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The hallmark of stroke injury is endothelial dysfunction leading to blood–brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of matrix metalloproteinase-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.
Ischemic stroke is a devastating disease of endothelial origin causing obstruction of oxygen and nutrient supply to the brain (Rosamond et al. 2008). The obstruction causes necrotic neuronal death early in the core; however, even greater apoptotic neuronal damage occurs later in the penumbra, especially in and around the cerebral vessel walls (Lo et al. 2003; Gursoy-Ozdemir et al. 2004; Yemisci et al. 2009). An effective therapy for stroke patients, other than thrombolysis by tissue plasminogen activator, which suffers a short window of treatment (~3 h), is not available mainly because of limited understanding of the cross-talk between the multiple pathways involved in the injury (Moskowitz et al. 2010; Chen et al. 2011). Delineation of these mechanisms is complicated partly because of excessive production of reactive oxygen/nitrogen species (ROS/RNS) not only in the brain cells but also in the endothelial cells (the entire neurovascular unit). These reactive species are implicated in blood–brain barrier (BBB) disruption, inflammation, neuronal cell death, and neurological deficits following ischemia reperfusion (IR) injury (Gursoy-Ozdemir et al. 2004). ROS/RNS alter the metabolic and signaling functions of multiple enzymes and worsen neurovascular injury following stroke. One of the highly deleterious ROS/RNS produced following IR is peroxynitrite, a product of a diffusion-limited instantaneous reaction between superoxide and nitric oxide (NO). This reaction occurs when the levels of superoxide exceed the levels of NO. In IR, therefore, this conversion of NO to peroxynitrite not only decreases the bioavailability of NO but also compromises its function. Once formed, peroxynitrite causes endothelial dysfunction and life-threatening edema by compromising BBB integrity (Pacher et al. 2007). BBB is a dynamic integral part of the neurovascular unit, and it remains open from minutes to weeks after IR (Strbian et al. 2008). Its disruption is associated with edema and inflammation. Our previous studies show that down-regulating inflammation using anti-inflammatory drugs such as GSNO improves overall outcomes after IR (Khan et al. 2005, 2006), spinal cord injury (Chou et al. 2011), and traumatic brain injury (TBI) (Khan et al. 2009, 2011).
GSNO is formed in vivo by oxidation of glutathione (GSH) and NO in the presence of oxygen (Singh et al. 1996; Kluge et al. 1997). It is an endogenous component of the human body and executes its action via S-nitrosylation of target proteins, a comparatively newly characterized mechanism (Foster et al. 2009). S-Nitrosylated proteins (PSNO) and GSNO are in dynamic equilibrium in the human body, and their dysregulation hampers cellular functions. In stroke pathology, the levels of GSNO and PSNO are believed to decrease because of four major reasons: (i) Decreased oxygen supply under ischemic/hypoxic condition reduces GSNO biosynthesis; (ii) Excessive superoxide formed during reperfusion instantaneously reacts with nitric oxide synthase (NOS)-derived nitric oxide NO, forming peroxynitrite and thus reducing NO bioavailability for GSNO biosynthesis; (iii) Decreased biosynthesis of GSNO as a result of reduced levels of GSH (redox imbalance) and NO because of its reaction with superoxide under IR conditions. Furthermore, NO reacts slowly with GSH as compared with superoxide; and (iv) In an environment of inflammation, the GSNO-degrading enzyme, GSNO reductase (GSNOR), is activated (Que et al. 2005), resulting in reduced levels of GSNO. Therefore, GSNO supplementation therapy was investigated in this study to determine whether it would maintain the dynamic equilibrium of S-nitrosylation and thereby lead to BBB protection.
