Interactions between integrin α9ß1 and VCAM-1 promote neutrophil hyperactivation and mediate poststroke DVT.

ABSTRACT: Venous thromboembolic events are significant contributors to morbidity and mortality in patients with stroke. Neutrophils are among the first cells in the blood to respond to stroke and are known to promote deep vein thrombosis (DVT). Integrin α9 is a transmembrane glycoprotein highly expressed on neutrophils and stabilizes neutrophil adhesion to activated endothelium via vascular cell adhesion molecule 1 (VCAM-1). Nevertheless, the causative role of neutrophil integrin α9 in poststroke DVT remains unknown. Here, we found higher neutrophil integrin α9 and plasma VCAM-1 levels in humans and mice with stroke. Using mice with embolic stroke, we observed enhanced DVT severity in a novel model of poststroke DVT. Neutrophil-specific integrin α9-deficient mice (α9fl/flMrp8Cre+/-) exhibited a significant reduction in poststroke DVT severity along with decreased neutrophils and citrullinated histone H3 in thrombi. Unbiased transcriptomics indicated that α9/VCAM-1 interactions induced pathways related to neutrophil inflammation, exocytosis, NF-κB signaling, and chemotaxis. Mechanistic studies revealed that integrin α9/VCAM-1 interactions mediate neutrophil adhesion at the venous shear rate, promote neutrophil hyperactivation, increase phosphorylation of extracellular signal-regulated kinase, and induce endothelial cell apoptosis. Using pharmacogenomic profiling, virtual screening, and in vitro assays, we identified macitentan as a potent inhibitor of integrin α9/VCAM-1 interactions and neutrophil adhesion to activated endothelial cells. Macitentan reduced DVT severity in control mice with and without stroke, but not in α9fl/flMrp8Cre+/- mice, suggesting that macitentan improves DVT outcomes by inhibiting neutrophil integrin α9. Collectively, we uncovered a previously unrecognized and critical pathway involving the α9/VCAM-1 axis in neutrophil hyperactivation and DVT.

CD14 Blockade Does Not Improve Outcomes of Deep Vein Thrombosis Following Inferior Vena Cava Stenosis in Mice.

Background: Neutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) promote deep vein thrombosis (DVT). CD14, a co-receptor for toll-like receptor 4 (TLR4), is actively synthesized by neutrophils, and the CD14/TLR4 signaling pathway has been implicated in proinflammatory cytokine overproduction and several aspects of thromboinflammation. The role of CD14 in the pathogenesis of DVT remains unclear. Objective: To determine whether CD14 blockade improves DVT outcomes. Methods: Bulk RNA sequencing and proteomic analyses were performed using isolated neutrophils following inferior vena cava (IVC) stenosis in mice. DVT outcomes (IVC thrombus weight and length, thrombosis incidence, neutrophil recruitment, and NETosis) were evaluated following IVC stenosis in mice treated with a specific anti-CD14 antibody, 4C1, or control antibody. Results: Mice with IVC stenosis exhibited increased plasma levels of granulocyte colony-stimulating factor (G-CSF) along with a higher neutrophil-to-lymphocyte ratio and increased plasma levels of cell-free DNA, elastase, and myeloperoxidase. Quantitative measurement of total neutrophil mRNA and protein expression revealed distinct profiles in mice with IVC stenosis compared to mice with sham surgery. Neutrophils of mice with IVC stenosis exhibited increased inflammatory transcriptional and proteomic responses, along with increased expression of CD14. Treatment with a specific anti-CD14 antibody, 4C1, did not result in any significant changes in the IVC thrombus weight, thrombosis incidence, or neutrophil recruitment to the thrombus. Conclusion: The results of the current study are important for understanding the role of CD14 in the regulation of DVT and suggest that CD14 lacks an essential role in the pathogenesis of DVT following IVC stenosis.

Poststroke venous thromboembolism and neutrophil activation: an illustrated review.

