Novel PMVs/ZIP4/Zinc/Prelamin A Axis Promotes Nuclear Dysmorphism and Vascular Aging in Humans and Rodents Post‐Injury: Effective Treatment With Platelet Membrane‐Coated ZIF‐8 Nanoparticles
Tengzhi Ma, Han Bao, Zhijue Xu, He Ren, Wenhao Tian, Jiahe Chen, Zhongqian Liu, Xinwu Lu, Fan Lv, Qingping Yao, Yingxin Qi, Kai Huang
TL;DR
This study shows that vascular injury causes nuclear shape changes and aging, which can be treated with special nanoparticles.
Contribution
The discovery of a new PMVs/ZIP4/Zinc/prelamin A pathway causing vascular aging and a novel nanoparticle treatment.
Findings
PMVs reduce zinc levels, impairing prelamin A processing and causing nuclear dysmorphism.
ZIP4 deficiency contributes to zinc depletion and vascular aging.
Platelet membrane-coated Zn-MOF nanoparticles effectively treat nuclear dysmorphism and aging.
Abstract
Interventional therapy and surgery play important roles in the treatment of various diseases, but they cause varying degrees of vascular injury. Currently, the side effects are often overlooked. Here, we observed abnormal nuclear morphology (nuclear dysmorphism) and vascular aging in injured human and rodent arteries. Platelet‐derived microvesicles (PMVs) adhere to injured blood vessels, leading to nuclear dysmorphism and cell senescence in vascular smooth muscle cells (VSMCs). This occurs because PMV adherence reduces intracellular Zn2+ levels, which impairs Zn2+‐dependent processing of prelamin A by the enzyme ZMPSTE24. Consequently, prelamin A accumulates in VSMCs, contributing to the observed nuclear dysmorphism and cell senescence. RNA sequencing and loss‐of‐function assays revealed that Zinc transporter solute carrier family 39 member 4 (SLC39A4, also called ZIP4) deficiency…
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FIGURE 8- —National Natural Science Foundation of China10.13039/501100001809
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Taxonomy
TopicsExtracellular vesicles in disease · Cell Adhesion Molecules Research · Protease and Inhibitor Mechanisms
Introduction
1
Clinical surgery and interventional therapy are both commonly used medical treatments to address a variety of health conditions. Interventional therapy typically involves minimally invasive procedures to diagnose and treat a wide range of conditions, including heart disease (Calabro et al. 2021), vascular disorders (Davidson et al. 2022), and cancer (Li et al. 2020). Clinical surgery is frequently used to treat conditions that cannot be addressed with interventional therapies alone, such as tumor resection (Eom et al. 2024) and organ transplantation (Guo et al. 2023). However, both interventional therapy and clinical surgery can cause varying degrees of vascular damage to patients. These pathological vascular changes are often overlooked.
In eukaryotic cells, the nucleus serves as both the repository of genetic information and the control center for the genome. Although physiological nuclear morphology is roughly conservative with a round or oval shape, it can be modified by various signals and stimuli including biochemical inputs and mechanical forces (Heckenbach et al. 2022; Kalukula et al. 2022; Shah et al. 2021). Based on super‐resolution microscopy, live cell imaging, and high‐throughput sequencing, it has been demonstrated that nuclear dysmorphisms, such as wrinkling, irregularities, blebs, and invaginations, affect chromatin organization, genomic instability, and cell signaling involved in transcriptional regulation, thereby determining cell fate (Heckenbach et al. 2022; Navarro‐Lerida et al. 2015; Papathanasiou et al. 2023; Perez‐Hernandez et al. 2022). Clinically, nuclear dysmorphism has been used as a hallmark of various tumors to determine tumor grade and predict patient prognosis (Mi et al. 2021; Schmitz et al. 2021; Zink et al. 2004). Very recently, nuclear area and volume have been shown to be effective in identifying cardiomyocyte ploidy (Yao et al. 2024). However, the role of nuclear dysmorphism in vascular aging and the intrinsic molecular mechanism are still unclear in injured arteries.
The nuclear morphology and architecture are orchestrated by proteinaceous nuclear lamins, a class of V‐type intermediate filament proteins that underlie the inner nuclear membrane (Ho et al. 2013; Turgay et al. 2017). Lamins perform diverse biological functions, including maintaining nuclear structure, stabilizing and organizing chromatin, regulating the cell cycle, modulating spatiotemporal gene expression and DNA repair, and mediating mechanotransduction (Dittmer and Misteli 2011; Evangelisti et al. 2020). Besides, the expression, modification and maturation of Lamins contribute to the stability and function of the Lamin networks (Karoutas et al. 2019; Kochin et al. 2014). Accumulation of the immature Lamin A precursor, prelamin A, has been reported to occur during vascular aging and to promote VSMCs senescence and dysfunction (Ho et al. 2025; Liu et al. 2013; Revechon et al. 2025). However, the upstream mechanisms that lead to prelamin A accumulation in vascular cells remain unclear. Our previous work showed that nuclear A‐type Lamins, especially Lamin A, are significantly repressed in both endothelial cells (ECs) and VSMCs which subsequently induce pathological vascular remodeling via binding to specific DNA motifs (Bao et al. 2020; Han et al. 2015; Qi et al. 2016). In summary, lamins contribute to nuclear architecture and genome integrity (Karoutas et al. 2019; Schreiber and Kennedy 2013). However, the roles of Lamins in vascular aging as well as the underlying mechanisms after injury need to be elucidated.
Herein, we validated that pMVs/ZIP4/zinc/prelamin A served as a novel signaling pathway accounting for nuclear dysmorphism and vascular aging. Zinc supplementation may effectively prevent injury‐induced nuclear dysmorphism and vascular aging.
Materials and Methods
2
Human Injured Arteries
2.1
Informed consent was obtained from all patients participating in this study, and the investigation conformed to the principles outlined in the Declaration of Helsinki. The study involving human femoral artery and posterior tibial artery samples was approved by the Ethics Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine (approval ID: SH9H‐2021‐TK32‐1).
Eighteen clinical arteries (Table S1) samples were collected from patients who underwent percutaneous transluminal angioplasty (PTA) and subsequent amputation for reasons unrelated to arterial injury, as well as from control subjects who underwent amputation due to distal trauma. The inclusion criteria for the clinical samples were as follows: age over 18 years, absence of manifest cardiovascular diseases, absence of diabetes, and nonpregnant status.
Elastic Van Gieson Staining
2.2
Elastic Van Gieson (Verhoeff's Van Gieson, EVG, VVG) staining was performed as we previously published (Huang, Narumi, et al. 2021; Huang et al. 2022). Sections were deparaffinized by sequential washes in xylene (2×), descending ethanol from 100%, 90%, 75%, 50%, and distilled water. Sections were stained in Verhoeff's solution for 70 min, followed by differentiation in 2% ferric chloride for 90 s. Then, incubated with 5% sodium thiosulfate for 60 s, followed by counterstaining with Van Gieson's solution. Then, sections were subjected to dehydration with 95% and 100% alcohol, washed in xylene. Images were captured using an Olympus IX71 microscope.
Nuclear Morphology Measurements
2.3
DAPI‐stained nuclei in vascular cross‐sectional or VSMC images were imported into CellProfiler software (v4.2.5) (Carpenter et al. 2006) for automated nuclei identification. The typical object diameter was set between 5 ‐ 100 pixels (1 pixel = 0.236 μm) to exclude small debris and large non‐nuclear structures. Objects falling outside this range were excluded from subsequent analysis. Nuclei touching the image borders were discarded to avoid including incomplete nuclei in the analysis, ensuring accurate measurement of fully contained nuclei. To separate nuclei from the background under varying illumination and staining intensity, an adaptive thresholding strategy was applied using the Minimum Cross‐Entropy method. A smoothing scale of 0.1 and a threshold correction factor of 0.5 were applied, with an allowable threshold range set between 0.5 and 1.0. An adaptive 20‐pixel window was used to account for local intensity variations, and a logarithmic transformation was applied before thresholding to enhance contrast in dim regions.
To resolve overlapping or clumped nuclei, shape‐based declumping was employed, using a shape method to draw dividing lines between clumped objects. The size of the smoothing filter for local maxima detection was automatically calculated based on the spatial distribution of intensity peaks within the nuclei clusters. Local maxima suppression was performed to remove local maxima that were closer than 50 pixels. Additional parameters included automatic calculation of the minimum allowed distance between local maxima, ensuring proper segmentation of clustered nuclei. The resolution of images was adjusted to optimize the detection of local maxima, especially in low‐resolution images. The method used a smoothing filter size of 1 for declumping and 20 pixels for the adaptive window size, with further suppression of closely positioned local maxima (less than 50 pixels apart). This combination of settings enhanced the accuracy and precision of nuclei identification. Final segmentation results were manually verified to ensure accurate identification and measurement of individual nuclei. The final data was outputted for further morphological analysis, including nuclear size, shape, and intensity measurements.
Compactness, Eccentricity, Formfactor, Solidity and Zernike shape features (Figure S2A) were determined using the “Measure Object size Shape” and “MeasureTexture” modules.
Compactness describes the roundness of the cell nucleus, with values greater than 1; Formfactor is also used to describe the roundness of the cell nucleus while the value is less than 1; Eccentricity is the ratio of the distance between the foci of the ellipse and its major axis length with the value between 0 and 1; Solidity represents the proportion of the pixels in the convex hull that are also in the object, with the value less than 1.
TEM Analysis
2.4
For conventional chemical fixation, the procedure began with primary fixation using 2.5% glutaraldehyde in a 0.1 M PBS; vascular tissues should be excised rapidly and trimmed into blocks, while cell samples were fixed as a pellet. Following three 15‐min washes in PBS to remove excess aldehydes, the specimens undergo secondary fixation (post‐fixation) in 1% osmium tetroxide (OsO_4_) for 1–2 h to stabilize lipids and enhance ultrastructural contrast. The samples are then subjected to graded dehydration through a series of ethanol concentrations (30%–100%) and optionally transitioned into acetone. Finally, the dehydrated specimens are processed through resin infiltration using increasing ratios of resin to solvent (Epon 812), embedded in molds, and placed in a 60°C oven for 48 h to achieve full polymerization for ultramicrotomy.
