Junhao Yan

Early inhibition of HIF-1α with small interfering RNA reduces ischemic–reperfused brain injury in rats

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1alpha siRNA can protect the brain from ischemic damage by inhibiting HIF-1alpha induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1alpha siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1alpha mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1alpha, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1alpha positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1alpha siRNA may protect the ischemic-reperfused neurons in vivo via inhibition of HIF-1alpha, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke.

Multiple effects of 2ME2 and D609 on the cortical expression of HIF-1α and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model

Despite 2‐methoxyestradiol (2ME2) and tricyclodecan‐9‐yl‐xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down‐regulate hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF). We hypothesize HIF‐1α plays an essential role in cerebral ischemia as a pro‐apoptosis regulator; 2ME2 and D609 decrease the levels of HIF‐1α and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague–Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF‐1α and VEGF (enzyme‐linked immunosorbent assay), depress the expression of HIF‐1α, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF‐1α positive immunoreactive materials are co‐localized with BNIP3 and terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7‐triphenyltetrazolium chloride) and blood–brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF‐1α expression, attenuating the superfluous expression of VEGF to avoid blood–brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.

Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusion-induced focal ischemia rat model.

RNA interference appears to have a great potential not only as an in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. We hypothesize that MMP-9 siRNA can be effective as an MMP-9 protein inhibitor in a rat focal ischemia model. Male Sprague-Dawley rats (156) were subjected to 2 h of middle cerebral artery occlusion (by using the suture insertion method) followed by 24 h of reperfusion. In the treatment group, 5 microl MMP-9 siRNA was administrated by intracerebroventricular injection within 60 min after 2 h of focal ischemia. The siRNA transfection was demonstrated by fluorescence conjugated siRNA. Treatment with MMP-9 siRNA produced a significant reduction in the cerebral infarction volume, brain water content, mortality rate and accompanying neurological deficits. The followings were recorded: Evans blue and IgG extravasation were reduced; the expression of MMP-9 mRNA and protein were significantly silenced; and immunohistochemistry and Western blot analysis revealed that the expression of MMP-9 and VEGF were reduced while occludin and collagen-IV were up-regulated in brain tissues. Our findings provide evidence that a liposomal formulation of siRNA might be used in vivo to silence the MMP-9 gene and could potentially serve as an important therapeutic alternative in patients with cerebral ischemia.

The role of p53 in brain edema after 24 h of experimental subarachnoid hemorrhage in a rat model

Our previous study demonstrated that p53 plays an orchestrating role in the vasospasm and apoptotic cell death after subarachnoid hemorrhage (SAH). We now hypothesize that p53 also plays an important role in brain edema by up-regulating the expression of MMP-9 via the NF-kappaB molecular signaling pathway. Adult male rats (300-350 g) were divided into five groups (n=20 each): Sham, SAH treatment with DMSO or PFT-alpha (0.2 mg/kg and 2.0 mg/kg), intraperitoneally. The monofilament puncture model was used to induce SAH and animals were subsequently sacrificed at 24 h. The blood-brain barrier (BBB) disruption, brain water content, MMP-9 activity, immunohistochemistry, treble fluorescence labeling, Western blot, and ultra-structural observations were performed. Evans blue extravagation, BBB diffuse leakage of IgG protein and brain water content were significantly reduced by PFT-alpha treatment; and the expression of p53, NF-kappaB and MMP-9 were significantly increased. The tight junction protein (Occludin) in endothelia cells and Collage IV in basal lamina were decreased in the brain of SAH rats, and were also modified by PFT-alpha treatment. Ultra-structural changes included disruption of endothelial tight junction and widening of the inter-endothelial spaces. Treble labeling showed p53 colocalized with NF-kappaB and MMP-9 in cerebral endothelia cells. We thus conclude that the level of p53 in cerebral microvasculature significantly affects the BBB permeability and brain edema after 24 h of SAH in rats. This can be at least partially ascribed to p53 inducing a significant up-regulation of MMP-9 via NF-kappaB in the endothelium, which in turn opened the tight junction by degrading Occludin and disrupting the basal lamina by degrading collagen IV.

