Hypoxia-inducible factors (HIFs) are the master regulators of angiogenesis, a process that is impaired in patients with diabetes mellitus (DM). The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF1β) has been implicated in the development and progression of diabetes. Angiogenesis is driven primarily by endothelial cells (ECs), but both global and EC-specific loss of ARNT-cause are associated with embryonic lethality. Thus, we conducted experiments in a line of mice carrying an inducible, EC-specific ARNT-knockout mutation (ArntΔEC, ERT2) to determine whether aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. Mice were first fed with a high-fat diet to induce diabetes. ArntΔEC, ERT2 mice were then adminstrated with oral tamoxifen to disrupt Arnt and peripheral angiogenesis was evaluated by using laser-Doppler perfusion imaging to monitor blood flow after hindlimb ischemia. The ArntΔEC, ERT2 mice had impaired blood flow recovery under both non-diabetic and diabetic conditions, but the degree of impairment was greater in diabetic animals. In addition, siRNA-mediated knockdown of ARNT activity reduced measurements of tube formation, and cell viability in human umbilical vein endothelial cells (HUVECs) cultured under high-glucose conditions. The ArntΔEC, ERT2 mutation also reduced measures of cell viability, while increasing the production of reactive oxygen species (ROS) in microvascular endothelial cells (MVECs) isolated from mouse skeletal muscle, and the viability of ArntΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with an ROS inhibitor. Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production.

Background Approximately 1 in 6 adolescents report regular binge alcohol consumption, and we hypothesize it affects heart growth during this period. Methods and Results Adolescent, genetically diverse, male Wistar rats were gavaged with water or ethanol once per day for 6 days. In vivo structure and function were assessed before and after exposure. Binge alcohol exposure in adolescence significantly impaired normal cardiac growth but did not affect whole‐body growth during adolescence, therefore this pathology was specific to the heart. Binge rats also exhibited signs of accelerated pathological growth (concentric cellular hypertrophy and thickening of the myocardial wall), suggesting a global reorientation from physiologic to pathologic growth. Binge rats compensated for their smaller filling volumes by increasing systolic function and sympathetic stimulation. Consequently, binge alcohol exposure increased PKA (protein kinase A) phosphorylation of troponin I, inducing myofilament calcium desensitization. Binge alcohol also impaired in vivo relaxation and increased titin‐based cellular stiffness due to titin phosphorylation by PKCα (protein kinase C α). Mechanistically, alcohol inhibited extracellular signal‐related kinase activity, a nodal signaling kinase activating physiology hypertrophy. Thus, binge alcohol exposure depressed genes involved in growth. These cardiac structural alterations from binge alcohol exposure persisted through adolescence even after cessation of ethanol exposure. Conclusions Alcohol negatively impacts function in the adult heart, but the adolescent heart is substantially more sensitive to its effects. This difference is likely because adolescent binge alcohol impedes the normal rapid physiological growth and reorients it towards pathological hypertrophy. Many adolescents regularly binge alcohol, and here we report a novel pathological consequence as well as mechanisms involved.

Background: HIF pathway is quickly activated during myocardial ischemia after myocardial infarction(MI), and cardiac microvascular leakage contributes to heart tissue damage. HIF2α isprofoundly expressed in cardiac endothelial cells (ECs) and the embryonic deletion of HIF2Aresults in increased vascular permeability and aberrant ECs behavior. However, the direct roleof endothelial cell-specific HIF2α (ecHIF2α) in ischemic heart disease is not known. Wehypothesized that ecHIF2α expression in response to myocardial infarction (MI) is protectiveagainst heart failure through the reduction of cardiac ECs apoptosis and inflammation. Methods and Results: To address our hypothesis, we generated EC-specific inducible-HIF2α knockout mice (ecHIF2α -/- ) by crossing Hif2a flox/flox mice with Cre ERT2 mice. To assess the functional role of HIF2α inischemic heart injury, we ligated the proximal left anterior descending coronary artery to induceMI using the same age and gender-matched ecHIF2α -/- and control ( Hif2a flox/flox ) mice. Cardiacfunction was determined by echocardiography after two and four weeks of ligation. Analysis ofechocardiography revealed worsened heart function, and Masson’s Trichrome stain displayedincreased fibrosis in ecHIF2α -/- mice. In vitro , ECIS analysis of isolated cardiac microvascularendothelial cells showed decreased endothelial barrier function in ecHIF2α -/- cells. In addition,hypoxic stimulation reduces the tube formation capacity in ecHIF2α-/- cells and is sensitive tohypoxia-induced early-stage apoptosis. Deletion of HIF2α, as well as its binding partner ARNT,increased the expression of several inflammatory genes, including IL-6. Interestingly,overexpression of ARNT alone abolishes the HIF2α deletion-induced inflammatory geneexpression. IL-6 protein levels in HIF2α deleted human aortic endothelial cells (HAoEC) show asignificant reduction (n=3-5, p<0.001) in ARNT overexpressed ECs. Conclusion: Collectively our data revealed an essential role of endothelial HIF2α/ARNT in maintaining cardiacfunctions by increasing endothelial barrier function and decreasing inflammation. Therefore,HIF2A/ARNT could provide a potential therapeutic target for the treatment of ischemic heartdisease.