For high‐pressure freezing‐based TEM preparation, VSMCs were cryo‐fixed using a Leica EM ICE high‐pressure freezer (Leica Microsystems, Germany), ensuring millisecond vitrification and optimal preservation of ultrastructural integrity. VSMCs were harvested, filtered to form a paste, and cryoimmobilized by high‐pressure freezing. The samples were stored in liquid nitrogen until freeze substitution. The freeze substitution process was conducted to facilitate the dehydration and fixation of high‐pressure frozen specimens. The protocol was initiated with a primary incubation at −90°C for 48 h, a duration that may be optimized (8–72 h) based on the specific dimensions and nature of the biological tissue. Following this, a controlled warming ramp was implemented: the temperature was raised to −60°C over 6 h and maintained for 24 h, then further increased to −30°C and −20°C, with holding periods of 8 h at each stage to ensure stable fixative stabilization. The samples were subsequently transitioned to 4°C over a 2 h period. To eliminate residual fixatives such as OsO_4_, the specimens underwent three successive 15‐min washes using precooled pure acetone. Finally, the system was equilibrated to room temperature over 15 min, transitioning the samples for subsequent resin infiltration and embedding. Ultrathin sections (~70 nm) were cut using an EM UC7 ultramicrotome (Leica Microsystems, Germany) equipped with a diamond knife and collected on copper grids. Sections were sequentially contrasted with 3% uranyl acetate and 2.7% lead citrate to enhance electron density. Ultrastructural imaging was performed using a Tecnai G2 Spirit BioTwin transmission electron microscope (Thermo Fisher Scientific, USA) operated at an accelerating voltage of 120 kV. Images were acquired at low and high magnifications to evaluate nuclear morphology and chromatin organization.
Rat Carotid Artery Intimal Injury Model
2.5
The animal care and experimental protocols were conducted following Animal Management Rules of China (55, 2001, Ministry of Health, China), and the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The study was approved by the Animal Research Committee of Shanghai Jiao Tong University.
Male Sprague–Dawley (SD) rats (8–10 weeks old, 320 ± 20 g) were anesthetized by 2% isoflurane at 2 L/min oxygen flow using an isoflurane vaporizer (MATRX VIP 3000, USA) (Huang et al. 2017). The left carotid arteries were exposed, and a percutaneous transluminal angioplasty balloon dilatation catheter (2 F, 0.67 mm, Edwards Lifesciences, USA) was used to establish vascular intimal injury (Guo et al. 2015). The arteries were harvested after 1, 3, 7, and 14 days post‐injury, respectively. The uninjured right carotid artery served as the self‐control. In all experiments, rats were euthanized by CO_2_/O_2_ inhalation.
RNA Sequencing
2.6
Total RNA was extracted using the TRIzol reagent according to the manufacturer's protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using TruSeq Stranded Total RNA with Ribo‐Zero Gold (illumina, Cat. No. RS‐122‐2301).
The RNA libraries were then sequenced on an Illumina HiSeq X Ten platform, and 150 bp paired‐end reads were generated. Sequencing reads were mapped to the human genome (GRCh38) using HISAT2. FPKM of each gene was calculated using Cufflinks, and the read counts of each gene were obtained by HTSeq‐count. Differential expression analysis was performed using the DESeq (2012) R package. p value < 0.05 was set as the threshold for significantly differential expression.
β‐Galactosidase Assay
2.7
For frozen sections of artery samples, rewarming was carried out and washed with PBS. The sections were treated with 4% paraformaldehyde for 30 min and stained overnight at 37°C in the dark using a senescence β‐galactosidase staining kit (Beyotime, China).
VSMCs were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, washed with PBS three times, and stained overnight at 37°C in the dark using a senescence β‐galactosidase staining kit. The optical density (OD) of SA‐β‐gal staining was measured after background subtraction. The mean OD from multiple fields per sample was calculated and expressed as arbitrary units (AU).
Immunofluorescence Staining
2.8
Paraffin sections were deparaffinized, permeabilized with 0.3% Triton X‐100 for 30 min, and washed three times with PBS. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) and boiling by microwave for 15 min. After cooling at room temperature, the sections were washed by PBS and immersed in 10% goat serum for 30 min at room temperature to block nonspecific binding. Subsequently, the sections were incubated with the primary antibodies against CD41 (1:100, Abcam, Cambridge, UK), Lamin A (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), prelamin A (1:200, Sigma‐Aldrich, USA), Tri‐Methyl Histone 3 lysine 9 (H3K9me3) (1:200, Abcam, USA), Acetyl‐Histone H3 lysine 9 (H3K9ac) (1:200, Cell Signaling Technology, USA), p16 (1:500, Proteintech, China), p21 (1:500, Proteintech, China), and phosphorylated p53 (P‐p53; 1:1000, Cell Signaling Technology, USA) at 4°C overnight. After incubating with a secondary antibody (1:1000. Abcam, England) for 2 h, DAPI was used for nuclei staining for 15 min at room temperature. For cellular staining, paraformaldehyde was used to fix VSMCs for 15 min, permeabilized with 0.3% Triton X‐100 for 15 min, and then staining was performed as described above.
Imaging was performed using a confocal laser scanning microscope (Olympus, Japan, Confocal‐FV1000). For tissue sections and cultured cells utilized in nuclear morphology analysis, Z‐series (XYZ scanning) were conducted and analyzed using ImageJ software. DAPI‐stained nuclei in vascular cross‐sectional or VSMC images were imported into CellProfiler software (v4.2.5) 28 for automated nuclei identification. The typical object diameter was set between 5 and 100 pixels (1 pixel = 0.236 μm) to exclude small debris and large non‐nuclear structures. Objects outside this range were automatically discarded. Nuclei touching the image borders were discarded to avoid including incomplete nuclei in the analysis, ensuring accurate measurement of fully contained nuclei. To separate nuclei from the background under varying illumination and staining intensity, an adaptive thresholding strategy was applied using the Minimum Cross‐Entropy method. A smoothing scale of 0.1 and a threshold correction factor of 0.5 were applied, with an allowable threshold range set between 0.5 and 1.0. An adaptive 20‐pixel window was used to account for local intensity variations, and a logarithmic transformation was applied before thresholding to enhance contrast in dim regions. To resolve overlapping or clumped nuclei, shape‐based declumping was employed, using a shape method to draw dividing lines between clumped objects. The size of the smoothing filter for local maxima detection was automatically calculated based on the spatial distribution of intensity peaks within the nuclei clusters. Local maxima suppression was performed to remove local maxima that were closer than 50 pixels. Additional parameters included automatic calculation of the minimum allowed distance between local maxima, ensuring proper segmentation of clustered nuclei. The resolution of images was adjusted to optimize the detection of local maxima, especially in low‐resolution images. The method used a smoothing filter size of 1 for declumping and 20 pixels for the adaptive window size, with further suppression of closely positioned local maxima (less than 50 pixels apart). This combination of settings enhanced the accuracy and precision of nuclei identification. Final segmentation results were manually verified to ensure accurate identification and measurement of individual nuclei. The final data was outputted for further morphological analysis, including nuclear size, shape, and intensity measurements.
Quantification of the fluorescence intensity was performed using ImageJ. The mean fluorescence intensity was measured after background subtraction. Data are expressed as arbitrary units (AU). Analyses were performed in a blinded manner.
Western Blotting
2.9
Five milligrams frozen tissue was homogenized in 50 μL RIPA lysis buffer. VSMCs were gently washed with cold PBS and lysed at 4°C for 5 min with RIPA lysis buffer. The lysis buffer was centrifuged at 10,000 g for 10 min at 4°C, and the protein concentrations were assayed using a BCA kit.
Lysates were subjected to electrophoretic separation with 10% SDS‐PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Western blot analysis was performed using antibodies directed against Lamin A/C (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), Lamin A (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), Lamin C (1:500, Abcam, Cambridge, UK), Lamin B1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), Lamin B2 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), prelamin A (1:500, Sigma‐Aldrich, USA), γ‐H2AX (1:500, Abcam, Cambridge, UK), and GAPDH (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA), respectively. After incubation with HRP‐conjugated secondary antibodies (1:2000, Proteintech Group, China), the bands were detected via enhanced chemiluminescence (ECL), and densitometric analysis was performed using Quantity One software.
Platelet Collection and PMVs Treatment
2.10
Whole blood from the heart of anesthetized rats was collected into syringes with 100 μL/mL anticoagulant (2.94% sodium citrate, 136 mM glucose [pH = 6.4]), and transferred to 5 mL 0.9% NaCl containing 5 mM EDTA, 0.1 g/mL PGE1, and 1 U/mL apyrase. Then, platelet‐rich plasma was obtained by centrifugation at 300 g for 15 min. As previously described (Bao et al. 2018, 2017; Ren et al. 2024), platelets were prepared from platelet‐rich plasma by centrifugation at 2100 g for 10 min and resuspended with modified Tyrode solution (12 mM NaHCO_3_, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 2 mM MgCl_2_, 0.42 mM NaH_2_PO_4_, and 10 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid [pH 7.4]).
Platelets were activated with two methods. For the Transwell assay, a total of 2 × 10^5^ VSMCs were seeded in six‐well plates. Transwell inserts (1.0 μm pore inserts, Corning, USA) were incubated with 1 mL collagen I and put into the six‐well plates. The platelets were then added to the upper chamber for activation at 37°C for 24 h. The pMVs that derived from platelets migrated to the lower chamber to stimulate the VSMCs. For pMVs direct treatment assay, collagen I (1 μL/mL) was added into platelets, incubated in a 37°C water bath for 1 h, and centrifuged at 20,500 g for 90 min to obtain pMVs. The collected pMVs were suspended in DMEM for further VSMC treatment in a concentration of 10^9^/mL or suspended in normal saline for further testing (Bao et al. 2021).
