Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds.

We have ex ed the effects of pregnancy and sex hormones on calciumdependent and m-independent nitric oxide synthas (NOSs) in the guinea pig. Pregnancy (near term) caused a >4-fold iase in the activity of cacium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebeflum. The increase in NOS activity in the cerebellum during pregnanc was nhibited by the estn-receptor antagonittam len. Treatment with a ol (but not p rone) also i ed calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone a calcium-dependent NOS only in the cerebeflum. No sign nt change in calcium-independent NOS acv was observed either during pregency or after the admiration f any sex hormone. Both pregan and estradol treatment increased the amount of mRNAs for NOS Isogymes eNOS and nNOS in skeletal muscle, 8 that the ireases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to rution by esten, an action that could explain some of the changes that occur during pegn and some gender differences in physiology and pa polg.Nitric oxide (NO) synthases (NOSs) constitute a family of isozymes that catalyze the oxidation of L-arginine to NO and citrulline. First identified in the vascular endothelium (1, 2), NO synthesis has subsequently been shown to play important roles in the regulation of vascular and gastrointestinal tone, in cell-mediated cytotoxicity against bacteria and tumors, and in a variety of central and peripheral nervous system activities (for review, see ref.3). NOSs can be divided into two functional classes based on their sensitivity to calcium (3). The cytokine-or bacterial product-inducible isoenzyme iNOS binds calmodulin tightly at resting intracellular calcium concentrations. The constitutive forms, isozymes eNOS (originally described in endothelial cells) and nNOS (originally described in neuronal tissue), bind calmodulin in a reversible and calcium-dependent fashion. The mechanisms by which their synthesis is controlled are unknown.The cDNA species encoding the rat, mouse, and human nNOS, the human and bovine eNOS, and iNOS from several species and cell types have been cloned and sequenced (4-17). ref. 28). This protection is lost with the onset of menopause or after surgical castration. This observation could be linked to reports that NO might slow the development ofatherosclerosis by inhibiting the proliferation of smooth muscle cells while stimulating endothelial cell proliferation (29,30).We therefore decided to investigate the hypothesis that sex hormones could regulate constitutive NOSs. We tested this in female and male guinea pigs by determining the effect of pregnancy on NOS activity and by measuring both NOS activity and NOS mRNA before and after sex hormone therapy. MATERIALS AND METHODSHartley guinea pigs of similar chronological age were obtained from a commercial breeder (Charles River Breeding Laboratories): males and nonpregnant females (600-700 g) and pregnant females of >5...

Background and Purpose-Neutrophils have been traditionally recognized as major mediators of a deleterious inflammatory response in acute ischemic stroke, but their potential as a therapeutic target remains unexplored. Recent evidence indicates that neutrophils may acquire different phenotypes and contribute to resolution of inflammation through the release of anti-inflammatory mediators. Thus, similar to M2 macrophages, neutrophils have been proposed to shift toward an N2 phenotype, a polarization that is peroxisome proliferator-activated receptor-γ dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-γ activation with rosiglitazone induces changes in neutrophilic mobilization and phenotype that might influence stroke outcome. Methods-Brain sections and cell suspensions were prepared from mice exposed to permanent distal middle cerebral artery occlusion. Double immunostaining with stereological counting of brain sections and flow-cytometry analysis of brain cell suspensions were performed. Results-Rosiglitazone accelerated neutrophil infiltration to the ischemic core, concomitantly to neuroprotection. Some neutrophils (≈31%) expressed M2 markers, namely Ym1 and CD206 (mannose receptor). After treatment with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone, most neutrophils (≈77%) acquired an N2 phenotype. Interestingly, rosiglitazone increased neutrophil engulfment by microglia/macrophages, a clearance that preferentially affected the N2 subset. Conclusions-We present the first evidence of neutrophil reprogramming toward an N2 phenotype in brain inflammation, which can be modulated by activation of the peroxisome proliferator-activated receptor-γ nuclear receptor. We also show that N2 polarization is associated with an increased neutrophil clearance, thus suggesting that this switch is a crucial event for resolution of inflammation that may participate in neuroprotection. (Stroke. 2013;44:3498-3508.)

Background and Purpose-Matrix metalloproteinase-9 (MMP-9) activity has been associated with hemorrhagic transformation (HT) in experimental models of cerebral ischemia. Our aim was to investigate the relationship between MMP-9 concentrations in blood within 24 hours of stroke onset and subsequent HT of cerebral infarction. Methods-We studied 250 patients with a hemispheric ischemic stroke of 7.8Ϯ4.5 hours' duration. Early CT signs of cerebral infarction were evaluated on admission. The HT and infarct volume were analyzed from the CT performed on days 4 through 7. MMP-9 levels were determined by enzyme-linked immunosorbent assay in blood samples obtained on admission. Results-HT was observed in 38 patients (15.2%): 24 (63.2%) had a hemorrhagic infarction, and 14 (36.8%) had a parenchymal hematoma. A total of 108 patients (43%) received anticoagulants before the second CT scan. Systolic and diastolic blood pressures, body temperature, frequency of early CT signs of ischemia (92% versus 22%), and treatment with anticoagulants (79% versus 37%) were significantly higher in the group with HT (PϽ0.001). Mean infarct volume was 126Ϯ60 cm 3 in the HT group and 90Ϯ68 cm 3 in the group without HT (Pϭ0.003). Median (quartiles) plasma MMP-9 concentrations were higher in the HT group (193 [163, 213] versus 62 [40, 93] ng/mL, PϽ0.001), even in the 24 patients seen within 3 hours of symptom onset (Pϭ0.014). MMP-9 levels Ն140 ng/mL had a positive and negative predictive value of HT of 61% and 97%, respectively. MMP-9 Ն140 ng/mL was associated with HT (odds ratio, 12; 95% confidence interval, 3 to 51; PϽ0.001) after adjustment for potential confounders and final infarct volume. Conclusions-High