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MT2A promotes angiogenesis in chronically ischemic brains through a copper–mitochondria regulatory mechanism

Journal of Translational Medicine volume 23, Article number: 162 (2025) Cite this article

Abstract

Background

Approximately half of patients with chronic ischemic cerebrovascular disease (CICD) exhibit poor revascularization. Metallothionein 2 A (MT2A) has a high affinity for metal ions and is potentially capable of chelating toxic copper ions to alleviate the impairment of angiogenesis. Therefore, we hypothesized that MT2A could promote angiogenesis in chronically ischemic brains by neutralizing excessive copper ions during copper overload (CPO).

Methods

We first collected dura matter (DM) samples from CICD patients and examined the expression of cuproptosis-related genes (DLAT, FDX1, and SDHB) to confirm the inhibitory effect of CPO on angiogenesis. Then, we treated human umbilical vein endothelial cells (HUVECs) with different concentrations of elesclomol and CuCl2 to determine the optimal concentration for inducing CPO. HUVEC activity and mitochondrial structure and function were detected to explore the ability of MT2A to alleviate CPO-induced damage. Finally, a rat model of 2-vessel occlusion plus encephalo-myo-synangiosis (2VO + EMS) with CPO was established to test the proangiogenic effect of MT2A through the copper–mitochondria regulatory mechanism in chronically ischemic brains.

Results

Compared with those from Matsushima grade A patients, DM samples from Matsushima grade C patients presented significantly greater DLAT and FDX1 expression and significantly lower SDHB expression. The optimal drug concentration for inducing CPO was subsequently determined, and in vitro experiments revealed that HUVEC activity was significantly decreased in the CPO group under hypoxic culture, accompanied by increased DLAT oligomerization, decreased SDHB expression, increased HSP70 expression. Moreover, significantly more common mitochondrial aberrations and significantly lower mitochondrial activity were detected in the CPO group compare with the control group. Additionally, MT2A overexpression alleviated CPO-induced mitochondrial dysfunction and cytotoxicity, improving HUVEC viability. In vivo, a CPO rat model was established, and CPO inhibited cerebral angiogenesis in 2VO + EMS model rats. Moreover, significantly greater CD31 expression, less DLAT accumulation, more mitochondria, and fewer mitochondrial abnormalities were observed in the CPOMT2A+ group than in the CPO group, accompanied by significantly improved cerebral blood perfusion and cognitive function.

Conclusion

MT2A can promote angiogenesis in chronically ischemic brains by neutralizing excessive copper ions and rescuing CPO-induced mitochondrial dysfunction.

Introduction

Chronic ischemic cerebrovascular disease (CICD) is a disease caused by various factors (such as atherosclerosis and moyamoya disease), which can induce stroke and cause disability or death [1]. Revascularization surgery is a routine surgical procedure for CICD patients and is reportedly able to improve cerebral blood perfusion (CBP) and relieve symptoms of cerebral ischemia; however, approximately half of CICD patients cannot achieve satisfactory revascularization results and fail to reduce the risk of stroke [2].

Metallothionein 2 A (MT2A) is a cysteine-rich protein in cells with a high affinity for heavy metal ions [3] and a highly conserved gene sequence [4]. MT2A is the most widely expressed member of the metallothionein (MT) family [5] and is involved in maintaining the balance of metal ions in cells to counter the toxic effects of copper, zinc, iron and other metal ions [6], playing important roles in antioxidant activity [7], immune defense [8], cell proliferation and cell differentiation [9]. Moreover, there is evidence that MT2A is involved in regulating the repair of vascular injury and angiogenesis in tumor tissues [10, 11].

Copper, a divalent metal ion that can be chelated by MT2A, plays an important regulatory role in various biological processes (mitochondrial respiration [12], angiogenesis [13] and regulation of the cell cycle [14]). Under physiological conditions, the concentration of intracellular copper is extremely low, and some copper is chelated and stored by MTs [15]. When copper is mobilized to regulate cell function, it detaches from MTs and binds to the corresponding functional proteins [16]. However, when the concentration of intracellular copper is abnormally elevated under pathological conditions, which is called copper overload (CPO), excessive copper causes an imbalance in intracellular copper homeostasis (CHI) and high cytotoxicity, even leading to cell death (cuproptosis) [17].Recent studies have shown that CPO mainly leads to cell impairment by interfering with mitochondrial respiration, which is called the copper-mitochondria regulatory (CMR) mechanism [18], and that these toxic effects of CPO are mostly observed in tumor cells. However, the cytotoxic effect of CPO on endothelial cell (EC) proliferation under hypoxia and strategies for neutralizing the toxic effects of CPO on EC proliferation and angiogenesis in chronically ischemic brains are still unclear.

On the basis of these previous results, we speculated that the CMR mechanism may be involved in the inhibition of EC proliferation under hypoxia and that MT2A may reverse this process by chelating excessive free copper ions in ECs. Therefore, to determine the relationship between CHI and cerebral angiogenesis, we first harvested dura mater (DM) samples from CICD patients to compare the expression of cuproptosis-related genes between patients with different degrees of angiogenesis. Then, human umbilical vein endothelial cells (HUVECs) under CPO conditions were used as a cell model to explore whether CPO has an inhibitory effect on mitochondrial respiration in HUVECs and whether elevated MT2A expression in HUVECs can reverse this effect by chelating excessive free copper ions. The above regulatory mechanism of MT2A was also tested in a 2-vessel occlusion plus encephalo-myo-synangiosis (2VO + EMS) rat model. These findings provide new targets for the regulation of EC proliferation and angiogenesis under hypoxia and new ideas for promoting the revascularization effect in CICD patients.

Materials and methods

Analysis of the DM samples

In CICD patients, a small part (20–40 mm2) of DM tissue just near the recipient artery (the M4 segment of the middle cerebral artery on the ipsilateral side) was harvested during surgical procedures. The samples were stored at -80 °C. During the follow-up, digital subtraction angiography (DSA) images were used to determine the Matsushima grade (grade A, more than two-thirds of the middle cerebral artery circulation was fulfilled; grade B, one-third to two-thirds fulfillment; and grade C, less than one-third fulfillment). The expression levels of cuproptosis-related genes (DLAT, FDX1 and SDHB) in DM samples harvested from patients with Matsushima grade A (n = 8) and Matsushima grade C (n = 8) disease were compared via western blotting (WB) and real-time quantitative PCR (qPCR) analysis.

HUVEC culture and subgrouping

HUVECs were obtained from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences, and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum and 100 U penicillin‒streptomycin at 37 °C in a Tri-gas incubator (Hypoxia, Directheat and air jacket, 637 × 768 × 869 mm, Heal Force). HUVECs were used for in vitro research and experiments until passage 6. HUVECs in the normoxic group were cultured at 37 °C in 21% O2 and 5% CO2. The cells in the hypoxia group were cultured at 37 °C in 1% O2, 5% CO2 and 94% N2. The subgroup information is shown in Table 1.