Identification of PMVs
2.11
Nanoparticle tracking analysis (NanoSight NS300, UK) was used to analyze the number and diameter of pMVs. For Transmission Electron Microscope (TEM, Talos L120C G2, Thermo Fisher Scientific, Waltham, MA, USA), the microvesicle suspension was dropped on the hydrophilic‐treated TEM grid, dried, washed three times with double‐distilled water, and stained with phosphotungstic acid for 1 min.
Flow cytometry (Cytoflex; Beckman Coutler Co.Ltd., Indianapolis, IN, USA) was further used to identify the collected microvesicles. CD41 (1: 100; Abcam, Cambridge, UK) and CD45 (1: 100; Abcam, Cambridge, UK) were used as markers of platelets and leukocyte respectively.
Cell Culture
2.12
Primary VSMCs were harvested from the thoracic aortas of male SD rats as we previously described (Bao et al. 2020; Qi et al. 2016; Ren et al. 2024). The media of the aorta was isolated surgically and minced into small pieces, which were plated onto 25‐cm^2^ culture flasks for culture in DMEM (Gibco, USA) containing 10% heat‐inactivated FBS (Gibco, Australia), 100 U/mL penicillin, and 100 μg/mL streptomycin, and were incubated at 37°C in a humidified incubator (95% air and 5% CO_2_). The VSMC monolayers were passaged every 3–4 days after trypsinization, and cells in passages 4–7 were used for experiments. Different rat‐derived VSMCs were used for each independent experiment.
Zmpste24 Knockout Mice
2.13
The Zmpste24 ^ +/− ^ mice (Cat. NM‐KO‐18011) were obtained from the Shanghai Model Organisms Center (Shanghai, China), Zmpste24 ^ −/− ^ mice were generated by crossing heterozygous animals, and Zmpste24 ^ +/+ ^ littermates were used as wild type controls. For mouse genotyping, genomic DNA was extracted from tails and subsequently detected with polymerase chain reaction (PCR) (Figure S1). The PCR primer sequences are shown in Table S2.
Prelamin A Protein Mass Spectrometry Identification
2.14
Mice arteries were collected, and the total proteins were subsequently separated using a 10% polyacrylamide gel. Bands with predicted location in the gel were cut and performed in‐gel trypsin digestion. Peptides were then analyzed using liquid chromatography–tandem mass spectrometry (LC‐MS/MS) on a Q‐Exactive Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher). Peptides (LLGNSSPRTQSPQNC) were identified from the MS data using SEQUEST (RRID:SCR_014594).
Zinc Measurements
2.15
Inductively coupled plasma mass spectrometry (ICP‐MS) was used to detect zinc in arteries. The arteries were washed in ultra‐pure water and dried at 60°C to constant weight. HNO_3_ was then added and microwave digestion was carried out. Digested samples were measured using a 7700× ICP‐MS device (Agilent Technologies) equipped with an ASX‐520 autosampler (Agilent Technologies). For each element analyzed, a five‐point calibration curve, including a blank. The quantity was normalized to μg/g by dividing by the corresponding carotid arteries weight.
VSMCs were incubated for 1 h in PBS containing 1 μM FluoZin‐3 AM (Molecular Probes; Invitrogen, Camarillo, USA) and then washed with PBS. Fluorescence intensity (494 nm excitation/516 nm emission) was measured using a microplate reader (SpectraMax; Molecular Devices).
Whole Genome Bisulfite Sequencing (WGBS)
2.16
Genomic DNA was extracted from rat injured carotid arteries and the respective controls. Then it was purified using the Qiagen DNeasy Kit and sample quality was determined by NanoDrop. DNA libraries were prepared by following the Accel‐NGS Methyl‐Seq DNA Library kit, and the fragmented DNA (350 bp) was purified and converted by bisulfite. Then the bisulfite‐converted DNA was denatured and ligated with an adaptor. After extension of the primer annealed to the first adapter, a dsDNA adapter was ligated to the other end of the copied DNA, and the products were performed indexing PCR to create the final DNA libraries. The libraries were quality‐controlled using a Bioanalyzer 2200 (Agilent, Santa Clara, CA) and sequenced by HiSeq X (Illumina, San Diego, CA) on a 150 bp paired‐end run.
Raw sequence reads were quality‐trimmed using TrimGalore. Then the clean data were aligned to rat genome (Rnor6, NCBI) using Bismark. Differences in methylation between two groups were measured using a logistic regression model built in methylKit, and the significantly differentially methylated regions (DMRs) were identified with at least 25% difference in methylation levels and a q value less than 0.01.
Gene Set Enrichment Analysis
2.17
Gene set enrichment analysis (GSEA) was performed to assess the functional enrichment of transcriptome data using GSEA version 4.1.0 software (http://software.broadinstitute.org/gsea/index.jsp). FDR < 0.05 was used as a cutoff.
Ingenuity Pathway Analysis (IPA)
2.18
The possible biological processes and functional classifications were obtained using IPA software (Qiagen) (https://www.qiagenbioinformatics.com/products/ingenuity‐pathway‐analysis content). IPA integrated the available knowledge on genes, drugs, chemicals, protein families, processes, and pathways based on the interactions and functions derived from the Ingenuity Pathways Knowledge Database Literature. IPA was used to understand the complex biological and chemical systems at the core of life science research based on lectures or predictive analysis.
Mice Carotid Artery Intimal Injury Model
2.19
Male C57BL6J mice (8–10 weeks old, 20 ± 2 g) were anesthetized by 1.5% isoflurane at 1.5 L/min oxygen flow using an isoflurane vaporizer (MATRX VIP 3000, USA). The left carotid artery was carefully dissected under a dissecting microscope. A catheter (0.38 mm in diameter) was introduced into the arterial lumen towards the aortic arch and withdrawn five times with a rotating motion (Fei et al. 2016). Following the catheter's extraction, the perforated site was closed off, and blood flow was restored in the common carotid artery. The arteries were harvested 14 days post‐injury. The uninjured right carotid artery served as the self‐control. In all experiments, mice were euthanized by an overdose of isoflurane inhalation (5%) followed by cervical dislocation.
ZMPSTE24 Activity Test
2.20
ZMPSTE24 activity was measured using a fluorogenic FRET substrate peptide (Abz‐MQPSTATAAPK(DNP)EKTSSEKKDNYIIKGVFWDPAC(Fr)‐OMe). Crude membrane proteins were isolated from mouse aortas by mechanical homogenization and lysis in buffer containing 100 mM NaCl, 6 mM MgCl_2_, 10 mM Tris‐HCl (pH 7.5), 1% Triton X‐100, 0.1% SDS, and protease inhibitors. After centrifugation at 500 × g for 10 min to remove debris, membrane fractions were collected by ultracentrifugation at 100,000 × g for 1 h, resuspended in 10 mM Tris–HCl, and quantified by BCA assay. For the enzymatic assay, 80 μL of membrane protein extract was incubated with 20 μL of FRET substrate (final concentration 30 μM) in 96‐well plates. Reactions were carried out at 37°C for 30 min, after which fluorescence was measured at 37°C using excitation/emission wavelengths of 320/420 nm.
High Zinc Diet
2.21
All mice (8–10 weeks old, 20 ± 2 g) were fed with a normal diet (30 ppM zinc ions, Dyets Inc., Bethlehem, PA, USA). After intimal injury, the high‐zinc content diet (150 ppM zinc, Dyets Inc., Bethlehem, PA, USA) was supplied. Carotid arteries were harvested 14 days post‐injury. The control group was continually fed with the normal diet.
Platelet‐Membrane Cloaked Zn‐MOF Nanoparticles
2.22
Zinc nitrate hexahydrate (Sigma‐Aldrich, final concentration 1 mg/mL) and 2‐methylimidazole (Sigma‐Aldrich, final concentrations 20 mg/mL) were vigorously mixed for 30 s and allowed to stand undisturbed for 3 h to obtain Zn‐MOF nanoparticles (Hu et al. 2015; Zhuang et al. 2020). TEM was used to detect the morphology of Zn‐MOF. To obtain the platelet membrane, the material inside the platelet was emptied through repeated freeze–thaw cycles and low‐speed (1000 g) centrifugation. The purified platelet membrane and Zn‐MOF nanoparticles were then exposed to ultrasound (42 kHz, 2 min) to obtain the platelet‐membrane‐coated Zn‐MOF nanoparticles.
Statistical Analysis
2.23
Statistical analysis was performed and figures were created by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). All values were expressed as the mean ± SD. Prior to analysis, the normality of the data was evaluated using the Shapiro–Wilk test. Data were considered normally distributed if the p value exceeded 0.05, in which case parametric tests were applied. For non‐normally distributed data, nonparametric tests were utilized. Comparisons between two groups: For paired experimental groups, a paired t‐test was employed. For unpaired experimental groups, an F‐test was first performed to assess variance equality. If variances were equal, an unpaired t‐test was applied; if variances were unequal, an unpaired t‐test with Welch's correction was used. For multigroup comparisons: For paired experimental groups, repeated measures one‐way ANOVA (RM one‐way ANOVA) was conducted, followed by Tukey's post hoc test for specific group comparisons. For unpaired experimental groups, the Brown‐Forsythe test was used to evaluate variance equality. If variances were equal, ordinary one‐way ANOVA was performed, followed by Tukey's multiple comparisons test. For unequal variances, Brown‐Forsythe and Welch ANOVA tests were applied, followed by Dunnett's T3 multiple comparisons test. Correlation Analysis: The normality of the data was first assessed using the Shapiro–Wilk test. For normally distributed data, Pearson's correlation coefficient (r) was used to evaluate the strength of associations. For non‐normally distributed data, Spearman's rank correlation coefficient (ρ) was applied. Non‐Parametric Tests: For comparisons between two groups, the Mann–Whitney test was employed. For comparisons among three or more groups, the Kruskal–Wallis test was used, followed by Dunn's post hoc test to correct for multiple comparisons. p < 0.05 was regarded as statistically significant. *, **, and *** indicated p values < 0.05, 0.01, and 0.001, respectively.
