SEARCH

SEARCH BY CITATION

Keywords:

  • 3-nitrotyrosine;
  • HARM ;
  • matrix metalloprotease;
  • peroxynitrite;
  • reperfusion injury

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Cerebral reperfusion injury may account for complications of thrombolysis and endovascular recanalization. Experimental studies have shown that brain matrix metalloproteinase (MMP) activity increases during reperfusion and is correlated with oxidative/nitrative stress. Increased plasma MMP levels have been reported in stroke, but no information is available for reperfusion-induced plasma MMP and 3-nitrotyrosine (3-NT, a marker of oxidative/nitrative stress) changes immediately after recanalization. We obtained plasma from 29 patients undergoing endovascular recanalization, 12 patients treated with thrombolysis, and six control patients having diagnostic angiogram before and 1,3, and 24 h after treatment to investigate the effect of cerebral reperfusion on plasma MMP gelatinolytic activity and 3-NT level. Hypoperfusion was shown distal to the stenotic artery in endovascular treatment patients. Presence of an occluded artery and recanalization was documented in thrombolysis patients. A significant increase was detected in plasma 3-NT levels 3 and 24 h after stenting/angioplasty. Plasma MMP-9 gelatinolytic activity rose more than 50% of the pre-treatment level in 12 of 29 patients. However, this was not statistically significant and not correlated with any of the clinical or radiological correlates of reperfusion injury (e.g., hyperperfusion and hemorrhage). After thrombolysis, a significant increase in plasma MMP-9 gelatinolytic activity at 3 and 24 h and the cleaved form of MMP-9 were detected. 3-NT levels increased by 44% and 62% at 3 and 24 h, which did not achieve statistical significance, but was highly correlated with admission NIH Stroke Scale (r = 0.930 p < 0.001). No change was detected in MMP-2 in all groups. In conclusion, these data suggest that the increased plasma MMP-9 levels is not a direct measure of MMP-9 activity in the reperfused brain but rather a consequence of tissue plasminogen activator infusion, whereas plasma 3-NT levels appear to originate from the reperfused brain vasculature. The changes in 3-NT levels may therefore be useful to monitor oxygen/nitrogen radical formation during reperfusion with serial measurements.

Abbreviations used
3-NT

3-nitrotyrosine

ACA

anterior cerebral artery

BBB

blood–Brain Barrier

eNOS

endothelial nitric oxide synthase

HARM

hyperintense acute reperfusion marker

iNOS

inducible nitric oxide synthase

MCA

middle cerebral artery

MMP

matrix metalloprotease

nNOS

neuronal nitric oxide synthase

NO

nitric oxide

ONOO

peroxynitrite

PCA

posterior cerebral artery

TCD

transcranial doppler

tPA

tissue plasminogen activator

One of the serious complications of cerebral revascularization therapies such as pharmacological or mechanical thrombolysis and endovascular stenting or angioplasty is reperfusion-induced vascular damage that may lead to brain hemorrhage, swelling, and hyperperfusion syndrome (Thomalla et al. 2007; van Mook et al. 2005; Wahlgren et al. 2007; Donnan et al. 2011). Studies in animal models of stroke consistently suggest that oxygen and nitrogen radicals generated in excess amounts during reperfusion play an important role in loss of blood–brain barrier (BBB) integrity (Wei et al. 1999; Gursoy-Ozdemir et al. 2000, 2004; Pacher et al. 2007; Yemisci et al. 2009; Fraser 2011). Constitutive isoforms of nitric oxide synthase (NOS) (neuronal and endothelial NOS) are activated by rises in intracellular calcium over the basal levels. Accordingly, unregulated, non-physiological levels of nitric oxide (NO) may be produced until intraendothelial and intraneuronal Ca+2 levels are normalized during reperfusion if sufficient amounts of l-arginine, cofactors and O2 are available (Ohta et al. 1997; Wei et al. 1999; Guix et al. 2005; Pacher et al. 2007; Fraser 2011). The Km values for O2 of eNOS and neuronal nNOS are four and 350 μM, respectively, so, the rate of NO production from eNOS is likely not limited by O2 concentration (which is around 150 μM in arterial blood) after reperfusion (Hall and Garthwaite 2009). Albeit indirect, the available evidence suggests that excess NO generated by eNOS in the endothelium significantly contributes to reperfusion-induced vascular injury probably because of also its location within vascular wall (Gursoy-Ozdemir et al. 2000, 2004; Pacher et al. 2007; Yemisci et al. 2009). Similarly, O2- radical formation increases during ischemia/reperfusion; mitochondria and over activity of enzymes such as NADPH oxidase and xanthine oxidase account for this increase (Adam-Vizi 2005; Kahles et al. 2007; Chrissobolis and Faraci 2008; Miller et al. 2009; Chen et al. 2011). Concomitant generation of NO and O2-, which have high affinity to each other, leads to formation of the strong oxidant peroxynitrite (ONOO-)(Pacher et al. 2007). One of the mechanisms of ONOO- toxicity is nitration of tyrosine residues on proteins (Pacher et al. 2007). Detection of 3-nitrotyrosine by chemical or immunohistochemical methods has clearly shown that ONOO- is generated in the ischemic brain, and attains higher concentrations on microvessels (Gursoy-Ozdemir et al. 2000, 2004; Nagashima et al. 2000; Pacher et al. 2007; Yemisci et al. 2009). Several studies have illustrated that 3-NT is not readily degraded by denitrases under pathological conditions, allowing its detection in pathological tissue specimens (Pacher et al. 2007; Abello et al. 2009). Considering the close contact of vascular wall with circulating blood and the immense surface area of cerebral microcirculation where the radicals are produced, it is likely that 3-NT plasma levels may be used as a reporter of oxygen/nitrogen radical generation in the brain although the dilution of brain-derived 3-NT in total plasma volume and plasma denitrases may diminish the detected levels.

