Akt also crosstalks with NO signaling pathways to promote vascularization through vascular endothelial growth factor (VEGF) activation [
35,
43]. Deficiencies of either H
2S or NO levels have been linked with increased risk of cerebral IR by vascular restriction [
44]. VEGF is a potent pro-angiogenic factor that promotes vascularization in ischemia and cancer through a variety of signaling pathways such as Akt and STAT3 [
32,
40,
45,
46]. In ischemic muscle addition of VEGF can result in reduced damage and improved function. Rats subjected to hind limb ischemia induced by unilateral external iliac and femoral artery and vein ligation, then injected with an alginate gel containing 3 µg of VEGF and/or IGF-1 demonstrated that either treatment resulted in improved vascularization measured by laser Doppler perfusion injury, reduced fibrosis, and improved muscle regeneration and function [
47]. VEGF and IGF-1 acted in synergy to improve ischemia response superior to either treatment alone [
47]. Administration of H
2S donor once or twice a day using a dose of .25–.05 mg/kg over 7 days resulted in increased blood flow to rat hind limbs following femoral artery ligation [
48]. Another study involved implanting muscle derived stem cells into mice with muscular dystrophy [
49]. It was found that while stem cells alone stimulated in vivo angiogenesis and muscle regeneration, responses were improved when the cells were transduced to overexpress VEGF. Cells expressing soluble forms-like tyrosine kinase-1 displayed significantly less vascularization and increased fibrosis [
49], demonstrating that VEGF is crucial to re-establishment of vascularization following IR. In addition to blood flow, VEGF also promotes innervation of damaged muscles. Introduction of VEGF containing gel into damaged human sternomastoid displayed 50 percent innervated motorend plates, compared to only 5 percent for blank gels [
47,
50]. VEGF administration also increased expression of nerve growth factor and glial-derived neurotrophic factor, improving axonal regeneration in damaged muscle. Inhibition of the nerve growth factors disrupted VEGF induced nerve repair [
50], showing that VEGF acts on a variety of vascular, neural, and cell growth signaling pathways to repair muscular damage and restore function. VEGF activity is augmented by eNOS, and VEGF also acts to upregulate eNOS expression in endothelial cells, forming a feedback loop [
51,
52]. Akt is involved in the eNOS-VEGF signaling pathway as an upstream regulator, and has been implicated in regulation of vascularization in many ischemic tissues [
35,
51,
53]. One experiment induced hind limb ischemia in rats by femoral artery ligation, followed by daily intraperitoneal NaHS injection (50 µmol/kg). It was found that Akt, VEGF, and VEGF receptor 2 activity all increased in endothelial cells near the ligation site, along with increased measured vascular flow [
54], demonstrating a role for Akt in VEGF induction. The study by Yong et al. showed that the 10 s administrations of NaHS resulted in increased expression of eNOS, suggesting that eNOS was critical to NaHS induction of angiogenesis [
41]. H
2S administration reduced cardiac failure induced by transverse aortic constriction by upregulation of eNOS that was dependent on CSE activity, as CSE knockout mice did not respond to H
2S donor administration [
55]. A similar study found that a large dose (100 µg/kg) of Na
2S restored eNOS activity in CSE knockout mice, but mice lacking phospho eNOS activity were unresponsive to H
2S donor treatment following IR [
56]. eNOS is critical to angiogenesis during ischemia, as eNOS knockout resulted in absence of vascularization following NaHS administration [
35]. Dietary H
2S sources can also induce Akt and eNOS induced vascularization, as demonstrated by a study done injecting mice with daillyl trisulfide, a component of garlic oil that contains H
2S. A daily injection of 500 µg/kg daillyl trisulfide over 10 days resulted in improved blood flow following hind limb ischemia, along with increased Akt and eNOS phosphorylation. Akt and eNOS knockout mice were not affected by the treatment, demonstrating that both are essential to vascular repair in IR [
57]. It has been shown that dietary and environmental sources (ozone, garlic, vitamin E) have been effective in promoting IR healing through free radical scavenging, and likely also vascular signaling [
13,
16,
20,
57‐
59]. The effects of H
2S in improving IR response through Akt-eNOS are not limited to muscle, as demonstrated by use of H
2S donors to improve angiogenesis following ischemia in intestine [
35] and brain [
53], suggesting a wide range of targets for H
2S directed therapies.
In addition to affecting vascularization through VEGF and eNOS, H
2S can also directly interact with NO to produce nitroxyl, which has a longer half-life than either NO or H
2S. Both H2S and NO presence are neccesary to produce nitroxyl, and they work synergestically to promote smooth muscle relaxation and portal vein vasodilation [
60]. Low nitroxyl concentrations have been shown to be beneficial to vasodilation, cardiac function, smooth muscle relaxation, and cGMP activity, although high levels can be neurotoxic and inflammatory [
60,
61]. More research is necessary to deduce the exact roles of H
2S and NO crosstalk on VEGF activity and vascularization in order to safely induce vascularization while avoiding adverse side effects.