Patients with acute ischemic stroke are at a high risk of venous thromboembolism (VTE), such as deep vein thrombosis (DVT), estimated to affect approximately 80,000 patients with stroke each year in the United States. The prevalence of symptomatic DVT after acute stroke is approximately 10%. VTE is associated with increased rates of in-hospital death and disability, with higher prevalence of in-hospital complications and increased 1-year mortality in patients with stroke. Current guidelines recommend the use of pharmacologic VTE prophylaxis in patients with acute ischemic stroke. However, thromboprophylaxis prevents only half of expected VTE events and is associated with high risk of bleeding, suggesting the need for targeted alternative treatments to reduce VTE risk in these patients. Neutrophils are among the first cells in blood to respond after ischemic stroke. Importantly, coordinated interactions among neutrophils, platelets, and endothelial cells contribute to the development of DVT. In case of stroke and other related immune disorders, such as antiphospholipid syndrome, neutrophils potentiate thrombus propagation through the formation of neutrophil-platelet aggregates, secreting inflammatory mediators, complement activation, releasing tissue factor, and producing neutrophil extracellular traps. In this illustrated review article, we present epidemiology and management of poststroke VTE, preclinical and clinical evidence of neutrophil hyperactivation in stroke, and mechanisms for neutrophil-mediated VTE in the context of stroke. Given the hyperactivation of circulating neutrophils in patients with stroke, we propose that a better understanding of molecular mechanisms leading to neutrophil activation may result in the development of novel therapeutics to reduce the risk of VTE in this patient population.

Elevated plasma sulfides are associated with cognitive dysfunction and brain atrophy in human Alzheimer's disease and related dementias.

Emerging evidence indicates that vascular stress is an important contributor to the pathophysiology of Alzheimer's disease and related dementias (ADRD). Hydrogen sulfide (H2S) and its metabolites (acid-labile (e.g., iron-sulfur clusters) and bound (e.g., per-, poly-) sulfides) have been shown to modulate both vascular and neuronal homeostasis. We recently reported that elevated plasma sulfides were associated with cognitive dysfunction and measures of microvascular disease in ADRD. Here we extend our previous work to show associations between elevated sulfides and magnetic resonance-based metrics of brain atrophy and white matter integrity. Elevated bound sulfides were associated with decreased grey matter volume, while increased acid labile sulfides were associated with decreased white matter integrity and greater ventricular volume. These findings are consistent with alterations in sulfide metabolism in ADRD which may represent maladaptive responses to oxidative stress.

Dysregulated Sulfide Metabolism in Multiple Sclerosis: Serum and Vascular Endothelial Inflammatory Responses.

Multiple sclerosis (MS) is a leading cause of neurodegenerative disability in younger individuals. When diagnosed early, MS can be managed more effectively, stabilizing clinical symptoms and delaying disease progression. The identification of specific serum biomarkers for early-stage MS could facilitate more successful treatment of this condition. Because MS is an inflammatory disease, we assessed changes in enzymes of the endothelial hydrogen sulfide (H2S) pathway in response to inflammatory cytokines. Blotting analysis was conducted to detect Cystathionine γ-lyase (CSE), Cystathionine beta synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST) in human brain microvascular endothelial apical and basolateral microparticles (MPs) and cells following exposure to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). CSE was increased in MPs and cells by exposure to TNF-α/IFN-γ; CBS was elevated in apical MPs but not in cells or basolateral MPs; MST was not significantly affected by cytokine exposure. To test how our findings relate to MS patients, we evaluated levels of CSE, CBS, and MST in serum samples from healthy control and MS patients. We found significantly decreased levels of CBS and MST (p = 0.0004, 0.009) in MS serum samples, whereas serum levels of CSE were marginally increased (p = 0.06). These observations support increased CSE and lower CBS and MST expression being associated with the vascular inflammation in MS. These changes in endothelial-derived sulfide enzymes at sites of inflammation in the brain may help to explain sulfide-dependent changes in vascular dysfunction/neuroinflammation underlying MS. These findings further support the use of serum samples to assess enzymatic biomarkers derived from circulating MPs. For example, "liquid biopsy" can be an important tool for allowing early diagnosis of MS, prior to the advanced progression of neurodegeneration associated with this disease.

The Role of the ACE2/MasR Axis in Ischemic Stroke: New Insights for Therapy.

