Effects of Tissue Plasminogen Activator and Annexin A2 Combination Therapy on Long-Term Neurological Outcomes of Rat Focal Embolic Stroke
Background and Purpose—Tissue-type plasminogen activator (tPA) in combination with recombinant annexin A2 (rA2) is known to reduce acute brain damage after focal ischemia. Here, we ask whether tPA-plus-rA2 combination therapy can lead to sustained long-term neurological improvements as well.
Methods—We compared the effects of intravenous high-dose tPA alone (10 mg/kg) versus a combination of low-dose tPA (5 mg/kg) plus 10 mg/kg rA2 in a model of focal embolic cerebral ischemia in rats. All rats were treated at 3 hours after embolization. Brain tissue and neurological outcomes were assessed at 1 month. Surrogate biomarkers for endogenous neurovascular remodeling in peri-infarct area were analyzed by immunohistochemistry.
Results—Compared with high-dose tPA alone, low-dose tPA-plus-rA2 significantly decreased infarction and improved neurological function at 1-month poststroke. In peri-infarct areas, tPA-plus-rA2 combination therapy also significantly augmented microvessel density, vascular endothelial growth factor, and synaptophysin expression.
Conclusions—Compared with conventional high-dose tPA alone, combination low-dose tPA plus rA2 therapy may provide a safe and effective way to improve long-term neurological outcomes after stroke.
Improving tissue-type plasminogen activator (tPA) thrombolytic therapy is a high priority in stroke research. The ability of tPA to convert plasminogen efficiently into clot-dissolving plasmin relies on an endogenous fibrinolytic assembly via a triple complex formation of tPA, annexin A2, and plasminogen.1 Annexin A2 is a cell-surface protein, which, in complex with its binding partner p11, forms a heterotetrameric (A22p112) receptor for both plasminogen, the inactive precursor of plasmin, and its activator, tPA. By assembling tPA, annexin A2, and plasminogen, this complex increases the catalytic efficiency of tPA, enabling it to convert plasminogen to plasmin more efficiently than the same amount of tPA alone.2 We hypothesize that low-dose tPA plus recombinant annexin A2 protein (rA2) improve reperfusion and neurological outcomes.2 Our previous experiments have shown that combining rA2 with low-dose tPA successfully achieved reperfusion and reduced acute infarct size when treated at 2 hours, and significantly decreased hemorrhagic transformation when treatment was delayed to 4 hours after focal embolic stroke in rats.2–4 The purpose of the present study was to extend these promising findings by asking whether the benefits of tPA plus rA2 combination therapy can be sustained for long-term neurological outcomes.
Materials and Methods
Focal Embolic Cerebral Ischemia in Rats
All experiments were performed following an institutionally approved protocol in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats (280–330 g) were subjected to focal embolic strokes as we previously described.5
Assessments of Neurological Function Deficits
Modified neurological severity score (NSS), foot fault test for motor coordination function, and adhesive tap-removal test for sensorimotor neurological deficits were assessed on days 1, 3, 7, 14, 21, and 28 after stroke by following standard methods.6–8
Measurements of Brain Infarction and Mortality
At 28 days after stroke, brain hematoxylin and eosin infarction volume was examined as we previously described and expressed as percentage of hemisphere.5 Within 28 days after stroke, dead animals were counted for mortality rates.
Immunohistochemistry and Quantification
At 28 days after stroke, immunohistochemistry was performed on the coronal sections at −0.8 and −2.8 mm from bregma (the maximal brain infarct area) by following standard methods.8 Primary antibodies against von Willebrand factor (Abbiotec, San Diego), vascular endothelial growth factor (VEGF; Santa Cruz biotechnology, Santa Cruz, CA), and synaptophysin (Chemicon, Temecula, CA) were used. Vessel density (von Willebrand factor-positive vessels on 3 fields per section in peri-infarct cortex) was quantified as percentage of von Willebrand factor-positive vessels area; VEGF expression (positive cells area on 3 fields per section in peri-infarct cortex) was quantitated as percentage of immunopositive area; and synaptophysin expression (positive signals on 8 fields per section in peri-infarct striatum) was quantitated as optical density.
