The authors report for the first time that first mechanically reducing LV work before coronary reperfusion with a transvalvular pump is necessary and sufficient to reduce infarct size and to activate a cardioprotective program that includes enhanced SDF-1α activity. Primary unloading further improved LV scar size and cardiac function 28 days after AMI.

Background-Ischemia/reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular wall stress with a percutaneous left atrial-to-femoral artery centrifugal bypass system while delaying coronary reperfusion limits myocardial injury in a model of acute MI. Methods and Results-MI was induced by balloon occlusion of the left anterior descending artery in adult male swine. In the MI group (n=4), 120 minutes of left anterior descending artery occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+unload; n=4), percutaneous left atrial-to-femoral artery bypass was initiated after 120 minutes of ischemia, and left anterior descending artery occlusion was prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were euthanized after reperfusion, and infarct size was quantified by triphenyltetrazolium chloride staining. Compared with baseline, mean left ventricular wall stress and stroke work were not changed at any point in the MI group but were decreased after reperfusion in the MI+unload group (mean left ventricular wall stress, 44 658 versus 22 963 dynes/cm 2 ; stroke work, 2823 versus 655 mm Hg·mL, MI versus MI+unload). Phosphorylation of reperfusion injury salvage kinase pathway proteins from noninfarcted left ventricular tissue was unchanged in the MI group but was increased in the MI+unload group. Compared with the MI group, total infarct size was reduced in the MI+unload group (49% versus 28%, MI versus MI+unload). Conclusions-These Kapur et al Reducing Wall Stress in AMI 329regulated kinase (ERK) and the serine/threonine kinase Akt. [8][9][10][11][12] Other approaches to limit ischemia/reperfusion injury include systemic hypothermia, which has also shown promise as a method to reduce total body metabolic demand 13 ; however, this approach does not specifically target myocardial injury in AMI. Alternatively, the generation of repeated periods of myocardial ischemia and reperfusion with balloon angioplasty, known as ischemic conditioning, has also been shown to promote activity of the RISK pathway. [14][15][16] Although promising, critical barriers to these cardioprotective strategies include the multifactorial nature of reperfusion injury, thereby limiting the impact of a single-target pharmacological strategy; the potential for coronary vascular injury (dissection or perforation) with ischemic conditioning; and the mandate for rapid coronary reperfusion and thus insufficient time for a drug to penetrate into myocardial injury zones. A need exists for improved cardioprotective strategies that broadly affect the multiple levels of reperfusion injury without causing further myocardial damage. In this study, we explore the central hypothesis that initially reducing left ventricular (LV) wall stress by mechanically reducing LV preload while delaying coronary reperfusion activat...

Background Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine, transforming growth factor beta-1 (TGFβ1), promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a co-receptor for TGFβ1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure. Methods and Results We first report that endoglin expression is increased in the left ventricle (LV) of human subjects with heart failure and determined that endoglin is required for TGFβ1 signaling in human cardiac fibroblasts using neutralizing antibodies and a siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves LV function, and improves survival in a mouse model of pressure-overload induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin (sEng), which disrupts TGFβ1 signaling in endothelium. We now demonstrate that sEng limits TGFβ1 signaling and Type I collagen synthesis in cardiac fibroblasts and further show that sEng treatment attenuates cardiac fibrosis in an in vivo model of heart failure. Conclusions Our results identify endoglin as a critical component of TGFβ1 signaling in the cardiac fibroblast and that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

