Introduction

Coronary heart disease (CHD) caused by coronary atherosclerosis is a common cause of heart attack. Clinically, patients with CHD exhibit symptoms such as angina pectoris induced by temporary myocardial ischemia. According to the China Cardiovascular Health and Disease Report 2022, there are an estimated 330 million people with cardiovascular diseases in China, among which 11.39 million cases are CHD.¹ The high incidence and mortality rate of CHD pose a high burden to society. Currently, the treatment of CHD mainly includes percutaneous coronary intervention, bypass surgery, and pharmacological therapy. However, many patients are not allowed to receive interventional procedures, and for those receiving treatment, the clinical outcomes are limited by myocardial damage and subsequent heart failure.² The exploration of novel effective therapeutic strategies is still in urgent need.

Endothelial dysfunction is closely associated with the onset of CHD.³ Endothelial progenitor cells (EPCs) can differentiate into mature endothelial cells and are considered as critical contributors to neovascularization and vascular repair, showing the potential for CHD therapy.⁴ Studies have demonstrated that EPCs facilitate the vascular remodeling of ischemic and damaged tissues in animal models and human cases of ischemia.^5,6 However, the transplantation of EPCs is challenged by the number, function, and aging.⁷ It is essential to develop effective strategies to improve the survival of EPCs in the recipient.

Long noncoding RNAs (lncRNAs) are noncoding transcripts over 200 nucleotides long and possess no protein-coding ability.^8,9 They are crucial regulators of gene expression at epigenetic, transcriptional, and post-transcriptional levels.¹⁰ The regulatory mechanisms of lncRNAs include chromatin remodeling, pre-mRNA splicing, mRNA translation, and microRNAs (miRNAs) sponging. Increasingly, studies have shown that numerous lncRNAs are aberrantly expressed in various diseases including cardiovascular diseases. For example, lncRNA uc003pxg.1 facilitates endothelial cell proliferative capacity and migration ability by regulating miR255p in coronary artery disease.¹¹ Long noncoding RNAs NEAT1 suppressed the survival of human coronary endothelial cells (HCAECs) and induced cell apoptosis via the miR-140-3p/MAPK1 axis in coronary atherosclerotic heart disease.¹² Growth arrest specific 5 (GAS5) is indicated to be related to the progression of cardiovascular diseases. For example, GAS5 is reported to protect against the norepinephrine-induced cardiomyocyte apoptosis by downregulating sema3a in myocardial infarction.¹³ Growth arrest specific 5 silencing is revealed to inhibit the proliferative capacity and senescence of endothelial progenitor cells by regulating the miR-223/NAMPT axis and the PI3K/AKT pathway.¹⁴ Growth arrest specific 5 also regulates the senescence of vascular smooth muscle cells by sponging miR-665 to upregulate SDC1.¹⁵ In the previous study, GAS5 was preliminarily found to participate in the progression of CHD by regulating the function of EPCs through the miR-495-3p/SIX1 and IGF2BP2/NRF2 dual-regulatory pathways.¹⁶ However, the specific molecular mechanism by which GAS5 regulates the function of EPCs still needs further exploration.

This study intended to investigate the role of the novel molecular mechanism of GAS5 in CHD. It was hypothesized that GAS5 affected CHD progression by regulating the function of EPCs via upregulating the expression of the ferroptosis inhibitor SLC7A11 and the glycolysis transcriptional regulator SIX1, which might provide novel insight into CHD therapy.

Methods

Patient Sample Collection

The atherosclerotic peripheral blood (2 mL) was collected from 30 CHD patients and 30 healthy individuals in ethylenediaminetetraacetic acid (EDTA) tubes in our hospital. The plasma was collected after centrifugation for 15 minutes at 3000 rpm at 4°C and then stored at −80°C for further analysis. All participants have signed the informed consent. The Ethics Committee of our hospital has approved this study.

Isolation and Incubation of Endothelial Progenitor Cells

Peripheral blood from CHD patients and healthy individuals were collected and diluted with phosphate buffer saline (PBS). Next, the isolation of peripheral blood mononuclear cells (PBMCs) was conducted with ficoll density gradient centrifugation, followed by washing and centrifugation to remove the platelet. The supernatant was removed and PBMCs were resuspended in culture medium to adjust a concentration at 1 × 10⁶. Next, cells were cultured on 6-well plates coated with fibronectin, and each well was added with 2 mL EGM-2 (LONZA, USA) and maintained at 37 °C with 95% air and 5% CO₂.

