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Supplemental Digital Content is available in the text., Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1−/−) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye–induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1−/− mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbβ3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5–13 mice/group or 7–10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbβ3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal–regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

The early restitution of blood flow to ischemic tissues is essential to halt the progression of cellular injury associated with decreased delivery of oxygen and metabolic substrates, and for removal of potentially harmful metabolic byproducts. However, it is now apparent that reperfusion also initiates a complex series of pathologic events that paradoxically injure tissues. The microcirculation appears to be particularly vulnerable to the deleterious effects of reperfusion in that microvascular dysfunction precedes the development of parenchymal cell injury. When blood flow to certain organs is reinstituted after ischemia, a large proportion of capillaries fail to reperfuse, a phenomenon referred to as capillary no-reflow. Leukocyte/capillary plugging has also been suggested to underlie development of capillary no-reflow in postischemic tissues. While this may contribute to the reduction in capillary perfusion during ischemia, physical impaction of leukocytes within the capillaries does not appear to play a role in the pathogenesis of no-reflow in skeletal muscle when normal perfusion pressures are established at reperfusion.

Hypertension is an established risk factor for subsequent cardiovascular diseases, with Ang II (angiotensin II) playing a major role in mediating thrombotic and inflammatory abnormalities. Although T cells and IL-6 (interleukin-6) play an important role in adaptive immune responses, little is known about their role(s) in the thromboinflammatory responses associated with Ang II. Here we show using intravital microscopy coupled with the light/dye injury model that Rag-1 deficient (Rag-1 −/− ) and IL-6 deficient (IL-6 −/− ) mice are afforded protection against Ang II–induced thrombosis. Blocking IL-6 receptors (using CD126 and gp130 antibodies) significantly diminished Ang II–mediated thrombosis and inflammatory cell recruitment in mice. Furthermore, the adoptive transfer of IL-6 −/− -derived T cells into Rag-1 −/− mice failed to accelerate Ang II–induced thrombosis compared with Rag-1 −/− mice reconstituted with wild-type–derived T cells, suggesting T cell IL-6 mediates the thrombotic abnormalities associated Ang II hypertension. Interestingly, adoptive transfer of WT T cells into Rag-1 −/ − /Ang II mice resulted in increased numbers of immature platelets, which constitutes a more active platelet population, that is, prothrombotic and proinflammatory. To translate our in vivo findings, we used clinical samples to demonstrate that IL-6 also predisposes platelets to an interaction with collagen receptors, thereby increasing the propensity for platelets to aggregate and cause thrombosis. In summary, we provide compelling evidence for the involvement of IL-6, IL-6R, and T-cell–dependent IL-6 signaling in Ang II–induced thromboinflammation, which may provide new therapeutic possibilities for drug discovery programs for the management of hypertension.

Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue., Graphical abstract fx1, Highlights • Reperfusion injury is implicated in a variety of human diseases and disorders. • Evidence implicating ROS in reperfusion injury continues to grow. • Several enzymes are candidate sources of ROS in post-ischemic tissue. • Inter-enzymatic ROS-dependent signaling enhances the oxidative stress caused by I/R. .

Background Stroke is the third leading cause of death and the leading cause of long-term disability in North America. On average, someone in the US has a stroke every 45 s, and worldwide, stroke claims 15 million lives each year. Therefore, reliable stroke models are vital to the production of effective new therapies for the treatment of this devastating cerebral vascular accident. New method Middle cerebral artery occlusion (MCAo) is considered to be the most clinically relevant surgical model of ischemic stroke, in which a variety of methods may be employed to block the MCA (the most common being through insertion of a monofilament). In this study, we have compared two different approaches that are currently used arbitrarily in various laboratories worldwide: one involving insertion of a monofilament via the common carotid artery (Koizumi et al.) and one via the external carotid artery (Longa et al.). Results and comparisons with existing methods We assessed various parameters, including: mortality rates, neurological scores, inflammation levels, cellular trafficking (using intravital microscopy) and infarct volumes in mice after using each of the two approaches. We found that the Longa method produced a greater, and robust, inflammatory response, versus the Koizumi method. Conclusions In conclusion, we suggest that the Longa method is superior for the study of both short and long-term outcomes of ischemic stroke. These results have considerable implications on stroke model selection for researchers.

