INTRODUCTION

Myocardial infarction (MI) represents one of the leading causes of death and disability worldwide.[1] Despite advancements in the treatment of the acute phase, the long-term prognosis after MI is severely affected by cardiac microvascular dysfunction.[2,3] Cardiac microvascular dysfunction not only limits blood supply to the myocardium but may also lead to heart failure.[4] Elevated homocysteine (Hcy) levels are closely associated with the occurrence of cardiovascular diseases;[5,6] thus, the damaging effect observed on the coronary microvasculature post-MI is notably important.[7] However, the specific mechanisms of Hcy-induced coronary microvascular dysfunction after MI have not been fully elucidated, and effective treatment strategies are currently lacking.[8,9]

The transcription factor SP1 plays an important role in various physiological and pathological processes, including cell proliferation, differentiation, and apoptosis.[10] The role of SP1 in the cardiovascular system has garnered increasing attention.[11] SP1 can affect cardiovascular function by regulating multiple signaling pathways, among which the signal transducer and activator of transcription 3 (STAT3), conductance calcium-activated potassium channel protein 4 (KCNN4, also known as KCa3.1), and endothelial nitric oxide synthase (eNOS) pathways are considered important mechanisms.[12-14] After a new dysfunction is induced by Hcy, the STAT3/eNOS signaling pathway becomes inhibited.[15] In addition, Hcy inhibits the activity of SP1.[16] STAT3 is a crucial signal transducer and activator of transcription involved in myocardial protection and angiogenesis.[17,18] KCa3.1 regulates endothelial cell membrane potential and calcium influx, which affects vasodilatory function.[19] eNOS produces nitric oxide (NO) and is an essential molecule for the maintenance of endothelial function.[20] SP1 may play a crucial role in the reversal of Hcy-induced coronary microvascular dysfunction post-MI by activating the STAT3/KCa3.1/eNOS pathway.

Although previous studies have highlighted the potential role of SP1 in the cardiovascular system, its specific mechanism in Hcy-induced coronary microvascular dysfunction post-MI remains unclear.[21,22] In particular, whether SP1 can reverse Hcy-induced microvascular dysfunction through activation of the STAT3/KCa3.1/eNOS pathway and the detailed molecular processes involved have not been adequately explored. Furthermore, effective therapeutic strategies targeting Hcy-induced microvascular dysfunction after MI are lacking. Therefore, in-depth research on the role of SP1 in this pathological process could not only elucidate the pathophysiological mechanisms of microvascular dysfunction following MI but also provide a theoretical basis for the development of new treatment strategies.

This study aimed to investigate the mechanism by which SP1 reverses Hcy-induced coronary microvascular dysfunction after MI through activation of the STAT3/KCa3.1/eNOS pathway. We conducted a series of in vivo and in vitro experiments to systematically explore the role and molecular mechanisms of SP1 in Hcy-induced microvascular dysfunction post-MI. Specifically, we assessed the effects of SP1 on microvascular function after MI, analyzed the role of the STAT3/KCa3.1/eNOS pathway in this process, and explored the interaction between SP1 and this pathway. In addition, we investigated the potential therapeutic role of SP1 in Hcy-induced microvascular dysfunction following MI. Through this study, we hope to reveal the mechanistic role of SP1 in microvascular dysfunction post-MI, provide theoretical support for the development of new therapeutic strategies, and offer fresh insights into the prevention and treatment of cardiovascular diseases.