The beneficial activities conferred by GSNO are not associated with conventional non-nitrosylating NO donors. We have previously reported that the neurovascular protective action of NO-modulating agents in stroke depends on the mechanistic and functional nature of the NO donor. It appears that peroxynitrite-reducing and cerebrovascular-protective activities of NO modulators relate to their ability to S-nitrosylate(Khan et al. 2006). Peroxynitrite is highly injurious and its reduction yields neuroprotection following experimental IR injury (Gursoy-Ozdemir et al. 2004; Thiyagarajan et al. 2004). Accordingly, 3-morpholino sydnonimine (SIN-1), a peroxynitrite-forming agent, is deleterious in animal models of TBI (Singh et al. 2007) and stroke (Khan et al. 2006; Pacher et al. 2007). Treatment with GSNO has been shown to decrease the levels of peroxynitrite following brain injury (Rauhala et al. 1998; Khan et al. 2006). It also inhibits endothelial cell activation (Prasad et al. 2007). However, the mechanisms through which GSNO reduces peroxynitrite, repairs BBB, and maintains endothelial function are not clear.
In this study, GSNO treatment of IR animals reduced not only the levels of peroxynitrite in the penumbra but also blocked BBB leakage and decreased edema formation. IR-mediated reduced NO bioavailability and the decreased expression of PSNO and the tight junction protein Zonula occludens-1 (ZO-1) were also restored by GSNO treatment. Furthermore, the expression of MMP-9 and E-selectin and the activity of nuclear factor kappa B (NF-κB) were down regulated by GSNO treatment. These results indicate that GSNO protects endothelial function in IR by its peroxynitrite-reducing and anti-inflammatory activities, likely via the novel mechanism of S-nitrosylation.
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Loss of BBB integrity is an early critical event in IR that contributes to the initiation of the inflammatory cascade, edema formation, and ultimately poor outcomes. Agents that reduce peroxynitrite levels and block BBB leakage are shown to protect the neurovascular unit (Chen et al. 2006). This BBB leakage is associated with endothelial cell activation and vascular peroxynitrite formation (Pacher et al. 2007). eNOS-derived peroxynitrite has been implicated in edema formation following brain trauma (Lundblad et al. 2009). We also observed increased levels of peroxynitrite in and around vessels (Fig. 3a–c) as well as BBB disruption and edema (Fig. 1a–c) early after IR, indicating that an approach to stroke therapy is needed that will simultaneously protect the vasculature and neurons. Therefore, we used the neurovascular-protective agent, GSNO (Khan et al. 2005, 2009), which also reduced the levels of peroxynitrite (Fig. 3a–c) and decreased BBB leakage and edema (Fig. 1a–c) following IR. These results have indicated that GSNO-mediated protective effects were related, at least in part, to the mechanisms of reducing peroxynitrite. On the basis of these observations, we hypothesized (Fig. 9) that excessive superoxide reacts with NO by a diffusion-limited reaction to form peroxynitrite early after reperfusion in endothelial cells. Peroxynitrite oxidizes and depletes the NOS cofactor tetrahydrobiopterin, leading to ‘uncoupled’ eNOS. Uncoupled (aberrant) eNOS produces both superoxide and NO. They react with each other to form peroxynitrite in the same compartment, thereby maintaining a sustained production of peroxynitrite, leading to BBB compromise, edema, and neurovascular injury (Gursoy-Ozdemir et al. 2004; Khan et al. 2006; Yemisci et al. 2009).Supporting this hypothesis that peroxynitrite is formed in endothelial cells where it inhibits NO-producing eNOS activity, a treatment of eNOS with peroxynitrite significantly decreased the ability of the enzyme to produce NO. Peroxynitrite increased superoxide in vessels from wild, but not in vessels from eNOS-deficient mice, suggesting that eNOS was the source of superoxide, and hence of peroxynitrite (Laursen et al. 2001; Kuzkaya et al. 2003). The observed increased peroxynitrite in and around the vessels (Fig. 3c) and