Results
3
Vascular Injury Promotes VSMC Nuclear Dysmorphism
3.1
Here, we observed abnormal nuclear morphology (nuclear dysmorphism) in injured human and rodent arteries and elucidated the molecular mechanism. Femoral arteries were collected from patients who underwent percutaneous transluminal angioplasty (PTA) and subsequent amputations unrelated to the PTA procedure (Figure 1A). Vascular remodeling including neointima formation and breakdown of elastic fibers was detected in the injured arteries by EVG (Figure 1B) staining. Following DAPI staining, a CellProfiler pipeline was used to identify and analyze nuclear morphology (Figure 1C). DAPI staining revealed that the nuclei of VSMCs in normal human femoral arteries were cigar‐shaped, while the nuclei of VSMCs in injured arteries were smaller with blebs and invaginations, indicating nuclear dysmorphism. CellProfiler software (v4.2.5) (Carpenter et al. 2006) was used to measure nuclear morphology (Figure 1D), the parameters included Compactness, Eccentricity, Formfactor, and Solidity (please refer to Figure S2A for a detailed explanation of these parameters). Briefly, the Compactness, Eccentricity, and Formfactor mainly reflected the nuclear roundness, while the Solidity presented the blebs and invaginations in the nuclear boundary. Quantitative analysis results demonstrated that the Compactness value was substantially decreased from 2.80 ± 0.53 in the nuclei of normal human femoral arterial VSMCs to 1.62 ± 0.25 in the nuclei of injured arterial VSMCs (Figure 1F); Eccentricity value was effectively reduced from 0.96 ± 0.03 to 0.85 ± 0.05 (Figure 1G); Formfactor value was robustly increased from 0.46 ± 0.06 to 0.73 ± 0.09 (Figure 1H); and Solidity value was significantly dropped from 0.87 ± 0.02 to 0.81 ± 0.07 (Figure 1I). Of note, this reduction in Solidity was consistently observed in VSMCs located in both the media and intima of injured vascular (Figure S2E).
*Vascular injury promotes VSMC nuclear dysmorphism. (A) Injured femoral arteries were harvested from patients who underwent PTA and later amputations. (B) Representative EVG images of injured femoral artery and the normal control. n = 9. (C) Schematic diagram of nuclear morphology acquisition process and analysis using CellProfiler software. (D) Representative DAPI‐stained nuclei showing cigar‐shaped nuclei in control arteries and irregular nuclei in injured arteries. (E) Representative TEM images of nuclei in VSMCs from patient femoral artery after PTA and control. (F–I) Quantification of nuclear morphology parameters including Compactness, Eccentricity, Formfactor, and Solidity. The data are shown as mean ± SD, ***p < 0.001 (Student's t‐test). n = 9. (J) Balloon injury was performed in the carotid arteries of rats. (K) Representative EVG images of rat injured carotid artery (2 weeks) and intact contralateral carotid artery (self‐control). n = 5. (L) Representative DAPI‐stained nuclei showing nuclear dysmorphism in VSMCs following injury. n = 5. (M) Heatmap and quantification of nuclear morphology parameters in rat samples, the data are shown as mean ± SD, **p < 0.001 (Student's t‐test). n = 5. (N) Representative TEM images of nucleus in VSMCs from rat injured carotid artery and self‐control. n = 5. L, lumen; N, nucleus.
Besides, nuclear dysmorphism was also verified in the injured rat carotid arteries following balloon injury (Figure 1J). EVG staining (Figure 1K) exhibited vascular remodeling including flattening and breakdown of elastic fibers and neointima formation after injury. DAPI staining and nuclear morphology analysis (Figure 1L) confirmed nuclear dysmorphism in VSMCs in the injured arteries of rats indicated by the parameters of Compactness, Eccentricity, Formfactor, and Solidity (Figure 1M). The distinct nuclear dysmorphism in injured arteries of both human and rat samples was further affirmed using transmission electron microscope (TEM) (Figure 1E,N). To further validate these observations in three dimensions, 30‐μm‐thick vascular tissue sections were prepared and subjected to DAPI staining followed by Z‐stack imaging and three‐dimensional reconstruction using Imaris software. Three‐dimensional analyses demonstrated that nuclei in normal arteries from rats consistently exhibited elongated, cigar‐shaped morphologies, whereas injured vascular displayed a marked increase in irregular and pleomorphic nuclear shapes (Figure S3). Notably, these three‐dimensional findings were fully consistent with the results obtained from conventional 5‐μm tissue sections, thereby providing independent and complementary confirmation of nuclear dysmorphism across species. Therefore, injury induces nuclear dysmorphism in both human and rodent arteries.
Nuclear Dysmorphism Reflects Vascular Aging
3.2
The above findings unveiled the characteristics of nuclear dysmorphism in the injured arteries. Herein, the relationship between nuclear dysmorphism and vascular aging was explored in a rat vascular injury model. The carotid arteries were harvested at days 1, 3, and 7, respectively, after injury. Elastic fibers were markedly flattened and disrupted in carotid arteries on days 1 and 3 and worsened with neointima formation on day 7 after injury (Figure S4A). Of note, nuclear morphology analysis (Figure 2A,B) revealed significantly dysmorphism as early as the first day after injury (Figure 2C–F). These data indicate that nuclear dysmorphism is initiated immediately after injury.
*Nuclear dysmorphism reflects vascular aging. (A) Rat carotid arteries were collected 1, 3, and 7 days respectively after vascular injury. Nuclei were stained with DAPI (gray). (B) Representative images of nuclear morphology were analyzed by CellProfiler. (C–F) The quantification of the four parameters including Compactness, Eccentricity, Formfactor, and Solidity in control and injured arteries. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns = Not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (G) Top 20 of Enriched functions by GO analysis based on the differentially expressed molecules between rat injured carotid artery and self‐control. (H) Heatmap showing the expression profiles of senescence‐associated genes. (I) Representative images of β‐galactosidase staining in the section of patient femoral arteries sample, indicating cell senescence after injury. (J) Representative images of β‐galactosidase staining of the rat injured carotid arteries. (K) Correlation analysis between nuclear morphological parameter (Solidity) and aging (β‐galactosidase staining). (L) Representative IF staining for p16 and p21 in normal and injured human arteries. Quantification of the fluorescence intensity is shown in the right panels. The data are shown as mean ± SD, **p < 0.01 Student's t‐test. n = 3. (M) Representative immunofluorescence staining for p16 and p21 in normal and injured rat arteries. Quantification of the fluorescence intensity is shown in the right panels. The data are shown as mean ± SD, **p < 0.001 (Student's t‐test). n = 3. L, Lumen; N, nucleus.
Whole‐transcriptome sequencing was performed to analyze the differentially expressed genes in injured arteries 2 weeks after surgery. The differentially expressed genes were enriched related to functions including vascular aging and cell senescence (Figure 2G,H and Figure S5). These senescence and aging‐associated signatures were subsequently validated in injured human femoral arteries and rat carotid arteries. β‐galactosidase staining revealed a marked increase in cellular senescence in injured human femoral arteries (Figure 2I). Consistently, indices of vascular aging were significantly elevated in injured rat carotid arteries (Figure 2J, Figure S5B). To further investigate the relationship between nuclear morphology and vascular aging, Pearson correlation analysis was performed between the nuclear morphological parameter Solidity and vascular aging indices. As shown in Figure 2K, the Solidity value, the reduction of which implies more blebs and invaginations in the nuclear boundary, was negatively correlated with vascular aging in injured arteries. In addition, immunofluorescence staining for the aging markers p16 and p21 demonstrated a pronounced increase in senescent cells in injured vascular from both human and rat (Figure 2L,M). These results suggest that nuclear dysmorphism is negatively associated with vascular aging.
These findings demonstrate that injury induces nuclear dysmorphism and vascular aging, where nuclear structural abnormalities serve as a hallmark of post‐injury vascular senescence.
PMVs Induced Prelamin A Accumulation Promotes Nuclear Dysmorphism and Cell Senescence
3.3
Our previous work elucidated that the activation of platelets and the release of pMVs are rapid and sustained inducer of vascular dysfunction after intima injury and hypertension (Bao et al. 2018, 2021; Li et al. 2021). Consistent with these findings, pMVs were observed to adhere to the vascular wall after vascular injury (Figure S6), pMVs were isolated (Figure 3A) and identified by TEM (Figure 3B). Nanoparticle tracking analysis (NTA) confirmed the size (Figure 3C) and flow cytometry (Figure 3D) validated the marker (CD41‐positive and CD45‐negative) of pMVs. The adhesion of CD41‐positive pMVs to VSMCs was observed 1 h after incubation (Figure 3F,G). Nuclear dysmorphism was detected in VSMCs after 24 h incubation with pMVs (Figure 3H). CellProfiler software outlined the nuclei shape and quantified the nuclear morphology (Figure 3I,J). Compactness was significantly increased while Formfactor and Solidity were effectively decreased after pMVs treatment (Figure 3J). TEM images further confirmed nuclear dysmorphism in pMVs treated VSMCs (Figure 3K, Figure S7).