Ischemic stroke remains the leading cause of neurologically based morbidity and mortality. Current stroke treatment is limited to two classes of FDA-approved drugs: thrombolytic agents (tissue plasminogen activator (tPA)) and antithrombotic agents (aspirin and heparin), which have a narrow time-window (<4.5 h) for administration after onset of stroke symptoms. While thrombolytic agents restore perfusion, they carry serious risks for hemorrhage, and do not influence damage responses during reperfusion. Consequently, stroke therapies that can suppress deleterious effects of ischemic injury are desperately needed. Angiotensin converting enzyme-2 (ACE2) has been recently suggested to beneficially influence experimental stroke outcomes by converting the vasoconstrictor Ang II into the vasodilator Ang 1-7. In this review, we extensively discuss the protective functions of ACE2-Ang (1-7)-MasR axis of renin angiotensin system (RAS) in ischemic stroke.

Human placenta mesenchymal stem cell protection in ischemic stroke is angiotensin converting enzyme-2 and masR receptor-dependent.

Thromboembolic stroke remains a major cause of neurological disability and death. Current stroke treatments (aspirin, tissue plasminogen activator) are significantly limited by timing and risks for hemorrhage which have driven researchers to explore other approaches. Stem cell-based therapy appears to be an effective option for ischemic stroke. Besides trans-differentiation into neural cells, stem cells also provide acute protection via paracrine signaling pathways through which releasing neuroprotective factors. We previously reported that intraperitoneal administration of human placenta mesenchymal stem cell (hPMSC) therapy upon reperfusion significantly protected the brain against middle cerebral artery occlusion (MCAO)-induced injury. In the present study, we specifically investigated the role of hPMSC-derived angiotensin converting enzyme-2 (ACE-2) in protection of MCAO-induced brain injury by measurement of brain tissue viability, cerebral blood flow, and neurological score. Here, we report for the first time that hPMSC expressing substantial amount of ACE-2, which mediates hPMSC protection in the MCAO model. Strikingly, we found that the protective effects of hPMSC in MCAO-induced brain injury could be attenuated by pretreatment of hPMSCs with MLN-4760, a specific inhibitor of ACE-2 activity, or by transfection of hPMSCs with ACE-2-shRNA-lentivirus. The hPMSC-derived ACE-2 specific protective mechanism was further demonstrated by administration of PD123319, an Angiotensin type-2 receptor antagonist, or A779, a MasR antagonist. Importantly, our study demonstrated that the protective effects of hPMSC in experimental stroke are ACE-2/MasR dependent and this signaling pathway represents an innovative and highly promising approach for targeted stroke therapy.

Plasma hydrogen sulfide: A biomarker of Alzheimer's disease and related dementias.

While heart disease remains a common cause of mortality, cerebrovascular disease also increases with age, and has been implicated in Alzheimer's disease and related dementias (ADRD). We have described hydrogen sulfide (H2 S), a signaling molecule important in vascular homeostasis, as a biomarker of cardiovascular disease. We hypothesize that plasma H2 S and its metabolites also relate to vascular and cognitive dysfunction in ADRD. We used analytical biochemical methods to measure plasma H2 S metabolites and MRI to evaluate indicators of microvascular disease in ADRD. Levels of total H2 S and specific metabolites were increased in ADRD versus controls. Cognition and microvascular disease indices were correlated with H2 S levels. Total plasma sulfide was the strongest indicator of ADRD, and partially drove the relationship between cognitive dysfunction and white matter lesion volume, an indicator of microvascular disease. Our findings show that H2 S is dysregulated in dementia, providing a potential biomarker for diagnosis and intervention.

Human placental mesenchymal stem cells improve stroke outcomes via extracellular vesicles-mediated preservation of cerebral blood flow.