The gastrointestinal (GI) circulation receives a large fraction of cardiac output and this increases following ingestion of a meal. While blood flow regulation is not the intense phenomenon noted in other vascular beds, the combined responses of blood flow, and capillary oxygen exchange help ensure a level of tissue oxygenation that is commensurate with organ metabolism and function. This is evidenced in the vascular responses of the stomach to increased acid production and in intestine during periods of enhanced nutrient absorption. Complimenting the metabolic vasoregulation is a strong myogenic response that contributes to basal vascular tone and to the responses elicited by changes in intravascular pressure. The GI circulation also contributes to a mucosal defense mechanism that protects against excessive damage to the epithelial lining following ingestion of toxins and/or noxious agents. Profound reductions in GI blood flow are evidenced in certain physiological (strenuous exercise) and pathological (hemorrhage) conditions, while some disease states (e.g., chronic portal hypertension) are associated with a hyperdynamic circulation. The sacrificial nature of GI blood flow is essential for ensuring adequate perfusion of vital organs during periods of whole body stress. The restoration of blood flow (reperfusion) to GI organs following ischemia elicits an exaggerated tissue injury response that reflects the potential of this organ system to generate reactive oxygen species and to mount an inflammatory response. Human and animal studies of inflammatory bowel disease have also revealed a contribution of the vasculature to the initiation and perpetuation of the tissue inflammation and associated injury response.

Microvascular dysfunction is well established as an early and rate-determining factor in the injury response of tissues to ischemia and reperfusion (I/R). Severe endothelial cell dysfunction, which can develop without obvious morphological cell injury, is a major underlying cause of the microvascular abnormalities that accompany I/R. While I/R-induced microvascular dysfunction is manifested in different ways, two responses that have received much attention in both the experimental and clinical setting are impaired capillary perfusion (no-reflow) and endothelial barrier failure with a transition to hemorrhage. These responses are emerging as potentially important determinants of the severity of the tissue injury response, and there is growing clinical evidence that they are predictive of clinical outcome following reperfusion therapy. This review provides a summary of animal studies that have focused on the mechanisms that may underlie the genesis of no-reflow and hemorrhage following reperfusion of ischemic tissues, and addresses the clinical evidence that implicates these vascular events in the responses of the ischemic brain (stroke) and heart (myocardial infarction) to reperfusion therapy. Inasmuch as reactive oxygen species (ROS) and matrix metalloproteinases (MMP) are frequently invoked as triggers of the microvascular dysfunction elicited by I/R, the potential roles and sources of these mediators are also discussed. The available evidence in the literature justifies the increased interest in the development of no-reflow and hemorrhage in heart and brain following reperfusion therapy, and suggests that these vascular events may be predictive of poor clinical outcome and warrant the development of targeted treatment strategies.

Background and Purpose— Inflammation and thrombosis currently are recognized as critical contributors to the pathogenesis of ischemic stroke. CD147 (cluster of differentiation 147), also known as extracellular matrix metalloproteinase inducer, can function as a key mediator of inflammatory and immune responses. CD147 expression is increased in the brain after cerebral ischemia, but its role in the pathogenesis of ischemic stroke remains unknown. In this study, we show that CD147 acts as a key player in ischemic stroke by driving thrombotic and inflammatory responses. Methods— Focal cerebral ischemia was induced in C57BL/6 mice by a 60-minute transient middle cerebral artery occlusion. Animals were treated with anti-CD147 function–blocking antibody (αCD147) or isotype control antibody. Blood–brain barrier permeability, thrombus formation, and microvascular patency were assessed 24 hours after ischemia. Infarct size, neurological deficits, and inflammatory cells invaded in the brain were assessed 72 hours after ischemia. Results— CD147 expression was rapidly increased in ischemic brain endothelium after transient middle cerebral artery occlusion. Inhibition of CD147 reduced infarct size and improved functional outcome on day 3 after transient middle cerebral artery occlusion. The neuroprotective effects were associated with (1) prevented blood–brain barrier damage, (2) decreased intravascular fibrin and platelet deposition, which in turn reduced thrombosis and increased cerebral perfusion, and (3) reduced brain inflammatory cell infiltration. The underlying mechanism may include reduced NF-κB (nuclear factor κB) activation, MMP-9 (matrix metalloproteinase-9) activity, and PAI-1 (plasminogen activator inhibitor-1) expression in brain microvascular endothelial cells. Conclusions— Inhibition of CD147 ameliorates acute ischemic stroke by reducing thromboinflammation. CD147 might represent a novel and promising therapeutic target for ischemic stroke and possibly other thromboinflammatory disorders.