For this translation study, all STAIR (stroke therapy academic industry roundtable) and RIGOR guidelines were followed in terms of randomization, blinding, and statistical powering.9,10 Two experimental groups: standard rat dose of tPA 10 mg/kg11 (Genentech Inc, San Francisco, CA) and combination of tPA 5 mg/kg plus rA2 (produced as previously described)3 10 mg/kg were given intravenously at 3 hours after embolization. Nontreatment saline control was not included in this study because of unacceptable high mortality (>50%) observed in our pilot study. Inclusion criteria were set as the following: (1) stable 50% or less regional cerebral blood flow of preischemic baseline for up to 1 hour after embolization; (2) NSS score at 3 hours after stroke is ≥8. Thirty rats per group were enrolled in this study.
Infarction volume and immunohistochemistry were analyzed by Student t test. Mortality rate was analyzed by 2-sided Fisher exact test. Neurobehavioral assessments were analyzed by repeated measures ANOVA followed by post hoc t test. The most conservative multiple-test correction was applied using the Bonferroni method. Differences with P<0.05 were considered statistically significant.
Neurological Function Deficits
There was a statistically significant better performance in combination group compared with tPA alone controls on all 3 neurobehavioral assessments, and a significant interaction effect between treatments (combination versus tPA alone) and time for neurological functional recovery (up to 28 days after focal stroke in rats) in all 3 neurobehavioral assessments (Figure 1).
Brain Infarction, Mortality, and Vessel Density
At 28 days after stroke, infarction volume in the tPA-plus-rA2 combination group was significantly smaller than the tPA alone group with reduction rate of 19.6% (Figure 2A and 2B); however, there was no significant difference in mortality between tPA alone group (13/30, 43%) and the combination group (6/30, 20%; P=0.095). Immunohistochemistry showed the combination-treated animals had significantly higher cerebrovascular density in the peri-infarct cortex (Figure 2C and 2D).
VEGF and Synaptophysin Expression in Peri-Infarct Areas
At 28 days after stroke, quantification of immunohistochemistry showed rats treated with the tPA-plus-rA2 combination had significantly higher expression of the proangiogenic factor VEGF in peri-infarct cortex (Figure 3A and 3B), and significantly higher synaptic vesicle protein synaptophysin expression in peri-infarct striatum compared with tPA alone–treated controls (Figure 3C and 3D).
Existing data suggest that combining rA2 with tPA may allow one to use lower doses of tPA for effective thrombolysis.2 However, acute neuroprotection may not always correspond to improvements in long-term outcomes. Hence, it was important to demonstrate that the beneficial effects of tPA-rA2 combination therapy would be sustained. Our present study showed that combination of low-dose tPA at 5 mg/kg plus rA2 10 mg/kg significantly reduced brain infarction and improved long-term neurological outcomes compared with standard high-dose tPA at 10 mg/kg alone.
During stroke recovery, neurovascular remodeling is induced in peri-infarct areas. The underlying mechanisms of these endogenous processes may involve matrix enzymes from the matrix metalloproteinase and plasminogen activator families.12 Therefore, it was also important to determine whether manipulating tPA actions with rA2 could influence these recovery events. In this study, we measured microvessel density, VEGF, and synaptophysin as representative surrogate markers of neurovascular remodeling in peri-infarct tissue. Along with infarct reduction and functional improvement, the combination of tPA-plus-rA2 did not dampen and, in fact, slightly augmented these indirect biomarkers of recovery. We speculate that in addition to the reduction in tPA-associated blood–brain barrier disruption, rA2–tPA complex might limit tPA brain penetration–associated neuronal excitotoxicity,13 and rA2 might bind and neutralize angiostatin (one of tPA-plasminogen converting products)-associated endothelial toxicity.14 Ultimately, improved blood flow and fewer neurovascular side effects of the combination should translate into improved long-term outcome.2 We acknowledge that stroke recovery involves complex cascades of neurovascular and gliovascular responses,12 and how full spectrum of molecular events correlating with neurological outcomes remains to be elucidated. Nevertheless, our initial findings here may be consistent with a sustained long-term benefit of tPA-plus-rA2 therapy.
Taken together, our results demonstrated that combination treatment with tPA-plus-rA2 may sustain early brain tissue protective effects and preserve long-term neurovascular remodeling and functional recovery. Further translational development of this tPA-plus-rA2 combination stroke therapy may be warranted.
Sources of Funding
This work was supported in part by the National Institutes of Health grant R01-NS065998 (to Xiaoying Wang).
Guest Editor for this article was Malcolm R. Macleod, PhD, FRCP.
- Received October 11, 2013.
- Accepted November 19, 2013.
- © 2013 American Heart Association, Inc.
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