Table 1 Subgrouping of HUVECs
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HUVEC transfection

The MT2A overexpression plasmid, small interfering RNA targeting MT2A, and negative control (NC) were constructed by Hanheng Biotechnology (Shanghai, CN). HUVECs were transfected with Lipofectamine 2000 (Invitrogen; Carlsbad, CA), following the manufacturer’s protocol. The transfection efficiency was determined via qPCR and WB, while the effects of MT2A transfection on HUVEC viability and mitochondrial function under hypoxia were examined via CCK-8 and JC-10. The subgroup information of MT2A-transfected cells is shown in Table 2.

Table 2 Subgrouping of HUVECs after transfection
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Real-time qPCR

Total RNA was extracted via TRIzol (Thermo Scientific Co., Ltd.) according to the manufacturer’s instructions, and the concentration of total RNA was measured via a NanoDrop spectrophotometer. RNA (1000 ng) from each sample was reverse transcribed into cDNA via a RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific Co., Ltd.) according to the manufacturer’s instructions. Finally, the cDNA was mixed with primers and 2X SYBR Green Fast qPCR Mix (ABclonal, Inc.), and the mRNA levels were determined via a qPCR instrument. The relative expression level of the target gene was normalized to that of β-actin. The primers spanned exons for each gene. The primer sequences are shown in Table S1.

Cell counting Kit-8 (CCK-8)

A CCK-8 (Beyotime, China) assay was utilized to quantitatively evaluate cell viability. HUVECs were seeded onto 96-well culture plates, incubated for 24 h and then pretreated with increasing concentrations of elesclomol (LGM Pharma, USA) (0 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, 1 mM, 10 mM, or 100 mM) and CuCl2 (0.1 µM, 1 µM, or 10 µM for all groups except the control group) for 48 h. Where specified, CuCl2 (1 µM) was added to the media 30 min before the addition of elesclomol. Then, CCK-8 reagent (10 µM) was added to each well, and the samples were incubated at 37 °C for 2 h. The absorbances at 450 nm were determined at 48 h after the elesclomol + CuCl2 treatment via a microplate reader (BioTek Instruments, USA). DMEM containing 10% CCK-8 reagent was used as a blank. Each experiment was repeated at least three times.

MitoTracker Red staining

The cells were stained with MitoSOX Red Mitochondrial Superoxide Indicator (Yeasen, China) and Hoechst 33,342 (Cell Signaling Technology, MA). The cells at 80 − 90% confluence were treated with elesclomol with (without) CuCl2 for 30 min. Next, the cells were incubated with the MitoSOX Red Mitochondrial Superoxide Indicator for 10 min at 37 °C. Then, the cells were washed with PBS (Beyotime, China) and stained with Hoechst 33,342 labeling solution (1:10000) for 10 min at room temperature. The cells were observed under a fluorescence microscope (Olympus IX71). Each experiment was repeated at least three times.

JC-10

The mitochondrial activity in cells was determined by JC-10. HUVECs were washed with PBS (Beyotime, China) and incubated with 5 µM JC-10 for 30 min in the dark. The fluorescence intensity of both the J-aggregate and monomeric forms of JC-10 was measured via a flow cytometer. Red fluorescence was monitored at excitation and emission wavelengths of 585 and 590 nm, and green fluorescence was monitored at excitation and emission wavelengths of 515 and 529 nm. After the blank values were subtracted, the red/green fluorescence ratios per cell were calculated. Each experiment was repeated at least three times.

Transmission electron microscopy (TEM)

Cell mitochondrial aberrations, such as mitochondrial swelling and the appearance of vacuoles, were observed via TEM. After treatment and trypsinization, the cells were centrifuged at 1200 rpm/min for 5 min and fixed with 4% glutaraldehyde at 4 °C for 2 h. The cells were then incubated at 4 °C with 1% osmium tetroxide for 1 h, dehydrated with alcohol and acetone, and embedded in Epon 816 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were prepared with a Leica ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with uranyl acetate and lead citrate. TEM images were obtained via a JEM-1400Plus transmission electron microscope (HEOL, Ltd. Tokyo, Japan). Mitochondria were counted manually and the mitochondrial minor axis length was measured by ImageJ software. Abnormal mitochondria were identified as mitochondria containing vacuoles and mitochondrial with an abnormal size. Each experiment was repeated at least three times.

Tube formation test

The ability of HUVECs to form tube-like structures was determined via a tube formation test. A 96-well plate was coated with 50 µl of Matrigel per well, and HUVECs were subsequently seeded in the Matrigel-coated 96-well plate at a density of 1.5 × 104 cells per well. After 6 h, pictures were taken with a light microscope (Olympus, Tokyo, Japan), and the Angiogenesis Analyzer plugin of ImageJ was used to determine the number of meshes, branches, and nodes in capillary-like structures in five random fields of view (×100). Each experiment was repeated at least three times.

Scratch wound assay

The migration ability of HUVECs was determined via a scratch wound assay. After the medium was replaced with serum-free medium, HUVECs in the 6-well plates were scratched with a micropipette tip and washed with PBS. After 24 h of incubation, the degree of wound closure was observed, and the wound width was measured via ImageJ software. The migration ability was evaluated by the percentage of wound closure. Each experiment was repeated at least three times.

Transwell migration assay

The migration ability of HUVECs was also determined via a Transwell migration assay. HUVECs (1 × 104) were resuspended in serum-free medium and inoculated onto the upper Transwell chamber layer. After 24 h of incubation, the cells in the lower layer were stained with crystal violet (Beyotime, China). The number of stained cells was determined via ImageJ software, which indicates the degree of HUVEC migration. Each experiment was repeated at least three times.2.12. Animals and subgrouping.

Adult male Sprague–Dawley (SD) rats (8–9 weeks of age, 300–320 g) were obtained from the Institute of Experimental Animals of Sun Yat-Sen University and were housed in temperature-controlled facilities with a 12:12 h light cycle. The rats had access to water and food ad libitum. Before intraventricular lentivirus injection, based on the materials we used during EMS, the rats were randomly divided into the following groups: (1) the Control group, (2) the 2VO + EMS group, and (3) the 2VO + EMS + CPO group. Based on the materials injected and transfected during EMS, the remaining rats were randomly divided into the following groups: (1) the control group (2VO + EMS), (2) the CPO group, (3) the CPOMT2A+ (NC) group, (4) the CPOMT2A+ group, (5) the CPOMT2A− (NC) group, and (6) the CPOMT2A− group. The experimental schedule is presented in Fig. 1. The animal experiment was performed in compliance with the ARRIVE guidelines and approved by the Animal Ethics Committee of Guangzhou Huateng Biopharmaceutical Technology Co., Ltd. (IACUC No. C202304-5). The subgroups of SD rats are shown in Tables 3 and 4.

