The role of calcium ions (Ca2+) in cell function is beginning to be unraveled at the molecular level as a result of recent research on calcium-binding proteins and particularly on calmodulin. These proteins interact reversibly with Ca2+ to form a protein . Ca2+ complex, whose activity is regulated by the cellular flux of Ca2+. Many of the effects of Ca2+ appear to be exerted through calmodulin-regulated enzymes.

Robust expression of TSP-1 and TSP-2, 2 major angiostatic factors, was noted in the ischemic brain with different temporal expression profiles from different cellular origins. The expression of these angiostatic factors, especially TSP-2, likely contributes to the spontaneous resolution of postischemic angiogenesis. Further studies are needed to explore the molecular mechanisms that regulate the balance of angiogenic and angiostatic factors in the ischemic brain.

Postischemic cerebral blood flow and blood volume changes have been associated with angiogenesis; nevertheless, the spatiotemporal changes in vascular permeability, vascular density, and vessel size have not been investigated. Here we report a prolonged increase in vascular permeability from day 3 to day 21 after ischemia, in particular in the reperfused outer cortical layers and leptomeninges. Increased cerebral blood volume (CBV) was observed from day 3 to day 14, whereas increased blood volume in small vessels, primarily capillaries, was noticed from day 7 to day 14 in the reperfused cortex. An initial decrease in vascular density and a reciprocal increase in vessel size were observed within the reperfused cortex at days 1 and 3 after ischemia. Immunohistological analysis confirmed a similar decrease in microvessel density and an increase in vessel size in vessels with a diameter greater than 30 microm. These large-sized vessels exhibited intense basic fibroblast growth factor and endothelial nitric oxide synthase immunoreactivity, suggesting the growth of collateral vessels. By contrast, a late increase in vascular density was noticed in the reperfused outer cortex at days 14 and 21 after ischemia. Together, these findings suggest that the early phase of CBV increase is likely because of the improvement in collateral circulation, whereas the late phase of CBV increase is attributed to the surge of angiogenesis.

Objective-Brain expresses abundant lipocalin-type prostaglandin (PG) D 2 (PGD 2 ) synthase but the role of PGD 2 and its metabolite, 15-deoxy-⌬ 12,14 PGJ 2 (15d-PGJ 2 ) in brain protection is unclear. The aim of this study is to assess the effect of 15d-PGJ 2 on neuroprotection. Methods and Results-Adenoviral transfer of cyclooxygenase-1 (Adv-COX-1) was used to amplify the production of 15d-PGJ 2 in ischemic cortex in a rat focal infarction model. Cortical 15d-PGJ 2 in Adv-COX-1-treated rats was increased by 3-fold over control, which was correlated with reduced infarct volume and activated caspase 3, and increased peroxisome proliferator activated receptor-␥ (PPAR␥) and heme oxygenase-1 (HO-1). Intraventricular infusion of 15d-PGJ 2 resulted in reduction of infarct volume, which was abrogated by a PPAR␥ inhibitor. Rosiglitazone infusion had a similar effect. 15d-PGJ 2 and rosiglitazone at low concentrations suppressed H 2 O 2 -induced rat or human neuronal apoptosis and necrosis and induced PPAR␥ and HO-1 expression. The anti-apoptotic effect was abrogated by PPAR␥ inhibition. Key Words: COX-1 Ⅲ 15d-PGJ 2 Ⅲ PPAR␥ Ⅲ apoptosis Ⅲ stroke P rostaglandin (PG) H synthase-1 (also known as cyclooxygenase-1 [COX-1]) is constitutively expressed in almost all mammalian cells. 1 It is a bifunctional enzyme with a cyclooxygenase activity that converts arachidonic acid to PG G 2 (PGG 2 ) and a peroxidase activity that converts PGG 2 to PGH 2 . 2 PGH 2 is converted to diverse prostanoids by specific enzymes. COX-1 plays an important role in maintaining physiological homeostasis and protecting brain tissues from ischemia-reperfusion (I/R) injury. COX-1 deleted mice are highly susceptible to ischemic brain infarction, 3 whereas COX-1 overexpression protects brain from I/R damage, which is abrogated by a selective COX-1 inhibitor. 4 COX-1 overexpression in ischemic brain augments the production of PGI 2 , PGD 2 , and PGE 2 , and suppresses leukotriene B 4 (LTB 4 ) and LTC 4 . As LTB 4 and LTC 4 have been shown to be detrimental to brain tissue, whereas PGI 2 is protective, 5-7 COX-1 overexpression tilts the eicosanoid balance toward tissue protection. PGD 2 is elevated in COX-1 overexpressed brain tissues but its role in brain I/R injury is unclear. Brain is enriched in lipocalin-type PGD synthase (L-PGDS), which catalyzes the formation of abundant PGD 2 . 8 The role of PGD 2 in I/R brain injury is unclear. As 15-deoxy-⌬ 12,14 ; PGJ 2 (15d-PGJ 2 ), a nonenzymatic product of PGD 2 , was shown to possess anti-inflammatory properties through activation of peroxisome proliferator activated receptor-␥ (PPAR␥), 9 -13 PGD 2 has been implicated in tissue protection. However, it has recently been argued that the tissue 15d-PGJ 2 level is too low to elicit an anti-inflammatory action in vivo, especially in vascular tissues. 14 In view of abundant expression of L-PGDS and PGD 2 in brain, we postulated that 15d-PGJ 2 contributes to cerebral protection. Our experimental findings show a considerable amount of 15d-PGJ 2 in ischemia brain, which...

