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Keywords:

  • BBB ;
  • endothelial dysfunction;
  • GSNO ;
  • ischemia reperfusion;
  • peroxynitrite;
  • S-nitrosylation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations used
BBB

blood–brain barrier

CAM

adhesion molecule

EB

Evan's blue

eNOS

endothelial nitric oxide synthase

GSH

glutathione

GSNO

S-nitrosoglutathione

IHC

immunohistochemistry

iNOS

inducible nitric oxide synthase

IR

ischemia-reperfusion

MCAO

middle cerebral artery occlusion

MMP

matrix metalloproteinase

NF-κB

nuclear factor kappa B

NO

nitric oxide

NOS

nitric oxide synthase

PSNO

S-nitrosylated proteins

RNS

reactive nitrogen species

ROS

reactive oxygen species

SCI

spinal cord injury

Sham

sham-operated animals

SIN-1

3-Morpholino-sydnonimine

SMA

smooth muscle cell actin

ZO-1

zonula occludens-1

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents

GSNO was purchased from World Precision Instruments (Sarasota, FL, USA). SIN-1 was obtained from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals and reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise.

Animals

Animals were male Sprague–Dawley rats (n = 123) weighing between 250 and 290 g at the time of surgery. All animals received humane care in compliance with the Medical University of South Carolina's (MUSC) guidance and the National Research Council's criteria for humane care. Animal procedures were approved by the institutional animal care and use committee of MUSC.

Experimental groups, drugs, and dose

The animals were randomly divided into four groups: (i) ischemia reperfusion treated with vehicle (IR), (ii) IR+GSNO treatment (GSNO), (iii) IR+SIN-1 treatment (SIN-1), and (iv) sham-operated treated with vehicle (Sham). The number of animals used in each experiment is indicated in figure legends. In the treatment groups, the rats were administered freshly prepared GSNO or SIN-1 (3.0 μmol/kg body weight), which was dissolved in sterile saline (~250 μL) and administered intravenously slowly at reperfusion or as stated. The same dose of GSNO or SIN-1 was repeated once every 24 h until the end point (maximum 48 h). The dosage (3.0 μmol/kg) of both GSNO and SIN-1 was based on a previously reported dose–response curve study in a rat model of IR (Khan et al. 2006). Physiological parameters did not alter after GSNO or SIN-1 treatment. Details of the study on physiologic parameters in vehicle-treated and GSNO- or SIN-1-treated IR rats have been reported earlier (Khan et al. 2006).

Middle cerebral artery occlusion (MCAO) rat model

Rats were anesthetized by ketamine hydrochloride (80 mg/kg body weight) and xylazine (10 mg/kg body weight) administered intraperitoneally. Stroke was induced for 60 min by left MCAO using an intraluminal filament as previously described (Khan et al. 2006; Matsuda et al. 2011). Regional cerebral blood flow was monitored during the occlusion and early reperfusion to ensure the obstruction of blood flow (Khan et al. 2006). Reperfusion was established by withdrawal of the filament. After specified time points, animals were killed with an overdose of nembutal.

Evaluation of BBB disruption by Evan's blue (EB) extravasation

BBB leakage was assessed as described (Hoda et al. 2009; Khan et al. 2009) by the method of Weismann and Stewart (Weissman and Stewart 1988), with slight modification. The rats received 100 μL of a 5% solution of EB in saline administered intravenously 4 h following IR. At 48 h, cardiac perfusion was performed under deep anesthesia with 200 mL of saline to clear the cerebral circulation of EB. The brain was removed and photographed. Later, they were mechanically homogenized in 1 mL of N, N-dimethylformamide. The suspension obtained was kept at 25°C in the dark for 72 h. It was centrifuged, and the supernatant was spectrofluorimetrically analyzed (λex 620 nm, λem 680 nm) to determine EB content.