MATERIAL AND METHODS MI model

The experimental animals were 10-12-week-old C57 wild-type mice weighing 22-28 g, purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a carbon dioxide (CO2) incubator at temperatures of 23-26°C, with humidity levels maintained at 50-55%, and a 12 h light/dark cycle. This study was approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, approval No. 2021-028. A total of 140 C57BL/6 mice were randomly divided into the following groups: sham, MI, hyperhomocysteinemia (HHcy)+MI, HHcy+MI+SP1, HHcy+MI+SP1+Stattic (10 mg/kg, HY-13818, MedChemExpress, Monmouth Country, NJ, USA),[23] and HHcy+MI+SP1+N(ω)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg, N5751, Sigma-Aldrich, St. Louis, MO, USA).[24] In accordance with the manufacturer’s instructions, mice in the MI+overexpression (OE)-normal control (NC) and MI+OE-SP1 groups received 100 µL tail-vein injections of OE-NC adenovirus or OE-SP1 adenovirus (5 × 108 plaque forming unit ( PFU)/mouse, provided by RiboBio, Guangzhou, China). Five days post-adenoviral injection, the MI model was induced. Myocardial ischemia was established through ligation of the left anterior descending (LAD) artery through a thoracotomy. The mice, positioned supine on the surgical table, were anesthetized with inhaled isoflurane (R510-22-10, RWD, Shenzhen, China), shaved at the precordial region, disinfected with povidone-iodine, and provided with oxygen for respiration. An incision was made perpendicular to the ribs, and the fourth intercostal space was exposed through blunt dissection of subcutaneous muscles. The heart was exteriorized to place a suture 2 mm below the left atrial appendage. Then, the heart was returned to the thoracic cavity, and the air was removed from the chest before the incision was temporarily cut off using a hemostatic clamp. The wound was sutured in layers using a 6-0 nylon thread. After closure, the anesthesia was stopped, and the mice were observed until they regained consciousness. The sham group underwent the same procedure without LAD ligation. The HHcy group was fed a diet supplemented with 1.8 g/L DL-Hcy (HY-101404, MedChemExpress, Monmouth County, NJ, USA).[25] All surgeries were performed on a clean bench with the aid of a surgical microscope (SZX16, Olympus Corporation, Tokyo, Japan). Assessment of postoperative cardiac function was completed using a cardiac ultrasound diagnostic instrument (Logic E9, GE HealthCare, Chicago, Illinois, USA). At the experimental endpoint, the mice were deeply anesthetized with 5% isoflurane in oxygen. Once deep anesthesia was confirmed by the absence of pedal reflexes, the mice were euthanized through cervical dislocation. Hearts were immediately excised and processed in accordance with experimental requirements.

Fluorescein isothiocyanate (FITC)-conjugated Lycopersicon esculentum lectin staining

To assess myocardial microvascular reperfusion, we used FITC-conjugated L. esculentum lectin (FL-1171, Vector Laboratories, San Francisco, CA, USA) as a tracer. LAD ligation was performed on the mice using a clean bench and observation using an inverted phase-contrast microscope (CKX41, Olympus Corporation, Tokyo, Japan). Immediately after surgery, 100 µL FITC-conjugated lectin was injected through the tail vein using a 1 mL sterile syringe. The tracer was allowed to circulate for 30 min. Following circulation, cross-sections of the heart were cut at the level of papillary muscles. Sections were prepared at a thickness of 5 µm using a cryostat (CM1950, Leica, Weztlar, Germany). The sections were then observed and analyzed using a fluorescence microscope (BX53, Olympus Corporation, Tokyo, Japan) for the evaluation of microvascular blood flow restoration. The fluorescence microscope was equipped with a FITC filter set (excitation wavelength: 495 nm, emission wavelength: 519 nm). Images were captured using a charge-coupled device camera (DP80, Olympus Corporation, Tokyo, Japan) and analyzed quantitatively (mean fluorescence intensity = IntDen/Area) using ImageJ software (v1.8.0.345, National Institutes of Health, Bethesda, MD, USA).

CD31 immunofluorescence staining

Cardiac tissue sections (5 µm thickness) from each group were prepared using a cryostat. The sections were processed routinely with xylene (534056, Sigma-Aldrich, St. Louis, MO, USA) and graded ethanol (459844, Sigma-Aldrich, St. Louis, MO, USA), washed with phosphate buffer saline (PBS, pH 7.4, P4417, Sigma-Aldrich, St. Louis, MO, USA), and immersed in 0.3% Triton X-100 solution (T8787, Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 15 min. After blocking with 5% goat serum (16210064, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, the sections were incubated overnight at 4°C with a specific CD31 monoclonal antibody (1:100, ab28364, Abcam, Cambridge, MA, USA). The following day, the sections were washed with PBS and incubated with FITC-conjugated goat anti-rabbit immunoglobulin G (1:500, A-11008, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature in the dark for 1 h. After each antibody incubation, the sections were washed with PBS. Nuclei were stained with 4’,6-diamidino-2’-phenylindole (DAPI, D1306, Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 10 min and mounted with antifade mounting medium (H-1000, Vector Laboratories, San Francisco, Ca, USA). Imaging was performed using a laser confocal microscope (FV3000, Olympus Corporation, Tokyo, Japan). All experimental procedures were conducted under dark or light-protected conditions to prevent fluorescence quenching. Image analysis was performed using ImageJ software.