The angiopoietin/Tie receptor system may contribute to angiogenesis and vascular remodeling by mediating interactions of endothelial cells with smooth muscle cells and pericytes. The temporal expression of angiopoietin-1 (Angpo-1), angiopoietin-2 (Angpo-2), Tie-1, and Tie-2 mRNA was studied in a focal cerebral ischemia model in rats. The cDNA fragments obtained from reverse transcription polymerase chain reaction amplification were cloned and used as a probe to detect individual genes. Northern blot analysis showed a delayed increase of a 4.4-kb Angpo-1 transcript for up to 2 weeks after ischemia, eightfold higher than the values of the sham-operated controls. A biphasic expression of a 2.4-kb Angpo-2 transcript was noted, peaking at 24 hours (6.4-fold) and 2 weeks (4.6-fold) after ischemia. The expression of Tie-2 mRNA (4.3 kb), a receptor for Angpo-1, and Tie-1 mRNA (4.3 kb) also increased starting 24 hours after reperfusion and remained elevated for up to 2 weeks after ischemia. The temporal profiles of the expression of these genes were different from those of other angiogenic genes such as basic fibrobast growth factor/fibroblast growth factor receptor and vascular endothelial growth factor/vascular endothelial growth factor receptor and proteolytic enzymes (tissue-type plasminogen activator and urokinase plasminogen activator) and their inhibitors (plasminogen activator inhibitor-1). The expression patterns of these genes could be related to progressive tissue liquefaction and neovascularization after ischemia in this stroke model. Differential expression of these angiogenesis genes suggests the involvement of complex regulatory mechanisms that remain to be characterized.

Background Thiazolidinediones (TZD) were reported to protect against ischemia-reperfusion (I/R) injury. Their protective actions are considered to be PPAR-γ (peroxisome proliferator-activated receptor γ)-dependent. However, it is unclear how PPAR-γ activation confers resistance to I/R. Methods and Results We evaluated the effects of rosiglitazone or PPAR-γ overexpression on cerebral infarction in a rat model and investigated the anti-apoptotic actions in N2-A neuroblastoma cell model. Rosiglitazone or PPAR-γ overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-γ siRNA. In mice with knockin of a PPAR-γ domain negative mutant, infarct volume was enhanced. Proteomic analysis reveals that brain 14-3-3ε was highly upregulated in rats treated with rosiglitazone. 14-3-3ε upregulation was abrogated by PPAR-γ siRNA or antagonist. Promoter analysis and chromatin immunoprecipitation reveal that rosiglitazone induced PPAR-γ binding to specific regulatory elements on 14-3-3ε promoter and thereby increased 14-3-3ε transcription. 14-3-3ε siRNA abrogated the anti-apoptotic actions of rosiglitazone or PPAR-γ overexpression while 14-3-3ε recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3ε enhanced binding of phosphorylated Bad, and protected mitochondrial membrane potential. Conclusions Ligand-activated PPAR-γ confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3ε transcription. 14-3-3ε upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

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Antisera to calmodulin, a Ca"-dependent modulator protein, and a heat-labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this