*PMVs induced prelamin A accumulation promotes nuclear dysmorphism. Extraction, isolation, and identification of pMVs. (A) Schematic diagram of pMVs extraction from rat blood. (B) Representative TEM image of isolated pMVs. (C) Nanoparticle tracking analysis (NTA) was used to determine the size of isolated pMVs. (D and E) Flow cytometry was used to identify pMVs, which were CD41 (green) positive and CD45 (red) negative. (F) Schematic diagram of VSMCs treated with pMVs. (G) Representative immunofluorescence staining of DAPI (gray), α‐SMA (green), and CD41 (red) in VSMCs after treatment with pMVs for 24 h. (H) Representative immunofluorescence staining of nucleus co‐stained with DAPI (gray) and Lamin A (red) in VSMCs after pMVs treatment for 24 h. Arrows indicate the deformed nuclei. (I) Representative images of nuclear morphology analyzed with CellProfiler in VSMCs after pMVs treatment. (J) Compactness, Eccentricity, Formfactor, and Solidity quantified the nuclear morphology of VSMCs without and with pMVs treatment respectively (nuclei numbers > 100). The data are shown as mean ± SD, ***p < 0.001, ns, not significant (Student's t‐test). n = 3. (K) Representative images of the nucleus captured by TEM, and nuclear dysmorphism was found in VSMCs treated with pMVs. (L) Western blot was performed to detect the expression of Lamin A, Lamin C, Lamin B1, and Lamin B2 in VSMCs treated by pMVs (n = 6). (M) Representative western blots of prelamin A expression in VSMCs after pMVs treatment. The data are shown as mean ± SD, ***p < 0.001 (Student's t‐test). n = 6. (N) Representative western blots of Lamin A and prelamin A in the arteries of Zmpste24 −/− mice and pMVs treating VSMCs. (O) Schematic diagram of C‐terminal amino acid sequence of prelamin A analyzed by mass spectrometry. (P) Representative immunofluorescence staining of prelamin A (red) and DAPI (gray) in VSMCs, indicating pMVs induced prelamin A accumulation in nucleus especially in the dysmorphic nucleus. Arrows indicate the deformed nuclei. (Q) Pearson correlation was used to analyze the relationship between nuclear dysmorphism and prelamin A expression, and heatmap indicated that prelamin A levels were correlated with increased nuclear dysmorphism. (R) Representative western blot of Lamin A/prelamin A expression in VSMCs after CRISPR‐Cas9‐mediated knockout of Zmpste24, indicating that Zmpste24 ablation induces prelamin A accumulation. The data are shown as mean ± SD, ***p < 0.001 (Student's t‐test). n = 6. (S) Representative immunofluorescence images of Lamin A (red) and DAPI (gray) in VSMCs with or without Zmpste24 knockout. Arrows indicate the deformed nuclei. (T) CellProfiler analyzed the nuclear morphology in Zmpste24 knockout VSMCs. (U) Compactness, Eccentricity, Formfactor, and Solidity quantified nuclear dysmorphism in Zmpste24 knockout VSMCs (nuclei numbers > 100). (V) Representative images of nuclear morphology in Zmpste24 knockout VSMCs examined by TEM. (W) Representative immunofluorescence images of senescence‐associated markers p16(green), p21(red), and P‐p53(red) in control and pMV‐treated VSMCs. Quantification of the fluorescence intensity (arbitrary units, AU) is shown in the lower panels. The data are shown as mean ± SD, **p < 0.01, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (X) Representative immunofluorescence images of p16, p21, and P‐p53 in control and Zmpste24‐KO VSMCs. Quantification of the fluorescence intensity (arbitrary units, AU) is shown in the lower panels. The data are shown as mean ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (Y) was performed to detect Lamin A expression in injured rat arteries. The data are shown as mean ± SD, **p < 0.001 (Student's t‐test). n = 5. L, Lumen; LA, Lamin A; LB1, Lamin B1; LB2, Lamin B2; LC, Lamin C; N, nucleus; pLA, prelamin A.
Lamins are essential for maintaining nuclear architecture, providing structural support and influencing cellular functions, and our previous findings illustrated the vital roles of Lamin A/C in both vascular remodeling and vascular functions (Bao et al. 2020; Qi et al. 2016). Herein, the expression levels and functions of nuclear lamins in VSMCs after pMVs treatment were investigated. The expressions of Lamin A, Lamin C, Lamin B1, and Lamin B2 remained unaltered (Figure 3L, Figures [Link], [Link], [Link], [Link]). Intriguingly, a distinctive band, indicated by an orange arrow in Figure 3L, positioned above the Lamin A band piqued our interest. A blotting band at the similar molecular weight (~74 KD) was detected using a specific antibody targeting prelamin A, a Lamin A precursor (Figure 3M). Besides, the band was aligned with the sample from the artery of Zmpste24 ^−/−^ (an enzyme known to cleave Lamin A, the deficiency of which leads to accumulation of prelamin A (Varela et al. 2005)) mice (Figure 3N). Therefore, we can reasonably assume that the band corresponds to prelamin A. Furthermore, mass spectrometry was used to confirm our hypothesis, and the result showed that there was an additional amino acid sequence in the C‐terminal (from sites 647 to 665) (Figure 3O) compared to Lamin A, which could be cleaved by ZMPSTE24 during Lamin A maturation; these data ensured that the band is prelamin A. Additionally, western blot (Figure 3L) and IF staining (Figure 3P) displayed elevation of prelamin A after pMVs incubation. Taken together, prelamin A was accumulated after pMVs treatment.
We further investigated the relationship between prelamin A accumulation and nuclear dysmorphism. Pearson correlation analysis demonstrated a positive relationship between the Compactness value and prelamin A expression, while the Solidity, Formfactor, and Eccentricity exhibited a negative correlation with prelamin A expression (Figure 3Q). To investigate the impact of prelamin A on nuclear dysmorphism, CRISPR–Cas9 mediated specific knockout of Zmpste24 was performed to induce a substantial accumulation of prelamin A in VSMCs (Figure 3R). IF staining (Figure 3S) combined with nuclear morphology analysis (Figure 3T,U) demonstrated that prelamin A accumulation induces nuclear dysmorphism. TEM (Figure 3V) data also corroborated the results. Beyond its impact on nuclear architecture, Zmpste24 ablation also induced functional hallmarks of cellular senescence. Zmpste24‐deficient VSMCs exhibited increased senescence‐associated phenotypes and elevated DNA damage responses (Figure S9A,B). Consistently, immunofluorescence analysis revealed increased expression of phosphorylated p53, p16, and p21 in both pMVs‐treated and Zmpste24‐deficient VSMCs, indicating activation of canonical senescence signaling pathways (Figure 3W,X). Together, these findings establish prelamin A accumulation as a key driver of nuclear dysmorphism and senescence in VSMCs, linking defects in nuclear lamina processing to vascular aging–associated cellular dysfunction. Importantly, western blot analysis further confirmed the accumulation of prelamin A in injured rat vascular, extending these observations from cultured cells to an in vivo injury model (Figure 3Y). Together, these findings establish prelamin A accumulation as a critical driver of nuclear dysmorphism and cellular senescence in VSMCs, linking defective nuclear lamina processing to vascular aging–associated pathological remodeling.
Zinc Deficiency Leads to Prelamin A Accumulation, Nuclear Dysmorphism and Cell Senescence
3.4
The maturation of prelamin A is a complex process that involves sequential proteolytic cleavage, with ZMPSTE24 being the key enzyme responsible for the final cleavage step, and zinc serves as a crucial cofactor for the activation of the enzymes in this process (Varela et al. 2005) (Figure 4A). Therefore, the concentrations of ions in injured and control carotid arteries were measured using inductively coupled plasma mass spectrometry (ICP‐MS). A notable decrease of zinc concentration was observed in the injured arteries (Figure 4B). Moreover, the intracellular concentration of zinc, as indicated by FluoZin‐3 AM, exhibited a significant reduction in VSMCs following pMVs treatment for 24 h (Figure 4C).
*Zinc deficiency leads to prelamin A accumulation and nuclear dysmorphism. (A) The diagram displayed the processes regarding Lamin A maturation, including the cleavage of prelamin A by ZMPSTE24. (B) ICP‐MS measured the concentration of various metal ions in injured and contralateral carotid arteries (n = 4). (C) The intracellular zinc of VSMCs with or without pMVs treatment was assessed using the specific zinc indicator FluoZin‐3 AM. The data are shown as mean ± SD, **p < 0.01 (Student's t‐test). n = 4. (D) Western blot was performed to examine the expression of Lamin A and prelamin A in VSMCs treated with pMVs, zinc supplementation, pMVs combined with zinc supplementation, TPEN (zinc remover) and respective control. The data are shown as mean ± SD, **p < 0.01, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 6. (E) Representative IF staining depicted the shape of nuclei in VSMCs treated with pMVs, zinc supplementation, pMVs combined with zinc supplementation, TPEN and respective control. (F) CellProfiler analyzed the nuclear morphology. (G) Quantification of nuclear dysmorphism by Compactness, Eccentricity, Formfactor, and Solidity in VSMCs (nuclei numbers > 100). The data are shown as mean ± SD, **p < 0.01, ***p < 0.001, ns = Not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. LA, Lamin A; LC, Lamin C; N, nucleus; pLA = prelamin A. (H) Representative images of the nucleus captured by TEM after pMVs, zinc supplementation, pMVs combined with zinc supplementation or TPEN (zinc remover) treatment. (I) Representative immunofluorescence images of senescence‐associated markers p16 and p21 in VSMCs under control, pMVs, zinc supplementation, pMVs combined with zinc supplementation or TPEN treatment. Quantification of the fluorescence intensity (arbitrary units, AU) is shown in the right panels. The data are shown as mean ± SD, **p < 0.001, ns = not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (J) Representative immunofluorescence images showing nuclear morphology and quantitative assessment of nuclear dysmorphism in KO VSMCs with or without zinc supplementation.
To explore the role of zinc concentration in prelamin A accumulation and nuclear dysmorphism, VSMCs were treated with zinc supplements or TPEN, which is an intracellular membrane‐permeable zinc ion chelator. Western blot (Figure 4D), IF (Figure 4E), nuclear morphology analysis (Figure 4F,G), and TEM images (Figure 4H) all indicated that zinc supplementation alleviated pMVs‐induced nuclear dysmorphism, while TPEN aggravated nuclear dysmorphism. Meanwhile, DNA damage (Figure S10A) and cell senescence (Figure 4I, Figure S10B) were attenuated by zinc supplementation while exacerbated by TPEN treatment. Importantly, in Zmpste24‐deficient VSMCs, zinc supplementation failed to rescue nuclear dysmorphism (Figure 4J), indicating that the protective effects of zinc are dependent on intact Zmpste24‐mediated prelamin A processing.
