The development of red blood cells (erythrocytes) is distinguished by high-level production of the oxygen carrier, haemoglobin A (HbA), a heterotetramer of alpha- and beta-haemoglobin subunits. HbA synthesis is coordinated to minimize the accumulation of free subunits that form cytotoxic precipitates. Molecular chaperones that regulate globin subunit stability, folding or assembly have been proposed to exist but have never been identified. Here we identify a protein stabilizing free alpha-haemoglobin by using a screen for genes induced by the essential erythroid transcription factor GATA-1 (refs 4, 5). Alpha Haemoglobin Stabilizing Protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free alpha-haemoglobin but not with beta-haemoglobin or haemoglobin A (alpha(2)beta(2)). Moreover, AHSP specifically protects free alpha-haemoglobin from precipitation in solution and in live cells. AHSP-gene-ablated mice exhibit reticulocytosis and abnormal erythrocyte morphology with intracellular inclusion bodies that stain positively for denatured haemoglobins. Hence, AHSP is required for normal erythropoiesis, probably acting to block the deleterious effects of free alpha-haemoglobin precipitation. Accordingly, AHSP gene dosage is predicted to modulate pathological states of alpha-haemoglobin excess, such as beta-thalassaemia.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient (−/−) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox−/− mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NOX and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS−/− and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox−/− mice, enhanced protection was noted in 1400W-treated p47phox−/− mice. Interestingly, SODTg mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Background Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. As anti-inflammatory therapies limit thrombosis and anti-thrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of two pro-resolving endogenous mediators: Annexin A1 N-terminal derived peptide (AnxA1Ac2–26) and aspirin (ASA) triggered lipoxin A4 (ATL, 15-epi-lipoxin A4), on the cerebral microcirculation following ischemia-reperfusion (I/R) injury. Furthermore, we tested whether the lipoxin A4 receptor, formyl-peptide receptor 2/3 (Fpr2/3, ortholog to human FPR2/ALX), evoked neuro-protective functions following cerebral I/R injury. Methods and Results Using intravital microscopy, we found that cerebral I/R injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate (NPA) formation within cerebral microvessels. Moreover, ATL activation of neutrophil Fpr2/3 regulated NPA formation in the brain, and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2–26 administration. Blocking Fpr2/ALX with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, who displayed an exacerbated disease severity, evidenced by increased infarct area, blood brain barrier dysfunction, increased neurological score and elevated levels of cytokines. Furthermore, ASA treatment significantly reduced cerebral leukocyte recruitment, and increased endogenous levels of ATL, effects again mediated by Fpr2/3. Conclusions In summary, Fpr2/ALX is a therapeutic target for initiating endogenous pro-resolving, anti-inflammatory pathways following cerebral I/R injury.

Background and Purpose-Although chemokines have been implicated in cardiovascular diseases, few studies have addressed the role of these inflammatory mediators in ischemic stroke. This study tested the hypothesis that RANTES (CCL5; regulated on activation, normal T-cell expressed and secreted) mediates the cerebral microvascular dysfunction, inflammation, and tissue injury induced by brain ischemia and reperfusion. Methods-After 60-minute middle cerebral artery occlusion and reperfusion, the adhesion of leukocytes and platelets in cerebral venules, infarct volume, and blood-brain barrier permeability were measured in wild-type mice (WT), RANTES-deficient mice (RANTES Ϫ/Ϫ ), WT mice transplanted with RANTES Ϫ/Ϫ bone marrow (RANTESϾWT), and control bone marrow chimeras (WTϾWT). The concentration of RANTES and several cytokines was also measured by enzyme-linked immunosorbent assay and a cytometric bead array. Results-The enhanced leukocyte and platelet adhesion, increased blood-brain barrier permeability, and tissue infarction elicited in WT and WTϾWT mice after middle cerebral artery occlusion and reperfusion were significantly blunted in RANTES Ϫ/Ϫ mice. Similar attenuation of the middle cerebral artery occlusion and reperfusion-induced responses were noted in RANTESϾWT chimeras. Although RANTES deficiency did not alter the changes in tissue cytokine levels elicited by middle cerebral artery occlusion and reperfusion, plasma concentrations interleukin-6, interleukin-10, and interleukin-12 were all reduced. Conclusions-These findings implicate blood cell-derived RANTES in the microvascular, inflammatory, and tissue injury responses of the brain to ischemia and reperfusion.