plasma (Khan et al. 2006), as well as decreased NO-producing eNOS activity (Fig. 7b) and NO levels in the penumbra (Fig. 7c) and plasma (Khan et al. 2006) indicate that peroxynitrite is formed in cerebral vessels/endothelial cells. Furthermore, the observed greater degree of loss of BBB integrity following treatment with the peroxynitrite-forming agent SIN-1 (Fig. 1) indicates a deleterious role of peroxynitrite. Studies have already shown that peroxynitrite alone is sufficient to induce BBB leakage, endothelial dysfunction, and neurodegeneration (Parathath et al. 2006; Phares et al. 2007). Therapeutic agents that reduce peroxynitrite levels have been shown not only to restore BBB integrity but also to prevent neuronal cell death (Parnham and Sies 2000; Gursoy-Ozdemir et al. 2004; Thiyagarajan et al. 2004).Based on these observations, it can be argued that whether eNOS-derived NO is good or bad depends on the concomitant production of superoxide anions in the microenvironment where NO is synthesized and released. In presence of high levels of superoxide anion as in IR, the bioactivity of NO and the mechanism of S-nitrosylation are not only diminished but also transformed into peroxynitrite. Therefore, reducing peroxynitrite by GSNO resulted in neurovascular protection. Decreased eNOS activity following GSNO treatment (Fig. 7b) indicates that GSNO, by inhibiting aberrant eNOS activity via S-nitrosylation, may be reducing peroxynitrite formation (Fig. 9). eNOS has been shown to be dynamically regulated by S-nitrosylation at Cys101 resulting in inhibition of eNOS activity (Ravi et al. 2004; Erwin et al. 2005).
Figure 9. Schematic showing the vicious cycle of peroxynitrite production in endothelial cell in an animal model of ischemia-reperfusion (IR). S-nitrosoglutathione (GSNO) was hypothesized to protect against neuroinflammatory secondary injury in IR by reducing peroxynitrite, thereby blocking the vicious superoxide/aberrant eNOS/peroxynitrite cycle.
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The decreased expression of PSNO was also observed in the penumbra at 4 h following IR, and the expression was normalized by GSNO treatment (Fig. 8a and b), indicating that reduced -SNO levels are associated with IR pathophysiology. The observation is novel and it is supported by the reported decreased bioavailability of NO and the associated poor outcomes in stroke patients (Rashid et al. 2003). Plasma S-nitrosothiols are also decreased in patients with endothelial dysfunction (Heiss et al. 2006). These reports and our results indicate that S-nitrosylation is reduced in IR; therefore, GSNO supplementation is a logical strategy for acute stroke therapy in humans. Via S-nitrosylation, GSNO exerts its anti-inflammatory action mainly through inhibition of NF-κB, TNF-α, and iNOS (Fortenberry et al. 2001; Marshall and Stamler 2001; Khan et al. 2005; Prasad et al. 2007).
Vascular inflammation is an intrinsic essential component of stroke injury and it is considered to be responsible for the accumulation of inflammatory cells in the injured brain (Barone and Feuerstein 1999; Berti et al. 2002). Reperfusion sets in motion a number of events, including endothelial cell activation, peroxynitrite formation, BBB leakage, and the activation of cell adhesion molecules (CAMs) and MMP-9. Peroxynitrite-mediated increased expression of MMP-9 has been reported to exacerbate BBB leakage (Gursoy-Ozdemir et al. 2004). Our study showing GSNO-mediated down-regulation of MMP-9 in endothelium (Fig. 5) indicates that GSNO blocked BBB leakage by suppressing endothelial peroxynitrite. GSNO treatment also restored the IR-mediated reduced expression of BBB integrity proteins ZO-1 (Fig. 2a and b). Preservation by GSNO of ZO-1 suggests that GSNO protects endothelial tight junctions and therefore keeps the BBB intact. Interestingly, ZO-1 has been reported to contain a trans-nitrosylation consensus motif at the cysteine amino acid residue 1718 (Stamler et al. 1997). However, whether 1718 cysteine in ZO-1 is S-nitrosylated, or the effect this S-nitrosylation would have on BBB integrity are not known.