Measurement of edema (brain water content)

At 24 h following IR, animals were euthanized to determine brain water content (edema) as described (Hoda et al. 2009; Khan et al. 2009). The cortices, excluding the cerebellum, were quickly removed, and the contralateral and ipsilateral hemispheres were separately weighed. Each hemisphere was dried at 60°C for 72 h. Water was calculated in the ipsilateral hemisphere as: water content (%) = (wet weight−dry weight)/wet weight × 100.

Immunohistochemistry (IHC)

Paraffin-embedded sections from the formalin-fixed brain tissues, processed at 4 h after reperfusion or as indicated, were stained using antibodies against 3-nitrotyrosine (3-NT), neuron-specific enolase (NSE) (Abcam, Cambridge, MA, USA), nitrosocysteine (A. G. Scientific, San Diego, CA, USA), CD34 (Vector Labs, Burlingame, CA, USA), E-selectin (BD Pharmingen, San Jose, CA, USA), MMP-9, p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and SMA clone 1A4 conjugated with Cy3 fluorophore (Sigma-Aldrich).Secondary anti-rabbit and mouse IgG, conjugated with Alexa Fluor 568 and 488, respectively, were incubated on slides for 60 min. S-nitrosocysteine sections were allowed to react with anti-mouse IgG conjugated to a peroxidase-labeled dextran polymer and then developed with diaminobenzidine substrate. All sections were examined for immunoreactivity in the penumbral area using an Olympus microscope as previously described (Khan et al. 2011).

For double labeling, sections were incubated first with an antibody of 3-NT or MMP-9 followed by specific cell marker antibodies (SMA, CD34, or NSE). For immunofluorescent double labeling, immune complexes were visualized with Texas Red-conjugated anti-rabbit IgG (1 : 100, Vector Labs) and FITC-conjugated anti-mouse IgG (1 : 100, Vector Labs). Rabbit polyclonal IgG was used as a control primary antibody. Slides were examined for immunofluorescence as described (Khan et al. 2011).

Three areas in the penumbra area (Fig. 8a) of each immunostained section were digitized using a 40X microscope objective with microscope and camera without visual field overlap. Semi-quantitative cell counting was performed using Scion Image software as described (Khan et al. 2011).

Western blot analysis

Western blot was performed in the penumbra area from the ipsilateral injured brain tissue using antibodies against ZO-1 (Invitrogen Corporation, Carlsbad, CA, USA), 3-NT (Abcam), and β-actin, as described earlier (Khan et al. 2005). Protein concentrations were determined using protein assay dye from Bio-Rad Laboratories (Hercules, CA, USA). Densitometry of protein expression was performed using a GS800 calibrated densitometer from Bio-Rad laboratories.

NO assay in brain

The level of NO (as nitrite) was determined using a Roche's test ‘Nitric oxide colorimetric assay’ kit. The estimation was based on the following reaction: nitrites + sulfanilamide + N-(1-naphthyl)-ethylene-diamine dihydrochloride and yields a reddish-violet diazo dye whose absorbance was measured in the visible range at 540 nm. The assay was performed as described by the manufacturer.

NOS activity assay in brain

NOS activity was determined by the conversion of L-[4,5-3H] arginine (American Radiolabeled, Inc., St. Louis, MO, USA) to L-[4,5-3H] citrulline in the presence or absence of the competitive NOS inhibitor L-NAME using a NOS assay kit from Cayman Chemicals and following the instructions therein. Total cNOS activity was determined by subtracting calcium-independent NOS activity. The nNOS inhibitor, 7-nitroindazole (50 mg/kg), and the NOS inhibitor, L-NAME (30 mg/kg), were used to differentiate between eNOS and nNOS activity as described (Coert et al. 2003). The activity was expressed as picomoles/milligram protein/minute

NF-κB assay in brain

Brain tissue (collected at 4 h of reperfusion)was minced and placed in a lysis buffer. Nuclear extracts were prepared using a nuclear extract kit from Active Motif (Carlsbad, CA, USA). Equal protein was loaded in each well, and NF-κB activity was measured using a TransAM NF-κB p65 kit from Active Motif according to the manufacturer's directions and as described (Khan et al. 2007).