There is growing evidence for an interdependence of inflammation, coagulation, and thrombosis in acute and chronic inflammatory diseases. Inflammatory bowel diseases (IBD) are associated with a hypercoagulable state and an increased risk of thromboembolism. Although the IBD-associated prothrombogenic state has been linked to the inflammatory response, the mediators that link these 2 conditions remain unclear. Recent evidence suggests that interleukin-6 (IL-6) may be important in this regard. The objective of this study was to more fully define the contribution of IL-6 to the altered platelet function that occurs during experimental colitis. The number of immature and mature platelets, activated platelets, and platelet-leukocyte aggregates were measured in wild-type and IL-6 mice with dextran sodium sulfate (DSS)-induced colonic inflammation. DSS treatment of WT mice was associated with significant increases in the number of both immature and mature platelets, activated platelets, and platelet-leukocyte aggregates. These platelet responses to DSS were not observed in IL-6 mice. Chronic IL-6 infusion (through an Alzet pump) in WT mice reproduced all of the platelet abnormalities observed in DSS-colitic mice. IL-6-infused mice also exhibited an acceleration of thrombus formation in arterioles, similar to DSS. These findings implicate IL-6 in the platelet activation and enhanced platelet-leukocyte aggregate formation associated with experimental colitis, and support a role for this cytokine as a mediator of the enhanced thrombogenesis in IBD.

Patients with inflammatory bowel disease are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have revealed thrombocytosis, alterations in platelet activation, enhanced platelet-leukocyte interactions, and elevated plasma levels of prothrombotic cytokines. This study was directed toward determining whether the thrombocytosis, altered platelet functions, and enhanced platelet-leukocyte interactions observed in patients with inflammatory bowel disease can be recapitulated in the dextran sodium sulfate and T-cell transfer models of murine colonic inflammation. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood of control mice and in mice with colonic inflammation. Platelets were identified by characteristic light scattering and membrane expression of CD41. Thiazole orange labeling was used to differentiate between immature and mature platelets. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin. The combination of CD41, CD45.2, Gr-1, F4/80, and isotype control antibodies was used to detect and quantify aggregates of leukocytes, neutrophils, and monocytes with platelets. Our results indicated that colonic inflammation is associated with thrombocytosis, leukocytosis, and the appearance of immature platelets. An increased number of circulating activated platelets was detected in colitic mice, along with the formation of aggregates of leukocytes (PLA), neutrophils (PNA), and monocytes (PMA) with platelets. Selectin blockade with fucoidin inhibited dextran sodium sulfate-induced PLA formation. The findings of this study indicate that many features of the altered platelet function detected in human inflammatory bowel disease can be reproduced in animal models of colonic inflammation.

The microcirculation both responds and contributes to an inflammatory response. The inflammatory bowel diseases (IBD) provide a useful example of the profound reactivity of all segments (arterioles, capillaries, venules) of the microvasculature to inflammation, and illustrate how changes in microvascular function can influence the intensity and progression of disease activity. This chapter describes the key alterations in gut microvascular function that accompany human and experimental IBD, and addresses the prevailing views on the cellular and molecular mechanisms that underlie the altered vascular function. Impaired blood flow regulation (impaired vasomotor function), an increased adhesion of leukocytes and platelets to the vessel wall, activation of the coagulation cascade, enhanced thrombus formation, increased vascular permeability, and an accelerated rate of vascular proliferation (angiogenesis) are characteristic features of the phenotypic changes that occur in the gut vasculature during inflammation. These changes have been linked to the activation of cells that normally circulate in blood (leukocytes, platelets) or reside in the vessel wall (endothelial cells, vascular smooth muscle) or in the perivascular space (macrophages, mast cells). The chemical messengers elaborated from these activated cells stimulate different signaling pathways that ultimately elicit the phenotypic microvascular responses that accompany inflammation. Pharmacological interventions directed towards preventing some of the microvascular alterations in inflammation (e.g., angiogenesis) provide novel and potentially effective targets for treatment of IBD.

Oxidant stress contributes to the initiation and progression of hypertension (HTN) by enhancing endothelial dysfunction and/or causing perturbations in nitric oxide homeostasis. Differences in mitochondrial function may augment this process and provide insight into why age of onset and clinical outcomes differ among individuals from distinct ethnic groups. We have previously demonstrated that variation in normal mitochondrial function and oxidant production exists in endothelial cells from individuals of Caucasian and African-American ethnicity and that this variation contributes to endothelial dysfunction. To model these distinct mitochondrial redox phenotypes, we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). We demonstrate that the absence of NNT in 6J cells led to distinct mitochondrial bioenergetic profiles and a pro-oxidative mitochondrial phenotype characterized by increased superoxide production and reduced glutathione peroxidase activity. Interestingly, we found that 6J animals have significantly higher systolic blood pressure compared to 6N animals, and this difference is exacerbated by angiotensin II treatment. The changes in pressure were accompanied by both mitochondrial and vascular dysfunction revealed by impaired respiratory control ratios and endothelial-dependent vessel dilation. All end points could be significantly ameliorated by treatment with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO demonstrating a critical role for the production of mitochondrial reactive oxygen species in the development of HTN in these animals. Taken together, these data indicate that the absence of NNT leads to variation in mitochondrial function and contributes to a unique mitochondrial redox phenotype that influences susceptibility to HTN by contributing to endothelial and vascular dysfunction.