One of the mechanisms thought to mediate the reperfusion-induced BBB disruption is activation of the matrix metalloproteinases (MMPs)(Rosenberg et al. 1996; Yang et al. 2007; Rosell and Lo 2008). MMPs are zinc-dependent endopeptidases, produced by all cells of the neurovascular unit and secreted as inactive zymogens to the extracellular space (Jian Liu and Rosenberg 2005; Zhao et al. 2007; Rosell and Lo 2008; Candelario-Jalil et al. 2009). In stroke models, MMP activation has been shown to cause BBB damage, which was more prominent in transient compared with permanent ischemia (Lu et al. 2008). Supporting the role of MMP activation in BBB damage, MMP inhibitors or knocking out the MMP-9 gene prevent BBB opening after ischemia/reperfusion (Yang et al. 2007; Sood et al. 2008; Candelario-Jalil et al. 2009). In addition to the animal studies, MMP-9 activation has been detected in postmortem brain obtained from stroke patients (Rosell et al. 2008). Moreover, an increase in plasma MMP-9 protein level was reported in ischemic stroke patients appearing soon after stoke onset and peaking at 24 h (Montaner et al. 2001). Although MMP activity was demonstrated in selected patients by gel zymography in some of these studies (Heo et al. 2003; Horstmann et al. 2003; Ning et al. 2006), the reported studies encompassing a substantial number of stroke patients, all have used MMP protein levels to study its association with several stroke-related parameters such as the size of ischemic lesion and hemorrhagic conversion (Montaner et al. 2001, 2003; Heo et al. 2003; Horstmann et al. 2003; Ning et al. 2006; Castellanos et al. 2007).

In this study, we aimed at detecting plasma 3-NT levels, and MMP-9 and MMP-2 gelatinolytic activities just before and after recanalization therapies to investigate whether reperfusion-induced oxidative/nitrative stress and MMP activation in ischemic/reperfused cerebral vasculature can be detected from the peripheral blood in patients whose occluded cerebral artery was reopened with tissue plasminogen activator (tPA) or severely stenotic artery was recanalized with stenting or angioplasty. Comparing tPA-treated stroke patients with angioplasty/stenting group provided an opportunity to separately investigate tPA's direct effects than those of the revascularization. An additional control group consisting of diagnostic angiography patients who were not revascularized, allowed us to differentiate the effects of intra-arterial instrumentation than those of recanalization. Moreover, we compared the plasma levels obtained after revascularization with the basal measurements taken just before the procedure in each patient to reduce the effect of individual variation.

Subjects

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Two groups of patients and one control group were prospectively included in this study. All subjects gave informed consent before peripheral venous blood obtained. This study was approved by the Institutional Ethics Committee (HEK 08/113). The first group (n = 12) consists of acute ischemic stroke patients admitted to hospital within the first 6 h after symptoms and treated with tPA. In four cases, tPA was combined with mechanic thrombolysis according to NINDS criteria. All cases had an occluded artery (middle cerebral artery, ACA, posterior cerebral artery, or basilar artery) shown with CT angiography (CTA) and/or transcranial Doppler (TCD). Peripheral venous blood samples were taken at three time points; prior to treatment, 3 and 24 h after the treatment. The second group (n = 29) consists of endovascular therapy patients meeting the following criteria: symptomatic critical stenosis at internal carotid or basilar artery, hypoperfusion at stenotic artery irrigation area (detected by TCD, perfusion MRI, or perfusion CT), or disturbed TCD vasomotor reactivity if there is no hypoperfusion. Peripheral venous blood samples were taken at four time points: prior to treatment, 1 h, 3 h, and 24 h after the treatment. Clinical assessment in both groups was made by NIH Stroke Scale (NIHSS) on admission to the hospital, on the first day of treatment and on discharge. The control group (n = 6) consists of diagnostic cerebral angiography patients who had no cerebral hypoperfusion. This group was included to the study to investigate the effect of contrast agent and intraarterial instrumentation on plasma 3-NT level and MMP gelatinolytic activity; blood samples were obtained before and 3 h after angiography.

Neuroimaging

All patients were evaluated clinically by the researcher neurologists 24 h after the treatment, and brain imaging (CT or MRI) was performed if there was any neurological worsening. MRI including Diffusion Weighted Imaging and Gradient Recalled Echo sequences was performed in all patients 12 h to 5 days after the treatment. Hyperintense Acute Reperfusion Marker MR sequence was used to evaluate the blood–brain barrier leakage (Cho et al. 2009) in endovascular treatment patients. All CT and MR images were analyzed by a neuroradiologist.