Statistical evaluation

Statistical analysis was performed as described (Jatana et al. 2006) using software Graphpad Prism 3.0. Unless otherwise stated, all the values are expressed as mean ± SD of n determinations or as mentioned. The results were examined by unpaired Student's t-test. Multiple comparisons were performed using the Kruskal–Wallis test, or using anova followed by the Bonferroni test, as appropriate. A p value less than 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

GSNO decreases and SIN-1 increases BBB leakage and edema following IR

BBB disruption and edema indicate pathology associated with endothelial dysfunction (Shlosberg et al. 2010). Peroxynitrite, through 3-NT formation, has been implicated in cerebral vascular dysfunction and the consequent BBB disruption (Gursoy-Ozdemir et al. 2004; Yemisci et al. 2009). In contrast, the S-nitrosylating agent GSNO has been shown to reduce BBB leakage and edema (Khan et al. 2009, 2011), and to provide protection to neurons and vessels (Rauhala et al. 1998; Khan et al. 2006). To compare the neurovascular-protective action of GSNO/S-nitrosylation and the deleterious action of SIN-1/3-NT, we examined the effect of GSNO and SIN-1 on BBB leakage and edema in IR. Extravasations of EB dye into the brain of IR were significantly higher than in the sham brain (Fig. 1a and b). Treatment of IR with GSNO up to 3 h of reperfusion significantly decreased the extravasation. Unlike the effect of GSNO, treatment with SIN-1 even at 0 h of reperfusion increased EB extravasations (Fig. 1a and b). Moreover, the SIN-1-mediated extravasations were significantly greater than IR (p < 0.01), indicating that peroxynitrite causes neurovascular oxidative injury. A study of edema at 24 h after IR showed increased levels of water content in the IR brain (Fig. 1c). While the treatment with GSNO decreased the water content, SIN-1 treatment showed a trend toward increase. This comparative study of GSNO and SIN-1 on BBB integrity indicates that GSNO provides neurovascular protection, and that peroxynitrite worsens injury. Similar to the effect on BBB, GSNO treatment of IR significantly reduced infarct volume compared with IR (61.1 ± 18.2 mm3 GSNO vs. 266.6 ± 42.6 mm3 IR) (Khan et al. 2006).

image

Figure 1. Effect of S-nitrosoglutathione (GSNO) and 3-Morpholino-sydnonimine (SIN-1) on blood–brain barrier (BBB) leakage and edema after ischemia-reperfusion (IR). Representative photographs showing extravasation of Evan's blue (EB) in brain starting at 4 h and measured at 48 h after IR (a). Spectrofluorimetric estimation of EB was performed in ipsilateral hemisphere (b). Edema was measured at 24 h after IR (c). Significant EB leakage was observed in IR brain, whereas EB extravasations were not observed in sham brain. While SIN-1-treatment increased, the treatment with GSNO of IR decreased the EB extravasation in ipsilateral brain up to 3 h after the injury. GSNO treatment of IR also decreased edema. Data are expressed as means ± SD from triplicate determinations of five different samples in each group. ***p < 0.001 and **p < 0.01 versus IR and SIN-1, ##p < 0.05 versus IR.

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IR and SIN-1 decreases and GSNO protects the expression of ZO-1

BBB integrity depends on the expression of tight junction proteins such as ZO-1 and occludin. The expression of tight junction proteins is reduced following IR (Yu et al. 2012). Therefore, using western blot, we investigated whether GSNO protects and peroxynitrite disrupts BBB via the modulation of ZO-1. The expression of ZO-1 was significantly decreased (p < 0.05) in the IR brain compared to sham at 24 h following IR (Fig. 2a). While the treatment with GSNO increased the expression of ZO-1, SIN-1 had the expression similar to that in IR (Fig. 2a and b), indicating the BBB-protective role of GSNO. These data are in agreement with GSNO-mediated increased expression of ZO-1 and occludin in a rat model of TBI (Khan et al. 2009).