Fig. 1
figure 1

Experimental schedule. (A) Schedule used for observing the effects of EMS on EC proliferation, CBP and cognitive function in 2VO + EMS rats. (B) Schedule used for observing the effects of elesclomol on EC proliferation, CBP and cognitive function in 2VO + EMS rats. (C) Schedule used for observing the effects of MT2A on EC proliferation, CBP and cognitive function in 2VO + EMS rats during CPO. EMS: encephalo-myo-synangiosis; EC: endothelial cell; CBP: cerebral blood perfusion; CPO: copper overload; LDF: laser Doppler flowmetry; CCA: common carotid artery; MWM: Morris water maze; IF: immunofluorescence; WB: western blotting; TEM: transmission electron microscopy; IVLI: intraventricular lentivirus injection

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Table 3 Subgrouping of SD rats for the animal experiments before IVLI
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Table 4 Subgrouping of SD rats for the animal experiments with IVLI
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Induction of CPO in SD rats

The rats were fed elesclomol to introduce CPO. Starting at postnatal week 9 (PNW9), the rats were treated with elesclomol or vehicle by gavage every 2 days until they were sacrificed. The body weights of the rats were recorded every day from PNW9 until sacrifice. Based on the individual rat weight, elesclomol (10 mg/kg) was dissolved in dimethyl sulfoxide (DMSO) and then mixed with 5% methyl cellulose solution (Sigma) to reach a 2% final concentration of elesclomol-DMSO. The vehicle control solution contained 0.5% methyl cellulose solution with 2% DMSO.

Induction of chronic cerebral ischemia (CCI)

A model of CCI was established via 2VO. Briefly, each rat was anesthetized by inhaling isoflurane (3% for induction, 1.5% for maintenance, 0.6 l/min oxygen flow; RWD, China) and placed on an animal operating table on which a heating pad was placed to maintain body temperature at 37 °C. The right common carotid artery (CCA) was exposed before ligation with two 3–0 silk wires. After a 7-day recovery period, the left CCA was exposed and ligated via the same surgical technique.

Cerebral blood flow (CBF) was measured with laser Doppler flowmetry (LDF) before and after surgery and is expressed as a percentage relative to the baseline values. The successful establishment of the 2VO model was based on the following: (I) the CBF decreased by at least 30% before surgery; and (II) no significant cerebral infarction was observed by the MRI-T2 sequence 1 day after 2VO surgery (Fig. 2).

Fig. 2
figure 2

Procedures used for modeling 2VO + EMS rats. (A, B) Procedures used for CCA ligation. The white arrows indicate the CCA. (C) Representative images showing a rat brain after opening the skull and dura. (D) Rats were subjected to IV lentivirus injection to regulate the expression level of MT2A. (E) Brain samples showing the covering of the TM in the ischemic brain (black arrow). (F) Representative graph of CBF changes before and after 2VO via LDF. 2VO: 2-vessel occlusion; EMS: encephalo-myo-synangiosis; CCA: common carotid artery; MT2A: metallothionein 2 A; TM: temporal muscle; CBF: cerebral blood flow; IV: intraventricular

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Procedures for EMS surgery in rats

Two days after 2VO, EMS was performed on the left cerebral hemispheres of the 2VO rats. Each rat was anesthetized by inhaling isoflurane, and the skin and temporal muscle (TM) were moved from the skull into a U shape. An electric drill was used to remove a skull section approximately 4 to 5 mm in diameter from the temporoparietal region. The DM was carefully cut with microscopic tweezers and scissors to prevent damage to the surface of the brain. Under the microscope, the arachnoid was opened in multiple places with a 1-mL syringe needle. The TM was stitched together with the DM, ensuring that the TM was in close contact with the ischemic brain surface, and then, the skin was stitched.

Intraventricular (IV) lentivirus injection

The lentiviruses expressing MT2A (LV-MT2A) and its shRNA (shRNA-MT2A) and the corresponding NC were designed and packaged by Applied Biological Materials (Richmond, Canada). The anesthesia procedure was the same as above. In accordance with previous methods (Hu et al., 2020), a 1 mm diameter hole was drilled in the skull (1.5 mm posterior and 1.0 mm lateral to bregma), and a 10 µl syringe was inserted for lentiviral vector infusion into the right lateral ventricle (3.5 mm below the skull) of each rat. Five microliters of LV-MT2A (1 × 109 TU/ml) or 5 µl of LV-NC (1 × 109 TU/ml) was injected into rat brain tissues to induce MT2A overexpression or act as an NC, respectively. Similarly, 5 µl of shRNA-MT2A (1 × 109 TU/ml) and 5 µl of shRNA-NC (1 × 109 TU/ml) were injected into rat brain tissues for the MT2A knockdown and NC groups, respectively. All the lentiviral vectors were injected following the instructions for the transfection process (GeneChem, Shanghai, China). All information on the lentiviruses used for the animal experiments is provided in the supplementary materials.

Harvest of the rat brain

Each rat was anesthetized with isoflurane, and its abdominal and thoracic cavities were incised to expose its liver and heart. The right atrial appendage was cut open, and normal saline (NS) was continuously injected into the left ventricle until the liver turned white. Tissue scissors were used to cut the skull at the foramen magnum of the occipital bone. As we continued to peel away the skull and gradually reveal the whole brain, small tweezers were used to remove the entire brain from the base of the skull. The samples used for immunofluorescence were fixed with 4% PFA (Beyotime, China) and 2.5% glutaraldehyde (Servicebio, China). For WB, the brain-tissue samples were immediately homogenized in RIPA buffer (Beyotime, Shanghai, China) when the samples were still fresh. Protein extraction, SDS‒PAGE and WB were conducted as described below. For TEM, the cerebral cortex tissues were immediately fixed by immersion in 2% glutaraldehyde solution buffered with 0.2 M cacodylate buffer and postfixed in osmium tetroxide before being embedded in epoxy resin. Ultrathin sections (under 1 mm) were prepared with a Leica ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with uranyl acetate and lead citrate.

Magnetic resonance imaging (MRI)

We compared the improvement in CBP in the different groups via MRI arterial spin labeling (MRI-ASL) sequence measurements. After being anesthetized by isoflurane inhalation, the brains and CBP of the rats were assessed via a 7.0-T MRI animal scanner (PharmaScan MRI with ParaVision 7 system, Bruker, Germany). The MRI parameters were set as follows: echo spacing = 1 ms, echo time (TE) = 48 ms, repetition time (TR) = 3,500 ms, TR/TE = 2,500/33 ms, matrix size = 256 × 256, field of view (FOV) = 35 × 35 mm2, scan time = 2 min 30 s, and section thickness = 0.8 mm without a gap. ASL parameters were set as follows: TR = 12,000 ms, TE = 4.95 ms, inversion times (TI) = 30, 100, 200, 300, 400, 500, 800, 1200, 3000, and 5000 ms, image size = 96 × 96, FOV = 40 × 40 mm2, slice thickness = 1.2 mm, and scan time = 10 min. The regions of interest (ROIs) were set within the brain cortex in close contact with the TM tissue after EMS, with an area of 10 mm2 per ROI. The CBP value (ml/100 g·min) of each ROI was assessed and calculated via ParaVision software, and the ratio of the CBP on the EMS side to that on the contralateral side was also calculated and set as the “perfusion ratio”, which was used to represent the improvement in CBP on the EMS side.

