Left ventricular (LV) hypertrophy commonly develops in response to chronic hypertension and is a significant risk factor for heart failure and death. The serine-threonine phosphatase, calcineurin (CnA), plays a critical role in the development of pathologic hypertrophy. Previous experimental studies in murine models show that estrogen limits pressure overload-induced hypertrophy; our purpose was to explore further the mechanisms underlying this estrogen effect. Wild type, ovariectomized female mice were treated with placebo or 17β-estradiol (E2), followed by transverse aortic constriction (TAC) to induce pressure overload. At two weeks, mice underwent physiologic evaluation, immediate tissue harvest, or dispersion of cardiomyocytes. E2 replacement limited TAC-induced LV and cardiomyocyte hypertrophy while attenuating deterioration in LV systolic function and contractility. These E2 effects were associated with reduced abundance of CnA. The primary downstream targets of CnA are the nuclear factor of activated T-cell (NFAT) family of transcription factors. In transgenic mice expressing a NFAT-activated promoter-luciferase reporter gene, E2 limited TAC-induced activation of NFAT. Moreover, the inhibitory effects of E2 on LV hypertrophy were absent in CnA knockout mice supporting that CnA is an important target of E2-mediated inhibition. In cultured rat cardiac myocytes, E2 inhibited agonist-induced hypertrophy while also decreasing CnA abundance and NFAT activation. Agonist stimulation also reduced CnA ubiquitination and degradation that was prevented by E2; all in vitro effects of estrogen were reversed by an ER antagonist. These data support that E2 reduces pressure overload induced hypertrophy by an ER-dependent mechanism that increases CnA degradation, unveiling a novel mechanism by which E2 and ERs regulate pathologic LV and cardiomyocyte growth.

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.

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Objective-Early recognition of an acute coronary occlusion (ACO) improves clinical outcomes. Soluble fms-like tyrosine kinase-1 (sFLT1) is an endothelium-derived protein induced by hypoxia. We tested whether sFLT1 levels are elevated in ACO. Methods and Results-Serum sFLT1 levels were measured by enzyme-linked immunosorbent assay in patients with ST-segment elevations and angiographically confirmed ACO, unstable angina/non ST-segment elevation myocardial infarction, and 2 control groups. To further explore sFLT1 release, a mouse model of ACO and in vitro human coronary artery endothelial cell injury were used. sFLT1 levels were increased in ACO compared with unstable angina/non-ST-elevation myocardial infarction, catheterized controls, or healthy volunteers (200.7Ϯ15.5 versus 70.7Ϯ44.0 versus 10.2Ϯ4.0 versus 11.7Ϯ1.7 pg/mL respectively, PϽ0.001 versus ACO). At presentation, all ACO patients had elevated sFLT1 levels (Ͼ15 pg/mL, 99th percentile in controls), whereas 57% had levels of the MB isoform of creatine kinase levels Ͼ10 ng/mL (PϽ0.01) and 85% had ultrasensitive troponin I levels Ͼ0.05 ng/mL (PϽ0.05). Within 60 minutes after symptom onset, sFLT1 was more sensitive than the MB isoform of creatine kinase or ultrasensitive troponin I for ACO (100% versus 20% versus 20% respectively; PՅ0.01 for each). Within 60 minutes of ACO in mice, sFLT1 levels were elevated. Hypoxia and thrombin increased sFLT1 levels within 15 minutes in human coronary artery endothelial cells. Key Words: acute coronary syndromes Ⅲ coronary artery disease Ⅲ coronary heart disease Ⅲ endothelium Ⅲ thrombosis A cute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. During acute coronary occlusion (ACO), a delay in diagnosis beyond 2 hours after symptom onset reduces the benefits of reperfusion therapy by Ϸ50%. 1-3 In most cases, ACO is clinically diagnosed based on electrocardiographic (ECG) evidence of ST-segment elevation (STE) and symptoms consistent with myocardial ischemia. However, STE alone is not specific to coronary occlusion. Conversely, patients with ACO may have no evidence of STE. 4 The difficulty of timely identification of ACO is increased further because currently used biomarkers for the diagnosis of AMI, 5 such as the MB isoform of creatine (CK) kinase (CK-MB), myoglobin, and standard or ultrasensitive troponin require cardiomyocyte damage and may not be specific to coronary occlusion. 6,7 Therefore, relying on the ECG or biomarkers of myocyte necrosis to distinguish ACO from noncardiac causes of chest pain may be accompanied by both false-positive and negative results.Human vascular endothelial growth factor receptor-1, also known as fms-like tyrosine kinase-1 (FLT1), was originally identified in human placental tissue and later characterized as a receptor for the angiogenic factors vascular endothelial growth factor-A, vascular endothelial growth factor-B, and placental growth factor. 8 FLT1 is highly expressed by endothelial cells, vascular smooth muscle cells, and monocytes. 9,10 A trunca...

