Ischemic stroke is a common clinical disease that is associated with high levels of disability and mortality and seriously threatens human life and health. In recent years, Gucy1a3, which encodes the α1 subunit of sGC, has been proposed to be tightly correlated with cerebrovascular diseases and coronary artery disease [15–17,24]. However, only a few studies have examined the effects of GUCY1A3 on ischemic stroke, and the mechanisms underlying these effects remain unclear. In the present study, Gucy1a3-KO mice were used to investigate the effects of GUCY1A3 on ischemic stroke. Our results indicated that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. Moreover, Gucy1a3-KO brains exhibited significantly lower microvessel densities than WT brains at 96 hours post-pMCAO, suggesting that Gucy1a3 deletion suppressed angiogenesis after ischemic stroke. Further analysis confirmed that loss of Gucy1a3 decreased VEGFA and HIF-1α expression levels, which may contribute to the decrease in angiogenesis in Gucy1a3-KO mice after pMCAO.
The impact of genetic variations in the Gucy1a3 gene on ischemic cardio-cerebrovascular diseases has been highlighted by previous studies. Variants of the Gucy1a3 gene are enriched in patients suffering from ischemic stroke and coronary artery disease [15,24,25]. A previous study has also shown that the infarct size was larger and the neurologic outcome was worse in sGCα1−/− mice than in WT mice subjected to 1 hour of MCAO and 23 hours of reperfusion, but the infarct volumes and neurological deficits were similar after 24 hours of permanent occlusion in both genotypes . That study further revealed that impaired vascular relaxation may underlie the more marked reperfusion deficit observed in sGCα1−/− mice than in WT mice after middle cerebral artery reperfusion . Interestingly, our results showed no significant difference in neurological deficit scores between WT and Gucy1a3-KO mice 24 hours after pMCAO, but neurological deficits and the infarct volume were significantly greater in Gucy1a3-KO mice at 96 hours after pMCAO. However, temporal changes in the effects of sGCα1 deficiency on infarct volume and neurological deficits following pMCAO cannot be fully explained by impaired vascular relaxation.
Previous studies have confirmed that angiogenesis is correlated with improved functional recovery and prolonged survival after ischemic stroke [6,26]. Krupinski et al.  reported that angiogenic activity occurred in ischemic stroke patients 3–4 days after stroke. In rodent stroke models, endothelial cells in the peri-infarct brain tissue start to proliferate as early as 12–24 hours following ischemic stroke; accordingly, vessel density significantly increases in the peri-infarct region 3 days after ischemia [27, 28]. In our study, after 4 days (96 hours) of pMCAO, the microvessel density was lower in Gucy1a3-KO mice than in WT mice. A larger infarct volume and worse functional outcome were also observed, which might have resulted from the decreased angiogenic activity in the peri-infarct region.
VEGFA is the most important angiogenesis-promoting factor and plays an important role in angiogenesis after focal cerebral ischemia . As a key oxygen concentration-dependent transcription factor, HIF-1α can regulate multiple target genes, including VEGFA, and thereby modulate angiogenesis after ischemia . As mentioned previously, sGC is the major cytosolic receptor for NO, catalyzing the conversion of guanosine-5'-triphosphate to cGMP. NO and hypoxia were reported to upregulate the VEGFA gene by enhancing HIF-1 activity [31,32]. We further examined whether the decreased angiogenesis in the Gucy1a3-KO ischemic brain could be explained by the decrease in HIF-1α/VEGFA expression. The results showed that, compared with the WT group, the VEGFA and HIF-1α protein levels were significantly decreased at 96 hours after ischemia in Gucy1a3-KO mice, suggesting that, mechanistically, the HIF-1α/VEGFA-dependent signaling may mediate the effects of GUCY1A3 on angiogenesis.
Several limitations of this study should be considered. First, in the current study, only silencing experiments were performed. In the next set of experiments, overexpression of the Gucy1a3 gene should be employed to investigate the impact of GUCY1A3 on angiogenesis and cerebral injury after ischemic stroke. Second, a previously published paper suggested that hypertension and the responsiveness to NO in sGCα1 knockout mice are gender-specific . Therefore, female Gucy1a3-KO mice should be investigated in the future. Third, compensatory mechanisms in the Gucy1a3-KO model, including a compensatory increase in GUCY1A2 expression in Gucy1a3-KO mice, were not evaluated in the present study. Nevertheless, other studies have confirmed that loss of one of the sGC isoforms was not compensated by an upregulation of the remaining one, indicating that deletion of the sGCα1 subunit is not compensated by upregulation of the other form [33,34].
In conclusion, we demonstrated that GUCY1A3 expression after ischemic stroke may play a significant role in neurological function recovery, which is related to angiogenesis in the peri-infarct region. The beneficial effects of GUCY1A3 might be mediated by the HIF-1α/VEGFA signaling pathway. This raises the possibility of targeting Gucy1a3 as a reasonable and novel therapeutic strategy for ischemic stroke.