To understand the mechanism of GSNO-mediated down-regulation of the expression of E-selectin and MMP-9, we investigated the effect of GSNO on IR-mediated NF-κB activation. Under physiological conditions, NF-κB consists of a p65/p50 heterodimer and is retained in cytoplasm by its association with IκB. After IR injury, the cytosolic NF-κB/IκB complex dissociates, and free p65/p50 translocates to the nucleus, where it regulates the transcription of various genes. Activation of NF-κB has been shown to be critical for the expression of genes involved in endothelial cell activation, including MMPs and CAMs. E-selectin is expressed mainly by activated endothelial cells. It mediates the initial low-affinity interaction between leukocytes and endothelial cells, promoting the recruitment of leukocytes along vessel walls. We observed intense expression of E-selectin around vessels 4 h after IR (Fig. 4). GSNO treatment of IR decreased the expression of E-selectin (Fig. 4a and b), indicating that GSNO may block BBB leakage by down-regulation of E-selectin. These results are in agreement with our previous observation showing that GSNO inhibits monocyte adhesion to the activated endothelial cell. This inhibitory activity of GSNO was found to be dependent on NF-κB through inhibition of DNA binding via the S-nitrosylation of p65 (Prasad et al. 2007). GSNO-mediated inhibition of NF-κB activity in this study (Fig. 6c) further indicates that the neurovascular-protective mechanisms invoked by GSNO are dependent on inhibition of p65 DNA-binding activity. Expectedly, GSNO had no effect on p65 nuclear translocation (Fig. 6a lower panel).
In clinical settings, GSNO is of great relevance to acute stroke therapy because it shows antiplatelet (Radomski et al. 1992) and vasodilatory properties(de Belder et al. 1994), in addition to its neuroprotective and anti-inflammatory properties (Khan et al. 2005, 2009), further established herein. It protects BBB and reduces edema not only in IR (Fig. 1) and TBI (Khan et al. 2009, 2011) but also in a cerebral malaria model (Zanini et al. 2012). Furthermore, via S-nitrosylation, GSNO shows potent cardioprotective activity in animal models of heart ischemia and reperfusion (Lima et al. 2009). Administration to animals of GSNO, unlike organic nitrates, does not induce tolerance, making it more suitable for therapeutic purposes (Zhang et al. 1995). Although reducing peroxynitrite shows BBB protection in stroke, GSNO has an advantage over other reported agents, such as melatonin (Chen et al. 2006), curcumin (Jiang et al. 2007),or metal-based synthetic scavengers (Sharma et al. 2004; Thiyagarajan et al. 2004), because GSNO is an endogenous mediator metabolized ultimately into beneficial S-nitrosylation. In short, these data show that S-nitrosylation is a novel approach to block peroxynitrite-mediated IR injury. The observation that the formation of peroxynitrite can be blocked by inhibiting the enzyme activity of aberrant/uncoupled eNOS (Fig. 8b), involved in the production of peroxynitrite substrates, is novel.
In conclusion, GSNO is an endogenous anti-inflammatory agent. Its exogenous use as treatment for IR maintains the homeostasis of the NO metabolome, which down-regulates the disruption of the BBB. Such multimechanistic functional abilities are not embedded in non-S-nitrosylatingNO donors (Khan et al. 2006), making GSNO an ideal candidate for acute stroke therapy. Exogenous administration of GSNO in humans, rats, and dogs has not shown toxicity or side effects (Molloy et al. 1998; Colagiovanni et al. 2011). In view of the safety profile in humans and observed beneficial effects of GSNO (Figs 1-8), this study opens a new treatment paradigm in stroke, introducing the concept that treating secondary neuroinflammatory injury using the mechanisms of S-nitrosylation at the vascular-wall level protects the entire neurovascular unit, leading to reduced disabilities in those suffering from stroke.