Detection of MMP gelatinolytic activity

Venous blood samples were taken in EDTA tubes; centrifuged at 2 800 g for 10 min. Plasma was frozen at −80°C until analysis. Plasma MMP-2 and MMP-9 activities were measured using gel zymography as previously described (Heo et al. 1999; Snoek-van Beurden and den Hoff 2005). Briefly, blood samples were centrifuged and plasma was separated, which was then diluted (1/10) in a working buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, and 0.02% NaN3). Proteins were separated by electrophoresis through 10% polyacrylamide zymogram gels containing gelatin (Invitrogen, Carlsbad, California, USA) under non-denaturating and non-reducing conditions. After washes in 2.5% Triton X-100 to remove sodium dodecyl sulfate and then in 50 mM Tris-HCl, pH 7.5, the gels were incubated overnight in developing buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 1 M ZnCl2, and 0.05% BRIJ-35) at 37°C. Gels were stained with 0.25% Coomassie blue G-250 in 20% methanol and 10% acetic acid, and then were washed in solution containing 20% methanol and 10% acetic acid before being photographed and analyzed by densitometry. A representative gel zymogram of one of the thrombolysis patients is shown in Fig. 1.

image

Figure 1. Gel zymography illustrates the matrix metalloprotease (MMP)-9 and MMP-2 gelatinolytic activity in a plasma sample from a tPA-treated patient (patient no. 4 in Table 2). Panels show the plasma MMP-9 and MMP-2 gelatinolytic activity just before and 3 and 24 h after tPA administration. Various MMP isoforms and the MMP-9/TIMP1 complex were identified on the gel by their molecular weight.

Download figure to PowerPoint

3-Nitrotyrosine detection

Plasma 3-nitrotyrosine levels were determined with a “sandwich” ELISA kit (Bioxytech Nitrotyrosine-EIA, OxisResearch, Portland, OR, USA) according to the manufacturers' protocol. Hundred microliters of 10 times diluted serum samples were put into each well and incubated at 25°C for 1 h. After washing wells with washing buffer four times, 100 μL of anti-nitrotyrosine antibody (Bioxytech Nitrotyrosine-EIA) was added to each well and incubated at 25°C for 1 h. Hundred microliters of streptavidin peroxidase (Bioxytech Nitrotyrosine-EIA) was added to washed wells and incubated at 25°C for 1 h. Then, 100 μL tetrametilbenzidine substrate was added to washed wells and incubated for 30 min in the dark. Wells were washed with washing buffer four times before each of the steps above. Finally, 100 μL of stop solution was added to wells and absorbance of the samples in wells was detected at 450 nM in SpectraMax M2 Microplate Reader (Molecular Devices, San Diego, CA, USA). Counter concentrations in nM of measured absorbances were calculated by calibration curves drawn by using SOFTmax Pro 3.1.1 program (Spectramax, San Diego, CA, USA). Readings were divided by the quantity of plasma protein in each sample determined with Bradford-Solution (Applichem, Darmstadt, Germany). 3-nitrotyrosine levels were given as nanomolar per each microgram protein.

Statistical methods

All values are given as “mean ± SD”. Wilcoxon's signed rank and Fisher's exact tests were used to compare groups in terms of numerical and categorical variables, respectively. Temporal changes of biomarkers were evaluated using both anova for repeated measures followed by Wilcoxon's signed rank test for all pairwise comparisons. Spearman's rho test was used for correlation analysis. A p-value lower than 0.05 was considered statistically significant. For multiple comparisons, p-values were corrected with least square differences method. spss® 13.0 (SPSS Inc, Chicago, IL, USA) and MedCalc® statistical package programs were used for the analyses.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Plasma was obtained from 29 patients undergoing endovascular treatment, 12 patients treated with thrombolysis and six control patients having diagnostic angiogram. Hypoperfusion was shown distal to the stenotic artery in all endovascular treatment patients. Presence of occluded artery and recanalization after thrombolysis was documented in all thrombolysis patients.

Endovascular treatment group

Demographic and clinical features of the endovascular treatment group are shown in Table 1. Twenty four of the patients were male and the mean age of the group was 64 (range 48–77). Internal carotid artery/ies were stenotic in 26 patients. One patient had basilar artery stenosis, one patient had middle cerebral artery stenosis, and in one patient both basilar and internal carotid artery were stenotic. All patients were symptomatic and on antiaggregant therapy before the procedure. Hypoperfusion was detected with TCD in 16, with perfusion CT in six, with both TCD and perfusion CT in five and with perfusion MRI in one patient, and by demonstrating a disturbed cerebral vasomotor reactivity with TCD in one patient. None of the patients had catheterization-related complications.

Table 1. Demographic, clinical and radiological features of endovascular treatment group
Patient no AgeSexRisk FactorSymptomatic arteryHypoperfusion evaluation method HyperperfusionNIHS score increase HeadacheDWI acuteHARM contrast enhancement
  1. M: Male; F: Female; CAD: coronary artery disease; HL: Hyperlipidemia; HT: Hypertension; DM: Diabetes mellitus; ICA: Internal carotid artery; MCA: Middle cerebral artery; CTP: computed tomography perfusion; TCD: Transcranial doppler; VMR: Vasomotor reactivity; MRP: Magnetic resonance perfusion.