Morris water maze (MWM)

The rats were subjected to behavioral testing for spatial memory in the MWM to analyze 2VO-induced cognitive impairment and cognitive improvement after EMS and lentiviral transduction. The maze consisted of a black circular pool 200 cm in diameter filled with water (temperature approximately 23 °C, 40 cm in depth) situated in a room with four noteworthy visual cues on the walls. The pool was conceptually divided into four quadrants and had four equidistant quadrants. A black platform 10 cm in diameter was submerged in the water (2 cm below the water surface) and placed on the center of the third quadrant. Edible melanin was added to the water so that the platform was invisible to the test rat. The first five days were the learning period, and a rat was released from the four quadrants facing the wall in random order. Each rat needed to find the underwater platform within 60 s, and the escape latency and swimming route were recorded. If a rat did not find the platform, it was guided to the platform, and its escape latency was recorded as 60 s. Each rat was allowed to stay on the escape platform for 10 s regardless of whether the platform was found. The sixth day was the test period, the platform was removed, the rats were placed in the opposite quadrant from the target quadrant, and the escape latency, time spent in the target quadrant, number of platform crossings, and swimming route were recorded. The escape latency, time spent in the target quadrant and number of platform crossings were measured and analyzed on a video surveillance system (SMART, Panlab SL, Barcelona, Spain).

Inductively coupled plasma mass spectrometry (ICP-MS)

For the detection of copper, ICP-MS was conducted via Keystone analysis. The brain tissue sample was weighed into a plastic tube, and then, a certain amount of nitric acid (4.5 times the sample weight (g)) was added to the tube and incubated at 65 °C for two hours. After digestion, the sample was diluted with pure water (the same amount as nitric acid). For the cell pellet sample, the sample was digested with 0.1 mL of nitric acid at 65 °C for an hour and then diluted with 0.1 mL of water. All treated samples (brain tissue and cell pellets) were spiked with 50 µL of indium (100 ng/mL) as an internal standard and mixed well by vortexing for pumping into the ICP-MS instrument. Multielement standard solutions containing Cu and a working internal solution of indium were prepared from individual element standard stock solutions obtained from LGC (Manchester, NH). An Agilent 7800 inductively coupled plasma mass spectrometer (Santa Clara CA) was used to measure the elements. Each experiment was repeated at least three times.2.21. WB analysis.

HUVECs or the brain tissues of the rats were homogenized and lysed in ice-cold radioimmunoprecipitation lysis buffer (Beyotime, China) for 30 min and then homogenized with an ultrasonic cell crusher for 2 min. The homogenate was subsequently centrifuged at 13,000×g at 4 °C for 30 min. The supernatants were collected in steps as follows. Total proteins were extracted and separated by 10% SDS‒PAGE and transferred to nitrocellulose membranes (Bio‒Rad). The membranes were probed with primary antibodies against DLAT (Abcam Ab172617, USA), MT2A (Abcam Ab192385, USA), SDHB (Abcam Ab175225, USA), HSP70 (Abcam Ab2787, USA) and CD31 (Abcam Ab9498, USA) overnight. Then, the sections were further probed with appropriate horseradish peroxidase-conjugated secondary antibodies. The blots were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) and quantified with Quantity 5.2 software (Bio-Rad, USA). Each experiment was repeated at least three times.

Immunofluorescence staining

HUVECs or paraffin sections from the brains of the rats were permeabilized with 0.3% Triton X-100 (Beyotime, China) in phosphate buffer solution (PBS, Beyotime, China) for 5 min and blocked with 10% goat serum (Beyotime, China) in PBS for 1 h. Coverslips were incubated overnight with primary antibodies at 4 °C and then incubated with secondary antibodies in blocking solution for 1 h at room temperature. The primary antibodies used targeted CD31 (Thermo Fisher Scientific, USA), DLAT (Thermo Fisher Scientific, USA) and VDAC1 (Thermo Fisher Scientific, USA). Next, the slices were incubated with secondary antibodies with Alexa Fluor 488 (Thermo Fisher Scientific, USA) and Alexa Fluor 555 (Thermo Fisher Scientific, USA) at room temperature for 1 h. The nuclei of HUVECs and paraffin-embedded sections were stained with DAPI (Thermo Fisher Scientific, USA). For the determination of mitochondrial function, the cells were incubated with 100 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, USA) for 30 min prior to fixation. Each experiment was repeated at least three times.

Data analysis

The analyses were performed via the Statistical Program for Social Science (SPSS) version 23.0. The data are reported as the means ± standard deviations (SDs). The MWM results were analyzed by repeated-measures one-way analysis of variance (ANOVA). The immunofluorescence staining, qPCR, WB, MWM test and MRI-ASL results were evaluated via two-tailed unpaired Student’s t test. Differences were considered significant if P < 0.05.

Results

Expression of cuproptosis-related genes differs among CICD patients with different Matsushima grades

The DM samples from CICD patients were analyzed by WB and qPCR (Fig. 3A, B). Compared with those in patients with Matsushima grade A (good angiogenesis), the expression of FDX1 and DLAT in CICD patients with Matsushima grade C (poor angiogenesis) was significantly greater and the expression of SDHB in CICD patients with Matsushima grade C was significantly lower (Fig. 3C, D, E). These findings indicated that the degree of revascularization in CICD patients may be affected by cuproptosis.

Fig. 3
figure 3

Expression of cuproptosis-related genes in DM samples. (A) DSA images showing Matsushima grade A (left) revascularization and Matsushima grade C (right) revascularization. (B) Intraoperative image showing the harvested DM samples. (C) Representative western blot showing the expression of DLAT, FDX1 and SDHB in different DM samples. (D) Densitometric analyses of DLAT, FDX1 and SDHB expression in different groups normalized to the expression ratio of DLAT to β-actin in Matsushima grade A samples. (E) mRNA levels of DLAT, FDX1 and SDHB in different groups. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05. DM: dura matter; SD: standard deviation

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CPO inhibits the activity of HUVECs

We used a CCK-8 assay to determine the suitable concentrations of elesclomol and CuCl2 to induce the CPO model in HUVECs. When the CuCl2 concentration was 1 µM and the elesclomol concentration was less than 1 mM, there was no significant difference in the activity of HUVECs between the Hyp group and the HypCPO group. At an elesclomol concentration of 10− 3 M, the HUVEC activity ratio of the HypCPO group to Hyp group was significantly lower than that at an elesclomol concentration of 10− 4 M (P < 0.001) (Fig. 4). Therefore, these concentrations (the CuCl2 concentration was 1 µM and the elesclomol concentration was 10− 3 M) were set as the basic modeling conditions of copper-induced cell toxicity for subsequent in vitro experiments.

Fig. 4
figure 4

Viability of HUVECs under CPO conditions. (A) Line graph showing the cell viability in the 48 h CCK-8 assay. (B) Line graph showing the relative multiplication of HUVEC viability in the HypCPO group compared with that in the Hyp group. The error bars represent the ± SDs. *: P < 0.05, **: P < 0.01. HUVEC: human umbilical vein endothelial cell; Nor: normoxia; Hyp: hypoxia; CPO: copper overload; SD: standard deviation

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CPO impairs the mitochondrial structure and function of HUVECs under hypoxia

We next investigated the effect of CPO on HUVECs. The WB results showed that greater oligomerization of DLAT (oligomer-DLAT), lower SDHB expression, and greater HSP70 expression were detected in the elesclomol + CuCl2 group than in the elesclomol group (Fig. 5A, B). In the immunofluorescence analysis, more DLAT protein aggregates were observed near the mitochondria in the elesclomol + CuCl2 group than in the elesclomol group (Fig. 5C, D). The TEM results revealed that the number of mitochondria in the elesclomol + CuCl2 group was lower than that in the elesclomol group, and the mitochondrial minor axis length in the elesclomol + CuCl2 group was greater than that in the elesclomol group. Additionally, the percentage of abnormal mitochondria in the elesclomol + CuCl2 group was greater than that in the elesclomol group (Fig. 5E, F). The JC-10 results revealed that the mitochondrial activity of HUVECs in the elesclomol + CuCl2 group was significantly lower than that in the elesclomol group (P < 0.001; Fig. 5G, H). These results indicated that CPO induces cuproptosis in HUVECs via mitochondrial dysfunction.