BACKGROUND: Besides long-term trans-differentiation into neural cells, benefits of stem cell therapy (SCT) in ischemic stroke may include secretion of protective factors, which partly reflects extracellular vesicle (EVs) released by stem cell. However, the mechanism(s) by which stem cells/EVs limit stroke injury have yet to be fully defined. METHODS: We evaluated the protection effect of human placenta mesenchymal stem cells (hPMSC) as a potential form of SCT in experimental ischemic stroke 'transient middle cerebral artery occusion (MCAO)/reperfusion' mice model. FINDINGS: We found for the first time that intraperitoneal administration of hPMSCs or intravenous hPMSC-derived EVs, given at the time of reperfusion, significantly protected the ipsilateral hemisphere from ischemic injury. This protection was associated with significant restoration of normal blood flow to the post-MCAO brain. More importantly, EVs derived from hPMSC promote paracrine-based protection of SCT in the MCAO model in a cholesterol/lipid-dependent manner. INTERPRETATION: Together, our results demonstrated beneficial effects of hPMSC/EVs in experimental stroke models which could permit the rapid "translation" of these cells into clinical trials in the near-term.

Uncovering sex-specific mechanisms of action of testosterone and redox balance.

The molecular and pharmacological manipulation of the endogenous redox system is a promising therapy to limit myocardial damage after a heart attack; however, antioxidant therapies have failed to fully establish their cardioprotective effects, suggesting that additional factors, including antioxidant system interactions with other molecular pathways, may alter the pharmacological effects of antioxidants. Since gender differences in cardiovascular disease (CVD) are prevalent, and sex is an essential determinant of the response to oxidative stress, it is of particular interest to understand the effects of sex hormone signaling on the activity and expression of cellular antioxidants and the pharmacological actions of antioxidant therapies. In the present review, we briefly summarize the current understanding of testosterone effects on the modulation of the endogenous antioxidant systems in the CV system, cardiomyocytes, and the heart. We also review the latest research on redox balance and sexual dimorphism, with particular emphasis on the role of the natural antioxidant system glutathione (GSH) in the context of myocardial infarction, and the pro- and antioxidant effects of testosterone signaling via the androgen receptor (AR) on the heart. Finally, we discuss future perspectives regarding the potential of using combing antioxidant and testosterone replacement therapies to protect the aging myocardium.

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation.

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 αIIbß3 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 (αIIbß3) 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.

The isothiocyanate sulforaphane modulates platelet function and protects against cerebral thrombotic dysfunction.

BACKGROUND AND PURPOSE: Platelet activation provides a critical link between inflammation and thrombosis. Sulforaphane (SFN), a naturally occurring isothiocyanate, has been shown to display both anti-inflammatory and anti-thrombotic actions in the systemic microvasculature. As inflammation promotes thrombosis and vice versa, in this study we investigated whether SFN is able to reduce inflammatory potentiation of thrombotic events, suppress platelet activation and thrombus formation in the cerebral microvasculature. EXPERIMENTAL APPROACH: Thrombosis was induced in the murine brain using the light/dye-injury model, in conjunction with LPS treatment, with and without SFN treatment. In vitro and in vivo platelet assays (aggregation, flow and other functional tests) were also employed, using both human and murine platelets. KEY RESULTS: SFN was found to reduce LPS-mediated enhancement of thrombus formation in the cerebral microcirculation. In tail-bleed experiments, LPS treatment prolonged bleeding time, and SFN treatment was found to protect against this LPS-induced derangement of platelet function. SFN inhibited collagen-mediated platelet aggregation in vitro and in vivo and the associated adhesion and impaired calcium signalling. Furthermore, glycoprotein VI was shown to be involved in the protective effects observed with SFN treatment. CONCLUSIONS AND IMPLICATIONS: The data presented here provide evidence for the use of SFN in preventing stroke in selected high-risk patient cohorts.

N-acetylcysteine attenuates systemic platelet activation and cerebral vessel thrombosis in diabetes.