After culturing for 4 days, to select EPCs, the plates were washed with PBS for removing the non-attached cells. The attached cells were collected and subject to flow cytometry analysis with for surface markers including CD34, CD31, and CD45.¹⁷

Cell Transfection

Two specific siRNAs targeting GAS5 (si-GAS5#1/#2) and negative control (si-NC), SIX1 and IGF2BP1 overexpression vector and pcDNA3.1 empty vector (oe-NC), miR-23a-3p mimic and NC mimic were designed and synthesized by GenePharma (Shanghai, China). The transfection vector or plasmid was conducted with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 48 hours.

Reverse Transcription-Quantitative Polymerase Chain Reaction

Total RNA was extracted using TRIzol reagent. A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) was applied for synthesizing cDNAs. Quantitative polymerase chain reaction was carried out with a LightCycler FastStart DNA MasterPLUS SYBR Green I mix (Roche, Germany) on the ABI-7500 platform. Gene expression was calculated with the 2^−ΔΔCt method, and β-actin and U6 served as internal references.

Western Blot

Radioimmunoprecipitation assay lysis buffer was used for total protein collection. A Bicinchoninic Acid Kit was adopted for the detection of protein concentration. Next, the protein sample was electrophoresed by SDS–polyacrylamide gel electrophoresis (Bio-Rad, USA) and electro-transferred to nitrocellulose membranes. Subsequently, 5% skim milk was applied for blocking these membranes, which were then incubated overnight with the primary antibodies at 4°C, with β-actin as a loading control. After rinsing with Tris-Borate-Sodium Tween-20 (TBST), the membranes were incubated with a secondary antibody for 60 minutes. The enhanced chemiluminescence (Pierce, USA) was used for the visualization of protein signals.

Fluorescent In Situ Hybridization

Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) was used to determine the subcellular localization of GAS5 following the producer’s protocol. Endothelial progenitor cells were seeded in slides, processed with 4% paraformaldehyde, rinsed with PBS, and processed using 0.5% Triton X-100. Subsequently, EPCs were hybridized with probes for GAS5 and IGF2BP1 (RiboBio, China) overnight at 37 °C. 4’,6-diamidino-2-phenylindole (DAPI) was applied to stain the nucleus. The images were captured by a confocal fluorescence microscope (Leica, Wetzlar, Germany).

Co-Immunoprecipitation

Endothelial progenitor cells were treated using lysis buffer, centrifuged, and the supernatant was incubated with anti-IgG or anti-SIX1 overnight at 4°C. Next, the mixture was captured by incubation with protein A/G agarose beads for 4 hours. After washing with ice-cold IP buffer, the samples were centrifuged at 1000 × g for 2 minutes to remove the supernatant. Subsequently, the proteins were separated from beads using an immunoblotting loading buffer. The supernatant was harvested and the level of binding proteins was subjected to Western blot.

RNA Immunoprecipitation

Endothelial progenitor cells were lysed and the lysate was incubated with anti-IgG (negative control) or anti-Ago2 antibody conjugated with magnetic beads at 4°C overnight. The beads were collected and washed, followed by proteinase K treatment. RNA extraction from the complex was conducted using phenol: chloroform: isoamyl alcohol solution. Immunoprecipitated RNAs from anti-IgG or anti-Ago2 were subject to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.

RNA Pull-Down

Biotinylated GAS5, miR-23a-3p, and control NC probes were provided by Ribobio. Endothelial progenitor cells were lysed and incubated overnight with biotinylated probes-conjugated with magnetic beads at 4°C. After collecting and washing beads, the RNA or protein in the complex was purified. The enrichment of IGF2BP1, miRNAs, or SLC7A11 was subject to western blot or RT-qPCR analysis.