Western blot analysis

After the heart was harvested, the atria and right ventricle were removed, and proteins were extracted from the left ventricular myocardium (including non-infarcted and scar areas). Protein extraction was performed using radioimmunoprecipitation assay lysis buffer (R0010, Solarbio, Beijing, China). After centrifugation, the samples were sonicated and heat denatured. Protein concentration was determined using a Bicinchoninic Acid Assay Protein Assay Kit (A045, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). A total of 20 µg protein lysates were electrophoresed and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel using a 164-5050 electrophoresis system (Bio-RAD, Hercules, CA, USA) and then transferred onto a polyvinylidene fluoride membrane (IPVH00010, Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk at 25°C for 1 h and then incubated overnight at 4°C with the following primary antibodies (Invitrogen, Carlsbad, CA, USA): STAT3 (1:1000, PA5-12038), phospho-STAT3 (1:1000, PA5-121277), KCa3.1 (1:1000, PA-33875), eNOS (1:1000, PA1-037), phosphor-Enos (1:1000, PA5-17917), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, PA1-988). Subsequently, the membrane was incubated with horse radish peroxidase (POD)-conjugated secondary antibodies (1:10000, 31460, Invitrogen, Carlsbad, CA, USA) at 25°C for 1 h. Protein bands were developed using an enhanced chemiluminescence kit (BL520b, Biosharp Life Science, Hefei, Anhui, China) under a chemiluminescence apparatus (Image Quant LAS4000, GE Healthcare, Chicago, IL, USA). Band quantification was performed with ImageJ software, and the results were expressed as values normalized to GAPDH levels.

Human coronary artery endothelial cell (HCAEC) Culture

HCAECs (FC-0032, Lifeline Cell Technology, Frederick, MD, USA) preserved in liquid nitrogen were immediately placed on dry ice and thawed in a 37°C water bath. The HCAECs were authenticated through short-tandem repeat profiling and tested negative for mycoplasma contamination. Once the cell suspension was melted, the cells were seeded into culture flasks and incubated in a CO2 incubator. The medium was changed to endothelial cell medium (#1001, ScienCell Research Laboratories, San Diego, CA, USA) the following day. The cells were passaged every 2-3 days, with passages 4-6 used for experiments. When the cells reached 70%-80% confluence, they were digested with trypsin (HY-129047, MedChemExpress, Monmouth Country, NJ, USA), and cell status was observed using an inverted phase-contrast microscope. After counting with an automatic microplate reader (BioTek Synergy LX, Palo Alto, CA, USA), cell density was adjusted to 8 × 104 cells/mL and seeded into 96-well plates (control wells with 100 µL cell-free medium) at 100 µL/well and with edge wells filled with 150 µL PBS, and then incubated at 37°C and 5% CO2. Treatments were added the following day. Stattic (STAT3 inhibitor) was administered at a concentration of 5 µM for 24 h. L-NAME was administered at a concentration of 1 mM for 24 h.

Cell hypoxia treatment

HCAECs were passaged and cultured using a specialized endothelial cell medium. When cells reached approximately 80% confluence, their status was observed using an inverted phase-contrast microscope. For the hypoxia treatment group, the cells were transferred to a tri-gas incubator (5% CO2, 1% oxygen, and 94% nitrogen N2) for a hypoxic culture to simulate a hypoxic microenvironment. The HCAECs maintained under normal culture conditions served as the normal group, and they were incubated at 37°C in a CO2 incubator.