Inflammation is an underlying feature of a variety of human diseases. An important manifestation of this pathophysiological response is microvascular dysfunction, which includes the activation of vascular endothelial cells, and circulating leucocytes and platelets. While endothelial cells and leucocytes are widely accepted as critical players in the microvascular alterations induced by inflammation, recent attention has focused on the modulatory role of platelets, which act both as effector and target cells in inflamed microvessels. Evidence is presented to demonstrate the capacity for 'cross-talk' between platelets and other cells (endothelial cells, leucocytes) that contribute to an inflammatory response, and to illustrate the pathophysiological consequences of these interactions of platelets with other cells within the microvasculature.

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.

BACKGROUND: Previous studies have shown that intra-abdominal hypertension (IAH) has detrimental effects on organ function and is associated with significantly increased morbidity and mortality. IAH has also been shown to increase intracranial pressure. The exact mechanism is not known. This study tests the effect of an acute increase in intra-abdominal pressure on the permeability of the blood-brain barrier (BBB) in mice. METHODS: Male CD-1 mice weighing 30 g to 38 g were used. Mice in experimental groups underwent either 4 hours of IAH or 4 hours of IAH followed by 1 hour of decompression (DC). A set of control mice were anesthetized for either 4 hours or 5 hours. Femoral artery cannulation was used for blood pressure monitoring. IAH was induced by intraperitoneal infusion of mineral oil to a pressure of 20 mm Hg. DC was performed through an incision in the anterior abdominal wall. BBB integrity was determined by extravasation of 2% Evans blue (EB) dye administered through the femoral vein 1 hour before the mice were killed. BBB permeability was quantified by the EB extravasation method. RESULTS: EB content in brain tissue was higher in the IAH 4-hour group (n = 12) compared with their control group (n = 4; p < 0.05), indicating increased permeability of BBB. In the IAH 4-hour + DC 1-hour group (n = 6), EB content in brain tissue was not significantly higher than their respective control group (n = 6). CONCLUSION: IAH of 20 mm Hg in mice for 4 hours caused increased BBB permeability. This endothelial barrier dysfunction is reversed by abdominal DC.

Objective— It has been shown that CD40-TRAF6 axis in leukocytes plays a significant role in neointimal formation after carotid ligation. Because CD40 and TRAF6 are expressed not only in leukocytes but also in vascular cells, we examined the role of CD40 contributed by vascular wall cells in neointimal formation after carotid ligation in an atherogenic environment. Methods and Results— Both CD40 and TRAF6 in medial smooth muscle cells (SMCs) was upregulated significantly at 3 days and more prominently at 7 days after injury in wildtype mice, but the TRAF6 upregulation was abolished in CD40 −/− mice. In vitro, TRAF6 expression was induced by cytokines (tumor necrosis factor -α, interleukin-1β) via a NF-κB–dependent manner in wildtype SMCs, but this induction was blocked in CD40-deficient SMCs. Bone marrow chimeras revealed a comparable reduction in neointimal formation and lumen stenosis in mice lacking either vascular wall- or bone marrow-associated CD40. Lacking vascular wall-associated CD40 resulted in a significant reduction in monocyte/macrophage accumulation, NF-κB activation, and multiple proinflammatory mediators (ICAM-1, VCAM-1, MCP-1, MMP-9, tissue factor). In vitro data confirmed that CD40 deficiency or TRAF6 knockdown suppressed CD40L-induced proinflammatory phenotype of SMCs by inhibition of NF-κB activation. Moreover, both in vivo and in vitro data showed that CD40 deficiency prevented injury-induced SMC apoptosis but did not affect SMC proliferation and migration. Conclusion— CD40 signaling through TRAF6 in vascular SMCs seems to be centrally involved in neointimal formation in a NF-κB–dependent manner. Modulating CD40 signaling on local vascular wall may become a new therapeutic target against vascular restenosis.

Background and Purpose— Phosphoinositide 3-kinase (PI3K)-γ is linked to inflammation and oxidative stress. This study was conducted to investigate the role of the PI3Kγ in the blood–brain barrier dysfunction and brain damage induced by focal cerebral ischemia/reperfusion. Methods— Wild-type and PI3Kγ knockout mice were subjected to middle cerebral artery occlusion (60 minutes) followed by reperfusion. Evans blue leakage, brain edema, infarct volumes, and neurological deficits were examined. Oxidative stress, neutrophil infiltration, and matrix metallopeptidase-9 were assessed. Activation of nuclear factor-κB and expression of proinflammatory and pro-oxidative genes were studied. Results— PI3Kγ deficiency significantly reduced blood–brain barrier permeability and brain edema formation, which were time-dependently correlated with preventing the degradation of the tight junction protein, claudin-5, and the basal lamina protein, collagen IV, and the phosphorylation of myosin light chain in brain microvessels. PI3Kγ deficiency suppressed ischemia/reperfusion-induced nuclear factor-κB p65 (Ser536) phosphorylation and the expression of the pro-oxidant enzyme NADPH oxidase (Nox1, Nox2, and Nox4) and proinflammatory adhesion molecules (E- and P-selectin, intercellular adhesion molecule-1) at different time points. These molecular changes were associated with significant inhibition of oxidative stress (superoxide production and malondialdehyde content), neutrophil infiltration, and matrix metallopeptidase-9 expression/activity in PI3Kγ knockout mice. Eventually, PI3Kγ deficiency significantly reduced infarct volumes and neurological scores at 24 hours after ischemia/reperfusion. Conclusions— Our results provide the first direct demonstration that PI3Kγ plays a significant role in ischemia/reperfusion-induced blood–brain barrier disruption and brain damage. Future studies need to explore PI3Kγ as a potential target for stroke therapy.