Fig. 5
figure 5

CPO inhibits HUVEC proliferation through a copper–mitochondria regulatory mechanism.(A) Representative western blot showing the expression of DLAT, SDHB and HSP70 in different groups normalized to the expression of β-actin. (B) Densitometric analyses of DLAT, SDHB and HSP70 expression in different groups normalized to the expression of β-actin. (C) Immunofluorescence staining showing DLAT (green) and VDAC1 (red). White scale bar = 20 μm. (D) Quantified immunostaining of DLAT foci in different groups. (E) TEM results showing the mitochondrial structure in the different groups. The white arrows indicate normal mitochondria. The orange arrows indicate swelling. The yellow arrows indicate vacuoles. Scale bar = 1.0 μm. Yellow scale bar = 0.25 μm. (F) Quantification of the mitochondrial number, abnormal mitochondrial percentage, and average mitochondrial minor axis length. (G) JC-10 results showing the mitochondrial function of HUVECs in the different groups. (H) Column chart showing the ratio of aggregated JC-10 to monomeric JC-10 in the different groups. The data are presented as the means ± SDs of three independent experiments. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01. CPO: copper overload; HUVEC: human umbilical vein endothelial cell; ES: elesclomol; TEM: transmission electron microscopy; SD: standard deviation

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MT2A alleviates impaired EC activity and mitochondrial dysfunction induced by CPO

We performed cell experiments to explore the ability of MT2A to alleviate CPO-induced impairment. HUVECs were transfected with pcDNA3.1 or siRNA to overexpress or knock down MT2A, and presented no difference in cell viability or mitochondrial function under hypoxia without CPO induction (Fig. S2). After CPO induction, the copper content of the HUVECs significantly increased, whereas there was no significant difference between any other two CPO-induced groups (Fig. 6A). The CCK-8 results revealed that the activity of HUVECs in the CPOMT2A+ group was significantly greater than that in the CPO group, and that in the CPOMT2A− group was significantly lower than that in the CPO group (Fig. 6B). The results of the tubule formation experiments revealed that the numbers of tubes, nodes, branches and meshes in the CPOMT2A+ group were significantly greater than those in the CPO group. In contrast, the CPOMT2A− group had significantly fewer tubes, nodes, branches and meshes than the CPO group did (Fig. 6C, D). Scratch wound assays revealed that the percentage of wound closure in the CPOMT2A+ group was significantly greater than that in the CPO group, whereas that in the CPOMT2A− group was lower than that in the CPO group (Fig. 6E, G). Transwell migration assays revealed that the number of migrated HUVECs in the CPOMT2A+ group was significantly greater than that in the CPO group, whereas that in the CPOMT2A− group was significantly lower than that in the CPO group (Fig. 6F, H). WB results revealed that the CPOMT2A+ group presented lower oligomer-DLAT and HSP70 expression and greater SDHB expression compare with than the CPO group did, whereas CPOMT2A− group presented greater oligomer-DLAT and HSP70 expression and lower SDHB expression than the CPO group did. (Fig. 7A, B). Immunofluorescence results revealed that DLAT aggregation around mitochondria in the CPOMT2A+ group was significantly lower than that in the CPO group. In contrast, in the CPOMT2A− group, DLAT aggregation around mitochondria was significantly greater than that in the CPO group (Fig. 7C, D). The TEM results revealed that the number of mitochondria in the CPOMT2A+ group was significantly greater than that in the CPO group, accompanied by a significantly shorter average mitochondrial minor axis length and a lower percentage of abnormal mitochondria. In contrast, the number of mitochondria was lower, the average mitochondrial minor axis length was longer, and the percentage of abnormal mitochondria was greater in the CPOMT2A− group than in the CPO group (Fig. 8A, B). The JC-10 results revealed that the mitochondrial activity of HUVECs in the CPOMT2A+ group was significantly greater than that in the CPO group, and that in the CPOMT2A− group was significantly lower than that in the CPO group (Fig. 8C, D). Collectively, these results indicated that MT2A decreased CPO-induced mitochondrial dysfunction and promoted HUVEC proliferation.

Fig. 6
figure 6

Effects of MT2A on the viability of HUVECs under CPO. (A) Column chart showing the copper content of HUVECs in different group. (B) Column chart showing the viability of HUVECs in different groups at 48 h via the CCK-8 assay. (C) Representative fields showing the results from the tube formation assays in different groups. Bar = 100 μm. The results were quantified by (D) the total number of tubes, nodes, branches and meshes. (E) Representative fields showing the results from the scratch wound assays at 48 h obtained for each group. Bar = 200 μm. (F) The results were quantified by the percentage of wound closure at 48 h. (G) Representative fields showing the results from the Transwell migration assays for each group. Bar = 50 μm. (H) The results were quantified by the invaded cells per field. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01, **: P < 0.001. HUVEC: human umbilical vein endothelial cell; CPO: copper overload; ES: elesclomol; MT2A: metallothionein 2 A; SD: standard deviation

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Fig. 7
figure 7

The effect of MT2A on the cuproptosis process induced by CPO. (A) Representative western blot showing the expression of DLAT, SDHB and HSP70 normalized to the expression of β-actin in different groups. (B) Densitometric analyses of DLAT, SDHB and HSP70 expression normalized to the expression of β-actin in different groups. (C) Immunofluorescence staining showing DLAT (green) and MitoTracker (red). Scale bar = 20 μm. (D) Quantitative analysis of immunostaining of DLAT foci in different groups. The error bars represent the ± SDs. *: P < 0.05, **: P < 0.01. HUVEC: human umbilical vein endothelial cell; CPO: copper overload; MT2A: metallothionein 2 A; SD: standard deviation

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Fig. 8
figure 8

MT2A relieves the impairment of mitochondrial structure and function caused by CPO in HUVECs. (A) TEM results showing changes in the mitochondrial structure in the different groups. The white arrows indicate normal mitochondria. The orange arrows indicate swelling. The yellow arrows indicate vacuoles. White scale bar = 1 μm. Yellow scale bar = 0.5 μm. (B) Quantification of the mitochondrial number, abnormal mitochondrial percentage, and average mitochondrial minor axis length. (C) JC-10 results showing the mitochondrial function of HUVECs in different groups. (D) Column chart showing the ratio of aggregated JC-10 to monomeric JC-10 in the different groups. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01. HUVEC: human umbilical vein endothelial cell; CPO: copper overload; TEM: transmission electron microscopy; MT2A: metallothionein 2 A; SD: standard deviation

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CPO inhibits cerebral angiogenesis in 2VO + EMS rats

To test the role of copper in angiogenesis after revascularization, we established a CPO combined with 2VO + EMS rat model. Compare with the 2VO + EMS alone, long-term elesclomol feeding significantly increased the copper content in rat brain tissue (Fig. 9A), and resulted in significantly greater oligomer-DLAT expression and less CD31 expression (Fig. 9B-D). The immunofluorescence results revealed that, in the 2VO + EMS + CPO group, the expression of DLAT (including both oligomers and nonoligomers) was significantly greater than that in the 2VO + EMS group, whereas the expression of CD31 in the 2VO + EMS + CPO group was significantly lower than that in the 2VO + EMS group (Fig. 9E, F). These findings illustrated that CPO inhibited angiogenesis in chronically ischemic brains after revascularization surgery.