Nasal Administration of Recombinant Osteopontin Attenuates Early Brain Injury After Subarachnoid Hemorrhage

Background and Purpose— Neuronal apoptosis is a key pathological process in subarachnoid hemorrhage (SAH)–induced early brain injury. Given that recombinant osteopontin (rOPN), a promising neuroprotectant, cannot pass through the blood–brain barrier, we aimed to examine whether nasal administration of rOPN prevents neuronal apoptosis after experimental SAH. Methods— Male Sprague–Dawley rats (n=144) were subjected to the endovascular perforation SAH model. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 and 72 hours after SAH induction. The expressions of cleaved caspase-3, phosphorylated focal adhesion kinase (FAK), and phosphorylated Akt were examined using Western blot analysis. Neuronal cell death was demonstrated with terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling. We also administered FAK inhibitor 14 and phosphatidylinositol 3-kinase inhibitor, Wortmannin, prior to rOPN to establish its neuroprotective mechanism. ELISA was used to measure rOPN delivery into the cerebrospinal fluid. Results— Cerebrospinal fluid level of rOPN increased after its nasal administration. This was associated with improved neurological scores and reduced brain edema at 24 hours after SAH. rOPN increased phosphorylated FAK and phosphorylated Akt expressions and decreased caspase-3 cleavage, resulting in attenuation of neuronal cell death within the cerebral cortex. These effects were abolished by FAK inhibitor 14 and Wortmannin. Conclusions— Nasal administration of rOPN decreased neuronal cell death and brain edema and improved the neurological status in SAH rats, possibly through FAK–phosphatidylinositol 3-kinase–Akt–induced inhibition of capase-3 cleavage.

2-methoxyestradiol reduces cerebral vasospasm after 48 hours of experimental subarachnoid hemorrhage in rats.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and antiproapoptotic activities. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF-1alpha). We hypothesized that hypoxia in the major cerebral arteries might activate a unique signaling pathway, hypoxia-inducible factor-1alpha (HIF-1alpha), to produce or enhance cerebral vasospasm after subarachnoid hemorrhage (SAH). Sprague-Dawley male rats (n = 70) were randomly divided into 5 groups: Sham operated, SAH without treatment, SAH treated with vehicle (DMSO), SAH treated with two HIF-1alpha inhibitors, 2ME2, and D609 (positive control of 2ME2). SAH model was produced by middle cerebral artery perforation. 2ME2 and D609 were administered intraperitoneal at 1 h after SAH; rats were sacrificed after 48 h of SAH. Thick blood clot was observed around basilar artery under arachnoids in all animals except Sham group; severe morphological vasospasm was observed in basilar arteries in SAH and SAH+DMSO rats, and the mild vasospasm in rats treated with 2ME2 and D609; 2ME2 and D609 reduced the activity of HIF-1alpha in the basilar arteries by HIF-1alpha DuoSet ELISA; reduce the expression of HIF-1alpha, VEGF, BNIP3 and PCNA in basilar arteries by Western blotting and immunohistochemical staining. In addition, it decreased the mortality and improved the neurological deficits. In conclusion, 2ME2 is a powerful agent to reduce cerebral vasospasm by inhibiting HIF-1alpha activity and the expression of VEGF as its downstream, suppressing endothelium and VSMCs apoptosis via BNIP3 pathway, and attenuating vasoproliferation.

Blood–brain barrier disruption following subarchnoid hemorrhage may be faciliated through PUMA induction of endothelial cell apoptosis from the endoplasmic reticulum

The blood-brain barrier (BBB) plays a vital role as both a physiologic and physical barrier in regulating the movement of water from the vasculature to the brain. During a subarachnoid hemorrhage (SAH), the BBB is disrupted by a variety of mediators, one of which can result in endothelial cell death. As a result, in the present study, we investigated the role of PUMA (p53 upregulated modulator of apoptosis) following SAH injury in rats. Specifically evaluating whether through the endoplasmic reticulum (ER), PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood-brain barrier integrity. One hundred twelve male Sprague-Dawley rats were randomly divided into 4 groups: sham, SAH, SAH+control siRNA, SAH+PUMA siRNA. Outcomes measured include mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used Western blotting techniques to measure the expression of key pro-apoptotic proteins such as BAX, BAK, and DRP1. PUMA siRNA treatment significantly reduced the mortality rate, cerebral edema, neurobehavioral deficits, and BBB disruption as measured by Evans blue assay following SAH injury. The T2WI images showed there was an increase in vasogenic edema in the brain following SAH, which could be alleviated by PUMA siRNA. Immunohistochemical staining and Western blot analysis demonstrated an increased expression of PUMA, BAX, BAK, GRP78 and DRP1 in the microvascular endothelial cells of the hippocampus, which was accompanied with endothelium apoptosis. This study showed that PUMA induced endothelial cell apoptosis may in fact play a significant role in BBB disruption following SAH and its mediation may be through the endoplasmic reticulum. By blocking the activity of PUMA using siRNA, we were able to prevent the accumulation of cerebral edema that occurs following BBB disruption. This translated into a preservation of functional integrity and an improvement in mortality.