The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (βs mice). Enhanced microvascular thrombosis in βs mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from βs mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in βs mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in βs mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Background and Purpose— The therapeutic potential of bone marrow stromal cells (BMSCs) has been demonstrated in different models of stroke. Although it is well established that BMSCs selectively migrate to the site of brain injury, the mechanisms underlying this process are poorly understood. This study addresses the hypothesis that selectins mediate the recruitment of BMSCs into the postischemic cerebral microvasculature. Methods— Focal ischemic stroke was induced by middle cerebral artery occlusion and reperfusion. Cell recruitment was monitored using either fluorescent- or radiolabeled BMSCs detected by intravital microscopy or tissue radioactivity. Mice were treated with either a blocking antibody against P- or E-selectin or with the nonselective selectin antagonist, fucoidin. The role of CD44 in cell recruitment was evaluated using BMSCs from CD44 knockout mice. Results— Middle cerebral artery occlusion and reperfusion was associated with a significantly increased adhesion of BMSCs in cerebral venules compared with sham mice. Immunoneutralization of either E- or P-selectin blocked the middle cerebral artery occlusion and reperfusion-induced recruitment of adherent BMSCs. An attenuated recruitment response in the postischemic hemisphere was also noted after fucoidin treatment or administration of CD44-deficient BMSCs. Conclusions— Cerebral vascular endothelium assume a proadhesive phenotype after ischemic stroke that favors the recruitment of BMSCs, which use both P- and E-selectin to home into the infarct site. CD44 may serve as the critical ligand for selectin-mediated BMSC recruitment.

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.

Angiotensin II (AngII) and AngII type-1 receptors (AT1r) have been implicated in the pathogenesis of hypertension and ischemic stroke. The objectives of this study was to determine if/how chronic AngII administration affects blood-brain barrier (BBB) function and blood cell adhesion in the cerebral microvasculature. AngII-loaded osmotic pumps were implanted in wild type (WT) and mutant mice. Leukocyte and platelet adhesion were monitored in cerebral venules by intravital microscopy and BBB permeability detected by Evans blue leakage. AngII (two week) infusion increased blood pressure in WT mice. This was accompanied by an increased BBB permeability and a high density of adherent leukocytes and platelets. AT1r (on the vessel wall, but not on blood cells) was largely responsible for the microvascular responses to AngII. Immunodeficient (Rag-1(-/-) ) mice exhibited blunted blood cell recruitment responses without a change in BBB permeability. A similar protection pattern was noted in RANTES(-/-) and P-selectin(-/-) mice, with bone marrow chimeras (blood cell deficiency only) yielding responses comparable to the respective knockouts. These findings implicate AT1r in the microvascular dysfunction associated with AngII-induced hypertension and suggest that immune cells and blood cell-associated RANTES and P-selectin contribute to the blood cell recruitment, but not the BBB failure, elicited by AngII.

Two of the principal functions of intestinal lymphatics are to assist in the maintenance of interstitial volume within relatively normal limits during alterations in capillary filtration (e.g., acute portal hypertension) and the removal of absorbed water and chylomicrons. The contribution of lymphatics to the prevention of interstitial overhydration or dehydration during alterations in transcapillary filtration is similar in the small intestine and colon. While the lymphatics of the small intestine contribute substantially to the removal of absorbed water (particularly at low and moderate absorption rates), the contribution of colonic lymphatics to the removal of the fluid absorbate is negligible. This difference is attributed to the relative caliber and location of lymphatics in the mucosal layer of the small and large intestines. In the small intestine, large lacteals lie in close proximity to transporting epithelium, while colonic lymph vessels are rather sparse and confined to the basal portion of the mucosa. In the small intestine, the lymphatics assume a more important role in removing absorbed water during lipid absorption than during glucose absorption.

This collaboration of two physiologists and a gastroenterologist provides medical and graduate students, medical and surgical residents, and subspecialty fellows a comprehensive summary of...

