Abstract only
Introduction:
Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a ...master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood
Hypothesis:
Aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes
Methods and Results:
We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation (ArntΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia (n=8-12, p<0.01 vs Control group), delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow (n=10, P<0.01vs Control) In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in 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 MVECs isolated from mouse skeletal muscle, and the viability of ArntΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor.
Conclusions:
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. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.
Abstract only 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.
Objective: Sepsis is a complex disease characterized by an inflammatory response and tissue hypoxia. Hypoxia-inducible factor 1α (HIF-1α) expression level is regulated by hypoxia and inflammation. ...This study aimed to explore the correlation between HIF-1α expression level and sepsis by bioinformatics analysis and clinical investigation. Methods: Bioinformatics tools were used to identify differentially expressed genes between sepsis and nonsepsis groups using the Gene Expression Omnibus data set. A clinical investigation was carried out to validate HIF-1α protein level in 54 nonseptic patients and 173 septic patients who were followed up for 28 days. Results: Bioinformatics analysis revealed that HIF-1α messenger RNA level was significantly different between septic and nonseptic patients ( P < 0.05). Consistent with the study hypothesis, higher HIF-1α levels in plasma were found in septic patients compared with those in nonseptic patients. The diagnostic accuracy for sepsis, as quantified by the area under the curve, was 0.926 (0.885-0.968) for HIF-1α expression level combined with oxygen saturation to fraction of inspired oxygen (SpO 2 /FiO 2 ), white blood cell, and blood urea nitrogen. The HIF-1α expression level was also significantly correlated with the severity of the disease. The results of the restricted cubic splines model indicated a U-shaped relationship between HIF-1α expression level and intensive care unit (ICU) mortality. Univariate and multivariate linear regression analyses indicated that septic patients with the elevated HIF-1α expression levels had shorter length of ICU stay versus those with the lower HIF-1α expression levels. Conclusion: Hypoxia-inducible factor 1α expression level can be used for diagnosing disease, assessing severity, and predicting length of ICU stay in septic patients.
Abstract only Introduction: The blood-brain barrier (BBB) normally maintains the CNS microenvironment. The effect of isoflurane, widely used in clinical anesthesia, on the BBB permeability has not ...been well studied. Matrix metalloproteinase 3 (MMP3) is implicated in the disruption of BBB. However, the direct role for MMP3 in regulating BBB and the underlying molecular mechanism has not yet been elucidated. We hypothesized that MMP3 plays a critical role in isoflurane mediated increase in BBB permeability. Methods: We used MMP3 deficient (MMP3 -/- ) mice and its littermate, wild-type (MMP3 +/+ ), as a control. Animals were assigned to either vehicle-air or isoflurane in 3 different groups: control, MMP3 -/- and control+MMP3 administration. We evaluated the anesthetic effect of isoflurane by the time of anesthesia induction, emergency time and volume of isoflurane. MMP3 levels were measured in different organs. We monitored the permeability of mouse BBB through in vivo (Evans blue dye extravasation, Sodium-FITC, and brain water content) and an in vitro model of BBB (Electrical cell impedance sensor assay and transwell permeability assay). To determine the mechanism of MMP3 on BBB permeability, we also used an ERK-inhibitor. Expression of the endothelial tight junctional protein was detected by both western blot and immunofluorescence staining. Results: The use of MMP3 increased the anesthetic sensitivity of isoflurane compared with the control group. In contrast, MMP3 KO mice displayed significantly longer induction time, higher isoflurane usage volume, and lower emergency time. Data showed that MMP3 increased BBB permeability in both in vivo and in vitro experiments, which is associated with a reduction in occluding, ZO-1 and claudin-5. We found mmp3 levels had negative correlations with tight junctional proteins and positive associations with p-ERK. Moreover, the use of an ERK-inhibitor abolished the effect of MMP3 on the junctional protein and in vitro BBB permeability. Conclusion: Our data suggest that MMP3 is required for modulation of isoflurane mediated BBB permeability by the reduction of tight junctional protein expression via the ERK pathway. Thus, mmp3 enhances the anesthetic effect of isoflurane, while the deletion of MMP3 protects against BBB disruption.