image

Figure 2. Effect of S-nitrosoglutathione (GSNO) and 3-Morpholino-sydnonimine (SIN-1) on the expression of zonula occludens (ZO)-1 in ipsilateral (penumbra) rat brain. Western blot (a) and densitometry (b) of ZO-1 in the penumbra at 24 h after ischemia-reperfusion (IR). Representative western blot (two samples) from three different sets of experiments showed reduced expression of ZO-1 in brain from IR and SIN-1-treated animals. GSNO-treated IR animals had an expression of ZO-1 similar to the sham animals. *p < 0.05 versus Sham and GSNO.

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Peroxynitrite is formed early in and around the vessels after IR, and GSNO treatment reduces the expression of peroxynitrite

We have previously reported that GSNO protects not only the neurovascular unit after IR and TBI (Khan et al. 2005, 2006, 2009), but also reduces the levels of peroxynitrite in plasma following IR (Khan et al. 2006). Therefore, we investigated whether GSNO also reduces the levels of peroxynitrite, measured as the expression of 3-NT in the IR brain. Because peroxynitrite is unstable, its detection through 3-NT expression is an index of the levels of peroxynitrite (Pacher et al. 2007). The expression of 3-NT was measured at 4 h following IR in the penumbra region using western blot (Fig. 3a and b) and IHC (Fig. 3c). GSNO treatment significantly reduced the IR-mediated increased expression of 3-NT (Fig. 3a and b). A similar increase of the expression of 3-NT in IR (Fig. 3c ix) and decrease in GSNO (Fig. 3c v) was also observed using IHC. The expression was remarkable around vessels as indicated by its colocalization with SMA (Fig. 3c iii, vii, xi), which labels pericytes. The extreme-right, magnified panel of Fig. 3c shows that that the vessels are surrounded by 3-NT expression. The expression of 3-NT at 4 h was not observed in other cell types, including neurons (data not shown).

image

Figure 3. Effect of S-nitrosoglutathione (GSNO) on the expression of 3-nitrotyrosine (3-NT) in ipsilateral (penumbra) rat brain [(a) western analysis; (b) densitometry; (c) IHC analysis]. Representative western blot [one from sham and two each from GSNO and ischemia-reperfusion (IR)] from three different sets of experiments at 4 h after IR showed enhanced reactivity of 3-NT in brain from IR animals. GSNO-treated animals had reduced expression of 3-NT. IHC of 3-NT (green) and smooth muscle actin (SMA, red) in ipsilateral (penumbra) rat brain at 4 h after IR. (i–iv) Sham-operated animal. (v–viii) GSNO-treated IR animal. (ix–xii) Veh-treated IR animal. (ix'–xii') Magnified view of i-l. Green fluorescence in IR sections indicates higher immunoreactivity for 3-NT (ix, ix') in vessels labeled with SMA (red fluorescence, x, x'). Yellowish color in xi and xi' sections indicates the colocalization of 3-NT with SMA. In the magnified sections, asterisk indicates lumen of vessel while arrow points to vessel. Sham- and GSNO-treated animals did not show any significant staining of 3-NT. The photomicrograph is representative of three samples in each group.*p < 0.05 versus GSNO.

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IR increases the expression of E-selectin in vessels in the penumbra and GSNO down-regulates the expression

E-selectin and other adhesion molecules mediate the endothelial dysfunction that facilitates BBB leakage, resulting in the transmigration of blood-derived immune cells in the brain and thereby maintaining persistent inflammation following IR (Stanimirovic et al. 1997). Therefore, we determined whether the IR-mediated, early-increased expression of E-selectin would be remarkably decreased following GSNO treatment, leading to reduced BBB leakage. The penumbra region had significantly increased expression of E-selectin in and around the vessels following IR. GSNO treatment down-regulated the expression of E-selectin. The sham-operated brain had no significant expression of E-selectin (Fig. 4a and b).

image

Figure 4. Effect of S-nitrosoglutathione (GSNO) on the expression of E-selectin in ipsilateral (penumbra) rat brain at 4 h after ischemia-reperfusion (IR). Photomicrographs (a) and graph determining E-selectin-positive area (b). Photomicrographs show enhanced reaction of E-selectin in the penumbra from IR compared with GSNO group. Sham brain does not show significant staining of E-selectin. Symbol V on sections indicates vessel. Photomicrographs are representative of three samples in each group. **p < 0.01 versus Sham and GSNO.