This collaboration of two physiologists and a gastroenterologist provides medical and graduate students, medical and surgical residents, and subspecialty fellows a comprehensive summary of...

This collaboration of two physiologists and a gastroenterologist provides medical and graduate students, medical and surgical residents, and subspecialty fellows a comprehensive summary of...

Chymase, a chymotrypsin-like protease, is a non-ACE angiotensin II (Ang II) generating enzyme. We determined if maternal chymotrypsin-like protease/chymase activity was increased in women with preeclampsia (PE).Maternal plasma was extracted from venous blood of healthy nonpregnant women, women with normal and preeclamptic pregnancies. Chymotrypsin-like protease/chymase activity was measured by a colorimetric assay. Maternal vessel chymotrypsin-like protease/chymase expression was examined by immunohistochemistry.Maternal plasma chymotrypsin-like protease/chymase activity was significantly higher in women with PE than in nonpregnant and normal pregnant women: 0.181 +/- 0.011 vs. 0.097 +/- 0.05 (p0.01) and 0.132 +/- 0.013 (p0.05) microkat/mL. Chymotrypsin-like protease/chymase activity was markedly reduced 24 hours postpartum in women with PE, p0.05. Enhanced chymotrypsin-like protease/chymase expression was observed in vascular endothelium in women with PE compared with those in normal pregnancies.Elevated maternal chymotrypsin-like protease/chymase activity and enhanced protease immunostaining in the maternal vessel endothelium may constitute the exacerbated inflammatory state and account for the increased vascular Ang II sensitivity in PE.

Ischaemia and reperfusion (I/R) elicits an acute inflammatory response that is characterized by the recruitment of inflammatory cells, oxidative stress, and endothelial barrier failure. Over the past three decades, much progress has been made in our understanding of the mechanisms that underlie the inflammatory response and microvascular dysfunction associated with I/R. This review is focused on the role of leucocytes (neutrophils and T-lymphocytes) and platelets, and their activation products, as mediators of I/R-induced endothelial barrier failure. The contributions of cytokines, chemokines, and oxidative stress to I/R-induced barrier dysfunction are also discussed. It concludes with an analysis of how risk factors for cardiovascular disease, i.e. hypertension, diabetes, hypercholesterolaemia, and obesity, influence the vascular permeability response to I/R. Areas of uncertainty and controversy in this field of investigation are also identified.

Inflammatory bowel diseases (IBDs) are associated with platelet activation and an increased risk for thromboembolism. While the mechanisms that underlie the altered platelet function and hypercoagulable state in IBD remain poorly understood, emerging evidence indicates that inflammation and coagulation are interdependent processes that can initiate a vicious cycle wherein each process propagates and intensifies the other. This review addresses the mechanisms that may account for the mutual activation of coagulation and inflammation during inflammation and summarizes evidence that implicates a role for platelets and the coagulation system in the pathogenesis of human and experimental IBD. The proposed link between inflammation and coagulation raises the possibility of targeting the inflammation-coagulation interface to reduce the morbidity and mortality associated with IBD.

Obesity is a worldwide epidemic that continues to grow at an alarming rate. This condition increases the morbidity and mortality associated with both acute and chronic diseases. Some of the deleterious consequences of obesity have been attributed to its induction of a low-grade chronic inflammatory state that arises from the production and secretion of inflammatory mediators from the expanded pool of activated adipocytes. This review focuses on the mechanisms that underlie the proposed link between obesity and inflammation, and it addresses how obesity-induced inflammation may account for increased morbidity and mortality that is associated with a diverse group of diseases.

Although clinical obesity is associated with increases in the morbidity and mortality of sepsis, little is known about the mechanisms that underlie the influence of obesity on sepsis. The objective of this study was to determine (a) whether obesity is associated with exaggerated inflammatory and thrombogenic responses in the intestinal microvasculature of septic mice and (b) whether these microvascular alterations are related to changes in the serum levels of cytokines that are produced by adipose tissue. Intravital microscopy was used to quantify leukocyte and platelet adhesion in intestinal postcapillary venules of lean wild-type (WT) mice, and two murine models of obesity, that is, ob/ob and db/db mice. Sepsis was induced by cecal ligation and perforation (CLP). Serum cytokine levels were measured using a cytometric bead assay, whereas adipokines were quantified using enzyme-linked immunosorbent assay. Cecal ligation and perforation elicited significant increases in the adhesion of leukocytes and platelets in venules of lean WT mice. These CLP-induced adhesive interactions were much more pronounced in the microvasculature of both ob/ob and db/db mice. Cecal ligation and perforation was associated with significant increases in serum cytokines in both WT and ob/ob mice, but such changes were not detected in db/db mice. However, db/db (but not WT or ob/ob) mice did exhibit significant increases in serum leptin and adiponectin levels after CLP. Sepsis promotes more intense inflammatory and thrombogenic responses in the gut microcirculation of obese mice than in their lean counterparts. The obesity-enhanced microvascular dysfunction in septic mice shows no consistent correlation with serum cytokines or adipokines.