SP1 OE processing

The coding sequence (CDS) sequence of the target gene SP1 was obtained from the National Center for Biotechnology Information website. Based on this sequence, the upstream primer was designed as 5'-CCGCTCGAGCTATGAGCGACCAAGATCACT-3' and the downstream primer as 5'-CGGAATTCTCAGAAGCCATTGCCACTG-3'. The primers were designed and synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The SP1 gene was amplified using SYBR Green Real-time polymerase chain reaction (PCR) Master Mix (QPK-201, TOYOBO, Osaka, Japan) for the PCR. The PCR program was set as follows: 95°C predenaturation for 5 min; 30 cycles of 95°C denaturation for 30 s, 57°C annealing for 30 s, and 72°C extension for 3 min and 30 s, followed by a 72°C final extension for 7 min. Amplification was performed using a real-time quantitative PCR instrument (QuantStudio 6, Thermo Fisher Scientific, Waltham, MA, USA). The PCR product was subjected to 1.2% agarose gel electrophoresis using a Bio-Rad 164-5050 electrophoresis system. The recovered product was digested with XhoI and EcoRI along with a pEGFP-C1 empty vector (Clontech, Mountain View, CA, USA) using the restriction enzymes obtained from Takara. The digested products were ligated with T4 DNA ligase (2011A, Takara, Kafu City, Yamanashi Prefecture, Japan) at 16°C for over 4 h. The ligated product was transformed into DH5α competent cells (EC0112, Invitrogen, Carlsbad, CA, USA), plated on LuriaBertani agar plates containing kanamycin, and incubated at 37°C for 10 h. Clones were selected and subjected to colony PCR using Taq polymerase (R001A, Takara, Kafu City, Yamanashi Prefecture, Japan) for amplification. Positive clones were selected and cultured, and plasmids were extracted using a Plasmid Mini Kit (12123, Qiagen, Dusseldorf, Germany). The extracted plasmids were verified through double-enzyme digestion with XhoI and EcoRI.

Methylthiazolyldiphenyl-tetrazolium (MTT) assay

Cells from the blank, control, and overexpression groups were washed with PBS and adjusted to a density of 1 × 105 cells/mL. Then, they were reseeded into six-well plates and incubated in a CO2 incubator for 72 h. Freshly prepared MTT bromide solution (0.5%, 20 µL/well, 250 mg Thiazolyl Blue, M1020, Solarbio Technology, Beijing) was added, and the cells were left to stand for 4 h before the removal of the medium. After PBS washing, 150 µL dimethyl sulfoxide solution (100 mL, D8371, Solarbio Technology, Beijing) was added to each well, and the plates were shaken for 15 min to fully dissolve the crystals, followed by a 2 h static incubation. The absorbance at 490 nm was measured using an automatic microplate reader. The experiment was repeated thrice to obtain an average value.

5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay

The processed HCAECs were adjusted to a density of 1 × 105 cells per well, seeded on coverslips in six-well plates, and cultured in a CO2 incubator for 24 h. An EdU Cell Proliferation Kit (C10337, Invitrogen, Carlsbad, CA, USA) was used, and the cells were incubated with a 10 µmol/L EdU solution for 2 h. They were then fixed with 4% paraformaldehyde PBS solution at room temperature for 15 min, followed by PBS washing. The cells were permeabilized with 0.5% Triton X-100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) for 10 min, washed with PBS, and incubated in the dark with DAPI (D1306, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. After another PBS wash, coverslips were mounted with an antifade mounting medium (H-1000, Vector Laboratories, San Francisco, CA, USA), and images were acquired using a laser confocal microscope (Olympus FV3000, Japan). Blue fluorescence represented cell nuclei, and green fluorescence indicated EdU-positive cells. Six fields of view were randomly selected to determine the total number of cells and EdU-positive cells, with the EdU-positive rate calculated as follows: EdU-positive rate (%) = (Number of EdU-positive cells/Total number of cells) × 100%.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay

The TUNEL apoptosis assay was performed using a TUNEL assay kit (11684795910, Roche, Basel, Switzerland). The TUNEL reaction mixture was prepared with 50 µL TdT enzyme + 450 µL fluorescein-labeled dUTP solution for the experimental group. For the control group, 50 µL fluorescein-labeled dUTP solution was added, and for the positive control group, 100 µL DNase1 (EN0521, Thermo Fisher Scientific, Waltham, MA, USA) was included in the study. Each sample was treated with 50 µL reaction mixture, covered with coverslips or sealing film, and incubated in a dark humidified chamber at 37°C for 60 min. The samples were washed thrice with PBS. A drop of PBS was added, and apoptotic cells were counted under a fluorescence microscope. After air drying, 50 µL converter-POD was added to the samples, covered with coverslips or sealing film, and incubated in a dark humidified chamber at 37°C for 30 min, followed by three washes with PBS. The samples were then mounted using an antifade mounting medium and observed under a fluorescence microscope. All procedures were conducted under sterile conditions. Experimental data were analyzed using ImageJ software and statistically processed using GraphPad Prism software. The apoptosis rate was calculated using the formula: Apoptosis rate (%) = (Number of TUNEL-positive cells/Total number of cells) × 100%.