OBJECTIVE: We previously demonstrated that diabetes exacerbates stroke-induced brain injury, and that this correlates with brain methylglyoxal (MG)-to-glutathione (GSH) status. Cerebral injury was reversed by N-acetylcysteine (NAC). Here we tested if the pro-thrombotic phenotype seen in the systemic circulation and brain during diabetes was associated with increased MG-glycation of proteins, and if NAC could reverse this. METHODS: The streptozotocin (STZ)-induced mouse model of type 1 diabetes was used. Thrombus formation in venules and arterioles (pial circulation) was determined by intravital videomicroscopy using the light-dye method. Circulating blood platelet-leukocyte aggregates (PLAs) were analyzed by flow cytometry 1 wk before other measurements. GSH and MG levels in platelets were measured by HPLC. MG-modified proteins, glutathione peroxidase-1 (GPx-1), and superoxide dismutase-1 (SOD1) levels were detected in platelets by western blot at 20 weeks. Proteins involved in coagulation were quantified by ELISA. NAC (2mM) was given in drinking water for 3 weeks before the terminal experiment. RESULTS: Thrombus development was accelerated by diabetes in a time-dependent manner. % PLAs were significantly elevated by diabetes. Plasma activated plasminogen activator inhibitor type 1 levels were progressively increased with diabetes duration, with tail bleeding time reduced by 20 wks diabetes. Diabetes lowered platelet GSH levels, GPx-1 and SOD-1 expression. This was associated with higher MG levels, and increased MG-adduct formation in platelets. NAC treatment partly or completely reversed the effects of diabetes. CONCLUSION: Collectively, these results show that the diabetic blood and brain become progressively more susceptible to platelet activation and thrombosis. NAC, given after the establishment of diabetes, may offer protection against the risk for stroke by altering both systemic and vascular prothrombotic responses via enhancing platelet GSH, and GSH-dependent MG elimination, as well as correcting levels of antioxidants such as SOD1 and GPx-1.

Integrin signaling in atherosclerosis.

Atherosclerosis, a chronic lipid-driven inflammatory disease affecting large arteries, represents the primary cause of cardiovascular disease in the world. The local remodeling of the vessel intima during atherosclerosis involves the modulation of vascular cell phenotype, alteration of cell migration and proliferation, and propagation of local extracellular matrix remodeling. All of these responses represent targets of the integrin family of cell adhesion receptors. As such, alterations in integrin signaling affect multiple aspects of atherosclerosis, from the earliest induction of inflammation to the development of advanced fibrotic plaques. Integrin signaling has been shown to regulate endothelial phenotype, facilitate leukocyte homing, affect leukocyte function, and drive smooth muscle fibroproliferative remodeling. In addition, integrin signaling in platelets contributes to the thrombotic complications that typically drive the clinical manifestation of cardiovascular disease. In this review, we examine the current literature on integrin regulation of atherosclerotic plaque development and the suitability of integrins as potential therapeutic targets to limit cardiovascular disease and its complications.

The protection conferred against ischemia-reperfusion injury in the diabetic brain by N-acetylcysteine is associated with decreased dicarbonyl stress.

Diabetes, a risk factor for stroke, leads to elevated blood methylglyoxal (MG) levels. This is due to increased MG generation from the high glucose levels, and because diabetes impairs the glutathione (GSH)-glyoxalase system for MG elimination. MG glycates proteins and causes dicarbonyl stress. We investigated the contribution of MG and GSH to stroke outcome. Cerebral ischemia/reperfusion was performed in chemical-induced (streptozotocin) and genetic Akita mouse models of Type 1 diabetes. Brain infarction and functions of the GSH-dependent MG elimination pathway were determined. Diabetes increased post-ischemia-reperfusion cerebral infarct area in association with elevated MG and diminished GSH levels. Infarct size correlated with brain MG-to-GSH ratio. Expression of glutamate-cysteine ligase catalytic subunit (GCLc) was increased in diabetic brain. GCL activity was unchanged. MG-adducts were elevated in the diabetic brain and, using immunoprecipitation, we identified one of the bands as glycated occludin. This was accompanied by increased blood-brain barrier permeability. Total protein carbonyls were elevated, indicative of oxidative/carbonyl stress. N-acetylcysteine (NAC) corrected MG-to-GSH ratio, and reduced diabetic brain infarct area, occludin glycation and permeability. In addition, protein carbonyls were decreased by NAC. We showed that the diabetic brain exhibited a lower GSH-dependent potential for MG elimination, which contributed to increased protein glycation, and oxidative/carbonyl stress. The consequence of these changes was aggravated post-stroke brain injury. NAC administration protected against the exacerbated brain damage via restored GSH generation and normalization of the MG-to-GSH ratio and possibly by attenuating oxidative/carbonyl stress. This treatment could contribute to the successful management of stroke risk/outcome in diabetes.