171MCADICACTPNoNoNoNoNot available
273FCAD/HLICATCDNoNoNoPresentNo
350MHT/DMICACTPNoNoNoNoNo
466MHT/CADICATCD-VMRNoNoNoPresentNo
556FHT/DM/HL/CADICATCDNoNoPresentPresentNo
653MCAD/HLICACTPPresentNoPresentPresentNo
757MHTICATCDPresentNoPresentPresentNo
860MHT/CAD/HLICATCD&CTPNoNoNoPresentPresent
970MHT/DM/HLICATCD&CTPPresent1 pointNoPresentNot available
1050FHT/DM/HL/CADICATCD&CTPNoNoNoPresentNot available
1159FHT/CAD/HLICATCD&CTPPresentNoNoNoPresent
1269MHTICACTPNoNoNoNoNo
1358MHT/DM/HLICATCDPresentNoPresentNoNo
1469MHT/DM/HLICACTPPresent4 pointsPresentPresentNo
1548FHT/DMICATCDPresent1 pointPresentNoNo
1659MHTICATCDNoNoNoNoNot available
1766MHT/DM/HL/CADICATCDPresentNoPresentPresentNo
1868MHT/DM/HL/CADICATCDNoNoNoNoNo
1975MHT/DM/HL/CADICATCDNoNoPresentPresentNo
2077MHT/HLICATCDNoNoNoNoNo
2166MHT/DM/CADICATCDNoNoNoNoNo
2273MCAD/HLBasillaryTCDNoNoNoPresentNo
2360MHT/DM/HLICACTPNoNoNoPresentNo
2468MHT/DM/HL/CADBasillaryTCDPresent4 pointsNoPresentPresent
2569MHT/HLICATCDNoNoNoPresentNo
2663MHT/DM/CADMCAMRPPresentNoNoPresentNo
2761MHT/DM/HLICATCDPresentNoNoNoNot available
2857MHTICATCDPresentNoPresentNoPresent
2975MHT/DM/HL/CADICATCD&CTPNoNoNoPresentNo

After recanalization of the stenotic artery, mild hyperperfusion occurred in 12 patients, which recovered approximately in 1 week. Diffusion Weighted Imaging showed revascularization-related small, millimetric, and sometimes multiple acute ischemic lesions in 17 patients. Sulcal contrast enhancement was observed in four of 24 patients evaluated with hyperintense acute reperfusion marker MRI sequence (Fig. 2). Clinical worsening was detected in five patients showing hyperperfusion; three had headache, four had acute ischemic lesions in MRI. A left frontal small hemorrhage was detected in one of them (Fig. 2), and one patient had asymptomatic millimetric basal ganglia hemorrhage detected with Gradient Recalled Echo MRI.

image

Figure 2. Endovascular revascularization-induced cerebral lesions in different patients. (a) Diffusion-weighted imaging showing multiple embolic infarctions (arrows). (b) CT image showing a small left frontal hemorrhage (arrow). (c,d) FLAIR images before and after contrast injection showing sulcal contrast enhancement (arrows) indicating blood-brain barrier disruption (HARM- hyperintense acute reperfusion marker).

Download figure to PowerPoint

Despite the above listed clinical and imaging evidence of reperfusion injury, there was no significant change in plasma MMP-9 and MMP-2 gelatinolytic activity in this group of patients after intervention. A statistically insignificant increasing trend in MMP-9 was observed such that the MMP-9 gelatinolytic activity rose more than 50% in 12 of 29 patients (Fig. 3). However, no correlation was found between the basal MMP-9 gelatinolytic activity or activity change after recanalization and any of the above clinical or radiological findings. Also, none of the 29 patients' zymograms exhibited the cleaved MMP-9 band. On the other hand, a heterogeneous (from 0.49 ± 0.21 to 0.66 ± 0.25 nano mole/mg protein) but significant increase was detected in plasma 3-NT levels at 3 and 24 h after revascularization (p < 0.05), while the significance was marginal at the 1st hour (p = 0.068)(Fig. 3).

image

Figure 3. Temporal percent changes of matrix metalloprotease (MMP)-9 and MMP-2 gelatinolytic activity and 3-nitrotyrosine (3-NT) level in patients who underwent angioplasty/stenting (endovascular recanalisation), diagnostic angiography (control) and treated by thrombolysis. Columns represent mean percent (%) changes from the baseline measurements taken as 100% (horizontal line). Error bars represent ± 1 standard deviation. Dots illustrate the normalized individual changes (%) from the pre-treatment values. For normalization, the difference between the test and basal value was divided by the basal value for each case. Statistical evaluation was performed by comparing absolute values (in arbitrary units for MMPs and nanomole/mg protein for 3-NT, not shown) but not percent changes at different time points. *p < 0.05, #p = 0.068.

