Mice in which eNOS has been genetically ablated develop normally, but display a severe form of critical limb ischemia in mouse hindlimb models , , together with impaired wound healing and defective angiogenesis . to metallic centres within metalloproteins, most notably haem proteins. The rules of the enzymes which generate NO, CO and H2S have been shown to be affected at both the transcriptional and post-translational levels by redox-dependent mechanisms, while the activity and bioavailability of the gasotransmitters themselves are also subject to oxidative changes. Within vascular cells, the family of nicotinamide adenine dinucleotide phosphate oxidases (NAPDH oxidases/Noxs) have emerged as functionally significant sources of controlled O2- and H2O2 production and accordingly, STF-31 direct associations between Nox-generated oxidants and the functions of specific gasotransmitters are beginning to become recognized. This review focuses on the current knowledge of the redox-dependent mechanisms which regulate the generation and activity of these gases, with particular reference to their functions in angiogenesis. and its binding to endothelial-expressed, plasma membrane-bound, tyrosine kinase receptors, Flt-1 (VEGFR-1) and primarily, Flk-1/KDR (VEGFR-2). VEGF binding to VEGFR-2 initiates its autophosphorylation, dimerization and the subsequent activation of its tyrosine kinase website . This in turn activates downstream signalling cascades, including the MEK-ERK1/2 pathway to support cell growth and proliferation  as well as the anti-apoptotic phosphoinositide 3-kinase- (PI3-K-)Akt pathway to promote cell survival  (Fig. 1). Open in a separate windows Fig. 1 A schematic illustration of hypoxia- and VEGF-mediated signalling in the endothelium leading to angiogenesis through the promotion of cell survival STF-31 and proliferation. In response to hypoxia, the upregulation of HIF-1 leads to increased expression of a number of pro-angiogenic factors including SDF-1, PDGF-B, angiopoietin, placenta growth factor and importantly VEGF. VEGF signals have been the best characterised and have been shown to cause the stimulation of VEGFR2 within the endothelium. In turn this activates downstream signalling pathways including P13K/Akt and MEK/MAPK to promote pro-angiogenic cellular responses. Increased VEGF-dependent signalling triggers the angiogenic response and therefore the control of VEGF expression is critical to the regulation of angiogenesis. In this regard, the transcriptional regulation of VEGF appears to play the pre-eminent role, and multiple transcription factors which are positive mediators of VEGF transcription have been identified, together with cellular brokers which stimulate their activity through diverse signalling pathways . An important stimulus for angiogenesis is usually tissue hypoxia and, accordingly, VEGF is usually a known direct transcriptional target of hypoxia-inducible factor 1 (HIF-1). Similarly, the expressions of other known pro-angiogenic factors including angiopoietin 1 and 2, stromal cell-derived factor-1 (SDF-1), placenta growth factor and platelet-derived growth factor B are also known Rabbit Polyclonal to TNF12 to be upregulated by HIF-1 , . These regulatory pathways, both upstream and downstream of the action of VEGF, have been STF-31 extensively studied and emerging data indicate the involvement of redox-dependent molecular signalling mechanisms at multiple stages . Further, angiogenic responses have increasingly been shown to be mediated in part by the biological actions of a small family of gases, termed gasotransmitters, which are enzymatically generated within vascular cells . The precise mechanisms of the regulation of action of these short-lived mediators, which comprise nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) are not currently fully comprehended. However, there is STF-31 growing evidence that their generation may be regulated in part by redox-dependent mechanisms, while their chemical nature in some cases makes them highly susceptible to oxidation. In this review we summarise the current knowledge of the biochemistry which links reactive oxygen species generation, redox signalling and the action of the gasotransmitters in angiogenesis. A more comprehensive understanding of these mechanisms would be of great potential benefit in identifying new therapeutic targets for both cancer and vascular diseases such as peripheral arterial disease (PAD) . 1.1. Reactive oxygen species and redox-signalling Reactive oxygen species are partial reduction products of molecular oxygen (O2) and include superoxide (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (?OH) (Fig. 2). Historically, they have been thought of as merely potentially detrimental by-products of aerobic metabolism in the mitochondria or the result of unregulated uncoupling of various O2-dependent enzymatic reactions . The harmful biological effects of these oxidants are countered by the actions of enzymatic and non-enzymatic antioxidants that collectively form the cellular antioxidant system. Enzymatic antioxidants include superoxide dismutase (SOD) that removes O2-, as well as catalase, peroxiredoxin and glutathione peroxidase (GPx) that metabolise H2O2 . Non-enzymatic antioxidants include vitamins C and E as well as the major redox buffer, glutathione (GSH). GSH is present at millimolar concentrations in the cell and scavenges both H2O2 and free radicals through the.