Nuclear autoantigenic sperm protein facilitates glioblastoma progression and radioresistance by regulating the ANXA2/STAT3 axis.

AIMS: Although radiotherapy is a core treatment modality for various human cancers, including glioblastoma multiforme (GBM), its clinical effects are often limited by radioresistance. The specific molecular mechanisms underlying radioresistance are largely unknown, and the reduction of radioresistance is an unresolved challenge in GBM research. METHODS: We analyzed and verified the expression of nuclear autoantigenic sperm protein (NASP) in gliomas and its relationship with patient prognosis. We also explored the function of NASP in GBM cell lines. We performed further mechanistic experiments to investigate the mechanisms by which NASP facilitates GBM progression and radioresistance. An intracranial mouse model was used to verify the effectiveness of combination therapy. RESULTS: NASP was highly expressed in gliomas, and its expression was negatively correlated with the prognosis of glioma. Functionally, NASP facilitated GBM cell proliferation, migration, invasion, and radioresistance. Mechanistically, NASP interacted directly with annexin A2 (ANXA2) and promoted its nuclear localization, which may have been mediated by phospho-annexin A2 (Tyr23). The NASP/ANXA2 axis was involved in DNA damage repair after radiotherapy, which explains the radioresistance of GBM cells that highly express NASP. NASP overexpression significantly activated the signal transducer and activator of transcription 3 (STAT3) signaling pathway. The combination of WP1066 (a STAT3 pathway inhibitor) and radiotherapy significantly inhibited GBM growth in vitro and in vivo. CONCLUSION: Our findings indicate that NASP may serve as a potential biomarker of GBM radioresistance and has important implications for improving clinical radiotherapy.

Blockade of Annexin A2 Prevents Early Microvasculopathy in Murine Models of Diabetic Retinopathy.

Purpose: We examined the role of annexin A2 (A2) in the development of diabetic retinal vasculopathy by testing the effect of Anxa2 gene deletion as well as administration of anti-A2 antibodies on pericyte dropout and retinal neovascularization in diabetic Akita mice, and in mice subjected to oxygen-induced retinopathy. Methods: We analyzed diabetic Ins2AKITA mice with or without global deletion of Anxa2, as well as Ins2AKITA mice that received intravitreal anti-A2 IgG or control antibody at 2, 4, and 6 months, for retinal pericyte dropout at 7 months of age. In addition, we assessed the effect of intravitreal anti-A2 on oxygen-induced retinopathy (OIR) in neonatal mice by quantifying retinal neovascular and vaso-obliterative area, and by enumeration of neovascular tufts. Results: Both deletion of the Anxa2 gene and immunologic blockade of A2 prevented pericyte depletion in retinas of diabetic Ins2AKITA mice. Blockade of A2 also reduced vaso-obliteration and neovascularization in the OIR model of vascular proliferation. This effect was amplified when a combination of antivascular endothelial growth factor (VEGF) and anti-A2 antibodies was used. Conclusions: Therapeutic approaches that target A2, alone or in combination with anti-VEGF therapy, are effective in mice, and may also curtail the progression of retinal vascular disease in humans with diabetes.

Annexin A2-mediated cancer progression and therapeutic resistance in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a head and neck cancer with poor clinical outcomes and insufficient treatments in Southeast Asian populations. Although concurrent chemoradiotherapy has improved recovery rates of patients, poor overall survival and low efficacy are still critical problems. To improve the therapeutic efficacy, we focused on a tumor-associated protein called Annexin A2 (ANXA2). This review summarizes the mechanisms by which ANXA2 promotes cancer progression (e.g., proliferation, migration, the epithelial-mesenchymal transition, invasion, and cancer stem cell formation) and therapeutic resistance (e.g., radiotherapy, chemotherapy, and immunotherapy). These mechanisms gave us a deeper understanding of the molecular aspects of cancer progression, and further provided us with a great opportunity to overcome therapeutic resistance of NPC and other cancers with high ANXA2 expression by developing this prospective ANXA2-targeted therapy.

