Decoding post-myocardial infarction coronary microvascular dysfunction: The SP1-driven STAT3/KCa3.1/eNOS protective mechanism

Figures

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  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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  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…

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  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…

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  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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  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…

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  Figure 6:. Blockage of the eNOS pathway reverses 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, with the results showing substantial reduction in branch points and segment numbers. Measurements were performed in triplicate. (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. eNOS: Nitric oxide synthase, HCAECs: Human coronary artery endothelial cells, ECM: Extracellular matrix, EdU: 5-ethynyl-2'-deoxyuridine, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.