Extracellular matrix (ECM) gel tube formation assay

A 24-well plate was coated with 300 µL Matrigel Matrix (354234, BD Pharmingen, Franklin Hoo, New Jersey, USA) and allowed to set at 37°C for 30 min. HCAECs were adjusted to a density of 1 × 105 cells/well, seeded into each well, and incubated in a CO2 incubator for 6 h. At the end of the experiment, images were captured using a Nikon inverted phase-contrast microscope (Eclipse Ti-S, Nikon, Tokyo, Japan) at 100x magnification. The total length of the tubules was calculated using Image-Pro Plus image analysis software (6.0, Media Cybernetics, Rockville, MD, USA). The experiment was repeated thrice.

Statistical methods

Data were analyzed using the Statistical Package for the Social Sciences 24.0 statistical software (IBM Corp., Armonk, NY, USA). Measurement data are presented as mean ± standard deviation (mean ± standard deviation [SD]). An independent sample t-test was used to analyze measurement data between two samples. One-way analysis of variance was used for multigroup comparisons, followed by least significant difference-t tests for pairwise comparisons. P < 0.05 was considered statistically significant. Figures and graphs were created using GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). All experiments were repeated at least thrice, and data are expressed as mean ± SD. The significance level for statistical analyses was set at α = 0.05, with P < 0.05 indicating a statistically significant difference.

RESULTS SP1 improves cardiac microvascular dysfunction and angiogenesis

To evaluate the effects of SP1 on cardiac microvascular function and angiogenesis after MI of HHcy mice, we conducted a series of experiments. Initially, FITC-labeled L. esculentum lectin was used to detect myocardial microvascular reperfusion. This lectin specifically binds to glycoproteins on the surface of microvascular endothelial cells and serves as a reliable indicator of microvascular function and density. Two weeks after MI, fluorescence microscopy revealed a substantial reduction in lectin staining intensity in the MI group compared with the sham-operated group (Sham), which indicates an impaired microvascular function due to MI. Staining intensity was further diminished in the HHcy+MI+NC group, which suggests exacerbated microvascular dysfunction post-MI due to HHcy. Notably, staining intensity in the SP1 treatment group (HHcy+MI+SP1) was significantly higher than that in the HHcy+MI+NC group and neared those of the Sham group levels, which indicates that SP1 effectively ameliorated microvascular dysfunction induced by HHcy post-MI [Figure 1a]. Quantitative analysis further confirmed this observation [Figure 1b]. To further assess the effect of SP1 on cardiac angiogenesis, we employed immunofluorescence labeling of CD31, an endothelial cell marker, to quantify cardiac vascular density. Confocal microscopy images showed a marked reduction in CD31-positive vessels in the MI groups compared with the Sham group, with a more pronounced reduction in the HHcy+MI+NC group. However, the SP1 treatment group (HHcy+MI+SP1) exhibited a significant increase in CD31-positive vessels, which approached those of the Sham group levels [Figure 1c]. Quantitative analysis of CD31 further confirmed this trend [Figure 1d].

Figure 1: SP1 improves cardiac microvascular dysfunction and angiogenesis post-MI in hyperhomocysteinemic mice. (a) Detection of myocardial microvascular perfusion using FITC-labeled Lycopersicon esculentum lectin 2 weeks post-MI. Observations were made under a fluorescence microscope to capture green fluorescence, which is indicative of microvascular endothelial cell immunostaining. Scale bar: 50 µm (200×). (b) Quantitative analysis of tomato lectin staining. (c) Immunofluorescence labeling of CD31 to capture cardiac vascular density, with green representing vessels and blue indicating the nuclei stained with DAPI. Representative confocal microscopy images show the colocalization of CD31 (green) in ventricular tissues. Scale bar: 50 µm, (200×). (d) Quantitative analysis of CD31. ✶✶P < 0.01. n = 6 for each group. MI: Myocardial infarction, FITC: Fluorescein isothiocyanate, DAPI: 4’,6-diamidino-2’-phenylindole.