Dual-Luciferase Reporter Assays

Growth arrest specific 5 and SLC7A11 3′UTR containing including wild-type (Wt) and mutated (Mut) binding sequence with miR-23a-3p were subcloned into pmirGLO vector (Promega, USA) for the construction of GAS5-wt/mut or SLC7A11-wt/mut vector. The vector was co-transfected in EPCs with miR-23a-3p/NC mimic for 48 hours. The luciferase activities were determined with a Promega dual-luciferase reporter kit.

Cell Viability

The viability of EPCs was detected using CCK-8 assays. Briefly, EPCs were seeded in 96-well plates (5 × 10³ cells in each well) and cultured for indicated time (1, 2, 3, 4 days). EPCs in each well were supplemented with 10 μL CCK-8 reagent and cultured for 120 minutes. The absorbance was analyzed at 450 nm.

Colony Formation Assay

Endothelial progenitor cells were seeded in 6-well plates at 500 cells per well. After a 2-week incubation, 4% polyformaldehyde was applied to fasten colonies, which were then dyed by 0.5% crystal violet for 1 hour. The colony number was finally calculated with a microscope.

Flow Cytometry Analysis

The apoptosis of EPCs was measured using an Annexin V-FITC/PI Apoptosis Kit (Mutisiences, China) following the producer’s guide. Endothelial progenitor cells after transfection were harvested, and stained with Annexin V-FITC and Propidium Iodide for 20 minutes at 4°C in darkness. Cell apoptotic rate was evaluated by FACScan Flow Cytometer, BD Biosciences, USA) and analyzed with FlowJo software.

Transwell Assays

The transwell assays were performed for evaluating the migration and invasion abilities of EPCs using Transwell chambers (Costar, USA) coated with or without matrigel (BD Biosciences, USA). Endothelial progenitor cells (5 × 10⁴) were suspended in culture medium (0.5 mL) containing 0.1% bovine serum albumin and added in the top Transwell chambers, and culture media with 20% FBS in the lower chambers. The incubation last for 24 hours. After PBS washing, and processing in 4% paraformaldehyde, EPCs were stained for 5 minutes with crystal violet solution. The migrated or invaded cells were counted and imaged with an inverted microscope.

Glucose Uptake Measurement

Endothelial progenitor cells were grown into 96-well plates at 1 × 10⁴ cells/well, and incubated at 37°C overnight. Next, EPCs were deprived of sugar for 2 hours. Then each well was supplemented with 10 μL 2-deoxy-d-glucose (2-DG) and cultured for 20 minutes. The glucose intake was determined at 412 nm wavelength.

Lactic Acid Production Assay

The production of lactic acid in EPCs was assessed using an L-lactic acid test kit (colorimetry). Endothelial progenitor cells were first grown in 96-well plates and cultured overnight at 37°C. After starvation for 120 minutes, the supernatant was harvested and lactic acid production was evaluated at 450 nm.

Extracellular Acidification Rate and Oxygen Consumption Rate Measurement

Endothelial progenitor cells were grown in 96-well plates added with 10% FBS and cultured at 37°C overnight. After measuring the baseline concentration, glucose, oligomycin, and 2-DG were supplemented into each well successively for extracellular acidification rate(ECAR) measurement. For oxygen consumption rate(OCR) measurement, oligomycin, FCCP, antimycin A, and rotenone were added successively. The results were analyzed using XF-96Wave software.

Malondialdehyde Detection

The malondialdehyde (MDA) content in the lysate of EPCs was detected using a Lipid Peroxidation Assay Kit (Abcam, UK) based on the manufacturer’s protocol. Briefly, EPCs after erastin treatment and indicated transfection were harvested and lysed, and subsequently centrifuged for 10 minutes at 13 000 × g for removing the insoluble material. Then thiobarbituric acid (TBA) solution was supplemented to well-containing samples and MDA standards, incubated at 95°C for 1 hour, cooled in an ice bath for 10 minutes, and transferred to wells of the microplate. The absorbance is measured at 532 nm with a microplate reader.

Glutathione (GSH)/oxidized glutathione (GSSG) Detection

The level of GSH/GSSG in EPCs was measured using a GSH and GSSG Assay Kit (Beyotime, China) based on the producer’s guide. The resulting mixture was measured using a microplate reader at 412 nm. The GSH level is calculated as GSH = Total Glutathione−GSSG × 2.