Therefore, zinc deficiency promotes prelamin A accumulation, nuclear dysmorphism, and ultimately cellular senescence.
Downregulation of Zinc Transporter ZIP4 Accounts for Zinc Deficiency and Prelamin A Accumulation
3.5
To interpret the potential mechanism of zinc deficiency in VSMCs, the mRNA expression of all reported zinc transporters in rat intact arteries and the injured arteries were detected using RNA sequencing. Among all zinc transporters, slc39a4 (encode ZIP4) was the one most significantly inhibited in injured carotid arteries compared to the controls (Figure 5A). ZIP4, a mediator of the influx of zinc into the cells (Figure 5B), was suppressed in injured arteries detected by IF staining (Figure 5C). Besides, western blot also showed a shortage of ZIP4 after pMVs stimulation in VSMCs (Figure 5D). In addition, pMVs or slc39a4‐siRNA transfection induced prelamin A accumulation in VSMCs (Figure 5E). Notably, *slc39a4‐*siRNA also triggered nuclear dysmorphism, which was similar to the effects of pMVs treatment, as observed by IF staining (Figure 5F), nuclear morphology analysis (Figure 5G,H), and TEM (Figure 5I).
*ZIP4 downregulation accounts for Zinc deficiency, prelamin A accumulation and nuclear dysmorphism. (A) Volcano plot showing mRNA expression of zinc transporters in rat injured vs. self‐control carotid arteries, highlighting downregulation of slc39a4 (encode ZIP4) (fold change > 2 and p value < 0.05). n = 4. (B) The diagram illustrated the way how ZIP4 transport Zinc. (C) Immunofluorescence staining examined the expression of ZIP4 in the rat with intact or injured arteries. (D) Western blot revealed the ZIP4 expression in VSMCs treated with pMVs or slc39a4 siRNA. The data are shown as mean ± SD, **p < 0.01, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 6. (E) Western blot showed the expression of prelamin A in VSMCs treated with pMVs or slc39a4 siRNA. The data are shown as mean ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 5. (F) Immunofluorescence staining of Lamin A (red) and DAPI (gray) in VSMCs after pMVs treatment or slc39a4 siRNA transfection. (G) CellProfiler software characterized nuclear morphology. (H) Compactness, Eccentricity, Formfactor, and Solidity quantified the nuclear dysmorphism in VSMCs (nuclei numbers > 100). The data are shown as mean ± SD, **p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (I) Representative TEM images of the cell nuclei, indicating nuclear dysmorphism after pMVs treatment or slc39a4 siRNA transfection. N, nucleus; LA, Lamin A; LC, Lamin C; pLA, prelamin A.
Taken together, downregulation of ZIP4 leads to the reduction of zinc concentration in VSMCs, which subsequently drives prelamin A accumulation and nuclear dysmorphism.
DNA Demethylation in Lamina‐Associated Domains (LADs) Is Associated With Vascular Aging and Nuclear Dysmorphism
3.6
Lamin A is crucial in both nuclear architecture and chromatin remodeling via binding with LADs which enrich DNA methylation and participate in regulating gene expression, chromatin structure and biological processes (Berman et al. 2011; Briand and Collas 2020; Cheedipudi et al. 2019). WGBS revealed that methylation levels across genome‐wide CpG, promoter, Genebody and Inergenic sequences were repressed in injured arteries (Figure 6A). Besides, a significant decrease of methylation levels within LADs was found in rat injured arteries (Figure 6B,C). Then, we analyzed the correlation between methylation status of CpG sites and the transcription of corresponding genes. The joint analysis of RNA‐Seq and WGBS elucidated that 67.3% of the CpG methylation sites within LADs were reduced accompanied with the increase of corresponding genes (Figure 6D). Functional annotation of these aberrantly activated genes revealed a strong enrichment in senescence‐related pathways, as indicated by GO analysis and Ingenuity Pathway Analysis (IPA) (Figure 6E,F). Overall, injury‐induced DNA demethylation in LADs activates genes that promote cellular senescence.
*Demethylation of Lamina‐associated domains (LADs) is associated with nuclear dysmorphism and vascular aging. (A) WGBS detected the DNA methylation in the injured arteries and the self‐control arteries (2 weeks) (n = 4). Smoothed scatter plots showed the correlation of methylation percentage between the control and the injured arteries throughout the Genome wide CpG, Promoter, Genebody, and Intergenic regions. (B) Schematic representation of the spatial organization of genes within LADs, highlighting the interactions between chromatin and Lamin A. (C) CpG methylation level within the LADs in the injured arteries (purplish red) was significantly repressed compared with that in the control group (black). (D) Integrating RNA‐seq and WGBS data to analyze the correlation between CpG methylation and gene expression levels in LADs. (E) Ingenuity Pathway Analysis (IPA, Qiagen, Venlo, Netherlands) showed that genes within LADs participate in cell senescence. (F) Immunofluorescence staining of Lamin A (red), H3K9me3 (green) and DAPI (blue) in VSMCs after pMVs treatment. Arrows indicate deformed nuclei. (G) Pearson correlation was used to analyze the relationship between nuclear dysmorphism and H3K9me3 expression, and heatmap indicated that H3K9me3 levels were correlated with increased nuclear dysmorphism. Quantification of mean fluorescence intensity of H3K9me3 is shown on the right. The data are shown as mean ± SD, p < 0.05 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (I) Representative TEM images showing nuclear ultrastructure in control and pMVs‐treated VSMCs, with quantitative analysis of peri‐laminar heterochromatin thickness. (J) Representative three‐dimensional SIM images of VSMC nuclei stained for Lamin A (red), H3K9me3 (green), and DAPI (blue).
LADs play an important role in heterochromatin rearrangement (Berman et al. 2011; Cheedipudi et al. 2019), to examine whether prelamin A accumulation is associated with heterochromatin disruption at senescence‐related loci, we analyzed a published H3K9me3 ChIP‐seq dataset generated from VSMCs overexpressing prelamin A (Ho et al. 2025). Senescence‐associated genes identified from our RNA‐seq analysis, including NOX4, HMOX1, AHR, HIPK2, and CDKN2A (Figure S11), were interrogated within this dataset. Cross‐referencing their epigenetic profiles revealed a pronounced loss of H3K9me3 occupancy at these loci, indicating erosion of heterochromatin in the context of prelamin A overexpression. Based on these findings, we next assessed global chromatin remodeling induced by pMVs treatment using the heterochromatin marker H3K9me3 and the euchromatin marker H3K9ac. IF staining showed that pMVs treatment induced nuclear dysmorphism accompanied by reduced H3K9me3 expression (Figure 6G). Pearson correlation analysis highlighted a negative correlation between H3K9me3 expression and nuclear dysmorphism (Figure 6H). However, H3K9ac was highly expressed in the blebs (Figure S12A) after pMVs treatment. At the ultrastructural level, quantitative analysis of TEM images showed a significant reduction in peri‐laminar heterochromatin thickness in pMVs‐treated VSMCs (Figure 6I). Consistently, three‐dimensional SIM reconstructions clearly demonstrated loss of H3K9me3 signal at regions exhibiting nuclear dysmorphism (Figure 6J). Moreover, immunofluorescence staining of injured human femoral arteries and rat carotid arteries revealed a marked decrease in H3K9me3 fluorescence intensity compared with normal vessels (Figure S12B). Taken together, these results indicate that injury‐induced DNA demethylation within lamina‐associated domains disrupts heterochromatin organization, leading to nuclear dysmorphism and ultimately promoting vascular aging.
Zmpste24 Knockout Accelerates Prelamin A Accumulation, Nuclear Dysmorphism, and Vascular Dysfunction
3.7
To further confirm the pathological effects of prelamin A accumulation and its impact on nuclear dysmorphism and vascular dysfunction, Zmpste24 ^+/−^ and Zmpste24 ^−/−^ mice were used to induce prelamin A accumulation. IF staining (Figure 7A), nuclear morphology analysis (Figure 7B–F), and TEM images (Figure 7G) all confirmed a significant nuclear dysmorphism in the arteries of both Zmpste24 ^+/−^ and Zmpste24 ^−/−^ mice in comparison with WT. Besides, prelamin A was gradually accumulated in Zmpste24 ^+/−^ and Zmpste24 ^−/−^ mice compared to WT mice (Figure 7H). Additionally, Zmpste24 ^ −/− ^ mice exhibited accelerated vascular aging, as evidenced by aging‐associated vascular phenotypes (Figure 7J), which coincided with pronounced accumulation of prelamin A and a significant reduction in H3K9me3 levels (Figure S14A), in contrast to WT and Zmpste24 ^ +/− ^ mice (Figure 7H). To further investigate whether zinc supplementation could modulate vascular pathology, vascular injury and zinc replenishment experiments were performed in Zmpste24 ^ +/− ^ mice. ICP‐MS analysis revealed that a high‐zinc diet significantly increased zinc content in injured arteries (Figure 7K). To directly assess the effect of zinc on ZMPSTE24 enzymatic activity, a fluorescence resonance energy transfer (FRET)‐based peptide substrate was synthesized and used for enzyme activity assays (Hsu et al. 2019) (Figure S13A,B), and the results demonstrated that zinc supplementation effectively enhanced ZMPSTE24 enzymatic activity in vivo (Figure S13C). Notably, in injured arteries from Zmpste24 ^ +/− ^ mice, zinc supplementation markedly reduced prelamin A accumulation (Figure S14B), attenuated DNA damage as indicated by decreased γ‐H2AX expression (Figure S14C), reduced P‐P53, P16, and P21 (Figure 7L–N), and significantly restored H3K9me3 levels (Figure S14D).