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SP1 protects cardiac microvascular function via the STAT3/KCa3.1/eNOS pathway

This study aimed to explore how the transcription factor SP1 improves HHcy-induced cardiac microvascular dysfunction post-MI through activation of the STAT3/KCa3.1/eNOS pathway. We evaluated cardiac microvascular function across various experimental groups: Sham, MI, HHcy+MI+NC, HHcy+MI+SP1, and HHcy+MI+SP1+Stattic. For microvascular function assessment 2 weeks after MI, FITC-labeled L. esculentum lectin was used to analyze myocardial microvascular reperfusion. Fluorescence microscopy revealed a significantly higher lectin staining intensity in the Sham and HHcy+MI+SP1 groups than in the MI and HHcy+MI+SP1+Stattic groups [Figure 2a], which indicates the effective improvement of myocardial microvascular function by SP1. Quantitative analysis of lectin staining further confirmed SP1’s protective role, which showed significantly higher staining intensity in the Sham and HHcy+MI+SP1 groups compared with the MI and HHcy+MI+SP1+Stattic groups. This finding demonstrates SP1’s protective effects on microvascular reperfusion [Figure 2b]. Subsequently, we employed immunofluorescence with CD31 labeling to capture cardiac vascular density, with DAPI used for nuclear labeling. Representative confocal images displayed the distribution of CD31-positive vessels among the groups, with quantitative analysis indicating a notably higher number of CD31-positive vessels in the Sham and HHcy+MI+SP1 groups compared with the MI and HHcy+MI+SP1+Stattic groups. These results further support SP1’s promoting effect on cardiac angiogenesis [Figure 2c and d]. Finally, Western blot analyses of p-STAT3, KCa3.1, and p-eNOS revealed significantly higher relative expression levels in the Sham and HHcy+MI+SP1 groups compared with the MI and HHcy+MI+SP1+Stattic groups [Figure 2e-h]. This finding suggests that SP1 promotes the restoration of cardiac microvascular function by upregulating the activity of this signaling pathway.

Figure 2: SP1 protects cardiac microvascular function through the STAT3/KCa3.1/eNOS pathway. (a) Detection of myocardial microvascular perfusion using FITC-labeled Lycopersicon esculentum lectin 2 weeks post-MI. Observations were made under a fluorescence microscope, and the green fluorescence indicative of microvascular endothelial cell immunostaining was captured. Scale bar: 50 µm, (200×). (b) Quantitative analysis of tomato lectin staining. (c) Immunofluorescence labeling of CD31 to capture cardiac vascular density, with green representing vessels and blue indicating the nuclei stained with DAPI. Representative confocal microscopy images show the colocalization of CD31 (green) in ventricular tissue. Scale bar: 50 µm, (200×). (d) Quantitative analysis of CD31. (e) Western blot analysis of p-STAT3, KCa3.1, and p-eNOS in hyperhomocysteinemic (HHcy) + MI mice post-SP1 treatment, or with concurrent SP1 and Stattic (STAT3 inhibitor) treatment. (f-h) Relative expression of p-STAT3/STAT3, KCa3.1, and p-eNOS/eNOS. ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. n = 6 for each group. MI: Myocardial infarction, FITC: Fluorescein isothiocyanate, DAPI: 4’,6-diamidino-2’-phenylindole, STAT3: Signal transducer and activator of transcription 3, p-STAT3: Phospho signal transducer and activator of transcription 3, eNOS: Nitric oxide synthase, p-eNOS: Phospho nitric oxide synthase, KCa3.1: Conductance calcium-activated potassium channel protein 4, HHcy: Hyperhomocysteinemia.

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L-NAME reverses SP1’s protective effect on cardiac microvascular function

This study focused on the investigation of SP1’s role in enhancing cardiac microvascular function in Hcy-induced MI mice through activation of the STAT3/KCa3.1/eNOS pathway. It particularly examined the effect of the eNOS inhibitor L-NAME on this protective effect. Results show that SP1 treatment considerably improved myocardial microvascular function. FITC-labeled L. esculentum lectin staining intensity and CD31-positive vascular density were markedly improved in the Sham and HHcy+MI+SP1 groups, with significantly higher staining intensity and positive vessel numbers observed compared with those of the MI and HHcy+MI+NC groups [Figure 3a-d]. When eNOS was inhibited using L-NAME (HHcy+MI+SP1+L-NAME group), this improvement was partially reversed, which indicates the crucial role of eNOS in SP1-mediated cardiac protection.