Fig. 9
figure 9

Effects of CPO on angiogenesis in the brains of 2VO + EMS rats. (A) Column chart showing the copper concentration in brain tissue from different groups. (B) Representative western blot showing the expression of DLAT and CD31 normalized to the expression of β-actin in different groups. (C) Densitometric analyses of oligomer-DLAT and CD31 expression normalized to the expression of β-actin in different groups. (D) Column chart showing the changes in DLAT and CD31 mRNA expression in brain tissue from rats. (E) Immunofluorescence staining showing DLAT (green) and CD31 (red) expression. Scale bar = 20 μm. (F) CD31 was segmented and quantified in each condition. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01. CPO: copper overload; 2VO: 2-vessel occlusion; EMS: encephalo-myo-synangiosis; SD: standard deviation

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MT2A alleviates the inhibitory effect of CPO on cerebral angiogenesis in 2VO + EMS rats

To prove the ability of MT2A to neutralize CPO and promote angiogenesis, we performed intraventricular lentivirus injection, and successful transfection was confirmed by WB and qPCR (Fig. 10A-C). The copper content of the brain tissue in the CPO group was significantly greater than that in the control group, while there was no significant difference between any other two CPO-induced groups (Fig. 10D). The immunofluorescence results revealed that the accumulation of DLAT in the CPOMT2A+ group was significantly lower than that in the CPO group, while the count of CD31(+) cells in the CPOMT2A+ group was significantly greater than that in the CPO group. In contrast, in the CPOMT2A− group, the accumulation of DLAT was significantly greater and the count of CD31(+) cells was significantly lower than that in the CPO group (Fig. 10E, F). The TEM results revealed that the number of mitochondria was significantly greater, the average mitochondrial minor axis length was significantly shorter, and the percentage of abnormal mitochondria was significantly lower in the CPOMT2A+ group than in the CPO group. In contrast, the mitochondrial number was significantly lower, the average mitochondrial minor axis length was significantly longer, and the percentage of abnormal mitochondria was significantly greater in the CPOMT2A− group compare with the CPO group (Fig. 10G, H). The results of the MRI-ASL sequence revealed that the perfusion ratio in the CPO group was significantly lower than that in the control group, but the perfusion ratio in the CPOMT2A+ group was significantly greater than that in the CPO group (1.69 ± 0.07 vs. 1.44 ± 0.14, P < 0.05). Compared with that in the CPO group, the perfusion ratio in the CPOMT2A− group was significantly lower (0.85 ± 0.04 vs. 1.44 ± 0.14, P < 0.01) (Fig. 11A-C). The results of the MWM test revealed that the rats in the CPOMT2A+ group spent significantly more time in the target quadrant than those in the CPO group did (42.03 ± 7.56 s vs. 25.52 ± 7.47 s, P < 0.01) and passed the platform significantly more frequently (2.33 ± 0.75 vs. 0.66 ± 0.51, P < 0.05). In addition, the escape latency on day 5 was significantly shorter in the CPOMT2A+ group than in the CPO group (14.32 ± 2.74 s vs. 29.66 ± 4.32 s, P < 0.01). In contrast, the CPOMT2A− group spent significantly less time in the target quadrant than the CPO group did (19.33 ± 3.50 s vs. 25.52 ± 7.47 s, P < 0.05), and the cross-platform time was significantly shorter than that of the CPO group was (0.24 ± 0.13 vs. 0.66 ± 0.51, P < 0.05), resulting in a significantly longer escape latency than that of the CPO group was (33.56 ± 5.42 s vs. 29.66 ± 4.32 s, P < 0.05). The swimming speed did not significantly differ among the groups, suggesting that the above differences were due to cognitive dysfunction rather than motor dysfunction (Fig. 11D-G). There results demonstrated that MT2A can alleviate the impairment of angiogenesis caused by CPO and promote improvements in CBP and cognitive function in rats.

Fig. 10
figure 10

Effects of MT2A on angiogenesis in the brains of 2VO + EMS rats with CPO. (A) Representative western blot showing the expression of MT2A normalized to the expression of β-actin in different groups. (B) Densitometric analyses of MT2A expression normalized to the expression of β-actin in different groups. (C) mRNA levels of MT2A in different groups. (D) Column chart showing the copper concentration in brain tissue from different groups. (E) Immunofluorescence staining showing DLAT (green) and CD31 (red) expression. Scale bar = 20 μm. (F) DLAT foci and CD31 were segmented and quantified in each condition (n = 5). (G) Representative fields of TEM images showing changes in the mitochondrial structure of ECs. White scale bar = 1 μm. Yellow scale bar = 0.5 μm. The white arrows indicate normal mitochondria. The orange arrows indicate swelling. The yellow arrows indicate vacuoles. (H) Quantification of the mitochondrial number, abnormal mitochondrial percentage, and the average mitochondrial minor axis length. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01.MT2A: metallothionein 2 A; 2VO: 2-vessel occlusion; EMS: encephalo-myo-synangiosis; CPO: copper overload; EC: endothelial cell; TEM: transmission electron microscopy

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Fig. 11
figure 11

Results of MRI-ASL and the MWM test in 2VO + EMS rats. (A) MRI-ASL films showing differences in CBP between the different groups. The EMS side is surrounded by a gray dotted line. (B) Column chart showing the perfusion ratios in different groups. (C) Representative swimming paths obtained from the different groups. (D) Average escape latency in the different groups. (E) Number of platform crossings in different groups. (F) Time spent in the target quadrant by the rats in different groups. (G) Column chart showing the swimming speed in different groups. The error bars represent the ± SDs. ns: P > 0.05, *: P < 0.05, **: P < 0.01. CBP: cerebral blood perfusion; MRI-ASL: magnetic resonance imaging-arterial spin labeling; MWM: Morris water maze; 2VO: 2-vessel occlusion; EMS: encephalo-myo-synangiosis; CPO: copper overload; ROI: region of interest; SD: standard deviation

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Discussion

The relationship between intracellular CHI and the regulation of EC proliferation remains unclear. In this study, we first found that the expression of cuproptosis-related genes (DLAT, FDX1 and SDHB) differed between DM samples (blood supplying tissue) from Matsushima grade C and Matsushima grade A patients, which suggested that cuproptosis might be related to the regulation of EC proliferation under hypoxia. Our results further revealed that CPO, a prerequisite of cuproptosis, could inhibit EC proliferation and angiogenesis under hypoxia by interfering with mitochondrial structure and function. Additionally, increasing the expression of MT2A effectively relieved the decline in EC activity caused by CPO and ultimately promoted EC proliferation and cerebral angiogenesis under hypoxia (Fig. 12). A detailed discussion is presented below.