Iron Assay

Intracellular ferrous iron (Fe²⁺) level in EPCs was detected using an iron assay kit (Abcam, UK) following the manufacturer’s protocol. Endothelial progenitor cells were grown in a culture plate and exposed to erastin for 12 hours. Then cells were harvested, washed, and homogenized in iron assay buffer, followed by for 10-minute centrifugation at 4°C. Next, the supernatant was harvested and supplemented iron reducer into each well, mixed, and incubated for 30 minutes at ambient temperature. Subsequently, the iron probe was supplemented and mixed, followed by a 1-hour incubation in darkness. Finally, a colorimetric microplate reader was applied for absorbance detection at 593 nm.

Statistical Analysis

GraphPad Prism software (GraphPad Software Inc., San Diego, CA) was used for statistical analysis. The experiments were independently conducted at least 3 times. The normality of data distribution was evaluated with the Shapiro–Wilk test. Data were presented as the mean ± SD. Two-tailed Student’s t test was used for comparison between 2 groups, and one-way analysis of variance followed by Tukey’s post hoc test or two-way analysis of variance followed by Bonferroni’s post hoc test was applied for comparison among multiple groups. Pearson correlation analysis was used to evaluate the expression correlation between genes. P value less than .05 indicated statistical significance.

Results

Growth Arrest Specific 5 is Downregulated in Coronary Heart Disease and Accelerates Endothelial Progenitor Cell Proliferation, Migration, and Invasion

The GAS5 level was determined in the blood samples and EPCs collected from CHD patients (n = 30) and healthy individuals (n = 30). The results of RT-qPCR analysis identified that GAS5 was downregulated in both blood samples and EPCs of CHD patients compared with healthy controls (Figure 1A and B), suggesting its association with CHD progression and behaviors of EPCs. Next, The effects of GAS5 on EPC growth were investigated. The si-GAS5#1/2 or pcDNA3.1/GAS5 vector was transfected into EPCs to establish GAS5-silenced or overexpressed EPCs. The knockdown and overexpression efficiency of GAS5 was validated by RT-qPCR analysis (Figure 1C and D). Cell Counting Kit-8 assays demonstrated that GAS5 knockdown significantly decreased the viability of EPCs while GAS5 overexpression evidently enhanced EPC growth (Figure 1E and F). Consistently, colony formation assays showed that GAS5 silencing reduced the colony number of EPCs while GAS5 overexpression showed the contrary effects (Figure 1G and H). Based on flow cytometry analysis, GAS5 silencing induced a significant increase in the apoptotic rate of EPCs, while GAS5 overexpression significantly inhibited EPC apoptosis (Figure 1I and J). Furthermore, transwell assays demonstrated that migration and invasion of EPCs were repressed in response to GAS5 silencing, and enhanced in response to GAS5 overexpression (Figure 1K-N). Overall, GAS5 contributes to EPC growth, migration, and invasion in vitro.

Growth Arrest Specific 5 is Located in the Cell Cytoplasm of Endothelial Progenitor Cells

To determine the subcellular localization of GAS5 in EPCs, Fluorescence in situ hybridization (FISH) assays were conducted, and the results demonstrated the main distribution of GAS5 in the cell cytoplasm of EPCs (Figure 2A). Consistently, the results of subcellular fractionation assays also revealed that GAS5 was located in the cell cytoplasm of EPCs (Figure 2B). Collectively, these results suggested that GAS5 might exert its regulatory function at the post-transcriptional level.

Growth Arrest Specific 5 Promotes Glucose Metabolism Reprogramming and Inhibits Ferroptosis of Endothelial Progenitor Cells Via the IGF2BP1/SIX1 Axis