Figure 3: L-NAME reverses the protective effect of SP1 on cardiac microvascular function. (a) Detection of myocardial microvascular perfusion using FITC-labeled Lycopersicon esculentum lectin in HHcy + MI mice post-SP1 treatment or with concurrent SP1 and L-NAME (eNOS inhibitor) treatment. Observations were made under a fluorescence microscope, and the green fluorescence indicative of microvascular endothelial cell immunostaining was captured. Scale bar: 50 µm, (200×). (b) Quantitative analysis of tomato lectin staining. (c) Immunofluorescence labeling of CD31 to capture cardiac vascular density, with green representing vessels and blue indicating the nuclei stained with DAPI. Representative confocal microscopy images show the colocalization of CD31 (green) in ventricular tissues. Scale bar: 50 µm, (200×). (d) Quantitative analysis of CD31. (e) Western blot analysis of p-STAT3, KCa3.1, and p-eNOS in HHcy + MI mice post-SP1 treatment, or with concurrent SP1 and L-NAME treatment. (f-h) Relative expressions of p-STAT3/STAT3, KCa3.1, and p-eNOS/eNOS. ns: Not significant, ✶✶P < 0.01, ✶✶✶P < 0.001. n = 6 for each group. L-NAME, N(ω)-nitro-L-arginine methyl ester, FITC: Fluorescein isothiocyanate, HHcy: Hyperhomocysteinemia, MI: Myocardial infarction, DAPI: 4’,6-diamidino-2’-phenylindole, p-STAT3: Phospho signal transducer and activator of transcription 3, KCa3.1: Conductance calcium-activated potassium channel protein 4, p-eNOS: Phospho nitric oxide synthase, STAT3: Signal transducer and activator of transcription 3, eNOS: Nitric oxide synthase.

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In Western blot analysis, although the relative expressions of p-STAT3 and KCa3.1 were significantly increased in the SP1-treated group, the phosphorylation level of eNOS was significantly reduced with L-NAME, which suggests that eNOS activity is an integral component of SP1’s protective effects. Overall, the findings indicate that SP1 can enhance myocardial microvascular function through activation of the STAT3/KCa3.1/eNOS signaling pathway and that inhibition of eNOS can reverse such protective effects [Figure 3e-h]. Thus, eNOS plays a crucial role in SP1-mediated cardiac microvascular protection, which provides important insights into potential therapeutic targets for cardiovascular disease treatment.

SP1 alleviates Hcy and hypoxia-induced cytotoxicity in HCAECs through the STAT3/KCa3.1/eNOS signaling pathway

To investigate the protective effects of SP1 on Hcy-induced damage in HCAECs, we conducted a series of experiments. Under conditions of 1 mMol Hcy and hypoxia, the cytotoxicity of HCAECs increased in a time-dependent manner, with cell viability declining from 0 h to 12 h [Figure 4a]. Subsequently, we assessed the protective effects of SP1 against Hcy and hypoxia-induced damage in HCAECs. MTT assay results reveal that Hcy (1 mMol) and hypoxia significantly reduced cell viability compared with the control group, whereas SP1 treatment effectively reversed this damage, which restored cell viability close to the control levels [Figure 4b]. EdU assay further confirmed the protective effects of SP1, with the results showing that the proliferation rate of SP1-treated cells was significantly higher than that of the Hcy and hypoxia-treated group and comparable to that of the control [Figure 4c and d]. TUNEL assay results indicate that SP1 significantly reduced the apoptosis rate induced by Hcy and hypoxia, although it did not fully restore it to control levels [Figure 4e and f].

Figure 4: SP1 mitigates Hcy and hypoxia-induced cytotoxicity in HCAECs through the STAT3/KCa3.1/eNOS Pathway. (a) Time-dependent cytotoxicity of HCAECs with concurrent Hcy and hypoxia treatment. (b) Protective effect of SP1 on HCAEC cytotoxicity. (c and d) EdU assay demonstrating the protective effect of SP1 on HCAEC cytotoxicity. Scale bar: 50 µm, (200×). (e and f) TUNEL assay demonstrating the protective effect of SP1 on HCAEC cytotoxicity. Scale bar: 50 µm, (200×). ✶✶P < 0.01, ✶✶✶P < 0.001. n = 3 for each group. Hcy: Homocysteine, HCAECs: Human coronary artery endothelial cells, STAT3: Signal transducer and activator of transcription 3, eNOS: Nitric oxide synthase, KCa3.1: Conductance calcium-activated potassium channel protein 4, EdU: 5-ethynyl-2'-deoxyuridine, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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Inhibition of the STAT3/KCa3.1/eNOS pathway blocks SP1’s protective effect