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IR increases the expression of MMP-9 in endothelial and neuronal cells in the penumbra and GSNO down-regulates the expression

MMP-9 is a critical player in BBB disruption (Barr et al. 2010), and its expression activity is up-regulated in stroke patients (Horstmann et al. 2003). Endothelial peroxynitrite has been shown to increase MMP-9, leading to BBB leakage (Gursoy-Ozdemir et al. 2004). Therefore, we determined whether GSNO protects the integrity of BBB by reducing the expression of MMP-9. The penumbra region had increased expression of MMP-9 (Fig. 5a–c) at 4 h following IR. The expression of MMP-9 colocalized with CD34, an endothelial cell marker (lower panels of Fig. 5a and b) as well as NSE, a neuronal cell marker(lower panels of Fig. 5c and d). GSNO treatment prevented the increase in the expression of MMP-9. The sham-operated brain had no significant expression of MMP-9.

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Figure 5. Effect of S-nitrosoglutathione (GSNO) on the expression of matrix metalloproteinase (MMP)-9 in ipsilateral (penumbra) rat brain at 4 h after ischemia-reperfusion (IR). Photomicrographs of MMP-9/CD34 (a) and MMP-9/NSE (c), and graphs determining MMP-9/CD34 (b) and MMP-9/NSE (d) positive areas. Photomicrographs of IHC show enhanced reaction of MMP-9 (green color) in IR compared with GSNO group (a, b). Sham brain does not show MMP-9 positive cells. Colocalization (yellowish, merged) of MMP-9 (green color) with either endothelial cell marker CD34 (red) (a) or neuronal marker NSE (red) indicates that both neurons and endothelial cells have significantly increased expression of MMP-9 even at 4 h after IR. Photomicrographs are representative of three samples in each group. **p < 0.01 versus Sham and GSNO.

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IR induces p65 translocation to nucleus and increases NF-κB activity in the penumbra and GSNO decreases the activity

NF-κB regulates the activity/expression of several genes, including adhesion molecules and MMP-9, implicated in BBB leakage. We previously observed the increased NF-κB activity within 4 h in IR rats (Khan et al. 2007), and the activity of NF-κB was inhibited by the treatment with GSNO in endothelial as well as inflammatory cells (Khan et al. 2005; Prasad et al. 2007). GSNO was reported to reduce the activity of NF-κB by inhibiting its DNA-binding activity (Marshall and Stamler 2001).Therefore, we determined whether the increased NF-κB translocation/activity was inhibited by GSNO treatment of IR using IHC and the measurement of the activity of NF-κB/p65 binding with DNA as previously described (Khan et al. 2007). The expression of p65 was observed in all the three groups at 4 h after reperfusion (Fig. 6a and b). However, the expression of p65 in sham was localized in the cytosol, whereas p65 translocated and localized in the nucleus in IR and GSNO groups (Fig. 6a, lower magnified panel). To examine that the activity was decreased in spite of p65 translocation to the nucleus in the GSNO group, the activity of NF-κB was measured in the nuclear fraction. Fig. 6c shows that IR had significantly higher activity in the penumbra 4 h after reperfusion than sham. The activity of NF-κB was significantly inhibited (p < 0.001) by GSNO treatment.