Download figure to PowerPoint

Thrombolysis group

Twelve patients who showed successful recanalization after thrombolysis were included in the study. Four of the patients were female, and the mean age of this group was 63 (range 39–83). Demographic and clinical features are given in Table 2. The presence of arterial occlusion was demonstrated with CT angiography at admission in all patients; eight patients had an occluded middle cerebral artery, three patients had basilar artery occlusion, and one had an occluded posterior cerebral artery. Only i.v. rtPA used in eight of them and, in four patients, i.v. rtPA was followed by mechanic thrombolysis. Recanalization was achieved in all patients. Accordingly, there was a significant reduction in NIHSS scores between admission and discharge (admission 18 ± 6, post-rtPA 13 ± 6, 24 h 9 ± 6, discharge 9 ± 7; p < 0.02) despite the fact that CT at 24th hour showed hemorrhage in six patients (Table 2).

Table 2. Demographic, clinical and radiological features of thrombolysis treatment group
Patient noAgeSexRisk FactorBasal BP mmHgBasal NIHSSOccluded arteryThrombolysis methodHemorrhage at CTHemorrhage definitionDischarge NIHSS
  1. M: Male; F: Female; CAD: coronary artery disease; HL: Hyperlipidemia; HT: Hypertension; AF: Atrial fibrillation; MCA: Middle cerebral artery; PCA: Posterior cerebral artery; GRE-MRI: Gradient Recalled Echo Magnetic Resonance Image.

154MHT/CAD/Smoking125/7513MCAi.v. rtPANo 6
252MNo100/5017MCAi.v. rtPA + mechanic thrombolysisPresentAt basal ganglia 3.5 × 1.5cm18
353MHT/CAD/Smoking140/10015MCAi.v. rtPANo 7
476MHT/HL/AF/Smoking180/9016PCAi.v. rtPAPresentAt PCA infarction area3.5 × 2.5 cm13
562MHT/AF120/8027Basillaryi.v. rtPA + mechanic thrombolysisNoAt GRE-MRI milimetric hemorrhaga at pons23
678FHT/HL/AF140/8017MCAi.v. rtPA + mechanic thrombolysisPresent0.6 × 0.8 cm at lentiform nucleus4
759FHT120/8010MCAi.v. rtPAPresent3.5 × 3.5 cm lentiform nucleus4
883FHT/CAD/AF240/11530Basillaryi.v. rtPAPresentAt PCA infarction area15
969MHL/AF/Smoking175/8520MCAi.v. rtPANo 2
1039FSmoking140/11020MCAi.v. rtPA + mechanic thrombolysisPresentAt basal ganglia 2 ×1 cm7
1168MHT/CAD110/6016Basillaryi.v. rtPANo 2
1267MHT/HL/CAD110/7026MCAi.v.rtPANo 6

There was a significant increase of MMP-9 gelatinolytic activity at 3 and 24 h by 51 and 30%, respectively, whereas no significant changes observed in MMP-2 and 3-NT levels after thrombolysis (Figs 1and 3). In contrast to the endovascular treatment group, seven of 12 patients' zymograms displayed the cleaved MMP-9 band (Fig. 1). 3-NT levels increased by 44% within 3 h and 62% at 24 h. This increasing trend did not achieve statistical significance possibly because of low number of samples. However, the 24 h values were highly correlated with admission NIHSS (r = 0.930, p < 0.001).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

We found that changes induced by recanalization therapies in plasma 3-NT level and MMP gelatinolytic activity could be monitored by peripheral blood assays. Interestingly, revascularization of the hypoperfused vascular territories caused an increase in plasma 3-NT but no significant change in MMP-9, whereas recanalization of the occluded cerebral artery with tPA induced robust plasma MMP-9 activation. No significant changes were observed in plasma MMP-2 in all groups. These findings suggest that reperfusion-induced oxidative/nitrative stress in the cerebral vasculature can be detected in the peripheral blood and that tPA rather than reperfusion leads to changes in plasma MMP-9 gelatinolytic activity.

Despite significant progress in understanding the mechanisms of reperfusion injury in experimental studies, translation of these findings to clinical practice is limited because of lack of reliable and easily accessible biomarkers. Detection of plasma MMP protein levels, especially of MMP-9, has recently been proposed as a potential marker of the volume of infarcted tissue, stroke severity, functional outcome, and importantly, of the hemorrhagic transformation after thrombolytic treatment (Montaner et al. 2001, 2003; Heo et al. 2003; Horstmann et al. 2003; Montaner 2006; Ning et al. 2006; Castellanos et al. 2007; Ramos-Fernandez et al. 2011). The signal for MMP increase in the plasma may arise from the ischemic vasculature through which blood can circulate (i.e., penumbra and reperfused tissue). Induction of MMP synthesis in white blood cells may be the source of this increase and, may indirectly reflect the ischemic stress in the brain (Kouwenhoven et al. 2001; Cuadrado et al. 2008; Rosell et al. 2008; Chou et al. 2011). However, protein measurements should be cautiously correlated with reperfusion-induced toxic mechanisms in the brain because they do not directly correspond to MMP activity as the MMP activity is strictly regulated by endogenous inhibitors to avoid uncontrolled activation like are the other proteases (Candelario-Jalil et al. 2009; Hadler-Olsen et al. 2011). Our study based on serial measurements from the same patient in the acute period after reperfusion shows that plasma MMP-9 can promptly increase and be activated after tPA-mediated recanalization. However, the major contributor to this appears to be tPA itself rather than reperfusion. This finding strongly supports the concerns that tPA may lead to hemorrhagic transformation by activating MMPs (Tsuji et al. 2005; Ning et al. 2006; Yepes et al. 2009). Of note, we observed the cleavage of the pro-MMP-9 to its active form in only seven of 12 thrombolysis patients. The increase in pro-MMP activity may be secondary to an increase in its plasma levels as well as to its allosteric activation via cystein-switch without cleavage of the pro domain (Hadler-Olsen et al. 2011). Signals (e.g., cytokines and radicals) originating from the ischemic/reperfused vasculature might also contribute to MMP-9 activation/induction in plasma in tPA-treated patients. Contrary to this idea, relatively faster reperfusion after angioplasty or stenting did not lead to robust MMP activation although it induced significant radical formation as suggested by elevated 3-NT levels. However, such an activation might have been difficult to detect in some patients because reperfusion of the oligemic tissue distal to a severely stenotic artery may not always induce a signal as strong as reperfusion of the ischemic tissue does. On the other hand, plasma MMP-2 gelatinolytic activity was unaltered after reperfusion obtained with either treatment approaches despite the fact MMP-2 is activated with similar signals to MMP-9, and it is reported to be activated in the reperfused brain especially in parenchymal cells (Yang et al. 2007). The reasons for this remain unclear, but might simply be as a result of stronger suppression of MMP-2 activity in the plasma (Hadler-Olsen et al. 2011).