Background Early life adversity (ELA) is a risk factor for development of gastrointestinal disorders later in life. The underlying mechanisms through which ELA and sex interact to influence disease susceptibility remains poorly understood. Methods Utilizing a porcine early weaning stress (EWS) model to mimic ELA, we investigated the long-term effects of EWS on functional diarrhea, ileal permeability, mast cell activity and relationship to enteric ganglia. Key Results Juvenile and adult EWS pigs exhibited chronic, functional diarrhea (EWS 43.6% vs LWC 4.8%, p<0.0001), increased intestinal permeability (2 fold increase EWS vs LWC, p<0.0001), and mast cell numbers (at 7 weeks and 20 weeks ~1.6 fold increase EWS vs LWC, p<0.05). Compared with EWS male castrates (Male-C), females EWS pigs exhibited more frequent diarrhea (58.8% vs 29.9%, p=0.0016), and increased intestinal permeability (1–2 fold higher in EWS females, p<0.001). Increased mast cell numbers and their enhanced co-localization with neuronal ganglia were observed in both Male-C and female EWS pigs; however, female pigs exhibited greater release of mast cell tryptase upon activation with c48/80 (~1.5 fold increase, p<0.05), compared with Male-C pigs. Conclusions and Inferences These data demonstrate that pigs exposed to ELA exhibit increased vulnerability to functional diarrhea, intestinal permeability and mast cell activity. Further, these studies also showed that EWS female and Male-C pigs exhibited dimorphic responses to EWS with female piglets exhibited greater susceptibility and severity of diarrhea, intestinal permeability and mast cell tryptase release. Together, these findings mimic some of the key pathophysiologic findings in human functional GI disorders (FGIDs) suggesting that the EWS porcine model could be a valuable preclinical translational model for FGID research associated with ELA.

BackgroundBiological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders.MethodsTwelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis.ResultsSexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of β-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis.ConclusionsThese results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0113-7) contains supplementary material, which is available to authorized users.

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability. Permalink https://escholarship.org/uc/item/8t3660m4 Journal Abstract 33 34 Background: Psychological stress and heightened MC activation are linked with important 35 immunological disorders including allergy, anaphylaxis, asthma, and functional bowel 36diseases, but the mechanisms remain poorly defined. We have previously demonstrated that 37 activation of the corticotropin releasing factor (CRF) system potentiates MC degranulation 38 responses during IgE-mediated anaphylaxis and psychological stress, via CRF receptor 39 subtype 1 (CRF 1 ) expressed on MCs. 40 41 Objective: In this study, we investigated the role of CRF receptor subtype 2 (CRF 2 ) as a 42 modulator of stress-induced MC degranulation and associated disease pathophysiology. 43 44 Methods: In vitro MC degranulation assays were performed with bone marrow derived MCs 45 (BMMCs) derived from WT and CRF 2 -deficient (CRF 2 -/-) mice and RBL-2H3 MCs transfected 46 with CRF 2 -overexpressing plasmid or CRF 2 -siRNA. In vivo MC responses and associated 47 pathophysiology in IgE-mediated passive systemic anaphylaxis (PSA) and acute 48 psychological restraint stress were measured in WT, CRF 2 -/-, and MC-deficient Kit W-sh/W-sh 49 knock-in mice. 50 51 Results: Compared with WT mice, CRF 2 -/exhibited heightened serum histamine levels and 52 exacerbated PSA-induced anaphylactic responses and colonic permeability. In addition, 53 CRF 2 -/mice exhibited increased serum histamine and colonic permeability following acute 54 restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF 2 55 expressed on MCs suppresses store-operated Ca 2+ entry (SOCE) signaling and MC 56 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 degranulation induced by diverse MC stimuli. Experiments with MC-deficient Kit W-sh/W-sh mice 57 systemically engrafted with WT and CRF 2 -/-BMMCs demonstrated the functional importance 58 of MC-CRF 2 in modulating stress-induced pathophysiology. 59 60 Conclusions: MC CRF 2 is a negative, global modulator of stimuli-induced MC degranulation 61 and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis. 62 63 Key messages 64 • Loss of CRF 2 function induces exacerbated MC degranulation, IgE-mediated 65 anaphylaxis and psychological stress-induced intestinal barrier dysfunction. 66 • MC-specific CRF 2 suppresses degranulation induced by diverse MC stimuli via 67 negative regulation of SOCE. 68 • Further characterization of the mechanisms by which CRF 2 negatively modulates MC 69 activation could lead to novel therapeutic approaches for stress-related immunological 70 disorders associated with MC hyperactivity. 71 72

Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient mice engrafted with CRF bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted mice. mice engrafted with CRF BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF activation did not directly induce MC degranulation, CRF signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca from intracellular stores. Taken together, our results revealed a prominent role for CRF signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.

Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1).MethodsAdult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis.ResultsBoth primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dtmax) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dtmax and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups.ConclusionThese data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.