*Zmpste24 knockout accelerates prelamin A accumulation, nuclear dysmorphism and vascular aging. (A) Nuclei in the artery of wild type (WT), Zmpste24 +/− and Zmpste24 −/− mice (8–10 weeks old, 20 ± 2 g), were co‐stained with Lamin A (red) and DAPI (gray). (B) Representative images of nuclear morphology analyzed by CellProfiler. (C–F) Compactness, Eccentricity, Formfactor, and Solidity parameters quantified nuclear dysmorphism in the artery of WT, Zmpste24 +/− and Zmpste24 −/− mice. The data are shown as mean ± SD, **p < 0.01, ns, not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 5. (G) Representative TEM images of nuclear morphology in arterial tissues. n = 5. (H) Western blot showed the expression of prelamin A in arteries from wild‐type (WT), Zmpste24 +/− and Zmpste24 −/− mice. The data are shown as mean ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 6. (I) Representative images of β‐galactosidase staining of the arteries in WT, Zmpste24 +/− and Zmpste24 −/− mice. Quantification of SA‐β‐gal staining is shown in the right panel. The data are shown as mean ± SD, **p < 0.01 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (J) Representative IF staining of senescence‐associated markers p16 (green), P‐p53 (red), and p21 (red) in arterial sections from WT and Zmpste24 −/− mice. Nuclei were counterstained with DAPI (blue). (K) Zinc concentration in control arteries, injured arteries, and injured arteries with zinc supplementation (Injury + Zn2+), measured by ICP–MS. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, **p < 0.001 (one‐way ANOVA with Bonferroni's multiple‐comparison post hoc test). (L–N) Representative immunofluorescence staining of senescence‐associated markers p16, p21 and P‐p53 in control arteries, injured arteries, and injured arteries with zinc supplementation. Nuclei were counterstained with DAPI. L, Lumen; LA, Lamin A; N, nucleus; pLA, prelamin A.
Therefore, prelamin A accumulation accelerates nuclear dysmorphism and vascular aging in vivo.
Zinc Supplementation Attenuates Nuclear Dysmorphism and Vascular Aging
3.8
To explore the role of zinc supplementation in alleviating nuclear dysmorphism and vascular aging in vivo, we introduced two types of supplementary strategies, including high zinc diet (Figure 8A) and platelet membrane‐cloaked Zn‐MOF (ZIF‐8, Figure 8B). Elastin remodeling and neointima formation after injury were attenuated by zinc supplementation, especially in the ZIF‐8 group (Figure 8C). IF staining (Figure 8D), nuclear morphological analysis (Figure 8E–I), and TEM images (Figure 8J) all proved that zinc supplementation mitigates nuclear dysmorphism. Notably, the high zinc diet attenuated nuclear dysmorphism as evaluated by the Eccentricity (Figure 8G) and the Solidity (Figure 8I), while ZIF‐8 further reduced nuclear dysmorphism as validated by Compactness, Eccentricity, Formfactor, and Solidity (Figure 8F–I). Besides, the induced expression of prelamin A after injury was reversed by the high zinc diet and further decreased by ZIF‐8 (Figure 8K). Besides, vascular aging (Figure 8L–N) was increased in injured arteries while reduced by the high‐zinc diet and further attenuated by ZIF‐8.
*Zinc supplementation attenuates nuclear dysmorphism and vascular aging. The diagrams illustrated the supplementation of high zinc diet (A) and the preparation of platelets membrane cloaked Zn‐MOF nanoparticles (ZIF‐8) (B). (C) Representative EVG staining of injured carotid arteries after zinc supplementation (2 weeks). (D) Representative immunofluorescence staining of Lamin A (red) and DAPI (gray). (E) Representative images of nuclear morphology were generated using CellProfiler. (F–I) Compactness, Eccentricity, Formfactor and Solidity quantified nuclear dysmorphism in the injured artery with or without zinc supplementation. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 4. (J) TEM images of cell nuclei in injured arteries with or without zinc supplementation. n = 4. (K) Western blot showed that prelamin A accumulation induced by injury was reversed after both high zinc diet and ZIF‐8 supplementation. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 4. (L) Representative images of β‐galactosidase staining showing that injury‐induced cell senescence was rescued after ZIF‐8 supplementation. n = 4. (M) Representative IF staining of the senescence marker p16 (green) in control arteries, injured arteries, injured arteries with zinc supplementation, and injured arteries treated with PC‐ZIF‐8. Nuclei were counterstained with DAPI (blue). Quantification of p16 fluorescence intensity is shown in the right panels. The data are shown as mean ± SD, **p < 0.01, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. (N) Representative IF staining of the senescence marker p21 (red) in control arteries, injured arteries, injured arteries with zinc supplementation, and injured arteries treated with PC‐ZIF‐8. Nuclei were counterstained with DAPI (blue). Quantification of p21 fluorescence intensity is shown in the left panels. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, **p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3. L, Lumen; LA, Lamin A; LC, Lamin C; N, nucleus. pLA, prelamin A.
To test the side effects of PC‐Zn‐MOF on important organs of the body, H&E staining of the heart, liver, spleen, lung, and kidney was performed. No obvious morphological changes were found after the treatment (Figure S14), indicating that zinc supplementation had no obvious side effects. These results indicated that zinc supplementations, especially the way using platelet membrane cloaked Zn‐MOF, were effective in preventing vascular dysfunction via inhibiting prelamin A accumulation.
Our findings reveal that the pMVs/ZIP4/zinc/prelamin A axis constitutes a novel signaling pathway regulating nuclear dysmorphism and vascular aging.
Discussion
4
The most significant findings of the present study are the demonstration of a novel pathway of pMVs/ZIP4/zinc/prelamin A in modulating nuclear dysmorphism and vascular aging, and the potential therapeutic strategies by targeting VSMC prelamin A (Graphical abstract). Nuclear dysmorphism is first identified in VSMCs in injured arteries due to pMVs/ZIP4/zinc/prelamin A axis activation. During vascular injury, platelets adhere to injured arteries and release pMVs, leading to ZIP4 downregulation, which subsequently reduces zinc concentration and causes prelamin A accumulation. Accumulated prelamin A increases nuclear dysmorphism and the demethylation of genes associated with LADs, which triggers vascular cell senescence. In our study, senescence markers and prelamin A accumulation persisted throughout the vascular remodeling phase following injury. Moreover, Zmpste24 deficiency induced similar nuclear dysmorphism and senescence phenotypes in the absence of injury. Platelet membrane‐cloaked Zn‐MOF is utilized to deliver zinc for treating nuclear dysmorphism and vascular aging, which offers a promising strategy to mitigate the adverse effects of platelet activation while exploiting the targeting capabilities as a precision therapeutic carrier.
Zinc, an indispensable micronutrient, plays a pivotal role in a myriad of vital biological processes, including cell proliferation and differentiation, cellular transport and maintaining homeostasis (Roh et al. 2012; Schisterman et al. 2020). Numerous studies revealed that zinc deficiency leads to various pathological processes such as DNA impairment (Liu et al. 2009), apoptosis (Chapman et al. 2019), and oxidative damage to cellular components by reactive oxygen species (ROS) (Feng et al. 2008), and subsequently triggers chronic heart diseases, cancer, neurodegenerative diseases, and other pathological conditions. In the cardiovascular system, individuals with cardiovascular diseases have lower zinc concentrations compared to healthy people, highlighting the role of zinc in coronary heart disease (Vlad et al. 1994), atherosclerosis (Choi et al. 2018), and hypertension (Betrie et al. 2021). In addition to its biological functions, zinc is a component of the next‐generation biodegradable scaffold materials, offering ideal in vivo degradation rates, comprehensive hemocompatibility, cytocompatibility, inhibition of VSMC proliferation, and excellent antibacterial properties (Bowen et al. 2013; Su et al. 2019), indicating its huge potential for clinical application. However, the intrinsic biological functions and molecular mechanisms of zinc have been overlooked. Herein, we elucidated a novel mechanism of zinc deficiency in inducing prelamin A accumulation, nuclear dysmorphism and vascular aging. This also provides a new perspective for the clinical application of zinc‐based metallic materials in vascular stents. Furthermore, we utilized platelet membrane‐cloaked Zn‐MOF to deliver zinc, which offers a potential strategy to diminish the adverse effects of platelet activation while leveraging their targeting ability as carriers for precision therapy (Zhuang et al. 2020). Our findings demonstrated that ZIF‐8 formed by encapsulating Zn‐MOF within platelet membranes can effectively replenish zinc in vivo, alleviating nuclear dysmorphism and vascular aging. This work verified the potential of degranulated platelet membranes in serving as targeted delivery systems for therapeutic agents in vascular pathologies.
Nuclear morphology changes are associated with several diseases, including cancer, and premature (Schibler et al. 2023). Recently, Andria et al. revealed that nuclear morphological alterations can serve as a deep learning predictor of senescence which is applicable across tissues and species and correlate with health outcomes in humans (Heckenbach et al. 2022). Yao et al. verified that the nuclear area is reliable for determining ploidy in isolated cardiomyocytes, while nuclear volume offers unparalleled accuracy as a ploidy evaluation metric in both isolated cardiomyocytes and adult mouse heart cryosections (Yao et al. 2024). Others and we have demonstrated that vascular remodeling and vascular dysfunction are important pathological processes in cardiovascular diseases such as vein graft failure (Fan et al. 2025; Huang et al. 2017), hypertension (Bao et al. 2018, 2020; Cui et al. 2025; Qi et al. 2016; Wang et al. 2017), and aortic aneurysms (Huang, Narumi, et al. 2021; Huang, Wang, et al. 2021; Huang et al. 2022; Zhang et al. 2020). However, the alteration and significance of nuclear morphology in injured aortas, vascular aging and vascular remodeling have not been systematically investigated. In the present study, notable changes in nuclear morphology following injury were detected through the assessment of Compactness, Eccentricity, Formfactor, and Solidity parameters. Notably, the Solidity parameter consistently maintained a decreased trend across various conditions, including injured aortas, pMVs treated VSMCs, and aortas from both Zmpste24 ^ +/− ^ and Zmpste24 ^ −/− ^ mice. Solidity value was negatively correlated with senescence in injured arteries and pMVs treated VSMCs.