Baseline plasma MMP gelatinolytic activities and 3-NT levels showed considerable variation among individuals possibly because of confounding factors such as smoking, associated diseases (e.g., hypertension, hyperlipidemia and diabetes), and medications used (Shishehbor et al. 2003; Bo et al. 2005; Peluffo and Radi 2007; Jin et al. 2011; Marchesi et al. 2012). We reduced the effect of this variation by comparing the plasma levels obtained soon after revascularization with the basal measurements taken just before the procedure in each patient. Therefore, the values obtained within the first 3 h are not expected to be significantly affected by uncontrolled variables because patients went through similar procedures during this period. We found a 45% increase in 3-NT levels 1 h after angioplasty or stenting, which was sustained at 3 and 24 h (43 and 49%, respectively). This seems to be unrelated to intraarterial instrumentation because no similar enhancement was observed in the diagnostic angiography group. Rapid reperfusion in large arterial supply areas (usually most of the internal carotid territory) may have led to sizable radical production than can be detected in the peripheral blood despite dilution in total plasma volume and degradation (Pacher et al. 2007). After tPA treatment, 3-NT levels increased by 44% within 3 h, which was also sustained at 24 h (62%). These increases did not achieve statistical significance possibly because of low number of samples. The slow and variable speed and completeness of recanalization as well as the total volume of the successfully reperfused tissue may have induced varying degrees of radical formation (Gursoy-Ozdemir et al. in this issue)(Yemisci et al. 2009). Moreover, some patients may have already had high basal 3-NT levels depending on the size of the partially perfused penumbral tissue before thrombolysis. Taken together, it appears so that 3-NT levels reflect the radical formation at cerebral vasculature and the plasma measurements may be informative even when working with even small number patients if baseline values are available for each subject to reduce the effect of variation. In ischemia/reperfusion studies, 3-NT shows measurable changes and seems to be preferable over the other available markers of oxidative stress because it also detects O2- radicals converted to ONOO in addition to the mechanistic importance of ONOO in the reperfusion injury.

In conclusion; our study shows that plasma equivalents of biomarkers identified in the ischemic brain in experimental studies may not always straightforwardly reflect the mechanisms in ischemic human brain. Our findings suggest that the plasma MMP-9 gelatinolytic activity is not a direct measure of MMP-9 activity in the reperfused brain but rather a consequence of tPA infusion. However, plasma MMP-9 may also be indirectly induced by signals (e.g., cytokines and radicals) originating from the ischemic vasculature, and hence, it may be indirectly correlated with some of the ischemic processes in the brain. The enhanced plasma 3-NT levels on the other hand appear to be originating from the ischemic/reperfused brain vasculature. The changes in 3-NT levels may therefore be useful to monitor radical formation during acute ischemia/reperfusion especially over time with serial measurements. However, it should be noted that the magnitude of the 3-NT change is probably not a simple function of the radical formation on the vasculature but may be modified by factors such as size of the circulated ischemic/reperfused area (which provides ONOO to plasma), pace of the reperfusion, and elimination kinetics of 3-NT. Moreover, other sources of 3-NT than ONOO should also be considered when making mechanistic associations (Pacher et al. 2007).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The authors thank to Drs. Saruhan Çekirge, Serdar Geyik, and Kıvılcım Yavuz from the neurointervention team for allowing us to obtain blood samples from patients, and to Drs. Atay Vural and Evren Erdener for their help with zymograms, and to Dr. Kıvılcım Kılıç for her help with figure 1.

