Diabetes mellitus is currently a major public health problem. A common complication of diabetes is cardiac dysfunction, which is recognized as a microvascular disease that leads to morbidity and mortality in diabetic patients. While ischemic events are commonly observed in diabetic patients, the risk for developing heart failure is also increased, independent of the severity of coronary artery disease and hypertension. This diabetes-associated clinical entity is considered a distinct disease process referred to as "diabetic cardiomyopathy". However, it is not clear how diabetes promotes cardiac dysfunction. Vascular endothelial dysfunction is thought to be one of the key risk factors. The impact of diabetes on the endothelium involves several alterations, including hyperglycemia, fatty acid oxidation, reduced nitric oxide (NO), oxidative stress, inflammatory activation, and altered barrier function. The current review provides an update on mechanisms that specifically target endothelial dysfunction, which may lead to diabetic cardiomyopathy.

No abstract

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: Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy. Methods and Results: Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT -/- ) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT -/- mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway. Conclusion: Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.

Introduction: Cardiac microvascular hyperpermeability is a key contributor to heart disease in patients with diabetes. Although the link between diabetes and microvascular barrier dysfunction is largely unknown, expression of the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) is significantly downregulated in the cardiac microvascular endothelial cells (CMECs) of both diabetic mouse hearts (n=5, p<0.01) and the explanted hearts of patients with diabetes mellitus (n=3, p<0.05). We hypothesize that cardiac microvascular permeability is limited by the expression of ARNT in the endothelium and, consequently that endothelial-cell ARNT (ecARNT) expression is essential for normal cardiac function. Methods and Results: We have recently generated tamoxifen-inducible, endothelial-cell-specific, VE-cadherin-Cre ETR2 ARNT-knockout mice (ecARNT -/- mice). ecARNT deletion is achieved by tamoxifen oral administration for two weeks. Littermates controls are either mice without tamoxifen chow or ARNT flox/flox treated with tamoxifen diet. Induction of the ecARNT -/- mutation led to vascular leakage (as determined via in-vivo endothelial permeability assay) (n=5, p<0.05), which occurred predominantly in the heart, and to increases in matrix metalloproteinase (MMP) expression (microarray analysis), including a 3.8-fold increase in the expression of MMP3 (n=3, p<0.01). Furthermore, MMP3 inhibition attenuated the increase in cell permeability observed in CMECs from ecARNT -/- mouse hearts (as measured via electrical cell-substrate impedance sensing), as well as cardiac vascular leakage in ecARNT -/- mice, while long-term studies indicated that ejection fractions were significantly lower (ecARNT -/- : 22.3±2.6%, Control: 33.2±1.8%;), and Left ventricular end-diastolic diameters were significantly greater (ecARNT -/- : 4.8±0.8 mm, Control: 3.6±0.6 mm; n=8,p<0.01) six months after ecARNT deletion (echocardiography). Conclusion: ARNT-mediated MMP3 downregulation is required for maintaining cardiac microvascular barrier integrity and preserving cardiac function; thus, modulation of the ARNT/MMP3 axis could be a novel approach for the treatment of cardiovascular diseases such as diabetic cardiomyopathy.