Low dose tPA plus annexin A2 combination attenuates tPA delayed treatment-associated hemorrhage and improves recovery in rat embolic focal stroke.

We previously have shown that tissue type plasminogen activator (tPA) in combination with its receptor annexin A2 (rA2) protein significantly improved tPA thrombolytic efficacy. In this study we aimed to examine the therapeutic effects of the combination when treated at delayed 4-hour window after stroke compared to standard conventional tPA alone in an embolic focal stroke rat model. We compared effects of intravenous tPA alone (10 mg/kg) versus a combination of low-dose tPA (5 mg/kg) plus 10 mg/kg rA2. Totally 152 rats were used. Our results showed that: (1) at 24 h after stroke, the combination slightly reduced brain infarction compared to saline (9.2% reduction), and tPA (7.4% reduction), although the reductions did not reach statistical significance; while the combination significantly reduced (22.2% reduction) the conventional tPA-elevated intracerebral hemorrhagic (ICH) transformation; (2) at 7 days after stroke, the combination significantly attenuated conventional tPA alone-elevated iron deposition at peri-lesion area (68.2% reduction); (3) at 28 days after stroke, the combination significantly improved performance of adhesive tape-removal test, which was accompanied by a significantly higher micro vessel density at peri- infarct areas compared to conventional tPA alone group.In conclusion, compared to conventional tPA alone, when treated at delayed 4-hour after stroke, the combination of low-dose tPA plus rA2 therapy provides a safer profile by lowering risk of ICH transformation and improves neurological function recovery after stroke.

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.

Anti-neuroinflammatory and neurotrophic effects of combined therapy with annexin II and Reg-2 on injured spinal cord.

The present study was designed to investigate the neuroprotective effects of Ca(2+)-dependent phospholipid-binding protein annexin II and a secreted protein Reg-2 (regeneration gene protein 2) in spinal cord injury (SCI) model produced by contusion SCI at T(9) using the weight drop method. The agents were delivered intrathecally with Alzet miniosmotic pumps. We found that annexin II and Reg-2 remarkably reduced neuronal death, attenuated tissue damage and alleviated detrimental inflammation in vivo; meanwhile, a significant increase in white matter sparing and myelination area was observed. The propriospinal axons and long-distance supraspinal pathways were protected by the treatments as revealed by retrograde tracing. Basso Beattie Bresnahan locomotor rating scores also revealed a measurable behavioral improvement. However, no evident behavioral improvements in locomotor performance were achieved by the combined treatment with annexin II and Reg-2, compared with the separate treatment with annexin II and Reg-2.

The annexin A2 system and vascular homeostasis.

Optimal fibrin balance requires precisely controlled plasmin generation on the surface of endothelial cells, which line the blood vessel wall. As a co-receptor for plasminogen and tissue plasminogen activator (tPA), which are key factors in plasmin generation, the annexin A2 (A2) complex promotes vascular fibrinolysis. The intracellular A2 complex is a heterotetramer of two A2 monomers and two copies of the associated protein, p11. In response to endothelial cell activation, A2 is phosphorylated by src-kinase, and translocated to the cell surface in a highly regulated manner. Over-expression of A2 is seen in acute promyelocytic leukemia during the early hemorrhagic phase, while high titer antibodies to A2, as in antiphospholipid syndrome or cerebral venous thrombosis, are associated with thrombosis. In experimental hyperhomocysteinemia, moreover, derivatization of A2 by homocysteine leads to intravascular fibrin accumulation and dysangiogenesis, features that phenocopy the Anxa2(-/-) mouse. Exogenous A2 may also offer a novel therapeutic approach to ischemic thrombotic stroke, as administration of A2 in conjunction with conventional tPA-based thrombolytic therapy improved outcome in an animal model. Here, we discuss the role of the A2 system in vascular homeostasis, the molecular interactions that regulate its profibrinolytic activity, and its potential role in the pathogenesis and treatment of vascular disease.

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.

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.