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation.

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation.

Oxidative stress stimulates nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We found that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidative stress-induced activation of NF-κB through the classic IκB kinase-dependent pathway. Depletion of Nck prevented oxidative stress induced by exogenous hydrogen peroxide or hypoxia/reoxygenation injury from activating NF-κB in endothelial cells, increasing the abundance of the proinflammatory molecules ICAM-1 (intracellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and recruiting leukocytes. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors but not those encoding antioxidants. Nck promoted oxidative stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of PECAM-1 (platelet endothelial cell adhesion molecule-1) to the activation of p21-activated kinase, which mediates oxidative stress-induced NF-κB signaling. Consistent with this mechanism, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide blocked leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidative stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury.

The impact of primary and persistent cytomegalovirus infection on the progression of acute colitis in a murine model.

Cytomegalovirus (CMV) infects 60-100% of the population worldwide. CMV has been implicated in many diseases through the induction of inflammation. Inflammatory bowel disease (IBD) affects over 1 million Americans annually. IBD, in particular ulcerative colitis, has been associated with CMV infection. Here we use a murine model to test if both primary and persistent CMV infections exacerbate colitis. C57Bl/6J mice were injected with Mock inoculum or murine CMV (mCMV) 4d (primary infection) or 6wks (persistent infection) before inducing colitis. Colitis was induced by administering 3% DSS (dextran sodium sulfate) in the drinking water for 6 days. Distilled water was given to controls. Disease activity index (DAI), derived from scores for stool consistency, body weight loss, occult blood, and rectal bleeding, was recorded daily. DAI increased early with DSS treatment in Mocks when compared with water-treated controls. This was accelerated by both primary and persistent mCMV and appeared to be primarily due to the earlier appearance of gross bleeding vs. their Mock controls. Mocks reached similar DAI values by day 6. Myeloperoxidase was modestly elevated in the mCMV 4d-DSS over the Mock 4d-DSS, however there was no such synergism in the 6wk groups. Histology was comparable in Mock and mCMV groups. Taken together our findings show that mCMV accelerated the development of acute colitis although a milder model of colitis may be needed to better delineate the impact of the virus on disease progression. Further work focusing on disruption of barrier function and bleeding may help determine the underlying mechanisms.

Platelets, acting in part via P-selectin, mediate cytomegalovirus-induced microvascular dysfunction.

Cytomegalovirus (CMV) infects a majority of the population worldwide. It has been implicated in cardiovascular disease, induces microvascular dysfunction, and synergizes with hypercholesterolemia to promote leukocyte and platelet recruitment in venules. Although platelets and platelet-associated P-selectin contribute to cardiovascular disease inflammation, their role in CMV-induced vascular responses is unknown. We assessed the role of platelets in CMV-induced microvascular dysfunction by depleting platelets and developing bone marrow chimeric mice deficient in platelet P-selectin. Wild-type and chimeric mice received mock or murine (m)CMV intraperitoneally. Five weeks later, some mice were switched to a high-cholesterol diet (HC) to investigate the synergism between mCMV and HC. Arteriolar vasodilation and recruitment of leukocytes and donor platelets in venules were measured at 11wk. mCMV with or without HC caused significant endothelial dysfunction in arterioles. Platelet depletion restored normal vasodilation in mCMV-HC but not mCMV-ND mice, whereas protection was seen in both groups for platelet P-selectin chimeras. Only mCMV + HC elevated leukocyte and platelet recruitment in venules. Leukocyte adhesion was reduced to mock levels by acute platelet depletion but was only partially decreased in platelet P-selectin chimeras. Platelets from mCMV-HC mice and, to a lesser extent, mCMV-ND but not mock-HC mice showed significant adhesion in mCMV-HC recipients. Our findings implicate a role for platelets, acting through P-selectin, in CMV-induced arteriolar dysfunction and suggest that the addition of HC leads to a platelet-dependent, inflammatory infiltrate that is only partly platelet P-selectin dependent. CMV appeared to have a stronger activating influence than HC on platelets and may represent an additional therapeutic target in vulnerable patients.