Growth arrest specific 5 can regulate cellular metabolism, which is closely associated with the fate of EPCs. Thus, the effects of GAS5 on glucose metabolic reprogramming of EPCs were further investigated. As revealed by immunofluorescence staining of 2-NBDG, the glucose uptake of EPCs was significantly suppressed by GAS5 knockdown (Figure 3A). Extracellular acidification rate and OCR are 2 indicators of cell glycolytic capacity. GAS5 silencing was found to reduce ECAR but elevate OCR in EPCs, suggesting that GAS5 regulated the glucose metabolism reprogramming of EPCs (Figure 3B and C). Meanwhile, GAS5 silencing was revealed to induce a significant decrease in glycolysis capacity, ATP level, and elevation in maximal respiration rate (Figure 3D-F). Consistently, glucose uptake testing with 2-DG also indicated that the glucose uptake of EPCs was decreased after silencing GAS5 (Figure 3G). Moreover, the level of lactate (lactic acid) production in GAS5-silenced EPCs was lower relative to the control cells (Figure 3H). Furthermore, GAS5 knockdown also caused the downregulation of PFKL, LDHA, and PKM2 in EPCs (Figure 3I). These results indicate that GAS5 reprograms metabolism to glycolysis. Additionally, GAS5 overexpression is indicated to repress the ferroptosis of hypoxic myocardial cells.¹⁸ For EPCs, GAS5 overexpression significantly reversed the erastin-induced elevation in Fe2+ level, MDA content, and ROS level and reduction in GSH/GSSG level (Figure 3J-M). The evidence indicates that GAS5 suppresses the ferroptosis of EPCs.

Growth Arrest Specific 5 Interacts with IGF2BP1 to Stabilize SIX1

The underlying mechanism of GAS5 to regulate glucose metabolism reprogramming and ferroptosis of EPCs was further investigated. As shown in Supplementary Figure 1A, the regulation of GAS5 on C-Myc, SIX1, and HIF-1 was explored, which are common transcription factors for metabolic reprogramming. The results showed that GAS5 knockdown significantly downregulated the expression of SIX1 in EPCs. Thus, the regulation of GAS5 on SIX1 in EPCs was explored. As predicted by the starbase database, IGF2BP1 was an RNA binding protein (RBP) binding to GAS5 and SIX1. RNA pulldown assays verified that IGF2BP1 was enriched in the complex pulled down by Bio-GAS5, which suggested that IGF2BP1 interacted with GAS5 in EPCs (Figure 4A). Meanwhile, RIP assays showed that GAS5 was immunoprecipitated by anti-IGF2BP1 in EPCs, which validated the binding between GAS5 and IGF2BP1 (Figure 4B). Moreover, FISH assays showed that GAS5 and IGF2BP1 were colocalized in the cytoplasm of EPCs (Figure 4C). As IGF2BP1 regulates the stability of various mRNAs, GAS5 was assumed to regulate SIX1 expression via IGF2BP1. Co-immunoprecipitation assays were conducted and showed that IGF2BP1 was immunoprecipitated by anti-SIX1 in EPCs (Figure 4D). Meanwhile, RIP assays revealed that SIX1 was precipitated by anti-IGF2BP1 antibody, while this binding was attenuated by GAS5 knockdown in EPCs, suggesting that GAS5 promoted the binding between IGF2BP1 and SIX1 in EPCs (Figure 4E). The effects of GAS5 silencing and IGF2BP1 overexpression on mRNA stability of SIX1 were evaluated in Actinomycin D (ActD)-treated EPCs. GAS5 knockdown significantly promoted the degradation of SIX1 mRNA, which was reversed by IGF2BP1 overexpression (Figure 4F), suggesting that GAS5 enhanced the mRNA stability of SIX1 via IGF2BP1. Consistently, RT-qPCR analysis revealed that SIX1 expression reduced by GAS5 silencing was partially restored by upregulating IGF2BP1 (Figure 4G). Moreover, the expression levels of SIX1 and GAS5 were positively correlated in blood samples of CHD patients (Figure 4H). Overall, these results indicated that GAS5 binds to IGF2BP1 to enhance the mRNA stability of SIX1.