Introduction: Impaired endothelial function leads to the progression of heart failure after Ischemia-reperfusion (IR). Kinin activation of bradykinin receptor 1 (B1R), a G protein-coupled receptor that has been found to induce capillary leakage, may serve as a critical mediator in cardiac microvascular barrier dysfunction. However, the underlying mechanisms are not clear. We found that B1R inhibition abolished IR-induced endothelial matrix metalloprotease (MMP3) expression and improved endothelial barrier formation. Thus, we hypothesized that B1R antagonist protects against cardiac IR injury through an MMP3 pathway. Methods and Results: MMP3-/- mice and their littermate controls (WT) were subjected to either cardiac IR or sham control. The baseline characteristics of these mice showed minimal phenotypes. Cardiac function was determined at 3, 7 and 24 days post-IR by echocardiography. The MMP3-/- mice displayed improved cardiac function compared to the control mice, as determined by fractional shortening (26% ± 1.1 MMP3-/- vs. 21% ± 0.9 WT, p<0.05, n=5) and ejection fraction (48% ± 1.9 MMP3-/- vs. 42% ± 2.8.1 WT, p<0.05, n=5), and treating with B1R antagonist (300 μg/Kg) significantly reduced serum MMP3 levels (p<0.01). Compared to the control mice, MMP3-/- mice had significantly less infarction/area at risk 24 hours post-IR demonstrated through TTC staining. In vitro studies revealed that cellular hypoxia-reoxygenation (HR) injury significantly increased both B1R and MMP3 expression levels in primary isolated cardiac mice microvascular endothelial cells (mCMVEC). MMP3 levels were measured via ELISA. Moreover, B1R agonist treatment (1uM) increased MMP3 levels, while the use of the antagonist attenuated the increase of MMP3 in response to HR. Finally, the use of B1R antagonist improved MMP3 induced endothelial barrier dysfunction, which was measured by the electric cell-substrate impedance sensing (ECIS) system. Taken together, the results demonstrated that B1R antagonist abolished IR induced MMP3 induction and that the deletion of MMP3 is protective of cardiac function upon IR injury. Conclusions: MMP3 is a critical regulator of cardiac microvascular barrier function, and B1R/MMP3 could potentially serve as a novel therapeutic target for heart failure in response to IR injury.

Rationale: Cardiac microvascular leakage and inflammation are triggered during myocardial infarction (MI) and contribute to heart failure. Hypoxia-inducible factor 2α (Hif2α) is highly expressed in endothelial cells (ECs) and rapidly activated by myocardial ischemia, but whether it has a role in endothelial barrier function during MI is unclear. Objective: To test our hypothesis that the expression of Hif2α and its binding partner aryl hydrocarbon nuclear translocator (ARNT) in ECs regulate cardiac microvascular permeability in infarcted hearts. Methods and Results: Experiments were conducted with mice carrying an inducible EC-specific Hif2α-knockout (ecHif2α-/-) mutation, with mouse cardiac microvascular endothelial cells (CMVECs) isolated from the hearts of ecHif2α-/- mice after the mutation was induced, and with human CMVECs and umbilical-vein endothelial cells transfected with ecHif2α siRNA. After MI induction, echocardiographic assessments of cardiac function were significantly lower, while measures of cardiac microvascular leakage (Evans blue assay), plasma IL6 levels, and cardiac neutrophil accumulation and fibrosis (histology) were significantly greater, in ecHif2α-/- mice than in control mice, and RNA-sequencing analysis of heart tissues from both groups indicated that the expression of genes involved in vascular permeability and collagen synthesis was enriched in ecHif2α-/- hearts. In cultured ECs, ecHif2α deficiency was associated with declines in endothelial barrier function (electrical cell impedance assay) and the reduced abundance of tight-junction proteins, as well as an increase in the expression of inflammatory markers, all of which were largely reversed by the overexpression of ARNT. We also found that ARNT, but not Hif2α, binds directly to the IL6 promoter and suppresses IL6 expression. Conclusions: EC-specific deficiencies in Hif2α expression significantly increase cardiac microvascular permeability, promote inflammation, and reduce cardiac function in infarcted mouse hearts, and ARNT overexpression can reverse the upregulation of inflammatory genes and restore endothelial-barrier function in Hif2α-deficient ECs.