Abstract only Introduction: Iron is essential for the activity of several cellular proteins, but excess free iron can cause cellular damage through production of reactive oxygen species (ROS). Iron ...accumulation in mitochondria, the major site of cellular iron homeostasis, leads to cardiomyopathy. However, it is not known whether a reduction in baseline mitochondrial iron (as opposed to iron in other cellular compartments) can protect against ischemia-reperfusion (I/R) injury in the heart. We hypothesized that since mitochondria are the major site of iron homeostasis and that mitochondrial iron can lead to oxidative damage, a reduction in mitochondrial iron at baseline would be sufficient to protect against I/R injury. Results: Transgenic (TG) mice with cardiomyocyte-specific overexpression of the mitochondrial iron export protein ATP-binding cassette (ABC)-B8 had significantly lower mitochondrial iron in the heart than nontransgenic (NTG) littermates at baseline, but their cardiac function and the expression of key antioxidant systems were similar to NTG littermates. In response to I/R, TG mice displayed significantly less apoptosis and lipid peroxidation products and better preserved cardiac function than NTG littermates, suggesting that a reduction in mitochondrial iron protects against I/R injury. To confirm these results, we next took a pharmacological approach to assess the effects of a reduction in mitochondrial vs cytosolic iron on the response to I/R using 2,2’-bipyridyl (BPD, a mitochondria-accessible iron chelator) and deferoxamine (DFO, an iron chelator that can only reduce cytosolic iron). Mice pretreated with BPD but not DFO are protected against I/R injury. In addition, BPD but not DFO treatment in rat cardiomyoblast H9C2 cells significantly lowered chelatable mitochondrial iron and protected against H2O2 induced cell death. These results suggest that a reduction in baseline mitochondrial, but not cytosolic, iron is sufficient to protect against I/R injury. Conclusions: Our findings demonstrate that selective reduction in mitochondrial iron is protective in I/R injury. Thus, targeting mitochondrial iron with selective iron chelators may provide a novel approach for treatment of ischemic heart disease.
Abstract only 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 its impact in microvascular endothelial barrier function during MI is unclear. Objective: To test our hypothesis that the expression of Hif2α in ECs regulates cardiac microvascular permeability in infarcted hearts, which is through its binding partner aryl hydrocarbon nuclear translocator. (ARNT). 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, ec Hif2α 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: Endothelial HIF-2a protects from hypoxia-induced cardiac microvascular barrier dysfunction, promotes inflammation damage and represents a potential therapeutic target for cardioprotection, and prevention of fibrosis following acute ischemic injury.
Abstract only Introduction: Hyperpermeability of the microvascular barrier and the resulting capillary leakage are hallmarks of sepsis. B1R has been proposed to be a therapeutic target for ...sepsis-induced microvascular hyperpermeability and multiorgan failure. However, its direct role in regulating of cardiac function during severe sepsis remains poorly characterized. Hypothesis: We hypothesized that B1R inhibition can protect against sepsis-induced cardiac dysfunction and improve survival. Methods and Results: To address the hypothesis, we pre-treated mice with either vehicle (control) or a specific B1R antagonist B6929via subcutaneous injection then subjected them to 40% cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Serum levels of IL-6 and TNFα increased significantly within 4 hours post-CLP in the controls. However, B1R antagonist treatment abolished CLP-induced expressions of IL-6 and TNFα by 70% and 30%, respectively. B1R antagonist also protected mice from sepsis-induced organ failure—as highlighted by the lowered increase in MSS score over the 24-h post-CLP—resulting in significant improvement in survival rate (P<0.05). In-vivo cardiac vascular permeability assessed using the Miles assay further demonstrated that B1R inhibition prevented sepsis-induced cardiac microvascular leakage and edema. Similar improvements were also observed in the liver and kidney. Further study revealed an early cardioprotective effect of B1R inhibitor, as evidenced by the improvement of EF%, FS%, and cardiac output in mice treated with B1R antagonist + CLP vs. CLP alone (p<0.01) from the echocardiogram analyses at 4-6 hours following CLP. Furthermore, mitochondrial H 2 O 2 measurement of the freshly isolated cardiac mitochondria from septic mice also showed that B1R inhibition partially abolished the production of H 2 O 2 . The opening of mPTP was also reduced with B1Ri treatment. Taken together, these findings indicated improvement in cardiac mitochondrial function following B1R inhibitor treatment. Conclusions: Our findings provide novel insights into the pathogenesis of sepsis-induced cardiac mitochondrial dysfunction and heart failure. Inhibition of B1R may be a novel therapeutic strategy for sepsis-associated cardiovascular disease.
