Inflammation modulates expression of laminin in the central nervous system following ischemic injury

Abstract

Background: Ischemic stroke induces neuronal death in the core of the infarct within a few hours and the secondary damage in the surrounding regions over a long period of time. Reduction of inflammation using pharmacological reagents has become a target of research for the treatment of stroke. Cyclooxygenase 2 (COX-2), a marker of inflammation, is induced during stroke and enhances inflammatory reactions through the release of enzymatic products, such as prostaglandin (PG) E2.

Methods: Wild-type (WT) and COX-2 knockout (COX-2KO) mice were subjected to middle cerebral artery occlusion (MCAO). Additionally, brain slices derived from these mice or brain microvascular endothelial cells (BMECs) were exposed to oxygen-glucose deprivation (OGD) conditions. The expression levels of extracellular matrix (ECM) proteins were assessed and correlated with the state of inflammation.

Results: We found that components of the ECM, and specifically laminin, are transiently highly upregulated on endothelial cells after MCAO or OGD. This upregulation is not observed in COX-2KO mice or WT mice treated with COX-2 inhibitor, celecoxib, suggesting that COX-2 is associated with changes in the levels of laminins.

Conclusions: Taken together, we report that transient ECM remodeling takes place early after stroke and suggest that this increase in ECM protein expression may constitute an effort to revascularize and oxygenate the tissue.

Figure 1

Neuronal death and Iba⁺microglia in ischemic brain following middle cerebral artery occlusion (MCAO). Sections at the indicated times after MCAO were stained with cresyl violet (B), 2,3,5-triphenyltetrazolium chloride (TTC) (C), and Iba-1 antibody (D). Contralateral sides were used as controls. Upper and lower sections are from the cortex and striatum, respectively. The sections were stained with cresyl violet; the areas of neuronal death were revealed by the absence of cresyl violet stain and are represented in the cartoons as gray shading (A). (E) Infarct volume was measured as described in the Methods section. Data were obtained from five animals per timepoint. Values are means ± SEMs of at least three independent experiments of each group unless otherwise indicated. Bars, 50 μm.

Figure 2

Impaired laminin expression in COX-2 knockout (COX-2KO) mice following ischemic injury. Lower magnification images of cresyl violet staining ((A), upper) on brain sections from wild-type (WT control) and COX-2KO, mice that were subjected to middle cerebral artery occlusion (MCAO). Sections shown were collected 12 h after MCAO and stained with cresyl violet. White dotted lines demarcate the infarct. *P <0.05 compared to WT control or WT vehicles. (B,C) Extracellular matrix (ECM) protein expression in extracts from ipsilateral sides after MCAO was analyzed for Toll-like receptor 9 (TLR9), CD14, fibrin(ogen), collagen IV (B), and laminin levels (α, β and γ subunits) (C) and was quantified using the ImageJ software, normalized against α-tubulin and graphed as fold difference in mean intensity. *P <0.01 compared to the contralateral WT and COX-2KO sides at 12 h (C12). Data were obtained from at least five animals in each group.

Figure 3

Laminin expression increased on blood vessels following transient middle cerebral artery occlusion (tMCAO). (A) At 6 h post MCAO, sections were stained with antibodies against anti-pan-laminin and occludin. Data are representative of results from at least five animals. Scale bar, 100 μm. (B) Brain microvascular endothelial cells (BMECs) were challenged with oxygen-glucose deprivation (OGD) and assayed for laminin expression. *P <0.01 compared to control. (C) RT-PCR for laminin subtypes at indicated times after MCAO.

Figure 4

Impaired laminin expression in cyclooxygenase 2 (COX-2)-inhibited cells and mice following ischemic injury. (A) Brain microvascular endothelial cells (BMECs) were treated with celecoxib (coxib, 2.5 and 5 μM) and subjected to oxygen-glucose deprivation (OGD). (B) Laminin expression was determined in cell extracts. α-Tubulin normalized protein loading. Bands were quantified by densitometry and plotted as mean intensity. The value of laminin expression in bar graph was averaged over all experiments for the expression of all laminin subunits; *P <0.01. (C) Lower magnification images of cresyl violet staining (upper) of brain sections from wild-type (WT) mice treated with vehicle alone (WT + vehicle) and the COX-2 inhibitor coxib (WT + coxib) prior to middle cerebral artery occlusion (MCAO). White dotted lines demarcate the infarct. Infarct volume was measured. *P <0.05 compared to WT vehicle (lower). (D) Sections shown were collected 12 h after MCAO and stained with pan-laminin antibodies. Data are representative of results from three independent experiments. Bars, 50 μm.

Figure 5

Decreased laminin expression in cyclooxygenase 2 (COX-2)-inhibited endothelial cells following oxygen-glucose deprivation (OGD). (A) Cells were pretreated with antagonists, subjected to OGD and western blot analysis. AH, AH6809 (1 μM; Ki = 350 nM), EP2 antagonist; L, L798,106 (1 nM; Ki = 0.3 nM), EP3 antagonist; A, AH23848 (1 μM; IC₅₀ = 0.26 μM), EP4 antagonist. (B) Laminin expression in COX-2-inhibited endothelial cells following OGD. Cells were pretreated with antagonists, subjected to OGD and western blot analysis. (C) Brain microvascular endothelial cells (BMECs) were pretreated with celecoxib (coxib) and antagonists for 15 minutes, then subjected to OGD and western blot analysis using antibodies against Toll-like receptor 9 (TLR9) and Mac-2. α-Tubulin was used to normalize protein loading. Quantification was performed using the ImageJ software, normalized against α-tubulin. *P <0.01 compared to control. Data are representative of results from at leas five experiments.

Figure 6

Laminin protein levels increase after treatment of endothelial cells with prostaglandin E₂(PGE₂). BMECs were treated with 0.1 and 1 μM PGE₂ for 1 or 2 days. At the end of the incubation period the cells were lysed and subjected to western blot analysis to reveal laminin protein levels. α-Tubulin was used to normalize protein loading. Data are representative of three experiments. The α, β and γ subunits are indicated by arrows. *P <0.01 compared to control.