Publisher Summary This chapter focuses on the role of the microvasculature in regulating the recruitment of leukocytes into the gastrointestinal (GI) tract and liver. The contributions of the GI and liver microcirculations to the assimilation of a meal, and other relationships between organ function and these vascular beds have also been discussed. An exciting and robust area of investigation related to GI and liver pathology is inflammation. There is a large body of evidence that implicates the microcirculation as a rate-determining component of the inflammatory cell recruitment and activation that occurs during inflammation of the GI tract and liver. Inflammatory diseases of the GI tract and liver are however characterized by a pronounced and persistent recruitment of inflammatory cells into these tissues, which, coupled with dysregulated activation of the infiltrating cells, ultimately results in tissue injury and organ dysfunction. Functional blood and lymph microcirculations in the intestine are essential for immune defense by controlling immune cell trafficking. Key elements of the trafficking of lymphoid cells through the intestine are summarized. Various types of leukocytes appear to contribute to the pathology of many different liver diseases. Depletion of leukocytes results in reduced injury in many of these disease states, however, it is worth noting that in some situations despite the accumulation of leukocytes, they may not be involved in injury. The leukocyte recruitment cascade as it pertains to the liver is summarized and the important and often controversial aspects of this work are highlighted.

Recent evidence implicating tissue factor and the protein C pathway in the hypercoagulable state associated with intestinal inflammation suggests that thrombin is likely to contribute to this response. The objective of this study was to assess the role of thrombin in the extraintestinal thrombosis associated with experimental colitis. Thrombus formation was quantified in microvessels of the cremaster muscle in mice with dextran sodium sulfate (DSS)-induced colonic inflammation. The light/dye endothelial injury model was used to elicit thrombus formation in DSS colitic mice treated with either hirudin, heparin, or antithrombin III. The initiation and propagation/stabilization phases of thrombus formation were quantified using the time of onset of the thrombus and time to blood flow cessation, respectively. Thrombus formation was accelerated in arterioles of DSS colitic mice, as exhibited by significant reductions in the time of thrombus initiation and propagation/stabilization. Colitic mice treated with hirudin, heparin, or antithrombin III did not exhibit a significant change in the time of onset of the thrombus compared with untreated colitic mice. However, all three antithrombin agents largely prevented the DSS-induced reduction in the time to flow cessation following light/dye injury, with hirudin offering complete protection. These findings indicate that thrombin plays a major role in the extraintestinal thrombus formation associated with experimental colitis. Thrombin appears to contribute to the propagation/stabilization, rather than initiation, phase of the colitis-associated thrombogenesis at the distant vascular site. The results support the therapeutic use of antithrombin agents for reducing the risk of thromboembolism in patients with inflammatory bowel disease.

Although hypercholesterolemia is known to impair endothelium-dependent vasodilation (EDV) long before the appearance of atherosclerotic plaques, it remains unclear whether the immune mechanisms that have been implicated in atherogenesis also contribute to the early oxidative stress and endothelial cell dysfunction elicited by hypercholesterolemia. EDV (wire myography), superoxide generation (cytochrome c reduction), and NAD(P)H oxidase mRNA expression were monitored in aortic rings from wild-type (WT) and mutant mice placed on either a normal diet or a cholesterol-enriched diet (HC) for 2 wk. WT mice on HC exhibited impaired EDV, enhanced superoxide generation, and increased expression of NAD(P)H oxidase subunit Nox-2 mRNA. The impaired EDV and increased superoxide generation induced by HC were significantly blunted in severe combined immunodeficient (SCID) mice and CD4+ T lymphocyte-deficient mice. These responses were also attenuated in HC mice genetically deficient in IFN-γ; however, adoptive transfer of WT-HC CD4+ T lymphocytes to IFN-γ-deficient recipients restored HC-induced responses. The HC-induced impaired EDV and oxidative stress were also attenuated in HC mice genetically deficient in Nox-2 (gp91phox−/−) and in WT→gp91phox−/−-HC chimeras. HC-induced gp91phoxmRNA expression was significantly blunted in mice deficient in CD4+ T cells or IFN-γ and was restored with adoptive transfer of WT-HC CD4+ T cells to IFN-γ-deficient recipients. These findings implicate the immune system in the early endothelial cell dysfunction associated with hypercholesterolemia and are consistent with a mechanism of impaired EDV that is mediated by CD4+ T cells and IFN-γ, acting through the generation of superoxide from vascular NAD(P)H oxidase.