Abstract only 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.
Abstract only Background: Myocardial ischemia occurs during myocardial infarction results in the stabilization of Hypoxia-inducible factors (HIFs). Hif2α expresses profoundly in vascular endothelial ...cells (EC), and its embryonic deletion increases vessel permeability. It has been shown that HIF2a is protective from renal and pulmonary injury. However, the direct role of ecHIF2α in ischemia heart disease is unknown. We hypothesized that ecHIF2a expression in response to myocardial infarction (MI) protects cardiac barrier dysfunction and against heart failure. Methods and Results: We generated the Inducible endothelial-specific knockout mice (ecHIF2a -/- ) by crossing Hif2a flox/flox mice with Cre ERT2 mice under the VE-cadherin promoter. Followed with MI, ecHIF2a -/- mice displayed worsened cardiac function determined by echocardiography, and they had increased mortality as compared to the controls. In vitro, we used primary mouse cardiac microvascular endothelial cells (mCMVEC) from ecHIF2a -/- mouse hearts. We found that under hypoxia condition or 1mM Dimethyloxalylglycine treatment, the deficiency of HIF2a in the mCMVEC increased endothelial permeability determined by trans-endothelial electrical resistance. The knocking down of HIF2a in HUVECs induced by a HIF2a siRNA led to impaired tube formation accessed by the significant reduction in total node counts, junctions, meshes, and full tube length compared to control-siRNA treated cells. HIF2α deletion and hypoxia both reduced endothelial cell migration, and interestingly, the retarded HIF2α-/- ECs migration seems to be independent of hypoxia. Moreover, apoptosis assay showed that ecHIF2a -/- ECs increased cell early apoptotic stage compared to WT in hypoxic conditions, but not in normoxia indicating the critical role of HIF2α in ECs survival during cardiac ischemia. Finally, several increased markers of inflammation, such as ICAM-1 and VCAM-1, are associated with HIF2a deletion. Conclusion: These data revealed an essential role of HIF2α in protecting cardiac remodeling in response to MI, which might through promoting endothelial cell migration, barrier function, as well as vascularization. Thus, HIF2α is a potential therapeutic target in the treatment of ischemic heart disease.
Abstract only
Introduction:
Iron is essential for the activity of several cellular proteins, but excess free iron can cause cellular damage through production of reactive oxygen species (ROS). Iron ...accumulation in mitochondria, the major site of cellular iron homeostasis, leads to cardiomyopathy. However, it is not known whether a reduction in baseline mitochondrial (as opposed to cytosolic) iron can protect against ischemia-reperfusion (I/R) injury in the heart. We hypothesized that since mitochondria are the major site of iron homeostasis and that mitochondrial iron can lead to oxidative damage, a reduction in mitochondrial iron at baseline would be sufficient to protect against I/R injury.
Results:
Transgenic (TG) mice with cardiomyocyte-specific overexpression of the mitochondrial iron export protein ATP-binding cassette (ABC)-B8 had significantly lower mitochondrial iron in the heart than nontransgenic (NTG) littermates at baseline, but their cardiac function and the expression of key antioxidant systems were similar to NTG littermates. In response to I/R, TG mice displayed significantly less apoptosis and lipid peroxidation products and better preserved cardiac function than NTG littermates, suggesting that a reduction in mitochondrial iron protects against I/R injury. To confirm these results, we next took a pharmacological approach to assess the effects of a reduction in mitochondrial vs cytosolic iron on the response to I/R using 2,2’-bipyridyl (BPD, a mitochondria-accessible iron chelator) and deferoxamine (DFO, an iron chelator that can only reduce cytosolic iron). Treating rat cardiomyoblast H9C2 cells with BPD but not DFO significantly lowered chelatable mitochondrial iron and protected against H
2
O
2
induced cell death, and pretreatment with BPD but not DFO protected mice against I/R injury and reduced ROS production, suggesting that a reduction in baseline mitochondrial, but not cytosolic, iron is sufficient to protect against I/R injury.
Conclusions:
Our findings demonstrate that selective reduction in mitochondrial iron is protective in I/R injury. Thus, targeting mitochondrial iron with selective iron chelators may provide a novel approach for treatment of ischemic heart disease.