Background and Purpose-Circulating blood cells have been implicated in the pathogenesis of cerebral ischemia/ reperfusion (I/R) injury and stroke. The objective of this study was to define the magnitude and molecular determinants of the platelet-and leukocyte-endothelial cell adhesive interactions induced by I/R in the mouse brain. Methods-Bilateral common carotid artery occlusion was induced for 1 hour in C57BL/6 mice, followed by either 40 minutes or 4 hours of reperfusion. Fluorescent platelets were administered intravenously, and the frontal brain surface was observed with intravital fluorescence microscopy. Leukocyte-endothelial cell adhesion was monitored with the use of rhodamine-6G. Results-Ischemia followed by 40 minutes of reperfusion resulted in the rolling (125.1Ϯ23.6/mm 2 ) and firm adhesion (109.5Ϯ25.8/mm2 ) of leukocytes but not platelets in venules. However, with 4 hours of reperfusion, rolling (138.8Ϯ24.6/mm 2 ) and firm adhesion (153.7Ϯ22.3/mm 2 ) of platelets were detected, and this was accompanied by a more intense recruitment of rolling (374.5Ϯ54.6/mm 2 ) and adherent (445.2Ϯ57.1/mm 2 ) leukocytes. In mice deficient in either P-selectin (P-selectin Ϫ/Ϫ ) or intercellular adhesion molecule-1 (ICAM-1) (ICAM-1 Ϫ/Ϫ ), the I/R-induced plateletendothelial cell (by 80% and 60%, respectively) and leukocyte-endothelial cell (by 84% and 78%, respectively) interactions were significantly blunted compared with those of wild-type mice. Conclusions-These

These findings suggest that the increased morbidity to sepsis in obesity may result from exaggerated microvascular inflammatory and thrombogenic responses that include the activation of endothelial cells with subsequent expression of adhesion molecules, such as P-selectin.

Abstract-Hypertension is associated with an increased risk of thrombosis that appears to involve an interaction between the renin-angiotensin system and hemostasis. In this study we determined whether angiotensin II-mediated thrombosis occurs in arterioles and/or venules and assessed the involvement of type 1 (AT 1 ), type 2 (AT 2 ), and type 4 (AT 4 ) angiotensin II receptors, as well as receptors for endothelin 1 and bradykinin 1 and 2 in angiotensin II-enhanced microvascular thrombosis. Thrombus development in mouse cremaster microvessels was quantified after light/dye injury using the time of onset of the thrombus and time to blood flow cessation. Wild-type and AT 1 receptor-deficient mice were implanted with an angiotensin II-loaded ALZET pump for 2 weeks. Angiotensin II administration in both wild-type and AT 1 receptor-deficient mice significantly accelerated thrombosis in arterioles. Genetic deficiency and pharmacological antagonism of AT 1 receptors did not alter the thrombosis response to angiotensin II. Isolated murine platelets aggregated in response to low (picomolar) but not high (nanomolar) concentrations of angiotensin II. The platelet aggregation response to angiotensin II depended on AT 1 receptors. Antagonism of AT 2 receptors in vivo significantly prolonged the onset of angiotensin II-enhanced thrombosis, whereas an AT 4 receptor antagonist prolonged the time to flow cessation. Selective antagonism of either endothelin 1 or bradykinin 1 receptors largely prevented both the onset and flow cessation responses to chronic angiotensin II infusion. Our findings indicate that angiotensin II induced hypertension is accompanied by enhanced thrombosis in arterioles, and this response is mediated by a mechanism that involves AT 2 , AT 4 , bradykinin 1, and endothelin 1 receptor-mediated signaling.

Experimental animals placed on a high-cholesterol diet for 2 or more weeks exhibit an inflammatory response in postcapillary venules. The aims of this study were to determine (1) whether superoxide mediates the hypercholesterolemia-induced inflammatory response and (2) whether leukocyte and/or vessel wall NAD(P)H oxidase contributes to this response. Intravital videomicroscopy was used to quantify leukocyte-endothelial cell adhesion in cremasteric postcapillary venules of wild-type (WT) mice, CuZn-superoxide dismutase transgenic (SOD TgN) mice, and mice heterozygous (p47(phox)+/-) or homozygous (p47(phox)-/-) for NAD(P)H oxidase placed on either a normal diet or high-cholesterol diet (HCD) for 2 weeks. The number of adherent and emigrated leukocytes in postcapillary venules of WT HCD mice was significantly higher than that detected in venules of their normal-diet counterparts. However, the HCD-induced recruitment of adherent and emigrated leukocytes was not observed in SOD TgN mice. Whereas hypercholesterolemic p47(phox)+/- and WT mice exhibited similar inflammatory responses, p47(phox)-/- mice did not. Bone marrow chimeras were developed to selectively delete p47(phox) from either the vessel wall or circulating leukocytes. Whereas WT marrow transplanted into WT mice produced a normal inflammatory response of venules to HCD, chimeric mice with p47(phox) deficiency in either the vessel wall or leukocytes exhibited an attenuated inflammatory response to HCD that was comparable with that observed in p47(phox)-/- HCD mice. Our findings indicate that enhanced superoxide production is a critical event that initiates the leukocyte-endothelial cell adhesion in postcapillary venules of HCD mice. NAD(P)H oxidase appears to be an important source of this superoxide.