Conflicts of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The authors declare no conflict of interest. Dr. T. Dalkara's work is supported by the Turkish Academy of Sciences.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  • Abello N., Kerstjens H. A., Postma D. S. and Bischoff R. (2009) Protein tyrosine nitration: selectivity, physicochemical and biological consequences, denitration, and proteomics methods for the identification of tyrosine-nitrated proteins. J. Proteome Res. 8, 32223238.
  • Adam-Vizi V. (2005) Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid. Redox Signal. 7, 11401149.
  • Bo S., Gambino R., Guidi S., Silli B., Gentile L., Cassader M. and Pagano G. F. (2005) Plasma nitrotyrosine levels, antioxidant vitamins and hyperglycaemia. Diabet. Med. 22, 11851189.
  • Candelario-Jalil E., Yang Y. and Rosenberg G. A. (2009) Diverse roles of matrix metalloproteinases and tissue inhibitors of metalloproteinases in neuroinflammation and cerebral ischemia. Neuroscience 158, 983994.
  • Castellanos M., Sobrino T., Millan M. et al. (2007) Serum cellular fibronectin and matrix metalloproteinase-9 as screening biomarkers for the prediction of parenchymal hematoma after thrombolytic therapy in acute ischemic stroke: a multicenter confirmatory study. Stroke 38, 18551859.
  • Chen H., Yoshioka H., Kim G. S. et al. (2011) Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid. Redox Signal. 14, 15051517.
  • Cho A. H., Suh D. C., Kim G. E., Kim J. S., Lee D. H., Kwon S. U., Park S. M. and Kang D. W. (2009) MRI evidence of reperfusion injury associated with neurological deficits after carotid revascularization procedures. Eur. J. Neurol. 16, 10661069.
  • Chou S. H., Feske S. K., Simmons S. L. et al. (2011) Elevated peripheral neutrophils and matrix metalloproteinase 9 as biomarkers of functional outcome following subarachnoid hemorrhage. Transl. Stroke Res. 2, 600607.
  • Chrissobolis S. and Faraci F. M. (2008) The role of oxidative stress and NADPH oxidase in cerebrovascular disease. Trends Mol. Med. 14, 495502.
  • Cuadrado E., Ortega L., Hernandez-Guillamon M., Penalba A., Fernandez-Cadenas I., Rosell A. and Montaner J. (2008) Tissue plasminogen activator (t-PA) promotes neutrophil degranulation and MMP-9 release. J. Leukoc. Biol. 84, 207214.
  • Donnan G. A., Davis S. M., Parsons M. W., Ma H., Dewey H. M. and Howells D. W. (2011) How to make better use of thrombolytic therapy in acute ischemic stroke. Nat. Rev. Neurol. 7, 400409.
  • Fraser P. A. (2011) The role of free radical generation in increasing cerebrovascular permeability. Free Radical Biol. Med. 51, 967977.
  • Guix F. X., Uribesalgo I., Coma M. and Munoz F. J. (2005) The physiology and pathophysiology of nitric oxide in the brain. Prog. Neurobiol. 76, 126152.
  • Gursoy-Ozdemir Y., Bolay H., Saribas O. and Dalkara T. (2000) Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia. Stroke 31, 19741980 ; discussion 1981.
  • Gursoy-Ozdemir Y., Can A. and Dalkara T. (2004) Reperfusion-induced oxidative/nitrative injury to neurovascular unit after focal cerebral ischemia. Stroke 35, 14491453.
  • Hadler-Olsen E., Fadnes B., Sylte I., Uhlin-Hansen L. and Winberg J. O. (2011) Regulation of matrix metalloproteinase activity in health and disease. FEBS J. 278, 2845.
  • Hall C. N. and Garthwaite J. (2009) What is the real physiological NO concentration in vivo? Nitric Oxide 21, 92103.
  • Heo J. H., Lucero J., Abumiya T., Koziol J. A., Copeland B. R. and del Zoppo G. J. (1999) Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J. Cereb. Blood Flow Metab. 19, 624633.
  • Heo J. H., Kim S. H., Lee K. Y., Kim E. H., Chu C. K. and Nam J. M. (2003) Increase in plasma matrix metalloproteinase-9 in acute stroke patients with thrombolysis failure. Stroke 34, e4850.
  • Horstmann S., Kalb P., Koziol J., Gardner H. and Wagner S. (2003) Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients: influence of different therapies. Stroke 34, 21652170.
  • Jian Liu K. and Rosenberg G. A. (2005) Matrix metalloproteinases and free radicals in cerebral ischemia. Free Radic. Biol. Med. 39, 7180.
  • Jin H., Webb-Robertson B. J., Peterson E. S., Tan R., Bigelow D. J., Scholand M. B., Hoidal J. R., Pounds J. G. and Zangar R. C. (2011) Smoking, COPD, and 3-nitrotyrosine levels of plasma proteins. Environ. Health Perspect. 119, 13141320.
  • Kahles T., Luedike P., Endres M., Galla H. J., Steinmetz H., Busse R., Neumann-Haefelin T. and Brandes R. P. (2007) NADPH oxidase plays a central role in blood-brain barrier damage in experimental stroke. Stroke 38, 30003006.
  • Kouwenhoven M., Carlstrom C., Ozenci V. and Link H. (2001) Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke. J. Clin. Immunol. 21, 365375.
  • Lu A., Clark J. F., Broderick J. P., Pyne-Geithman G. J., Wagner K. R., Ran R., Khatri P., Tomsick T. and Sharp F. R. (2008) Reperfusion activates metalloproteinases that contribute to neurovascular injury. Exp. Neurol. 210, 549559.