Fig. 12
figure 12

Schematic showing the mechanism through which MT2A relieves the mitochondrial impairment caused by copper overload

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CPO may affect cerebral angiogenesis in CICD patients

Copper is considered a key factor in promoting angiogenesis, which can upregulate the expression of HIF-1α and VEGF, and stimulate the eNOS pathway [19, 20]. However, different concentrations of copper ions have different proangiogenic effects [21]. Recently, Tsvetkov proposed “cuproptosis”, a new mode of cell death, that occurs through the induction of protein lipoylation in the tricarboxylic acid (TCA) cycle during mitochondrial respiration [18]. These findings suggested that the toxic effect of high concentrations of copper on ECs may be due to cuproptosis, and highlighted the potential role of maintaining CHI in EC proliferation. In this study, DM tissues (blood supplying tissue for indirect revascularization surgery) from CICD patients with either Matsushima grade A or Matsushima grade C revascularization were obtained for a comparative analysis of cuproptosis-related genes. DM tissues from Matsushima grade C patients presented greater FDX1 and DLAT expression and lower SDHB expression, indicating that Matsushima grade C presented a graeter level of cuproptosis in DM tissue, witch caused poor revascularization. FDX1 is an upstream regulator of cuproptosis. This protein promotes DLAT lipoylation and inhibits iron‒sulfur cluster proteins by reducing Cu2+ to Cu+, which promotes cuproptosis [22]. Copper can directly bind to DLAT and induce oligomer-DLAT formation, and an increase in insoluble oligomer-DLAT leads to cytotoxic stress, which induces cell death [23]. A study by Jae Ho Seo showed that the SDHB protein is related to mitochondrial activity [24]. Research on EC cuproptosis has not yet been reported; whereas, the upregulation of FDX1 and DLAT and downregulation of SDHB in the DM tissues of Matsushima grade C patients initially suggested that this CPO-induced cell death inhibits EC proliferation under hypoxia. These results led us to further explore the potential regulatory mechanism of CPO on angiogenesis and the neutralizing strategies of CPO.

The CMR mechanism is involved in regulating EC activity and proliferation

Most of the published literature relies on the removal of copper ions to verify the importance of copper ions for biological functions, and few studies have clarified the effects of excessive copper ions on angiogenesis [25]. Methods for establishing cell and animal CPO models have been sporadically reported in recent years [26,27,28]; for example, Tsvetkov established CPO models of various tumor cells in vitro via elesclomol and CuCl2 and discovered cuproptosis [18]. However, the optimal concentrations of elesclomol and CuCl2 and the exact CPO modeling method for ECs are not well established.

Previous studies on CPO in tumor cells often used a CuCl2 concentration of 1 µM plus different concentrations of elesclomol [18, 29]. Considering the bidirectional effect of copper on EC, we additionally studied the effects of a high CuCl2 concentration (10 µM) [30] and a low CuCl2 concentration (0.1 µM). The results showed that a high CuCl2 concentration (10 µM) presented excessive cytotoxicity, and a low CuCl2 concentration (0.1 µM) did not induce a decrease in HUVEC viability (Fig. S1). However, when the CuCl2 concentration was set at 1 µM, 10− 3 M elesclomol reduced HUVEC viability for the first time (Fig. 4); therefore, these concentrations were used to establish a CPO model of ECs in vitro. Given that cuproptosis is a mode of cell death based on oligomerization of protein lipoylation in the TCA cycle in mitochondria and that oligomer-DLAT is a signature manifestation of cuproptosis, we conducted additional cell experiments aimed at determining mitochondrial function and the expression and location of DLAT. According to the WB results, the ECs under CPO conditions had more oligomer-DLATs, which was consistent with the results of Tsvetkov and Shengqi Huo [18, 30]. Immunofluorescence also revealed that the accumulation of DLAT mainly occurred near the mitochondria. Since the accumulation of DLAT may lead to acute proteotoxic stress, we then examined the expression of SDHB and HSP70. The SDHB protein is an important protein in the inner mitochondrial membrane [31]. In a hypoxic environment, SDHB is beneficial for reducing damage to mitochondrial complex II in ECs [32]. Therefore, the downregulation of SDHB in this study suggests the occurrence of mitochondrial damage. HSP70 is a classical intracellular inflammatory protein and is expressed at extremely low levels in normal ECs. The induction of HSP70 indicates acute proteotoxic stress in cuproptosis [33]. These results suggest that CPO induces a decrease in EC activity in a hypoxic environment through a mitochondrial mechanism.

Furthermore, our results suggest that the impact of CPO on mitochondria in HUVECs is reflected in both their structure and function. Evidence has indicated that changes in mitochondrial structure and function may lead to changes in cell activity [34,35,36]. In vitro, the mitochondrial structure was obviously altered in HUVECs with CPO, as indicated by swelling and the appearance of vacuoles, and this change was accompanied by a decrease in mitochondrial number. Additionally, according to JC-10 staining, mitochondrial function was significantly decreased. These findings indicate that CPO impacts EC activity mainly through mitochondrial mechanisms. On the basis of the above evidence, we speculate that the CMR mechanism is involved in regulating HUVEC activity and proliferation, including interference with the TCA cycle and damage to mitochondrial structure and function.

Interestingly, our study revealed that a low concentration of copper ions was beneficial to HUVEC viability, which is consistent with previous publications on ECs and cardiomyocytes [37]. Our results showed that HUVEC activity was increased when elesclomol was administered alone. We believe that this result occurred because the copper ions present at an extremely low concentration in the medium were transported into the cells by elesclomol, promoting cell activity. A similar phenomenon was also observed in Jung Jin Hwang’s study [38]. Therefore, when studying the effects of CPO on EC activity, it is more appropriate to use the elesclomol group as the control group.

MT2A promotes angiogenesis by countering CPO

MT2A is a protein rich in cysteine that can bind excessive amounts of free heavy metal ions and reduce the damage caused by heavy metal ions to cells [6]. A few previous studies have indicated the necessity of MT2A in EC proliferation. However, the underlying mechanism remains unclear [4, 39, 40]. We hypothesized that MT2A can alleviate the mitochondrial damage caused by CPO by chelating excessive copper ions in ECs. In vitro, we transfected HUVECs with the MT2A overexpression plasmid and siRNA, and confirmed the success of transfection via WB and qPCR to demonstrate that potential off-target effects did not affect our results. We compared the activity of HUVECs between groups with different expression levels of MT2A and found that the activity of HUVECs under CPO conditions significantly increased when MT2A expression was upregulated. WB and immunofluorescence analysis revealed that HUVECs with higher MT2A levels presented lower oligomer-DLAT expression and less DLAT-positive staining around the mitochondria. The upregulation of SDHB by MT2A overexpression indicated that the inhibition of the TCA cycle by CPO seems to be reduced. HSP70 downregulation also revealed that acute proteotoxic stress from copper ions was weakened by MT2A overexpression, which may promote EC proliferation. Additionally, normal mitochondrial function depends on structural integrity [41]. To determine whether MT2A is involved in maintaining normal mitochondrial function, we used TEM and JC-10. The TEM and JC-10 staining results revealed that the HUVEC group with higher expression of MT2A had fewer abnormal mitochondria than did the CPO group and that mitochondrial function was improved in the HUVEC group with higher expression of MT2A compared with the CPO group. In addition, high expression of MT2A promoted an increase of the number of mitochondria in HUVECs with CPO. However, without inducing CPO, MT2A overexpression had no effect on HUVEC viability or mitochondrial function. These results indicated that MT2A can promote EC proliferation by rescuing the mitochondrial damage caused by CPO.

