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

Abstract

Mushfiquddin Khan,1 Tajinder S Dhammu,1 Fumiyo Matsuda,1,2 Mauhammad Baarine,3 Tejbir Singh Dhindsa,1 Inderjit Singh,1 Avtar K Singh3,4 1Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA; 2School of Health Sciences, Kagoshima University, Kagoshima, Japan; 3Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA; 4Ralph H Johnson VA Medical Center, Charleston, SC, USA Background: For stroke patients, stimulating neurorepair mechanisms is necessary to reduce morbidity and disability. Our previous studies on brain and spinal cord trauma show that exogenous treatment with the S-nitrosylating agent S-nitrosoglutathione (GSNO) – a nitric oxide and glutathione metabolite of the human body – stimulates neurorepair and aids functional recovery. Using a rat model of cerebral ischemia and reperfusion (IR) in this study, we tested the hypothesis that GSNO invokes the neurorepair process and improves neurobehavioral functions through the angiogenic HIF-1α/VEGF pathway.Methods: Stroke was induced by middle cerebral artery occlusion for 60 minutes followed by reperfusion in adult male rats. The injured animals were treated with saline (IR group, n=7), GSNO (0.25 mg/kg, GSNO group, n=7), and GSNO plus the HIF-1α inhibitor 2-methoxyestradiol (2-ME) (0.25 mg/kg GSNO + 5.0 mg/kg 2-ME, GSNO + 2-ME group, n=7). The groups were studied for either 7 or 14 days to determine neurorepair mediators and functional recovery. Brain capillary endothelial cells were used to show that GSNO promotes angiogenesis and that GSNO-mediated induction of VEGF and the stimulation of angiogenesis are dependent on HIF-1α activity.Results: IR injury increased the expression of neurorepair mediators HIF-1α, VEGF, and PECAM-1 and vessel markers to a limited degree that correlate well with significantly compromised neurobehavioral functions compared with sham animals. GSNO treatment of IR not only remarkably enhanced further the expression of HIF-1α, VEGF, and PECAM-1 but also improved functioning compared with IR. The GSNO group also had a higher degree of vessel density than the IR group. Increased expression of VEGF and the degree of tube formation (angiogenesis) by GSNO were reduced after the inhibition of HIF-1α by 2-ME in an endothelial cell culture model. 2-ME treatment of the GSNO group also blocked not only GSNO’s effect of reduced infarct volume, decreased neuronal loss, and enhanced expression of PECAM-1 (P<0.001), but also its improvement of motor and neurological functions (P<0.001).Conclusion: GSNO stimulates the process of neurorepair, promotes angiogenesis, and aids functional recovery through the HIF-1α-dependent pathway, showing therapeutic and translational promise for stroke. Keywords: GSNO, IR, HIF-1α, VEGF, motor function, subtle behavior, neuroprotection, neurorepair, angiogenesis, S-nitrosylation, stroke