Xiang Fan

Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke

Recent studies showed that soluble annexin A2 dramatically increases tissue plasminogen activator (tPA)-mediated plasmin generation in vitro, and reduces thrombus formation in vivo. Here, we hypothesize that combining annexin A2 with tPA can significantly enhance thrombolysis efficacy, so that lower doses of tPA can be applied in ischemic stroke to avoid neurotoxic and hemorrhagic complications. In vitro activity assays confirmed tPA-specific amplification of plasmin generation by recombinant annexin A2. In a rat focal embolic stroke model, combination therapy with tPA and recombinant annexin A2 protein at 2 h post-ischemia decreased the effective dose required for tPA by four-fold and reduced brain infarction. Combining annexin A2 with tPA also lengthened the time window for thrombolysis. Compared with tPA (10 mg/kg) alone, the combination of annexin A2 (5 mg/kg) plus low-dose tPA (2.5 mg/kg) significantly enhanced fibrinolysis, attenuated mortality, brain infarction, and hemorrhagic transformation, even when administered at 4 h post-ischemia. Combination with recombinant annexin A2, the effective thrombolytic dose of tPA can be decreased. As a result, brain hemorrhage and infarction are reduced, and the time window for stroke reperfusion prolonged. Our present findings provide a promising new approach for enhancing tPA-based thrombolytic stroke therapy.

Neuroglobin-overexpression Alters Hypoxic Response Gene Expression in Primary Neuron Culture Following Oxygen Glucose Deprivation

Neuroglobin (Ngb) is a tissue globin specifically expressed in neurons. Our laboratory and others have shown that Ngb overexpression protects neurons against hypoxia/ischemia, but the underlying mechanisms remain poorly understood. Recent studies demonstrate that hypoxia/ischemia induces a multitude of spatially and temporally regulated responses in gene expression, and initial evidence suggested that Ngb might function in altering biological processes of gene expression. In this study, we asked how Ngb may help regulate genes responsive to hypoxia. Expression of hypoxic response genes following oxygen-glucose deprivation (OGD) was examined using mRNA arrays in neuroglobin-overexpressing transgenic (Ngb-Tg) and wild type (WT) mouse neurons. From a total of 113 genes on the microarray, mRNA expression of 65 genes was detected. Under rest condition, 14 genes were downregulated in Ngb-Tg neurons compared to WT. In WT neurons, after 4-h OGD followed by 4-h reoxygenation (O4/R4), 20 genes were significantly downregulated, and only Fos mRNA was significantly increased. However, out of the 20 downregulated genes in WT neurons, 12 of them were no longer significantly changed in Ngb-Tg neurons: Add1, Arnt2, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Il6st, Khsrp, Mars and Rara. Among these 12 genes, 8 (Add1, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Khsrp) were already reduced in Ngb-Tg neurons compared to WT under rest conditions. Additionally, three genes that initially showed no changes in WT neurons (Ctgf, Egfr and Pea15) were downregulated after OGD in the Ngb-Tg neurons. These findings suggest that Ngb overexpression modulates mRNA expression of multiple hypoxic response genes in the early phase after OGD/reoxygenation. Further studies on these gene networks and interactions may lead to better understanding of Ngb in signaling pathways that contribute to neuroprotection.

A Rat Model of Studying Tissue-Type Plasminogen Activator Thrombolysis in Ischemic Stroke With Diabetes

Background and Purpose— Poststroke hyperglycemia and diabetes mellitus are associated with lower thrombolytic efficacy and an increased risk of postischemic cerebral hemorrhage. We aimed to develop a rodent model of thrombolysis in diabetic stroke that mimics the clinical situation. Method— Male 6-week Type I diabetic rats (14 weeks old) were subjected to embolic focal stroke and treated with tissue-type plasminogen activator at 1.5 hours. Reperfusion and 24-hour neurological outcomes were measured and compared with nondiabetic control rats. Results— Diabetic rats exhibited resistance to thrombolytic reperfusion, larger infarction volumes, and increased intracerebral hemorrhage. Conclusions— This animal model would be relevant to future studies investigating pathophysiological mechanisms and in developing new therapeutic approaches to enhance the efficacy of tissue-type plasminogen activator thrombolysis in stroke patients with diabetes or poststroke hyperglycemia.

Effects of Minocycline Plus Tissue Plasminogen Activator Combination Therapy After Focal Embolic Stroke in Type 1 Diabetic Rats

Background and Purpose— Poststroke hyperglycemia is associated with resistance to tissue plasminogen activator (tPA) reperfusion, higher risk of intracerebral hemorrhage, and worse neurological outcomes. In this study, we asked whether minocycline combined with intravenous tPA may ameliorate inflammation and brain injury after focal embolic stroke in type 1 diabetic rats. Methods— Type 1 diabetic rats were subjected to a focal embolic stroke. Three treatment groups were used: (1) saline at 1.5 hours after stroke; (2) tPA alone at 1.5 hours after stroke; (3) combined minocycline (intravenously) at 1 hour plus tPA at 1.5 hours, and second treatment of minocycline (intraperitoneally) at 12 hours after stroke. Acute brain tissue damages were assessed at 24 hours after stroke. Inflammatory biomarkers interleukin-1&bgr; and matrix metalloproteinases 2 and 9 were examined in plasma. Neutrophil infiltration, microglia activation, matrix metalloproteinase activation, and degradation of the tight junction protein claudin-5 were examined in the brain. Results— Compared with saline or tPA alone treatments, minocycline plus tPA combination therapy significantly reduced brain infarction, intracerebral hemorrhage, and hemispheric swelling at 24 hours after stroke. The combination also significantly suppressed the elevated plasma levels of matrix metalloproteinase-9 and interleukin-1&bgr; up to 24 hours after stroke. At 16 hours after stroke, neutrophil infiltration, microglia activation, matrix metalloproteinase-9, and tight junction protein claudin-5 degradation in the peri-infarct brain tissues were also significantly attenuated by the combination therapy. Conclusions— Combination therapy with minocycline plus tPA may be beneficial in ameliorating inflammation and reducing infarction, brain swelling, and hemorrhage after ischemic stroke with diabetes mellitus/hyperglycemia.

