Genetic Deletion or Pharmacological Inhibition of Cyclooxygenase-2 Reduces Blood-Brain Barrier Damage in Experimental Ischemic Stroke

It is your doctor and hospital responsibility to get this followed up by testing in HUMAN PATIENTS.  Or you could let incompetence reign and allow them to DO NOTHING.

 

Genetic Deletion or Pharmacological Inhibition of Cyclooxygenase-2 Reduces Blood-Brain Barrier Damage in Experimental Ischemic Stroke

Changjun Yang¹, Yi Yang², Kelly M. DeMars¹, Gary A. Rosenberg² and Eduardo Candelario-Jalil^1*

• ¹Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, United States
• ²Department of Neurology, Center for Memory and Aging, University of New Mexico Health Sciences Center, Albuquerque, NM, United States

Cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 are two crucial mediators contributing to blood-brain barrier (BBB) damage during cerebral ischemia. However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in ischemic stroke. In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of ischemic stroke. Male COX-2 knockout (COX-2^−/−) and wild-type (WT) mice were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Genetic deletion of COX-2 or post-ischemic treatment with CAY10404, a highly selective COX-2 inhibitor, significantly reduced BBB damage and hemorrhagic transformation, as assessed by immunoglobulin G (IgG) extravasation and brain hemoglobin (Hb) levels, respectively. Immunoblotting analysis showed that tight junction proteins (TJPs) zonula occludens (ZO)-1 and occludin as well as junctional adhesion molecule-A (JAM-A) and the basal lamina protein collagen IV were dramatically reduced in the ischemic brain. Stroke-induced loss of these BBB structural proteins was significantly attenuated in COX-2^−/− mice. Similarly, stroke-induced loss of ZO-1 and occludin was significantly attenuated by CAY10404 treatment. Ischemia-induced increase in MMP-9 protein levels in the ipsilateral cerebral cortex was significantly reduced in COX-2^−/− mice. Stroke induced a dramatic increase in MMP-9 enzymatic activity in the ischemic cortex, which was markedly reduced by COX-2 gene deficiency or pharmacological inhibition with CAY10404. Levels of myeloperoxidase (MPO, an indicator of neutrophil infiltration into the brain parenchyma), neutrophil elastase (NE), and lipocalin-2 (LCN2, also known as neutrophil gelatinase-associated lipocalin), measured by western blot and specific ELISA kits, respectively, were markedly increased in the ischemic brain. Increased levels of markers for neutrophil infiltration were significantly reduced in COX-2^−/− mice compared with WT controls following stroke. Altogether, neurovascular protective effects of COX-2 blockade are associated with reduced BBB damage, MMP-9 expression/activity and neutrophil infiltration. Our study shows for the first time that MMP-9 is an important downstream effector contributing to COX-2-mediated neurovascular damage in ischemic stroke. Targeting the COX-2/MMP-9 pathway could represent a promising strategy to reduce neuroinflammatory events in order to preserve the BBB integrity and ameliorate ischemic stroke injury.

Introduction

Prostaglandin-endoperoxide synthases (PTGS), also known as cyclooxygenases (COXs), are key players in inflammation, and are targets of the widely used non-steroidal anti-inflammatory drugs (NSAIDs) (1). COX exists in three forms: COX-1,−2, and−3, where COX-1 and COX-3 (a splice variant of COX-1) are encoded by the same PTGS1 gene, and COX-2 is encoded by the PTGS2 gene (1). COX-1 is constitutively expressed in most tissues and largely contributes to normal physiological functions. COX-2 is an inducible enzyme that is mostly associated with pathophysiological functions in response to proinflammatory stimuli or growth factors (1), whereas COX-3 is not functional in humans (2).

COX-2 inhibition is an attractive pharmacological target since the metabolism of arachidonic acid (AA) through the COX pathway produces large amounts of proinflammatory prostanoids, such as prostaglandin E₂ (PGE₂), which are key inflammatory mediators (3, 4). In ischemic rat brain, a dramatic increase in COX-2 expression has been reported starting at 30 min and lasting up to 15 days after stroke (5–8). Previous findings from our group and others have demonstrated that pharmacological inhibition of COX-2 confers neuroprotection in ischemic brain injury (9–14). COX-2 knockout (COX-2^−/−) mice display a dramatic reduction in the susceptibility to excitotoxicity and ischemic brain injury (15, 16), whereas neuronal overexpression of COX-2 increases infarct volume associated with a dramatic increase in PGE₂ levels in the ischemic brain (17). The detrimental effect of COX-2 after cerebral ischemia is attributed to the production of PGE₂ rather than to the generation of oxidative stress (18). Similarly, we previously found that the increase in PGE₂ formation in the ischemic cortex correlates with the evolution of cerebral infarct in this brain region, and the accumulation of PGE₂ in the ischemic cerebral cortex paralleled the substantial increase in blood-brain barrier (BBB) breakdown and leukocyte infiltration (12). These findings are in line with a previous report showing that PGE₂ produces a marked BBB breakdown when administered intracerebrally in rats (19).

There is increasing evidence indicating that COX-2 derived PGE₂, matrix metalloproteinase (MMP)-9 production and BBB disruption are important components of neuronal death in the ischemic penumbra, a potentially salvageable tissue surrounding the infarct core (6, 17, 20, 21). Active MMP-9 degrades the basal lamina around blood vessels and vascular endothelial tight junction proteins (TJPs), leading to disruption of the BBB (22–24). This opening of the BBB is associated with vasogenic edema and infiltration of polymorphonuclear leukocytes, which exacerbate brain injury. In non-neural cell types, such as tumor cells, COX-2-derived PGE₂ increases MMP-9 expression and activity. It has been demonstrated that tumor cells invasion or metastasis are dramatically reduced by COX-2 inhibitors, and this effect is mediated by a significant reduction in PGE₂-mediated MMP-9 expression/activity (25–28). However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in ischemic stroke.

In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of ischemic stroke. We found that gene deficiency of COX-2 or post-ischemic treatment with the COX-2 inhibitor, CAY10404, significantly reduced BBB damage and hemorrhagic transformation following transient focal cerebral ischemia, and these neurovascular protective effects were associated with a significant decrease in MMP-9 activity, reduced neutrophil infiltration, and a preservation of structural proteins composing the neurovascular unit.