Dynamics and implications of circulating anti-angiogenic VEGF-A165b isoform in patients with ST-elevation myocardial infarction

Figures

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  Figure 1. Flow chart showing the enrollment protocol of STEMI patients (A) and blood sampling (B). PCI, percutaneous coronary intervention; CMR, cardiac magnetic resonance; STEMI, ST-segment elevation myocardial infarction.

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  Figure 2. Temporal changes in levels of serum VEGF-A165b in STEMI patients and control subjects. Data are from controls (n = 20) and STEMI patients (n = 20) before reperfusion (time = 0 h) and at 24 h, 96 h and 1 month after primary percutaneous coronary intervention. Violin plots shows median values, interquartile range and 95% confidence intervals. ***p < 0.001, **p < 0.01, *p < 0.05 vs control subjects.

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  Figure 3. VEGF-A165b levels in STEMI patients are associated with larger infarct size and reduced left ventricular ejection fraction (LVEF). (A) Circulating VEGF-A165b levels (B) total VEGF-A levels and (C) VEGF-A165b/VEGF-A ratio in STEMI patients (n = 50) at 24 h after reperfusion and in control subjects (n = 23). Box plots show median and range values. *p < 0.05. (D) Spearman test correlations between VEGF-A165b and infarct size and (E) LVEF in STEMI patients.

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  Figure 4. Immunohistochemistry analysis of VEGF-A165b expression in heart tissue from autopsies of patients with previous history of myocardial infarction. (A) (I) representative images of picrosirius staining. (II) Myocardial sections were incubated with a mouse anti-human VEGF-A165b antibody (5 μg/ml) and specific labeling was detected with a biotin-conjugated goat anti-mouse secondary antibody. (III) and (IV) Myocardial sections were incubated with a mouse anti-human VEGF-A165b antibody (5 μg/ml) and immunoreactivity was visualized using Alexa Fluor 594 (VEGF-A165b, red) secondary antibodies. Nuclei were stained with DAPI (blue). Bars = 500 μm. (B) Densitometric analysis of VEGF-A165b immunofluorescent staining. Images from infarct and control sections were captured and digitized (Axio Observer A1, Carl Zeiss) and then analyzed with Image-Pro Plus analysis software (Media Cybernetics). Scoring was performed blinded on coded slides. Data represent mean ± SD of optical density in arbitrary units (a.u.). *p < 0.05 vs control. (C) Representative images showing colocalization of CD31/VEGF-A165b or VEGFR-2/VEGF-A165b in infarct myocardial tissue. Immunoreactivity was visualized using Alexa…

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  Figure 5. VEGF-A165b inhibits VEGF-A165-induced angiogenesis of human coronary artery endothelial cells (HCAEC). (A) Western blotting of VEGFR2 expression in HCAEC. HCAEC were treated with vehicle (PBS), human recombinant VEGF-A165 (30 ng/mL), recombinant VEGF-A165b (30 ng/mL) or their combination. (B) The number of tube-like structures was determined after 24 h. Data represent the mean ± SD of the number of tubes in 5 low-magnification (×100) fields (n = 6 independent experiments performed in triplicate). *p < 0.01 vs vehicle; †p < 0.05 vs VEGF-A165-treated cells. Right panels show representative images of endothelial cell differentiation on Matrigel. Bars = 300 μm. (C) Effect of VEGF-A165b on proliferation. Results are expressed as percentage of proliferating endothelial cells analyzed by BrdU incorporation. Data represent the mean ± SD (n = 5 independent experiments). *p < 0.01 vs vehicle; †p < 0.05 vs VEGF-A165-treated cells. (D) Effect of VEGF-A165b on migration. Results are expressed as percentage of cell migration analyzed by the wound healing assay. Data represent the mean ± SD. (n = 4 independent experiments) *p < 0.01 vs vehicle; †p < 0.05 vs VEGF-A165-treated cells. (E)…

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  Figure 6. Blocking serum VEGF-A165b from STEMI patients with a neutralizing antibody induces angiogenesis. HCAEC were incubated with diluted serum (10%) from STEMI patients (n = 50) or controls (n = 23). Samples were incubated in the presence of a mouse monoclonal anti-human VEGF-A165b blocking antibody (10 µg/mL) or irrelevant isotype- and concentration-matched IgG. (A) Phase contrast images were taken after 24 h and the number of tube-like structures was counted. Data represent mean ± SD of the number of tube-like structures in 5 low-magnification (×100) fields. Bars = 300 μm. Upper panels show representative images of endothelial cell differentiation on Matrigel. *p < 0.05 **p < 0.01. (B) Correlation between VEGF-A165b levels and the number of tubes induced by serum (+IgG control) from STEMI patients. (C) Percentage of proliferating endothelial cells were analyzed by BrdU incorporation. Data represent the mean ± SD. *p < 0.05 **p < 0.01. (D) Endothelial cell migration (%) was analyzed by a wound healing assay. Data represent the mean ± SD. *p < 0.05 **p < 0.01.