The Involvement of Programmed Cell Death 5 (PDCD5) in the Regulation of Apoptosis in Cerebral Ischemia/Reperfusion Injury

Programmed Cell Death 5 (PDCD5) is a protein that accelerates apoptosis in different types of cells in response to various stimuli and is down‐regulated in many cancer tissues. We hypothesized in this study that down‐regulating PDCD5 can protect the brain from ischemic damage by inhibiting PDCD5‐induced apoptotic pathway.

Role of SCH79797 in Maintaining Vascular Integrity in Rat Model of Subarachnoid Hemorrhage

Background and Purpose— Plasma thrombin concentration is increased after subarachnoid hemorrhage (SAH). However, the role of thrombin receptor (protease-activated receptor-1 [PAR-1]) in endothelial barrier disruption has not been studied. The aims of this study were to investigate the role of PAR-1 in orchestrating vascular permeability and to assess the potential therapeutics of a PAR-1 antagonist, SCH79797, through maintaining vascular integrity. Methods— SCH79797 was injected intraperitoneally into male Sprauge-Dawley rats undergoing SAH by endovascular perforation. Assessment was conducted at 24 hours after SAH for brain water content, Evans blue content, and neurobehavioral testing. To explore the role of PAR-1 activation and the specific mechanism of SCH79797’s effect after SAH, Western blot, immunoprecipitation, and immunofluorescence of hippocampus tissue were performed. A p21-activated kinase-1 (PAK1) inhibitor, IPA-3, was used to explore the underlying protective mechanism of SCH79797. Results— At 24 hours after SAH, animals treated with SCH79797 demonstrated a reduction in brain water content, Evans blue content, and neurobehavioral deficits. SCH79797 also attenuated PAR-1 expression and maintained the level of vascular endothelial-cadherin, an important component of adherens junctions. Downstream to PAR-1, c-Src–dependent activation of p21-activated kinase-1 led to an increased serine/threonine phosphorylation of vascular endothelial-cadherin; immunoprecipitation results revealed an enhanced binding of phosphorylated vascular endothelial-cadherin with endocytosis orchestrator &bgr;-arrestin-2. These pathological states were suppressed after SCH79797 treatment. Conclusions— PAR-1 activation after SAH increases microvascular permeability, at least, partly through a PAR-1-c-Src-p21-activated kinase-1-vascular endothelial-cadherin phosphorylation pathway. Through suppressing PAR-1 activity, SCH79797 plays a protective role in maintaining microvascular integrity after SAH.

Transmembrane protein 166 regulates autophagic and apoptotic activities following focal cerebral ischemic injury in rats.

Transmembrane protein 166 (TMEM166) is a lysosomal/endoplasmic reticulum-associated protein found in various species where it acts as a regulator of programmed cell death, mediating both autophagy and apoptosis. In the present study, we investigated the role of TMEM166 following MCAO injury in rats to determine whether the structural damages following injury were orchestrated in part by TMEM166. One hundred and fifty six male Sprague-Dawley rats were randomly divided into 4 groups: Sham, MCAO, MCAO+control siRNA, MCAO+TMEM166 siRNA. Outcomes were measured including mortality rate, brain edema, BBB disruption, and neurobehavioral testing. Western blotting techniques measured the expression of key pro-autophagic and apoptotic proteins such as TMEM166, Beclin-1, cleaved casepase-3 and Bcl-2/Bax. The study found that TMEM166 siRNA treatment significantly reduced the mortality rate, cerebral edema, neurobehavioral deficits, and BBB disruption as measured by Evans blue assay following MCAO injury. Immunohistochemical staining and western blotting analysis demonstrated an increased expressions of TMEM166, Beclin-1, LC3, cleaved casepase-3 and Bcl-2/Bax in the infarcted areas. This study suggests that TMEM166 induces autophagy and apoptosis may in fact play a significant role in cell death following MCAO injury and its mediation may be through the crosstalk of Bcl-2. By blocking the activity of TMEM166 using siRNA, we were able to prevent the cell loss that occured following cerebral ischemia injury. This translated into a preservation of functional integrity and an improvement in mortality.