To further confirm the importance of the STAT3/KCa3.1/eNOS pathway in SP1’s protective effect on HCAECs, we conducted a series of experiments. First, we evaluated the angiogenic ability of HCAECs using tube formation assay on an ECM gel [Figure 5a]. Results show that 1.0 mmol/L Hcy and 6 h hypoxia considerably inhibited the tube formation abilities of HCAECs compared with those of the control group. SP1 treatment effectively reversed this inhibition, which restored tube formation ability close to control levels. However, the protective effect of SP1 was markedly diminished when the STAT3 inhibitor Stattic was added, which reduced the tube formation ability to levels similar to those observed in the Hcy and hypoxia treatment groups. Quantitative analysis of HCAEC network formation further confirmed this observation [Figure 5b]. In comparison with the Hcy and hypoxia treatment groups, the numbers of branch points and segments were substantially increased in the SP1-treated group and reached levels close to those of the control. However, in the SP1 and Stattic co-treatment groups, these parameters decreased again to levels similar to those of the Hcy and hypoxia treatment groups. These results indicate the crucial role of STAT3 activation in exerting SP1’s protective effects. To delve deeper into the molecular mechanism, Western blot analysis was performed [Figure 5c]. Results indicate that Hcy and hypoxia treatments considerably lowered the phosphorylation levels of STAT3, KCa3.1, and eNOS. SP1 treatment effectively restored the phosphorylation levels of these proteins. However, when the STAT3 inhibitor Stattic was introduced, the phosphorylation of STAT3 was inhibited, and the levels of KCa3.1 and eNOS were also remarkably reduced. Quantitative analysis confirmed this finding and revealed that the relative expression levels of p-STAT3/STAT3, KCa3.1, and p-eNOS/eNOS were significantly higher in the SP1-treated group than in the Hcy and hypoxia-treated group and comparable to that of the control. Meanwhile, in the SP1 and Stattic co-treatment groups, these ratios decreased again to levels similar to those in the Hcy and hypoxia treatment groups [Figure 5d-f]. These findings collectively indicate that the STAT3/KCa3.1/eNOS pathway plays a critical role in the protective effects of SP1 against Hcy and hypoxia-induced damage in HCAECs. SP1 enhanced the HCAECs’ function and angiogenic capability by activating STAT3, which, in turn, activated KCa3.1 and eNOS. Inhibition of STAT3 considerably diminished SP1’s protective effects, which influenced not only the phosphorylation levels of STAT3 but also the downstream activation of KCa3.1 and eNOS and cellular function.

Figure 5: Inhibition of the STAT3/KCa3.1/eNOS pathway blocks the protective effect of SP1 on HCAECs. (a) Assessment of angiogenic capacity of HCAECs through tube formation assay on ECM gel. Images show the network formation observed after 6 h of incubation. Scale bar: 100 µm, (100×). (b) Quantitative analysis of HCAEC network formation, which displays the significant reduction in branch points and segment numbers compared with those in the H + hypo group. Measurements were performed in triplicate. (c) Western blot analysis of p-STAT3, KCa3.1, and p-eNOS in myocardial cells induced by Hcy and hypoxia post-SP1 treatment or with concurrent SP1 and STAT3 inhibitor or eNOS inhibitor treatment. (d-f) Relative protein expressions of p-STAT3/STAT3, KCa3.1/KCa3.1, and p-eNOS/eNOS analysis by Western Blot. ✶✶P < 0.01, ✶✶✶P < 0.001. n = 3 for each group. STAT3: Signal transducer and activator of transcription 3, KCa3.1: Conductance calcium-activated potassium channel protein 4, eNOS: Nitric oxide synthase, HCAECs: Human coronary artery endothelial cells, Hcy: Homocysteine, ECM: Extracellular matrix, p-STAT3: Phospho signal transducer and activator of transcription 3, p-eNOS: Phospho nitric oxide synthase.

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Blocking the eNOS pathway reverses SP1’s protective effect on HCAECs

To further validate the importance of the eNOS pathway in SP1’s protective effect on HCAECs, we conducted a series of experiments. We initially assessed the angiogenic capability of HCAECs using tube formation assay on ECM gel [Figure 6a]. Results indicate that 1.0 mmol/L Hcy and 6 h hypoxia treatment significantly inhibited the tube formation ability of HCAECs compared with the control group. SP1 treatment effectively reversed this inhibition, which restored tube formation ability close to control levels. However, SP1’s protective effect was markedly diminished after the addition of the eNOS inhibitor L-NAME, which reduced tube formation ability to levels si