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Figure 6. Effect of S-nitrosoglutathione (GSNO) on the expression of p65 of nuclear factor kappa B (NF-κB) and activity of NF-κB in ipsilateral (penumbra) rat brain at 4 h after ischemia-reperfusion (IR). Photomicrographs (a), graph determining p65-positive area (b) and NF-κB activity (c). While the expression of P65 in sham was localized mainly in cytosol, both IR and GSNO groups had expression present mainly in nucleus (a). NF-κB activity in the ipsilateral brain tissue was measured as described in Methods. The activity was significantly high in IR compared with sham and GSNO groups. Values are mean ± SD of triplicate determinations from three different experiments. ***p < 0.001 versus Sham and GSNO.

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Effect of GSNO on constitutive NOS (endothelial and neuronal) activities at -h reperfusion and NO levels at 4-h reperfusion in the penumbra following IR

Decreased NO levels in IR, because of its conversion to peroxynitrite, are implicated in endothelial dysfunction and its consequent BBB leakage. Therefore, the activity of cNOS and eNOS and the levels of NO were determined following IR. The contribution by iNOS was not studied because its induction was not observed until 6 h after reperfusion in the MCAO rat model. cNOS activity was significantly increased in IR at 1-h reperfusion (Fig. 7a). GSNO treatment of IR inhibited this activity (Fig. 7a). NO-producing eNOS activity was low in IR compared with sham (Fig. 7b). eNOS activity was further reduced by GSNO (Fig. 7b). Levels of NO in the penumbra decreased significantly in IR compared with sham measured at 4 h following reperfusion (Fig. 7c). These results are in agreement with the previously reported reduced levels of NO in cerebral tissues following acute brain injury (Irmak et al. 2003). Treatment with GSNO maintained the levels of NO in the penumbra (Fig. 7c).

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Figure 7. Effect of S-nitrosoglutathione (GSNO) on the activity of constitutive nitric oxide synthase (NOS) (a) and endothelial NOS (b) at 1 h and the levels of NO [c in ipsilateral (penumbra) brain] at 4 h after ischemia-reperfusion (IR). While activity of constitutive NOS is increased, endothelial NOS is decreased in the IR group. NO levels are also decreased in the IR group. GSNO and sham groups show similar activity of constitutive NOS and levels of NO. However, eNOS activity is decreased in the GSNO group compared with both sham and IR groups. The activity of cNOS and eNOS is expressed as pmol/mg protein/min and data are presented as mean ± SD, n = 3. ***p < 0.001,**p < 0.01 versus GSNO and Sham,+p < 0.05 versus Sham, ++p < 0.05 versus IR.

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GSNO enhances the expression of S-nitrosocysteine in the penumbra

Deficient S-nitrosylation is observed in several neurodegenerative disease processes (Rauhala et al. 1996; Ju et al. 2005; Schonhoff et al. 2006). Therefore, we determined whether the overall expression of S-nitrosylated (-SNO) proteins was decreased following IR and whether GSNO, an S-nitrosylating agent (Foster et al. 2009), increased -SNO expression. We observed the decreased expression of -SNO in the penumbra early at 4 h in IR brain, and this expression was normal in GSNO and sham groups (Fig. 8a and b), indicating that reduced –SNO expression is associated with IR pathophysiology.

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Figure 8. Effect of S-nitrosoglutathione (GSNO) on the expression of S-nitrosocysteine in ipsilateral (penumbra) rat brain at 4 h after ischemia-reperfusion (IR). Photomicrographs (a) and graph determining S-nitrosocysteine-positive area (b). The expression of S-nitrosocysteine was determined using immunohistochemistry (IHC). Photomicrographs show reduced reaction of S-nitrosocysteine in the IR compared with sham and GSNO groups. Photomicrographs are representative of n = 5 in each group (magnification x400). ***p < 0.001 versus Sham and GSNO.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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).

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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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

These studies were supported by grants from NIH (NS-72511, NS-22576 and NS-37766) and Veteran Administration merit awards. This work was also supported by the NIH, Grants C06 RR018823 and No C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources. We are grateful to Dr. Tom Smith from the MUSC Writing Center for his valuable editing and correction of the manuscript. The authors have no conflict of interest and have not received grants etc. from any commercial body.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References