  • Marchesi C., Dentali F., Nicolini E. et al. (2012) Plasma levels of matrix metalloproteinases and their inhibitors in hypertension: a systematic review and meta-analysis. J. Hypertens. 30, 316.
  • Miller A. A., Drummond G. R., De Silva T. M., Mast A. E., Hickey H., Williams J. P., Broughton B. R. and Sobey C. G. (2009) NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2. Am. J. Physiol. Heart Circ. Physiol. 296, H220225.
  • Montaner J. (2006) Stroke biomarkers: can they help us to guide stroke thrombolysis? Drug News Perspect. 19, 523532.
  • Montaner J., Alvarez-Sabin J., Molina C., Angles A., Abilleira S., Arenillas J., Gonzalez M. A. and Monasterio J. (2001) Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment. Stroke 32, 17591766.
  • Montaner J., Fernandez-Cadenas I., Molina C. A. et al. (2003) Safety profile of tissue plasminogen activator treatment among stroke patients carrying a common polymorphism (C-1562T) in the promoter region of the matrix metalloproteinase-9 gene. Stroke 34, 28512855.
  • van Mook W. N., Rennenberg R. J., Schurink G. W., van Oostenbrugge R. J., Mess W. H., Hofman P. A. and de Leeuw P. W. (2005) Cerebral hyperperfusion syndrome. Lancet neurol. 4, 877888.
  • Nagashima T., Wu S., Ikeda K. and Tamaki N. (2000) The role of nitric oxide in reoxygenation injury of brain microvascular endothelial cells. Acta Neurochir. Suppl. 76, 471473.
  • Ning M., Furie K. L., Koroshetz W. J. et al. (2006) Association between tPA therapy and raised early matrix metalloproteinase-9 in acute stroke. Neurology 66, 15501555.
  • Ohta K., Graf R., Rosner G. and Heiss W. D. (1997) Profiles of cortical tissue depolarization in cat focal cerebral ischemia in relation to calcium ion homeostasis and nitric oxide production. J. Cereb. Blood Flow Metab. 17, 11701181.
  • Pacher P., Beckman J. S. and Liaudet L. (2007) Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315424.
  • Peluffo G. and Radi R. (2007) Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc. Res. 75, 291302.
  • Ramos-Fernandez M., Bellolio M. F. and Stead L. G. (2011) Matrix metalloproteinase-9 as a marker for acute ischemic stroke: a systematic review. J. Stroke Cerebrovasc. Dis. 20, 4754.
  • Rosell A. and Lo E. H. (2008) Multiphasic roles for matrix metalloproteinases after stroke. Curr. Opin. Pharmacol. 8, 8289.
  • Rosell A., Cuadrado E., Ortega-Aznar A., Hernandez-Guillamon M., Lo E. H. and Montaner J. (2008) MMP-9-positive neutrophil infiltration is associated to blood-brain barrier breakdown and basal lamina type IV collagen degradation during hemorrhagic transformation after human ischemic stroke. Stroke 39, 11211126.
  • Rosenberg G. A., Navratil M., Barone F. and Feuerstein G. (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J. Cereb. Blood Flow Metab. 16, 360366.
  • Shishehbor M. H., Aviles R. J., Brennan M. L. et al. (2003) Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. JAMA 289, 16751680.
  • Snoek-van Beurden P. A. and Von den Hoff J. W. (2005) Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 38, 7383.
  • Sood R. R., Taheri S., Candelario-Jalil E., Estrada E. Y. and Rosenberg G. A. (2008) Early beneficial effect of matrix metalloproteinase inhibition on blood-brain barrier permeability as measured by magnetic resonance imaging countered by impaired long-term recovery after stroke in rat brain. J. Cereb. Blood Flow Metab. 28, 431438.
  • Thomalla G., Sobesky J., Kohrmann M. et al. (2007) Two tales: hemorrhagic transformation but not parenchymal hemorrhage after thrombolysis is related to severity and duration of ischemia: MRI study of acute stroke patients treated with intravenous tissue plasminogen activator within 6 hours. Stroke 38, 313318.
  • Tsuji K., Aoki T., Tejima E. et al. (2005) Tissue plasminogen activator promotes matrix metalloproteinase-9 upregulation after focal cerebral ischemia. Stroke 36, 19541959.
  • Wahlgren N., Ahmed N., Davalos A. et al. (2007) Thrombolysis with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST): an observational study. Lancet 369, 275282.
  • Wei G., Dawson V. L. and Zweier J. L. (1999) Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim. Biophys. Acta 1455, 2334.
  • Yang Y., Estrada E. Y., Thompson J. F., Liu W. and Rosenberg G. A. (2007) Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J. Cereb. Blood Flow Metab. 27, 697709.
  • Yemisci M., Gursoy-Ozdemir Y., Vural A., Can A., Topalkara K. and Dalkara T. (2009) Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat. Med. 15, 10311037.
  • Yepes M., Roussel B. D., Ali C. and Vivien D. (2009) Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic. Trends Neurosci. 32, 4855.
  • Zhao B. Q., Tejima E. and Lo E. H. (2007) Neurovascular proteases in brain injury, hemorrhage and remodeling after stroke. Stroke 38, 748752.