Exposure to Stress Alters Cardiac Gene Expression and Exacerbates Myocardial Ischemic Injury in the Female Murine Heart.

Mental stress is a risk factor for myocardial infarction in women. The central hypothesis of this study is that restraint stress induces sex-specific changes in gene expression in the heart, which leads to an intensified response to ischemia/reperfusion injury due to the development of a pro-oxidative environment in female hearts. We challenged male and female C57BL/6 mice in a restraint stress model to mimic the effects of mental stress. Exposure to restraint stress led to sex differences in the expression of genes involved in cardiac hypertrophy, inflammation, and iron-dependent cell death (ferroptosis). Among those genes, we identified tumor protein p53 and cyclin-dependent kinase inhibitor 1A (p21), which have established controversial roles in ferroptosis. The exacerbated response to I/R injury in restraint-stressed females correlated with downregulation of p53 and nuclear factor erythroid 2-related factor 2 (Nrf2, a master regulator of the antioxidant response system-ARE). S-female hearts also showed increased superoxide levels, lipid peroxidation, and prostaglandin-endoperoxide synthase 2 (Ptgs2) expression (a hallmark of ferroptosis) compared with those of their male counterparts. Our study is the first to test the sex-specific impact of restraint stress on the heart in the setting of I/R and its outcome.

Protocol for the induction of hindlimb ischemia and isolation of muscle endothelial cells in mice.

A mouse model of hindlimb ischemia is an important tool for studying diverse therapeutic approaches for vascularization with high surgical success and low mortality rates. Here, we present a protocol for the induction of hindlimb ischemia in mice, including the surgery procedure and steps to analyze blood perfusion in the ischemic area using a laser speckle contrast analyzer. We also detail the isolation of endothelial cells from thigh muscles using flow cytometry after ischemic surgery. For complete details on the use and execution of this protocol, please refer to Park et al. (2016).1.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

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.

A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Angiotensin II (Ang-II)-induced hypertension is associated with accelerated thrombus formation in arterioles and leukocyte recruitment in venules. The mechanisms that underlie the prothrombotic and proinflammatory responses to chronic Ang-II administration remain poorly understood. We evaluated the role of CD40/CD40 ligand (CD40L) signaling in Ang-II-mediated microvascular responses and assessed whether and how soluble CD40L (sCD40L) contributes to this response. Intravital video microscopy was performed to analyze leukocyte recruitment and dihydrorhodamine-123 oxidation in postcapillary venules. Thrombus formation in cremaster muscle arterioles was induced by using the light/dye endothelial cell injury model. Wild-type (WT), CD40-/-, and CD40L-/- mice received Ang-II for 14 d via osmotic minipumps. Some mice were treated with either recombinant sCD40L or the VLA5 (very late antigen 5; α5ß1) antagonist, ATN-161. Our results demonstrate that CD40-/-, CD40L-/-, and WT mice that were treated with ATN-161 were protected against the thrombotic and inflammatory effects of Ang-II infusion. Infusion of sCD40L into CD40-/- or CD40L-/- mice restored the prothrombotic effect of Ang-II infusion. Mice that were treated with ATN-161 and infused with sCD40L were protected against accelerated thrombosis. Collectively, these novel findings suggest that the mechanisms that underlie Ang-II-dependent thrombotic and inflammatory responses link to the signaling of CD40L via both CD40 and VLA5.-Senchenkova, E. Y., Russell, J., Vital, S. A., Yildirim, A., Orr, A. W., Granger, D. N., Gavins, F. N. E. A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke.

Stroke continues to be a leading cause of death and disability worldwide, yet effective treatments are lacking. Previous studies have indicated that stem-cell transplantation could be an effective treatment. However, little is known about the direct impact of transplanted cells on injured brain tissue. We wanted to help fill this knowledge gap and investigated effects of hematopoietic stem/progenitor cells (HSPCs) on the cerebral microcirculation after ischemia-reperfusion injury (I/RI). Treatment of HSPCs in I/RI for up to 2 wk after cerebral I/RI led to decreased mortality rate, decreased infarct volume, improved functional outcome, reduced microglial activation, and reduced cerebral leukocyte adhesion. Confocal microscopy and fluorescence-activated cell sorting analyses showed transplanted HSPCs emigrate preferentially into ischemic cortex brain parenchyma. We isolated migrated HSPCs from the brain; using RNA sequencing to investigate the transcriptome, we found metallothionein (MT, particularly MT-I) transcripts were dramatically up-regulated. Finally, to confirm the significance of MT, we exogenously administered MT-I after cerebral I/RI and found that it produced neuroprotection in a manner similar to HSPC treatment. These findings provide novel evidence that the mechanism through which HSPCs promote repair after stroke maybe via direct action of HSPC-derived MT-I and could therefore be exploited as a useful therapeutic strategy for stroke.-Smith, H. K., Omura, S., Vital, S. A., Becker, F., Senchenkova, E. Y., Kaur, G., Tsunoda, I., Peirce, S. M., Gavins, F. N. E. Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke.

IL-6 Mediates the Intestinal Microvascular Thrombosis Associated with Experimental Colitis.

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.

Interleukin-6 mediates enhanced thrombus development in cerebral arterioles following a brief period of focal brain ischemia.