Growth Arrest Specific 5 Promotes Glucose Metabolism Reprogramming by Upregulating SIX1

Whether SIX1 is involved in GAS5-mediated metabolic reprogramming of EPCs was further explored. SIX1 was overexpressed in EPCs, and the overexpression efficiency was verified by RT-qPCR analysis as well as western blot analysis (Figure 5A). As revealed by CCK-8 assays, SIX1 overexpression rescued the inhibition induced by GAS5 deficiency on the viability of EPCs (Figure 5B). The GAS5 silencing-induced reduction in ECAR and elevation in OCR was also significantly restored by SIX1 upregulation (Figure 5C and D). The results of immunofluorescence staining revealed that the glucose uptake of EPCs reduced by GAS5 knockdown was counteracted by overexpressing SIX1 (Figure 5E). Consistently, the GAS5 knockdown-induced reduction in glycolysis capacity, ATP level as well as elevation in maximal respiration rate of EPCs was significantly restored by SIX1 overexpression (Figure 5F-H). Meanwhile, the glucose uptake with 2-DG as well as the lactate production was significantly reduced in response to GAS5 silencing, while SIX1 overexpression reversed this inhibitory effect (Figure 5I and J). Moreover, western blot analysis demonstrated that SIX1 overexpression counteracted the GAS5 deficiency-caused downregulation in protein expression of PFKL, LDHA, and PKM2 in EPCs (Figure 5K). Overall, these results indicate that GAS5 regulates the glucose metabolism reprogramming of EPCs via SIX1.

Growth Arrest Specific 5 Binds to miR-23a-3p to Modulate SLC7A11 Expression

The mechanism of GAS5 to regulate ferroptosis of EPCs was further explored. Substantial literature has revealed that lncRNAs can also function as ceRNAs to regulate gene expression. Based on bioinformatics analysis, the candidate miRNA targets of GAS5 were explored on the ENCORI and lncbase databases. A total of 22 candidate miRNAs were screened by the 2 databases (Supplementary Figure 2A). The level of candidate miRNAs in EPCs from CHD patients and healthy controls was detected using RT-qPCR analysis. Only miR-23a-3p, miR-136-5p and miR-188-5p were found upregulated in the EPCs collected from CHD patients (Supplementary Figure 2B). Furthermore, the RNA pulldown assays showed that only miR-23a-3p was pulled down by bio-GAS5 probes in EPCs, indicating the interaction between miR-23a-3p and GAS5 (Supplementary Figure 2C). Meanwhile, the expression of miR-23a-3p and GAS5 was found to be negatively correlated in the EPCs from CHD patients (Figure 6A). RIP assays showed that GAS5 and miR-23a-3p were abundantly enriched in the precipitates of anti-Ago2, which indicated miR-23a-3p bound to GAS5 in EPCs (Figure 6B). Then miR-23a-3p was overexpressed in EPCs, and the overexpression efficiency was verified using RT-qPCR analysis (Figure 6C). The binding sequence between miR-23a-3p and GAS5 was predicted by the Encyclopedia of RNA Interactomes (ENCORI) database, and Wt or Mut luciferase reporter vector was constructed for assessing the interaction between miR-23a-3p and GAS5. The results demonstrated that miR-23a-3p overexpression induced the reduction in luciferase reporter activity of GAS5-wt luciferase reporter vector while showed no significant influence on the Mut group, suggesting that GAS5 bound to miR-23a-3p (Figure 6D and E). Meanwhile, RT-qPCR analysis showed evident upregulation of miR-23a-3p in GAS5-silenced EPCs (Figure 6F). Moreover, the downstream targets of miR-23a-3p was further investigated. MiR-23a-3p has also been revealed to promote the ferroptosis of cardiomyocytes by targeting ferroptosis mediator SLC7A11.¹⁹ Based on Pearson correlation analysis, the level of miR-23a-3p was found to be negatively correlated with SLC7A11 level in EPCs from CHD patients (Figure 6G). Furthermore, RNA pulldown assays demonstrated the enrichment of SLC7A11 in the RNA complexes pulled down by the bio-miR-23a-3p probe in EPCs (Figure 6H). Consistently, miR-23a-3p overexpression decreased the luciferase reporter activities in the wt group while not significantly altering those in the Mut group, suggesting that miR-23a-3p targeted SLC7A11 in EPCs (Figure 6I and J). Additionally, miR-23a-3p overexpression also caused a significant downregulation of SLC7A11 mRNA and protein levels in EPCs (Figure 6K). Then whether GAS5 regulated the miR-23a-3p/SLC7A11 axis in EPCs was investigated. The expression of GAS5 and SLC7A11 was in positive correlation in the EPCs from CHD patients (Figure 6L). SLC7A11 mRNA and protein expression also showed a significant decrease in GAS5-silenced EPCs (Figure 6M). Importantly, GAS5 overexpression induced an upregulation of SLC7A11 mRNA and protein, which was significantly reversed by overexpressing miR-23a-3p (Figure 6N and O). Overall, GAS5 binds to miR-23a-3p to upregulate SLC7A11 in EPCs.

