Promoting endothelial function by S-nitrosoglutathione through the HIF-1α/VEGF pathway stimulates neurorepair and functional recovery following experimental stroke in rats

Figures & data

Figure 1 Photomicrographs of immunoreactivity (immunohistochemistry) and Western blots of HIF-1α, VEGF, and PECAM-1 in the cortical peri-infarct area at 14 days after IR.

Notes: GSNO treatment of IR animals was performed for 14 days. While IR increased the expression of HIF-1α (A and B and graphs of cell counts [b] and densitometry [b]), VEGF (C and D and graphs of cell counts [b] and densitometry [b]), and PECAM-1 (E and F and graphs of cell counts [b] and densitometry [b]), GSNO treatment significantly increased the expression further. Data are presented as mean ± standard deviation (n=7). *P<0.05, **P<0.01 versus Sham; ⁺⁺⁺P<0.001 versus IR. Label IR represents IR group and label GSNO represents GSNO group.
Abbreviations: GSNO, S-nitrosoglutathione; IR, ischemia–reperfusion; Sham, sham-operated animals; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1 alpha.

Figure 2 Photomicrographs of immunohistochemistry of blood vessel markers and cell proliferation marker at 14 days after IR.

Notes: Photomicrographs show that IR and GSNO treatment of IR increased the vessel density ([A] laminin [a] and laminin-positive cell counts [b], [B] GSL-1 [a] and GSL-1-positive cell counts [b]) in the peri-infarct area and (C) cell proliferation marker Ki67 (a) and Ki67-positive cell counts (b) in the peri-infarct area compared with Sham. GSNO treatment significantly bolstered the expression compared with IR. Data are presented as mean ± standard deviation (n=3). *P<0.05, **P<0.01,***P<0.001 versus Sham; ⁺⁺⁺P<0.001 versus IR. Label IR represents IR group and label GSNO represents GSNO group.
Abbreviations: GSNO, S-nitrosoglutathione; IR, ischemia–reperfusion; Sham, sham-operated animals; GSL-1, Griffonia simplicifolia lectin 1.

Figure 3 Effect of GSNO on improvement of neurobehavioral functions at 14 days after IR.

Notes: Walking speed (rotarod) (A), body swing score (B), and body weight (C) were significantly increased/improved in the GSNO group compared with the IR group. mNSS (D) was also improved in the GSNO group compared with the IR group. Data are presented as mean ± standard deviation (n=7). *P<0.05, ***P<0.001 versus IR. Label IR represents IR group and label GSNO represents GSNO group.
Abbreviations: GSNO, S-nitrosoglutathione; IR, ischemia–reperfusion; mNSS, modified neurological severity score; Sham, sham-operated animals.

Figure 4 Effect of inhibition of HIF-1α by 2-ME on GSNO-mediated protective effects at 7 days after IR.

Notes: While GSNO treatment blunts the deleterious effects of IR, 2-ME treatment reverses the effects of GSNO on brain infarctions (TTC [A]; infarct volume [B]), neuronal viability (Nissl staining [C]; viable neuron counts [D]), neurobehavioral functions (neurological score [E]; walking time on rotarod [F]), expression of HIF-1α (Western blot [G]; densitometry [H]), and expression of PECAM-1 (immunohistochemistry [I]; cell counts [J]) in the peri-infarct area. GSNO treatment of IR animals was performed for 7 days. Treatment with 2-ME was initiated 24 hours after IR and continued until day 7. Black arrows indicate neuronal degeneration and blue arrows show viable neurons on Nissl staining (C). Data are presented as mean ± standard deviation (n=7). ***P<0.001 versus IR and/or GSNO + 2-ME. Label IR represents IR group, label GSNO represents GSNO group and label GSNO+2-ME represents GSNO+2-ME group.
Abbreviations: 2-ME, 2-methoxyestradiol; GSNO, S-nitrosoglutathione; IR, ischemia–reperfusion; Sham, sham-operated animals; TTC, 2,3,5-triphenyltetrazolium chloride; HIF-1α, hypoxia-inducible factor-1 alpha.

Figure 5 Effects of GSNO on expression of HIF-1α and VEGF in HIF-1α-silenced endothelial (bEnd3) cells.

Notes: Cells were seeded in 12-well plates. After 24 hours of culture, cells were transfected with two different HIF-1α siRNAs (A: SC-44308 and B: SC-35562) using the bEnd3 Standard Transfection protocol (CRL-2299; Altogen Biosystems, Las Vegas, NV, USA) for 72 hours. Transfected cells were treated with GSNO 100 μM for 24 hours. Cells were harvested and homogenized using RIPA buffer. (A) Western blot for HIF-1α, VEGF, and β-actin. (B) Densitometry of HIF-1α and VEGF normalized with β-actin. Densitometry data are presented as mean ± standard deviation (n=5). ***P<0.001 versus Untr; ⁺⁺⁺P<0.001 versus siRNA A or siRNA B; ^$$$P<0.001 versus GSNO.
Abbreviations: GSNO, S-nitrosoglutathione; Scr, scrambled; Untr, untreated; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1 alpha; RIPA, radioimmunoprecipitation assay.

Figure 6 Effect of GSNO on formation of capillary-like proangiogenic structures on endothelial cells.

Notes: bEnd3 endothelial cells were plated in a six-well Matrigel plate (BD Biocoat Matrigel Matrix, 354432). The cells were seeded at a density of 60,000 cells per well in 2 mL medium. Two hours after seeding on Matrigel, the cells were treated with GSNO, 2-ME, or GSNO followed by 2-ME (30 minutes later). The cells were observed under microscope for formation of capillary-like structures at 3, 18, and 48 hours. Photographs are presented for the 48-hour study (A). Capillary-like structures (pink arrow) were counted (B), and data are presented as mean ± standard deviation of % tube formation (n=5). ***P<0.001 versus Untr and 2-ME; ⁺⁺⁺P<0.001 versus GSNO.
Abbreviations: 2-ME, 2-methoxyestradiol; GSNO, S-nitrosoglutathione; Untr, untreated.

Figure 7 Schematic showing GSNO-mediated events leading to neuroprotection and neurorepair as well as functional recovery.

Notes: S-nitrosylation by GSNO of HIF-1α causes its stabilization, translocation to nucleus, and dimerization with HIF-1β, facilitating recruitment of p300/CBP and transcribing neurorepair-related genes such as PECAM-1 and VEGF. They, in turn, stimulate the neurorepair process, leading to functional recovery.

Abbreviations: GSNO, S-nitrosoglutathione; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1 alpha; -SNO, S-nitrosocysteine proteins.