OBJECTIVE: The cerebral microvasculature is rendered more vulnerable to thrombus formation following a brief (5.0 min) period of focal ischemia. This study examined the contribution of interleukin-6 (IL-6), a neuroprotective and prothrombotic cytokine produced by the brain, to transient ischemia-induced thrombosis in cerebral arterioles. APPROACH & RESULTS: The middle cerebral artery of C57BL/6J mice was occluded for 5 min, followed by 24h of reperfusion (MCAo/R). Intravital fluorescence microscopy was used to monitor thrombus development in cerebral arterioles induced by light/dye photoactivation. Thrombosis was quantified as the time of onset of platelet aggregation on the vessel wall and the time for complete blood flow cessation. MCAo/R in wild type (WT) mice yielded an acceleration of thrombus formation that was accompanied by increased IL-6 levels in plasma and in post-ischemic brain tissue. The exaggerated thrombosis response to MCAo/R was blunted in WT mice receiving an IL-6 receptor-blocking antibody and in IL-6 deficient (IL-6(-/-)) mice. Bone marrow chimeras, produced by transplanting IL-6(-/-) marrow into WT recipients, did not exhibit protection against MCAo/R-induced thrombosis. CONCLUSIONS: The increased vulnerability of the cerebral vasculature to thrombus development after MCAo/R is mediated by IL-6, which is likely derived from brain cells rather than circulating blood cells. These findings suggest that anti-IL-6 therapy may reduce the likelihood of cerebral thrombus development after a transient ischemic attack.

Selectin-mediated recruitment of bone marrow stromal cells in the postischemic cerebral microvasculature.

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.

Modulation of circulating cell-endothelial cell interaction by erythropoietin in lean and obese mice with cecal ligation and puncture.

OBJECTIVE: Septic shock related morbidity is increased further in obese patients. Obese mice have exaggerated inflammatory response compared to lean mice when exposed to cecal ligation and puncture (CLP). The current study employs use of erythropoietin (EPO), an anti-inflammatory agent in obese and lean mice with CLP to study inflammation. METHOD: Intravital videomicroscopy was used to quantify CLP-induced platelet (PA) and leukocyte (LA) adhesion in cerebral microcirculation of WT (C57Bl/6) and ob/ob (B6.V-Lepob/J) mice with or without EPO (500units/kg) treatment. P-selectin expression (P-sel) in various tissues, blood-brain barrier (BBB) and behavioral function and serum tumor necrosis factor (TNF) levels were quantified in these animals. LA, PA, BBB function and P-sel were measured in TNF monoclonal antibody (mAb) pre-treated ob/ob mice. RESULTS: EPO attenuated the LA, PA, BBB and behavioral dysfunctions and serum TNF levels 4h after CLP in both WT and ob/ob mice, and P-sel in ob/ob CLP mice. Furthermore, TNF mAb and EPO decreased the LA, PA, BBB dysfunction and P-sel in the ob/ob CLP animals to the same degree. CONCLUSION: EPO exerts significant anti-inflammatory actions in obese and lean septic mice. Modulation of P-selectin expression via attenuation of TNF production is a suggested underlying mechanism.

Obesity and sepsis.

Obesity has reached epidemic proportions over the last few decades. Obesity is associated with increased morbidity and mortality in hypertension, cardiovascular diseases, stroke, and cancer and is feared to decrease overall life expectancy over the next few decades. There is a growing body of evidence suggesting that obesity is a chronic inflammatory disease. Obesity is becoming a cause of concern in critically ill patients as well. Sepsis is the number one cause of morbidity and mortality in noncoronary artery disease critical care units all over the world and is associated with a high cost of care. An increase in morbidity in obese septic patients compared with lean people is a cause of growing concern. Laboratory evidence suggests that there is exaggeration in the inflammatory and prothrombogenic phenotype assumed by obese compared with lean septic animals. The exact mechanisms underlying this phenomenon are unknown. This article reviews some of the pathophysiological processes responsible for the underlying inflammation in obesity and sepsis and reviews the literature for the association of the two.

Glucocorticoids inhibit the cerebral microvascular dysfunction associated with sepsis in obese mice.

OBJECTIVES: Obesity is associated with increased morbidity and mortality in critically ill patients. It is unclear whether this increase is due to exaggerated inflammatory response alone or due to lack of response to therapeutic agents used. The objective of this study was to determine whether low-dose steroid therapy, which has proven effective in clinical setting, affords any benefit in the increased morbidity to sepsis in genetically obese (ob/ob) mice. METHODS: Intravital videomicroscopy was used to monitor and quantify the adhesion of platelets and leukocytes in the brain microcirculation of lean (WT) and ob/ob mice subjected to cecal ligation and puncture (CLP) with or without dexamethasone 4 mg/kg within 15 min of surgery. The dual radiolabeled monoclonal antibody method was used to measure P-selectin expression in the microcirculation, while the sepsis-induced behavioral deficit was quantified using a multicompartment chamber test. RESULTS: Dexamethasone completely prevented the accumulation of adherent leukocytes and platelets observed at 4 h after CLP in both WT and ob/ob mice. The steroid also prevented the CLP-induced upregulation of P-selectin in the brain and other vascular beds, and it attenuated the behavioral deficit in ob/ob, but not in lean, mice. CONCLUSION: Low-dose glucocorticoid therapy is beneficial in experimental sepsis in obese animals compared to lean animals.