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Background Enteric nervous system (ENS) abnormalities have been implicated in delayed gastric emptying but studies exploring potential treatment options are limited by the lack of an experimental animal model. We examined the ENS abnormalities in the mouse stomach associated with aging, developed a novel model of gastroparesis, and established a new approach to measure gastric emptying. Methods A modified gastric emptying assay was developed, validated in nNOS −/− mice, and tested in mice at multiple ages. Age‐related changes in ENS structure were analyzed by immunohistochemistry. Gastric aganglionosis was generated in Wnt1‐iDTR mice using focal administration of diphtheria toxin (DT) into the anterior antral wall. Key Results Older mice (>5 months) exhibit hypoganglionosis in the gastric antrum and a decreased proportion of nNOS neurons as compared to younger mice (age 5‐7 weeks). This was associated with a significant age‐dependent decrease in liquid and solid gastric emptying. A novel model of gastric antrum hypoganglionosis was established using neural crest‐specific expression of diphtheria toxin receptor. In this model, a significant reduction in liquid and solid gastric emptying is observed. Conclusions & Inferences Older mice exhibit delayed gastric emptying associated with hypoganglionosis and a reduction in nNOS‐expressing neurons in the antrum. The causal relationship between antral hypoganglionosis and delayed gastric emptying was verified using a novel experimental model of ENS ablation. This study provides new information regarding the pathogenesis of delayed gastric emptying and provides a robust model system to study this disease and develop novel treatments.

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Transforming growth factor beta-1 (TGFb1) promotes cardiac fibrosis. The transmembrane co-receptor Endoglin (Eng; CD105) facilitates TGFb1 signaling via SMAD effector proteins. In contrast, a circulating form of soluble endoglin (sEng) inhibits TGFb1 signaling in vascular endothelium. We recently reported that increased sEng levels in human serum correlate with clinical indices of heart failure severity. Therefore, we tested the hypothesis that Eng and sEng mediate opposing effects on cardiac fibrosis in heart failure. In male, wild-type mice (WT), Eng expression increased in the left ventricle (LV) after 2, 4, and 10 weeks of thoracic aortic constriction (TAC) accompanied by progressive LV fibrosis and hypertrophy. In contrast to WT mice, Eng haploinsufficient (Eng +/− ) mice had preserved LV function (FS%: 78±4 vs 22±16, Eng +/− vs WT, p<0.01) and improved survival [88%(7/8) vs 50%(4/8), Eng +/− vs WT, p<0.001) after 10 weeks of TAC. Reduced LV fibrosis was observed in Eng +/− mice, while LV mass, cardiomyocyte hypertrophy, and calcineurin, SerCA, and bMHC expression were comparable to WT after TAC. Capillary density was significantly higher in Eng +/− mice after TAC compared to WT. LV SMAD phosphorylation (pSMAD) after TAC was studied and a preferential increase was observed in pSMAD1/5/8 expression in Eng +/− mice as compared to WT mice, in which cardiac pSmad2/3 expression was increased. The dependence of TGFb1 induced collagen synthesis on Eng expression was tested in vitro using human cardiac fibroblasts (hCF). Neutralizing antibodies and siRNA against Eng each attenuated TGFb1 induced collagen synthesis. In contrast, conditioned media from cells transfected with an adenovirus over-expressing sEng attenuated pSMAD2/3 expression and TGFb1 induced collagen synthesis in hCF. Treatment of hCF with recombinant sEng blocked TGFb1 induced collagen synthesis in a dose-dependent manner, confirming an inhibitory role of sEng. These results indicate that Eng and sEng mediate opposite effects on TGFb1 induced collagen synthesis. Reduced Eng expression uncouples cardiac fibrosis from cardiomyocyte hypertrophy, promotes SMAD1/5/8-signaling, and enhances angiogenesis. Eng may represent a novel therapeutic target to improve survival in heart failure.