Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR , Cx40 , and Nox4 levels were decreased in CECs from diabetic and HuR -KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40 -KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.

Background Abnormal endothelial function in the lungs is implicated in the development of pulmonary hypertension; however, there is little information about the difference of endothelial function between small distal pulmonary artery (PA) and large proximal PA and their contribution to the development of pulmonary hypertension. Herein, we investigate endothelium‐dependent relaxation in different orders of PAs and examine the molecular mechanisms by which chronic hypoxia attenuates endothelium‐dependent pulmonary vasodilation, leading to pulmonary hypertension. Methods and Results Endothelium‐dependent relaxation in large proximal PAs (second order) was primarily caused by releasing NO from the endothelium, whereas endothelium‐dependent hyperpolarization (EDH)–mediated vasodilation was prominent in small distal PAs (fourth–fifth order). Chronic hypoxia abolished EDH‐mediated relaxation in small distal PAs without affecting smooth muscle–dependent relaxation. RNA‐sequencing data revealed that, among genes related to EDH, the levels of Cx37 , Cx40 , Cx43 , and IK were altered in mouse pulmonary endothelial cells isolated from chronically hypoxic mice in comparison to mouse pulmonary endothelial cells from normoxic control mice. The protein levels were significantly lower for connexin 40 (Cx40) and higher for connexin 37 in mouse pulmonary endothelial cells from hypoxic mice than normoxic mice. Cx40 knockout mice exhibited significant attenuation of EDH‐mediated relaxation and marked increase in right ventricular systolic pressure. Interestingly, chronic hypoxia led to a further increase in right ventricular systolic pressure in Cx40 knockout mice without altering EDH‐mediated relaxation. Furthermore, overexpression of Cx40 significantly decreased right ventricular systolic pressure in chronically hypoxic mice. Conclusions These data suggest that chronic hypoxia‐induced downregulation of endothelial Cx40 results in impaired EDH‐mediated relaxation in small distal PAs and contributes to the development of pulmonary hypertension.

No abstract

Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

Recent reports demonstrate that diabetic patients with coronary microvascular disease (CMD, also known as non‐obstructive coronary artery disease) exhibit higher cardiac mortality than diabetic patients without CMD. However, the molecular mechanisms in which diabetes leads to CMD are not well understood. In this study, we examine the role of gap junction intercellular communication in the development of CMD in diabetes. We used type 2 diabetic (T2D) mice induced by a single injection of low‐dose streptozotocin (75 mg/kg) with a high‐fat diet (60 % kcal). Coronary flow velocity reserve (CFVR) was measured in mice to assess coronary microvascular function and T2D mice showed significant decrease in CFVR compared to control mice, suggesting that T2D mice are suffering from CMD. Mouse coronary endothelial cells (MCECs) isolated from T2D mice and human coronary endothelial cells (HCECs) treated with high‐glucose exhibited the reduction of gap junction activity compared to their controls. Gap junction is composed of connexins (Cxs) and coronary endothelial cells express Cx37, Cx40, Cx43, and Cx45. Among those Cxs, Cx40 mRNA level was significantly decreased in MCECs isolated from T2D mice. Furthermore, Cx40 protein levels were significantly decreased in MCECs from inducible T2D mice, MCECs from spontaneous T2D mice (Tallyho mice), and HCECs from T2D patients compared to those controls. Cx40 knockout mice exhibited lower CFVR which was accompanied by decreased capillary density in the left ventricle (LV), when compared to the wild‐type mice. The potential causes of decreased capillary density include attenuated endothelial migration/proliferation and increased endothelial apoptosis. In ex vivo, high‐glucose treatment impaired capillary network formation in HCECs and Cx40 overexpression restored the level of capillary network in high‐glucose treated HCECs. We also found that Cx40 overexpression in T2D mice increased capillary density in the LV and CFVR. These data suggest that the downregulation of Cx40 decreases capillary density in the heart and leads to CMD in diabetes. Therefore, Cx40 overexpression in ECs could be the potential therapy for CMD in diabetic patients. Support or Funding Information This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL142214 and HL146764 to A. Makino).