activation of BK channels prevented diabetes-induced osteopenia by regulating mitochondrial Ca²⁺ and SLC25A5/ANT2-PINK1-PRKN-mediated mitophagy (19). This study was also the first to show that BK channel agonists regulate autophagy, which affects the secretion of MVs. Autophagy breaks down proteins and organelles through the lysosomal degradation pathway, which is recognized as a source of nutrients (37,38). This process is essential for cell survival, differentiation, and development (39,40). Two independent groups have provided new insights into the function of BK in non-excitatory cells, and designated BK as a lysosomal potassium channel (41,42), suggesting a potential connection between BK and autophagy. In non-excitatory cells, BK currents mostly exist in lysosomes, and are difficult to detect on the plasma membrane (42). In addition, lysosomes are important for BK function. The lysosomal membrane potential seems to be positive, and is equipped with the voltage-gated sodium channel TPC1. Under certain conditions, the activation of TPC1 can depolarize the lysosomal membrane, which is beneficial for lysosomal BK activation (43,44).

More importantly, lysosomes store calcium in cells. Due to its calcium sensitivity and single-channel conductivity, BK is considered an ideal sensor and reactor for lysosomal calcium release. Studies have confirmed that lysosomal BK can significantly change the lysosomal potential and affect lysosomal calcium signal transduction (41,42). Cao et al. conjectured that there was positive feedback between TRPML1 and BK in the lysosome. The calcium ions released from TRPML1 activate BK, and the subsequent K⁺ flux provides counter-positive ions, ensuring that sufficient calcium ions are released through TRPML1 (41). Some research suggests that lysosomal BK deficiency can cause lysosomal dysfunction (42,45). The function of the BK channel in the lysosome has yet to be determined; however, it is undoubtedly important and necessary for the lysosome and is a potential target for lysosomal diseases. Since the lysosome is the endpoint of the autophagy process, the BK channel is most likely to affect autophagy. As Figures 4,5 show, the generation of autophagic vesicles and the content of LC3II in the NS1619 and BK overexpression groups significantly increased, suggesting a significant increase in autophagy. This finding is consistent with our speculation. Future studies are warranted to further confirm the causality of BK-autophagy-MVs axis.

This study had certain limitations. First, all the experiments were conducted using the same samples. Second, the current data set is limited to mRNA and protein expression in mouse models due to the small sample quantity. BK probably affects VC by potential alternative mechanisms due to the lack of knockdown or antagonist experiments; BK channels probably interact with other known calcification pathways. Thus, it is important to determine how BK affects VC in human tissue in future studies. Additionally, although off-target effects are an unavoidable reality, further studies need to be conducted to develop VSMC-targeted agents for activating BK channels to treat VC.

Conclusions

This study was based on the scientific hypothesis that “BK may inhibit MV secretion which is potentially achieved by activating autophagy, thereby affecting VC”. This study found: (I) the difference in the expression of BK in VC; (II) the effect of the BK channel agonist NS1619 on VC; and (III) the inhibitory effect of NS1619 on the secretion of MVs and its specific mechanism. These findings enrich the understanding of the mechanism of VC.

Acknowledgments

We thank Dr. Adrianna Othmani (John Paul II Hospital, Institute of Cardiology, Collegium Medicum of the Jagiellonian University in Kraków, Poland) for the critical comments and valuable advice on this study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-86/rc

Data Sharing Statement: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-86/dss

Peer Review File: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-86/prf

Funding: This work was supported by grants from the Shanghai Municipal Health Commission (No. 202040105 to Q.Y.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-86/coif). Q.Y. reports a grant from the Shanghai Municipal Health Commission (No. 202040105). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All experiments were performed under a project license (No. 2017-03-YL-ZXM-01) granted by the Animal Experimentation Ethics Committee of Fudan University, in compliance with the Animal Management Rule of the Ministry of Health, China (documentation No. 55, 2001) as well as the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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