Growth Arrest Specific 5 Inhibits Ferroptosis of Endothelial Progenitor Cells Via SLC7A11

Further investigation was conducted on whether SLC7A11 was involved in GAS5-mediated ferroptosis of EPCs. The viability of EPCs reduced in response to erastin treatment was reversed after GAS5 overexpression, while SLC7A11 silencing attenuated the protective effects of GAS5 on EPCs, as evidenced by the reduced viability of EPCs (Figure 7A). The Fe²⁺ level and MDA content increased by erastin treatment was reversed by GAS5 overexpression and showed a significant increase by silencing SLC7A11 (Figure 7B and C). Growth arrest specific 5 overexpression also restored the erastin-caused reduction in GSH/GSSG ratio and elevation in elevation in ROS level, which was both counteracted by silencing SLC7A11 in EPCs (Figure 7D and E). Additionally, the lactate dehydrogenase (LDH) release elevated by erastin was reduced by overexpressing GAS5 and showed significant elevation after the co-transfection of si-SLC7A11 (Figure 7F). Moreover, the levels of ferroptosis-related markers were detected using western blot. The erastin-caused downregulation of SLC7A11 and GPX4, as well as upregulation of ACSL4, was reversed by overexpressing GAS5, while the co-transfection of SLC7A11 showed counteractive effects, as evidenced by significantly decreased SLC7A11 and GPX4 expression and increased ACSL4 level in EPCs (Figure 7G). Overall, these findings suggest that GAS5 inhibits the ferroptosis of EPCs by upregulating SLC7A11.

Discussion

Cardiovascular diseases remain a major cause of death globally, taking nearly 18 million lives annually, among which CHD is responsible for an estimated 7.4 million death cases.²⁰ However, the therapeutic interventions for CHD are still limited, and patients suffer from complications with unfavorable outcomes. Thus, it is essential to explore effective therapeutic targets to improve the treatment of CHD.

LncRNAs are closely associated with the progression of cardiovascular diseases by regulating diverse biological processes including inflammation, cell proliferation, apoptosis, as well as senescence.²¹ Functionally, lncRNAs are known as regulators for mRNA decay or sponging miRNAs to prevent the inhibitory effects of miRNAs on the stability or the translation of their targets at post-transcriptional levels.^22,23 Amount of research has shown that GAS5 regulates the proliferation, migration, invasion as well as apoptosis of tumor cells.^24-26 In cardiovascular diseases, GAS5 is indicated to play diverse roles. For example, some studies have shown that GAS5 knockdown shows protective effects against high glucose-induced cardiomyocyte damage, myocardial ischemia/reperfusion injury, as well as myocardial infarction-induced cardiomyocyte apoptosis.^27-29 However, there are also some studies that reveal that GAS5 alleviates endothelial cell injury in cardiovascular diseases. For example, GAS5 overexpression enhances the proliferation, migration, and angiogenesis abilities of cardiac microvascular endothelial cells and inhibits cell apoptosis.³⁰ Additionally, a study has revealed that GAS5 knockdown suppresses EPC proliferation and facilitates EPC senescence.¹⁴ Thus, GAS5 can also play cardioprotective roles by modulating the function of EPCs. This study revealed that GSA5 was downregulated in the blood sample and EPCs in CHD patients compared with healthy individuals. The effects of GSA5 on the function of EPCs were further investigated.