Background and Purpose— Although epidemiological studies reveal an increased incidence of obesity and an association between obesity and the prevalence/severity of ischemic stroke, little is known about the mechanisms that link obesity to ischemic stroke. This study tested the hypothesis that obesity exacerbates the cerebrovascular dysfunction and tissue injury induced by brain ischemia and reperfusion. Methods— The adhesion of leukocytes and platelets in cerebral venules, blood–brain barrier permeability, brain water content, and infarct volume were measured in wild-type, obese ( ob/ob ), and leptin-reconstituted ob/ob mice subjected to 30 minutes middle cerebral artery occlusion and reperfusion. Tissue and plasma cytokine levels were determined by cytometric bead array, and a role for monocyte chemoattractant protein-1 and interleukin-6 was assessed using blocking antibodies. Results— Compared with wild-type mice, ob/ob exhibited larger increases in leukocyte and platelet adhesion, blood–brain barrier permeability, water content, and infarct volume after middle cerebral artery occlusion–reperfusion. Reconstitution of leptin in ob/ob mice tended to further enhance all reperfusion-induced responses. Ob/ob mice also exhibited higher plasma levels of monocyte chemoattractant protein-1 and interleukin-6 than wild-type mice. Immunoneutralization of monocyte chemoattractant protein-1, but not interleukin-6, reduced infarct volume in ob/ob mice. Conclusions— Obesity worsens the inflammatory and injury responses to middle cerebral artery occlusion and reperfusion by a mechanism independent of leptin deficiency. monocyte chemoattractant protein-1 appears to contribute to the exaggerated responses to ischemic stroke in obese mice.

Inflammatory bowel diseases are associated with increased risk for thrombus formation both within the inflamed bowel and at distant sites. Although the increased propensity for distant organ thrombus development has been recapitulated in animal models of colitis and linked to interleukin-6 (IL-6), it remains unclear whether experimental colitis results in accelerated thrombus development within the inflamed bowel and whether IL-6 contributes to a local thrombogenic response. These issues related to thrombus formation within the inflamed bowel were addressed in mice with dextran sodium sulfate-induced colitis. Wild-type (WT) mice, IL-6 deficient (IL-6(-/-)) mice, and bone marrow chimeras (WT→WT and IL-6(-/-)→WT) were used. The effects of treatment with either an IL-6-blocking, IL-6Rα-blocking or gp130-blocking antibody were also evaluated. Disease activity index and colonic weight-to-length ratio (W/L) were used to monitor the development of colitis. Intravital videomicroscopy was used to study thrombus development (induced with the light/dye method) in mucosal vessels of the ascending colon. Thrombus development was significantly enhanced in WT colitic mice. Neither genetic deficiency nor immunoblockade of IL-6 significantly altered the disease activity index and W/L responses to dextran sodium sulfate treatment. However, colitis-induced thrombogenesis was attenuated in IL-6(-/-) mice and in WT mice treated with either the IL-6-blocking, IL-6Rα-blocking or gp130-blocking antibody. IL-6(-/-)→WT, but not WT→WT chimeras, exhibited a blunted thrombosis response to dextran sodium sulfate. These results indicate that experimental colitis is associated with accelerated thrombus development within the inflamed colon and that IL-6, derived from bone marrow-derived blood cells, is largely responsible for this response.

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1−/−) and wild-type (WT) mice were transplanted with WT (WT/COX-1−/−) or COX-1−/−(COX-1−/−/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1−/−/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1−/−chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.

Background/Aims Sepsis remains a leading cause of death in critically ill patients. Because endotoxemia is viewed as a key mediator of sepsis-induced inflammation, administration of bacterial endotoxin (LPS) is often used to simulate sepsis in experimental animals. This study tests the hypothesis that LPS is a critical determinant of the hepatic microvascular dysfunction in mice made septic by cecal ligation and puncture (CLP). Methods Intravital videomicroscopy was used to quantify sinusoidal perfusion, and platelet and leukocyte adhesion in terminal hepatic venules (THV) and sinusoids in LPS-sensitive and LPS-insensitive mice subjected to CLP or LPS (i.p.). mRNA expression of TLR-2, TLR-4, MyD-88, and Ly-96 was also assessed. Results While LPS-sensitive mice responded to both CLP and LPS challenges with elevated leukocyte and platelet adhesion in THV and sinusoids, and a reduced sinusoidal perfusion density, LPS-insensitive mice exhibited comparable blood cell adhesion and sinusoidal malperfusion following CLP, but not LPS. Hepatic mRNA of MyD-88 and TLR-2 was elevated in the CLP and LPS groups. Endotoxin was not detectable in the blood of LPS-sensitive mice after CLP, but was elevated after LPS administration. Conclusions These findings do not support a major role for LPS in the hepatic microvascular disturbances associated with polymicrobial sepsis.