Lamin A is an essential nuclear matrix protein located beneath the nuclear membrane which forms a tightly interwoven network of protein fibers in the perinuclear region and provides crucial structural support for the nucleus (Dechat et al. 2010). It is initially synthesized as a precursor protein that undergoes extensive post‐translational modifications, including proteolytic processing of its carboxy‐terminal to attain the mature functional form (Dechat et al. 2010). We found prelamin A accumulation in injured arteries and the accumulation was due to the inhibition of ZIP4 and subsequent zinc deficiency. Of note, Lamin A plays a crucial role in maintaining nuclear integrity and modulating genomic functions through its interaction with LADs on chromatin (Briand and Collas 2020). We examined the impact of prelamin A accumulation on gene expression in LADs. Accompanied by nuclear dysmorphism, the LADs genome in injured arteries undergoes DNA demethylation and activation of gene expressions. The aberrant activation of genes within the LADs contributes to vascular aging, which may provide novel therapeutic targets for treating vascular diseases and need further verification.
In summary, the findings of this study uncovered a novel pMVs/ZIP4/zinc/prelamin A axis in regulating nuclear dysmorphism and vascular aging after injury. Targeting prelamin A or zinc supplement, especially using platelet membrane‐coated Zn‐MOF nanoparticles, are potential ways in treating vascular aging post‐injury.
Author Contributions
T.M., H.B., Z.X., H.R., W.T., J.C., Z.L., and K.H. performed experiments. T.M., H.B., K.H., and Y.Q. analyzed data. Z.X., X.L., F.L., and Q.Y. provided advice, discussion, and critical materials. H.B., K.H., and Y.Q. conceived the project and designed experiments. K.H. and Y.Q. supervised the study. T.M., K.H., and Y.Q. wrote the manuscript with input from all authors.
Funding
This research was supported by grants from the National Natural Science Foundation of China, Nos. 12032003, 32471363, 12102261, and 12302409.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Figure S1: Zmpste24 knockout mice. Genotyping of Zmpste24 knockout mice. WT: wild‐type (563 bp), KO: knockout (339 bp).
Figure S2: Quantitative characterization of nuclear dysmorphism using CellProfiler‐based morphometric analysis. (A) Technical notes regarding 5 types of nuclear morphological parameters explained by CellProfiler. (B) Heatmap illustrated the changes in nuclear morphological parameters including Compactness, Eccentricity, Form Factor, Solidity, and Zernike shape features between injuryed femoral arteries and controls. (C) The p‐value of nuclear morphological parameters between intact and injured arteries (n = 9). (D) Comparison of Solidity between normal nuclei and dysmorphic nuclei based on analysis of all detected nuclei within the images. The fluorescence intensities of normal and dysmorphic nuclei were comparable, confirming correct cell segmentation Data are shown as mean ± SD; ns = not significant. (E) Comparison of nuclear Solidity values among VSMC nuclei from the media of control arteries, the intima of injured arteries, and the media of injured arteries, based on analysis of all detected nuclei. Data are shown as mean ± SD; ***p < 0.001; ns = not significant.
Figure S3: Three‐dimensional reconstruction reveals injury‐induced nuclear dysmorphism across species. Representative 3D reconstructions of nuclei from 30‐μm rat vascular sections under control and injury conditions (2 weeks after injury), displayed from frontal, inferior oblique, lateral, and superior oblique views. The reconstructions demonstrate pronounced alterations in nuclear organization and spatial morphology following vascular injury, compared with the elongated and aligned nuclear architecture observed in control.
Figure S4: Injury induces vascular remodeling and aging. (A) Representative EVG staining of rat carotid arteries after balloon injury for 1, 3, and 7 days, respectively. (B) Representative images of β‐galactosidase staining in rat carotid arteries 1, 3, and 7 days after injury. (C) Quantification of SA‐β‐gal staining (arbitrary units, AU) in carotid arteries at the indicated time points. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns = Not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3.
Figure S5: Transcriptome reveals cell senescence in injured rat carotid arteries. (A) GSEA of RNA‐seq data enriched the pathways related to cellular senescence. (B) Box plot showing increased aging scores derived from aging‐related disease genes in cardiovascular diseases in injured carotid arteries compared with controls. Data are presented as individual samples with median and interquartile range. *p < 0.05.
Figure S6: pMVs adhere to the vascular wall and deliver limited lamin‐related miRNAs. Representative IF image showing the adhesion of pMVs to the vascular wall 1 h after injury. α‐SMA (green) labels VSMCs, CD41 (red) labels pMVs, and nuclei are stained with DAPI (white). L, lumen.
Figure S7: pMVs induce nuclear dysmorphism without overt disruption of nuclear lamina ultrastructure. Representative TEM images of VSMC nuclei under control conditions and after pMVs treatment. Low‐magnification images (left) show overall nuclear morphology, while higher‐magnification views (right) highlight the nuclear periphery and chromatin organization. Despite pronounced nuclear dysmorphism following pMVs treatment, the nuclear lamina remains ultrastructurally intact, without obvious fragmentation or discontinuity. N, nucleus.
Figure S8: pMVs have no significant effect on lamins expressions. The quantification of lamins expression after pMVs treatment (Figure 3L in the manuscript). pMVs treatment had on effect on expressions of Lamin A (A), Lamin C (B), Lamin B1 (C), or Lamin B2 (D) in VSMCs (n = 6). The data are shown as means ± SD, ns = Not significant (Student's t‐test).
Figure S9: Zmpste24 knockout induces VSMC senescence and DNA damage. (A) Cell viability/proliferation assay measured by absorbance at 450 nm (OD 450 nm) in Control and Zmpste24 knockout (KO) VSMCs. The data are shown as mean ± SD, **p < 0.01 (Student's t‐test). n = 5. (B) Representative images of β‐galactosidase staining in VSMCs after CRISPER‐cas9 mediated knockout of zmpste24. Quantification of the percentage of SA‐β‐gal–positive cells is shown on the right. The data are shown as mean ± SD, ***p < 0.001 (Student's t‐test). n = 3. (C) Western blot was conducted to assess the expression of γ‐H2AX in VSMCs after CRISPER‐cas9 mediated knockout of zmpste24 (n = 4). Data were presented as means ± SD. ***p < 0.001 (Student's t‐test). n = 4.
Figure S10: Zinc replenishment alleviates abnormal senescence of VSMCs induced by pMVs. (A) Western blot examined the expression of γ‐H2AX in VSMCs under respective conditions. The data are shown as means ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 4. (B) β‐galactosidase staining detected cell senescence in VSMCs under respective conditions. Quantification of the percentage of SA‐β‐gal–positive cells is shown on the right. The data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns = Not significant (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 3.
Figure S11: Prelamin A‐associated loss of H3K9me3 at senescence‐related gene loci. Representative IGV tracks showing H3K9me3 ChIP‐seq signals at senescence‐related gene loci (NOX4, HMOX1, AHR, HIPK2,and CDKN2A). These genes were identified as significantly upregulated senescence‐associated genes in our RNA‐seq analysis of injured rat vascular tissues. H3K9me3 ChIP‐seq data were derived from a previously published dataset of VSMCs with prelamin A overexpression (pLA) and compared with WT VSMCs. Reduced H3K9me3 enrichment was observed at these loci in pLA VSMCs relative to controls, indicating heterochromatin loss associated with prelamin A accumulation.
Figure S12: pMVs induce nuclear dysmorphism accompanied by chromatin remodeling in VSMCs. (A) Representative IF images of VSMCs under control conditions and after pMVs treatment showing DAPI (blue), Lamin A (red), and H3K9ac (green) staining, with arrows indicating dysmorphic nuclei exhibiting increased H3K9ac signal. (B) Representative IF images of rat vascular sections showing DAPI (blue) and H3K9me3 (green) staining in control and injured tissues.
Figure S13: Zinc supplementation restores ZMPSTE24 enzymatic activity in injured vascular tissues. (A) HPLC chromatogram showing the purity of the synthesized FRET substrate peptide used for ZMPSTE24 activity assays. (B) Mass spectrometry analysis confirming the molecular weight and identity of the FRET substrate peptide. (C) ZMPSTE24 enzymatic activity measured by FRET assay in vascular membrane protein extracts from control, injured, and zinc‐supplemented injured tissues. n = 4. Data are presented as mean ± SD; *p < 0.05, ***p < 0.001.
Figure S14: Zinc supplementation attenuates injury‐induced prelamin A accumulation, DNA damage, and heterochromatin loss in Zmpste24 ^ +/− ^ mice. (A) Representative IF images of vascular sections from WT and Zmpste24^+^/^−^ mice (8–10 weeks old, 20 ± 2 g) showing DAPI (blue) and H3K9me3 (green) staining. (B) Western blot examined the expression of pre‐Lamin A in the injured carotid arteries with or without zinc supplementation in zmpste24 ^+/−^ mice. The data are shown as means ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 6. (C) Western blot detected the expression of γ‐H2AX in the injured carotid arteries with or without zinc supplementation in zmpste24 ^+/−^ mice. (D) Representative IF images showing DAPI (blue) and H3K9me3 (green) staining in control, injured, and zinc‐supplemented injured vascular tissues. The data are shown as means ± SD, ***p < 0.001 (one‐way ANOVA with Bonferroni's multiple comparison post hoc test). n = 4.
Figure S15: Zinc supplementation shows no overt systemic toxicity in major organs. Representative H&E‐stained sections of heart, liver, spleen, lung, and kidney from control, injured, zinc‐supplemented injured (Injury‐Zn^2+^), and Injury‐ZIF‐8 groups (2 weeks after injury). No obvious histopathological abnormalities were observed in major organs across all groups, indicating that zinc supplementation and ZIF‐8 treatment do not induce detectable systemic toxicity under the experimental conditions.
Table S1: Personal information of intimal injury patients and the controls. Table S2: The PCR primer for genotyping.
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