Then, we conducted animal experiments to further study the relieving effect of MT2A on CPO. 2VO + EMS rats were used to establish an angiogenic microenvironment after CCI, which could simulate the angiogenic environment after indirect revascularization surgery in the clinic [42, 43]. To determine whether MT2A can promote EC proliferation in chronically ischemic brains, we regulated the expression of MT2A in the rat brain by IV lentivirus injection and randomly tested the expression of MT2A in 3 rats per group, indicating successful transfection without off-target effects. We found that MT2A also alleviated the inhibition of EC proliferation caused by CPO. These results indicated that MT2A can promote angiogenesis in chronically ischemic brains by relieving the impairment of mitochondrial function caused by CPO, resulting in improvements in CBP and cognitive function.

In addition, MT2A has a high antioxidant capacity and may affect EC proliferation by scavenging free radicals and protecting EC from oxidative stress damage [44,45,46]. Mitochondria are the main sites of intracellular reactive oxygen species (ROS) production, and MT2A may also protect mitochondrial function by scavenging the ROS produced by mitochondria. The effect of ROS on EC proliferation is twofold and complex. In this study, the ability of MT2A to scavenge CPO-induced ROS may benefit EC proliferation and mitochondrial function. Without CPO, regulating the expression of MT2A under hypoxia had no effect on EC proliferation, which may also be involved in this complex regulatory network of ROS. Moreover, evidence has shown that MT2A can activate the Hippo [40, 47] and the PI3K-Akt-mTOR pathways [48, 49], which are potential pathways for studying angiogenesis.

Clinical translational application

For clinical application, we are performing a series of preliminary studies to develop a hydrogel patch for the ischemic brain, which contains exosomes that express high levels of MT2A [50]. Exosomes can deliver large amounts of growth-promoting factors, while hydrogels exhibit good tissue compatibility and slowly release exosomes to achieve long-term proangiogenic effects [51]. We believe that this application could benefit CICD patients, similar to the application of hydrogel patches in the treatment of wound healing [52] and myocardial injury [53].

Limitations

There are several limitations in this study. First, copper ions could not be completely removed from the HUVEC culture medium during the experiment, so the influence of the originally existing copper ions in the medium cannot be ruled out. To solve this problem, we chose the elesclomol group as the control group instead. Second, immunofluorescence could not distinguish oligomers or nonoligomers of DLAT. Third, our study could not clarify whether the proliferation of ECs was affected by the training of the rats during the MWM test. Fourth, we only established a 2VO rat model to explore the revascularization of CICD, and whether MT2A can promote angiogenesis in an acute ischemia model needs further study. Fifth, other mechanisms to chelate copper or improve mitochondrial function in promoting angiogenesis should be further investigated.

Conclusion

CPO inhibited EC proliferation and angiogenesis by affecting mitochondrial structure and function, whereas MT2A promoted EC proliferation and cerebral angiogenesis by neutralizing excessive copper ions from CPO. These findings suggested a new treatment approach for CICD.

Data availability

The data presented in this study are available on request from the corresponding author.

Abbreviations

2VO:

2-vessel occlusion

CCI:

Chronic cerebral ischemia

CICD:

Chronic ischemic cerebrovascular disease

CHI:

Copper homeostasis imbalance

CMR:

Copper–mitochondria regulatory

CPO:

Copper overload

DM:

Dura mater

DSA:

Digital subtract angiography

EC:

Endothelial cell

EMS:

Encephalo-myo-synangiosis

HUVEC:

Human umbilical vein endothelial cell

MT2A:

Metallothionein 2 A

MRI:

Magnetic resonance imaging

MWM:

Morris water maze

TEM:

Transmission electron microscopy

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Acknowledgements

The authors wish to thank Qingyang Liu for the excellent technical support. The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.

Funding

This research was supported by Guangzhou Key Research and Development Program under Grant No. 2023B03J0191; and by Research Project of Yuedong Hospital, the Third Affiliated Hospital of Sun Yat-sen University under Grant No. YDC2022001; and by Basic Research Project of Sun Yat-sen University under Grant No. b202408011053050001.

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Author notes

  1. Ni Mo MD, Chuyang Tai MD and Yang Yang MD contributed equally to this work.

Authors and Affiliations

  1. Department of Neurosurgery, Third Affiliated Hospital of Sun Yat-Sen University, No. 600 Tianhe Road, Guangzhou, Guangdong Province, PR China

    Ni Mo, Chuyang Tai, Cong Ling, Baoyu Zhang, Cian Yao, Hui Wang & Chuan Chen

  2. Department of Radiology, Third Affiliated Hospital of Sun Yat-Sen University, No. 600 Tianhe Road, Guangzhou, Guangdong, PR China

    Yang Yang

  3. Department of Neurology, Third Affiliated Hospital of Sun Yat-Sen University, No. 600 Tianhe Road, Guangzhou, Guangdong, PR China

    Lei Wei

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  1. Ni Mo
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  4. Cong Ling
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Contributions

Ni Mo: Writing– original draft, Investigation, Formal analysis, Methodology, Charting, Visualization, Validation; Chuyang Tai: Writing– original draft, Investigation, Formal analysis, Methodology, Visualization, Validation; Yang Yang: Writing– original draft, Formal analysis, Methodology; Cong Ling: Writing– review & editing, Data curation; Baoyu Zhang: Writing– review & editing, Data curation; Lei Wei: Writing– review & editing, Visualization; Ci’an Yao: Writing– review & editing; Hui Wang: Writing– review & editing, Conceptualization, Supervision; Chuan Chen: Writing– review & editing, Conceptualization, Supervision, Project administration, Funding acquisition.

Corresponding authors

Correspondence to Hui Wang or Chuan Chen.

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Ethics approval and consent to participate

The study was conducted following the Declaration of Helsinki; the Ethics Committee of the third affiliated hospital of Sun Yat-sen university, China, approved the study (II2024-091). Informed consent was obtained from all subjects involved in the study. The animal experiment was performed in compliance with the ARRIVE guidelines and approved by the Animal Ethics Committee of Guangzhou Huateng Biopharmaceutical Technology Co., Ltd. (IACUC No. C202304-5).

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All authors give consent for the publication of the manuscript in Journal of Translational Medicine.

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The authors declare that they have no competing interests.

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Mo, N., Tai, C., Yang, Y. et al. MT2A promotes angiogenesis in chronically ischemic brains through a copper–mitochondria regulatory mechanism. J Transl Med 23, 162 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06163-5

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Journal of Translational Medicine

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