Neuroprotective roles and mechanisms of neuroglobin

Abstract Objective: The objectives of this work were to update and summarize recent experimental works on neuroglobin, mainly focus on its neuroprotective effects and the mechanisms. Methods: The literature was reviewed using PubMed database, and some of the recent findings from our laboratory were included. Results: Neuroglobin is a recently discovered tissue globin with a high affinity for oxygen and is widely and specifically expressed in neurons of vertebrates central and peripheral nervous systems. Investigations in the past several years have advanced our knowledge on the functions and mechanisms of neuroglobin, but many issues remain unclear. Emerging reports have shown that overexpression of neuroglobin confers neuroprotection against neuronal hypoxia or ischemia-induced damage in cultured neurons and in cerebral ischemic animal models. Accumulating findings suggest several possible neuroprotective roles and mechanisms including ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function. Conclusion: Emerging experimental works suggest that neuroglobin is neuroprotective against hypoxic/ischemic insults, probably via ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function.

Annexin A2: A Tissue Plasminogen Activator Amplifier for Thrombolytic Stroke Therapy

Hemorrhagic transformation, incomplete reperfusion, neurotoxicity, and the short treatment time window comprise major challenges for thrombolytic therapy. Improving tissue plasminogen activator therapy has become one of the highest priorities in the stroke field. Recent efforts have been aimed at identifying new strategies that might enhance the thrombolytic efficacy of tissue plasminogen activator at the same time as reducing its associated complications related to hemorrhage and neurotoxicity. We believe that the combination of low-dose tissue plasminogen activator with recombinant annexin A2 (a tissue plasminogen activator and plasminogen coreceptor) might constitute a promising approach. Our pilot study using a focal embolic stroke model in rats supports this hypothesis.

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.

Cerebrovascular degradation of TRKB by MMP9 in the diabetic brain

Diabetes elevates the risk for neurological diseases, but little is known about the underlying mechanisms. Brain-derived neurotrophic factor (BDNF) is secreted by microvascular endothelial cells (ECs) in the brain, functioning as a neuroprotectant through the activation of the neurotrophic tyrosine kinase receptor TRKB. In a rat model of streptozotocin-induced hyperglycemia, we found that endothelial activation of MMP9 altered TRKB-dependent trophic pathways by degrading TRKB in neurons. Treatment of brain microvascular ECs with advanced glycation endproducts (AGE), a metabolite commonly elevated in diabetic patients, increased MMP9 activation, similar to in vivo findings. Recombinant human MMP9 degraded the TRKB ectodomain in primary neuronal cultures, suggesting that TRKB could be a substrate for MMP9 proteolysis. Consequently, AGE-conditioned endothelial media with elevated MMP9 activity degraded the TRKB ectodomain and simultaneously disrupted the ability of endothelium to protect neurons against hypoxic injury. Our findings demonstrate that neuronal TRKB trophic function is ablated by MMP9-mediated degradation in the diabetic brain, disrupting cerebrovascular trophic coupling and leaving the brain vulnerable to injury.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity.

Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

Early Insulin Glycemic Control Combined With tPA Thrombolysis Reduces Acute Brain Tissue Damages in a Focal Embolic Stroke Model of Diabetic Rats

Background and Purpose— Therapeutic effects of early insulin glycemic control for poststroke hyperglycemia in combination with tissue-type plasminogen activator (tPA) thrombolytic therapy have not yet been studied but are of great clinical interest. In this study, we tested the effects of insulin plus tPA combination in a model of focal embolic stroke in Type I diabetic rats. Methods— Streptozotocin was used to produce Type I diabetes in male Wistar rats for 6 weeks and then embolic focal strokes were induced. All rats were treated with insulin or saline at 1 hour followed by tPA or saline at 1.5 hour after stroke. Mortality, infarction, hemispheric swelling, hemorrhagic transformation, and perfusion defects were examined at 24 hours after stroke. Total plasma plasminogen activator inhibitor-1 antigen and activity levels were measured before stroke and 1.5, 3, and 6 hours after stroke by ELISA. Results— Early insulin glycemic control alone or tPA thrombolysis alone had no significant effects on ischemic infarction. However, early insulin glycemic control combined with tPA significantly reduced brain infarction and swelling, ameliorated tPA-associated hemorrhagic transformation, and improved plasma perfusion at 24 hours after stroke. We also found that the combination significantly decreased plasma plasminogen activator inhibitor-1 antigen level at 6 hours and plasminogen activator inhibitor-1 activity at 1.5 and 6 hours after stroke. Conclusions— Early insulin glycemic control may be beneficial in combination with tPA thrombolysis for ischemic stroke with diabetes mellitus or poststroke hyperglycemia.