Anti-intercellular adhesion molecule-1 antibody's effect on noise damage.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to investigate possible preventive effects of anti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab) on noise-induced cochlear damage as assessed by changes in auditory thresholds and cochlear blood flow. STUDY DESIGN: A controlled animal study. Pretreated rats with anti-ICAM-1 Ab or saline control, followed with exposure to 72 continuous hours of broad band noise (107 dB SPL), and 24 hours after noise exposure treated again with anti-ICAM-1 Ab or saline. METHODS: Eighteen healthy male Fischer rats (200-250 g) were used. Sixteen were randomly selected to study noise-induced temporary threshold shifts. The remaining two rats were used to study cochlear blood flow (CBF), using laser Doppler flowmetry and blood pressure measurements. RESULTS: Rats treated with anti-ICAM-1 Ab (1.875 mg/kg, intravenously) showed attenuated temporary threshold shifts (TTS) compared to controls. Both groups showed a partial threshold recovery 72 hours following noise exposure, normal for this noise exposure paradigm. Comparisons of baseline and post-treatment measurements of CBF and mean arterial blood pressure revealed no significant changes. Anti-ICAM-1 Ab animals displayed significantly lower mean auditory threshold shifts at all five test frequencies (P < .05) when compared to control. CONCLUSIONS: Blocking the cascade of reactive oxygen species (ROS) generation by using anti-ICAM-Ab protects against noise-induced hearing loss.

Efficacy of recombinant annexin 2 for fibrinolytic therapy in a rat embolic stroke model: a magnetic resonance imaging study.

Efficacy of recombinant annexin 2 (rAN II) in a rat model of embolic stroke was examined using a magnetic resonance imaging (MRI) and histology. The right middle cerebral artery of male Wistar rats was occluded by autologous clots under anesthesia. Four doses of rAN II (0.125, 0.25, 0.5 and 1.0 mg/kg, n=10 for each group) or saline (1 ml/kg, n=10) were administrated intravenously within 5 min before clot infusion. Serial changes in apparent diffusion coefficient (ADC) and relative blood flow (CBF) were measured with the use of MRI in half of the animals in each group. The remaining half of the animals in each group was evaluated for hemorrhage and final infarct size by histology at 48 h after embolization. At 3 h after embolization, lesion volumes with ADC were abnormality and CBF in the peripheral lesion was improved in groups treated with 0.25, 0.5 and 1.0 mg/kg, but not 0.125 mg/kg, of rAN II in comparison with the saline-treated group (P<0.05). Histological analyses were consistent with MRI findings. More importantly, no hemorrhagic transformation was documented in rats treated with 0.125 and 0.25 mg/kg of rAN II, whereas it was observed at higher doses. We concluded that rAN II at 0.25 mg/kg significantly reduced infarct size and improved CBF without hemorrhagic complications. rAN II is a novel compound that has the potential to be a promising fibrinolytic agent to treat embolic stroke.

Extracellular annexin II.

Annexin II belongs to a family of calcium-dependent, phospholipid binding proteins. Annexin II was first identified as an intracellular protein and attributed intracellular functions. Although it lacks a signal peptide and its mechanism of secretion is unknown, extracellular annexin II has recently been found in several tissues as both soluble and membrane-bound protein. Cell-surface annexin II has been identified as a receptor for a number of polypeptide ligands. Extracellular annexin II may be important in several biological processes, such as fibrinolysis, cell adhesion, ligand-mediated cell signaling and virus infection. These processes provide several possibilities for therapeutic approaches targeting extracellular annexin II, and future research should further illuminate the biology of this molecule.

Nocturnal cluster headache associated with sleep apnea. A case report.

We describe a 49-year-old man with chronic cluster headache unresponsive to all medications. Following investigation in the sleep lab he was found to have obstructive sleep apnea (OSA) with associated oxygen desaturations during rapid eye movement (REM) sleep. He awakened during one of these episodes with a typical headache. Treatment with nasal CPAP abolished his OSA and desaturations, and largely abolished his headaches. He then developed central apneas during REM sleep. Further treatment with BiPAP, with a set backup rate, abolished both the apneas and the headaches. We conclude that there may be a link between nocturnal cluster headaches and sleep apnea.