In recent decades, the potential of endothelial progenitor cells has been noticed in the treatment of cardiovascular diseases. As a group of cells released from bone marrow into peripheral blood, EPCs effectively promote endothelial repair and neovasculogenesis, and are thus considered useful for the cell therapy of ischemic heart disease.³¹ Clinical studies have revealed that the proliferation, and migration abilities of EPCs are impaired and the apoptosis rate is elevated in acute coronary syndrome patients.^32,33 Moreover, restoring EPC function has been revealed to show protective effects against cardiovascular diseases. For example, miR-214 regulates EPC vasculogenesis and migration by targeting vascular endothelial growth factor (VEGF) in CHD.³⁴ G-protein-coupled receptor 4 (GPR4) overexpression promotes the neovessel formation of EPCs from patients with coronary artery disease by activating the STAT3/VEGFA pathway.³⁵ Sirtuin 1 alleviates mouse coronary atherosclerosis by facilitating EPC proliferation and migration.³⁶ In our work, GAS5 silencing represses the proliferative capacity, migration, and invasion ability, and elevates the apoptotic rate of EPCs. Growth arrest specific 5 overexpression was demonstrated to facilitate EPC growth, migration, invasion and suppress apoptosis, suggesting the beneficial effects of GAS5 by improving the function of EPCs against CHD.

Glucose metabolic reprogramming is induced by myocardial infarction. However, the changes cannot support cardiomyocyte proliferation to replace lost cells caused by the ischemic injury.³⁷ Metabolic reprogramming is indicated to promote cardiac regeneration following injury, while the underlying targets need further investigation. Wang et al³⁸ have found that PPARγ agonist promotes cardiac glucose metabolic reprogramming by enhancing glycolytic capacity and reducing mitochondrial reactive ROS production via HIF-1α to prevent hypoxia-induced cardiac dysfunction. A study reveals that VEGF-B reprograms myocardial metabolism to enhance cardiac function in ischemic heart disease.³⁹ In our study, GAS5 silencing was demonstrated to inhibit glucose metabolic reprogramming by reducing glycolysis capacity, glucose uptake, and lactate production. SIX1 is a critical transcription regulator of glycolysis. In our previous study, GAS5 was found to regulate glucose metabolism reprogramming of EPCs via the miR-495-3p/SIX1 and IGF2BP2/NRF2 dual-regulatory pathways.¹⁶ In this study, GAS5 was further found to directly bind to IGF2BP1 to enhance the mRNA stability of SIX1 in EPCs. Moreover, rescue assays proved that GAS5 promoted the glucose metabolic reprogramming of EPCs by upregulating SIX1.

Ferroptosis is a novel form of iron-dependent programmed cell death. Studies have shown that anti-ferroptosis strategies can alleviate cell injury in coronary heart disease. For example, SGK1 deficiency attenuates the erastin-caused ferroptosis of mouse aortic endothelial cells by reversing the erastin-caused decrease in SLC7A11, GPX4, and GSH/GSSG and elevation in lipid peroxidation as well as Fe accumulation.⁴⁰ SLC7A11 as an amino acid transporter plays a crucial role in regulating GSH synthesis and cellular ferroptosis. In our previous study, GAS5 was found to participate in the progression of CHD by regulating ferroptosis of EPCs through the miR-495-3p/SIX1 and IGF2BP2/NRF2 dual-regulatory pathways.¹⁶ The current study has confirmed for the first time that GAS5 can interact with miR-23a-3p to upregulate SLC7A11, thereby protecting EPCs from ferroptosis in CHD.

Conclusion

Growth arrest specific 5 is downregulated in EPCs collected from CHD patients, and overexpression of GAS5 promotes glucose metabolism reprogramming via the IGF2BP1/SIX1 axis and inhibits ferroptosis of EPCs via the miR-23a-3p/SLC7A11 axis. The findings of our study might provide novel therapeutic targets for EPC protection in CHD treatment.

Supplementary Materials

Footnotes

Ethics Committee Approval: This study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

Informed Consent: All participants had signed the informed consent.

Peer-review: Internally peer-reviewed.

Author Contributions: Conception – M.Z.; Design – M.Z., W.X., B.T.; Supervision – M.Z., Y.L.; Materials – W.X., B.T., Y.L.; Data Collection and/or Processing – W.X., B.T., Q.Z., Z.J.; Analysis and/or Interpretation – M.Z., Q.Z., Z.J.; Literature Review – Q.Z., Z.J.; Writing – M.Z.; Critical Review – Y.L. All authors read and approved the final manuscript.

Declaration of Interests: The authors have no conflicts of interest to declare.

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