Abbreviations used: BBB, blood-brain barrier; ECA, external carotid artery; ICA, internal carotid artery; MCAO, middle cerebral artery occlusion; MMP, matrix metalloproteinase; NBO, normobaric hyperoxia; PBS, phosphate-buffered saline. AbstractEarly blood-brain barrier (BBB) disruption resulting from excessive neurovascular proteolysis by matrix metalloproteinases (MMPs) is closely associated with hemorrhagic transformation events in ischemic stroke. We have shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 increase in the ischemic brain. The aim of this study was to determine whether NBO could attenuate MMP-9-mediated early BBB disruption following ischemic stroke. Rats were exposed to NBO (95% O 2 ) or normoxia (30% O 2 ) during 90-min middle cerebral artery occlusion, followed by 3-hour reperfusion. NBO-treated rats showed a significant reduction in Evan's blue extravasation in the ischemic hemisphere compared with normoxic rats. Topographically, Evan's blue leakage was mainly seen in the subcortical regions including the striatum, which was accompanied by increased gelatinolytic activity and reduced immunostaining for tightjunction protein, occludin. Increased gelatinolytic activities and occludin protein loss were also observed in isolated ischemic microvessels. Gel gelatin zymography identified that MMP-9 was the main enzymatic source in the cerebral microvessels. Incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. Inhibition of MMP-9 by NBO or MMP-inhibitor, BB1101, significantly reduced occludin protein loss in ischemic microvessels. These results suggest that NBO attenuates early BBB disruption, and inhibition of MMP-9-mediated occludin degradation is an important mechanism for this protection. Keywords: blood-brain barrier, matrix metalloproteinases, oxygen, stroke. hemorrhage, a major contributing factor to brain injury and mortality following ischemic stroke. Matrix metalloproteinases (MMPs), especially gelatinases (MMP-2 and 9), are up-regulated in cerebral ischemia and closely associated with BBB disruption (Rosenberg et al. 1998), edema formation (Pfefferkorn and Rosenberg 2003), and hemorrhagic transformation (Sumii and Lo 2002). Tight junctions are important structural components of the BBB, which span the apical region of the interendothelial clefts and restrict paracellular permeability (Wolburg and Lippoldt 2002). They are formed via complex interactions of cytoskeletal proteins and tight junction proteins, including claudins, occludin, zonula occludens, and cingulin (Wolburg and Lippoldt 2002). Among these tight junction proteins, the transmembrane protein, occludin, is critical for sealing the tight junctions (Hirase et al. 1997;Lacaz-Vieira et al. 1999;Persidsky et al. 2006), and disruption of occludin alone is enough to cause functional changes in the tight junctions (Tavelin et al. 2003). Accumulating evidence indicates that hypoxia/ischemia increases BBB permeability by disrupting BBB tight junctions, for which M...

Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly-(ADP-ribosyl)ation is one likely mechanism for the reported cocarcinogenic activities of arsenic in UVR-induced skin carcinogenesis.Arsenic is a naturally occurring element that is present in food, soil, and water (1, 2). Environmental or occupational exposures to arsenic are associated with both acute and chronic toxic effects in humans, including increased incidence of skin, lung, liver, and urinary tract cancers (3). Although human epidemiological data link inorganic arsenic in drinking water with an elevated risk of non-melanoma skin cancer (4), arsenic as a sole agent is not an effective skin carcinogen in animal models (5). However, arsenite enhances tumor development in animals pretreated with other carcinogens (6), chronically stimulated by growth factors (7), or co-treated with UV radiation (UVR) 2 (8). It has been reported that sodium arsenite concentration as low as 1.25 mg/liter (10 M) in drinking water enhances UVRinduced tumorigenicity in mice (8), but the mechanisms underlying this observation are not fully understood.Arsenite exposure generates reactive oxygen species (ROS), and we have directly demonstrated that the production of O 2. , H 2 O 2 , and ⅐ OH in arsenite-exposed keratinocytes is associated with DNA damage (9, 10). Similarly UVR, particularly UVA, generates ROS in the skin also leading to oxidative DNA damage (11). Oxidative stress plays a significant role in UVR-induced skin carcinogenesis (12...

Blood brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier, however they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein we employed oxygen-glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3-hours before co-culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8-D1A cells). Incubation of OGD-treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin-5 proteins as well as increased VEGF secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

Background and Purpose-A major limitation of tissue plasminogen activator (tPA) thrombolysis for ischemic stroke is the narrow time window for safe and effective therapy. Delayed tPA thrombolysis increases the risk of cerebral hemorrhage and mortality, which, in part, is related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). We recently showed that normobaric hyperoxia treatment reduces MMP-9 expression and blood-brain barrier disruption in the ischemic brain. Therefore, we hypothesized that normobaric hyperoxia could increase the safety of delayed tPA thrombolysis in stroke. Methods-Male Sprague-Dawley rats were exposed to normobaric hyperoxia (95% O 2 ) or normoxia (21% O 2 ) during 5-hour filament occlusion of the middle cerebral artery followed by 19-hour reperfusion. Thirty minutes before reperfusion, saline or tPA was continuously infused to rats over 1 hour. Outcome parameters were neurological score, mortality rate, brain edema, hemorrhage volume, and MMP-9. Hemorrhage was quantified with a hemoglobin spectrophotometry method. Edema was evaluated as hemispheric enlargement. MMP-9 was measured by gelatin zymography. Results-In normoxic rats, delayed tPA treatment at 4.5 hours after stroke onset resulted in high mortality, more severe neurological deficits, increased hemorrhage volumes, and augmented MMP-9 induction compared with saline. Rats treated with combined normobaric hyperoxia and tPA showed significantly reduced tPA-associated mortality, brain edema, hemorrhage, and MMP-9 augmentation as compared with tPA alone. Conclusions-Our results suggest that early normobaric hyperoxia treatment may represent an important strategy to increase the safety of delayed tPA thrombolysis in ischemic stroke.

Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA repair processes. Poly (ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA repair protein, which can promptly sense DNA strand breaks and initiate DNA repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/or UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite (≤2 μM) alone did not induce significant DNA strand breaks, but greatly enhanced the DNA strand breaks induced by UVR. Further studies showed that 2 μM arsenite effectively inhibited PARP-1 activity. Zinc supplementation of arsenite-treated cells restored PARP-1 activity and significantly diminished the exacerbating effect of arsenite on UVR-induced DNA strand breaks. Importantly, neither arsenite treatment, nor zinc supplementation changed UVRtriggered reactive oxygen species (ROS) formation, suggesting their effects upon UVR-induced DNA strand breaks is not through a direct free radical mechanism. Combination treatments of arsenite with PARP-1 inhibitor 3-aminobenzamide or PARP-1 siRNA demonstrate that PARP-1 is the target of arsenite. Together, these findings show that arsenite at low concentration exacerbates UVRinduced DNA strand breaks by inhibiting PARP-1 activity, which may represent an important mechanism underlying the co-carcinogenicity of arsenic.