Development Of Diabetic Macrovascular Complications Research Articles

462-P: DPP-4 Deficiency Suppresses the Progression of Atherosclerosis via Inducing M2 Macrophage Polarization

Background: Dipeptidyl peptidase-4 (DPP4) inhibitors suppressed progression of atherosclerosis in atherosclerotic mouse models. However, the role of DPP4 on the progression of atherosclerosis is not clear. We investigated the effect of DPP4 deficiency on the progression of atherosclerosis in DPP4 and apolipoprotein-E double knockout (DPP4/ApoE-DKO) mice. Methods: Both ApoE-KO mice and DPP4/ApoE-DKO mice were fed with normal chow until 20 weeks of age. Whole aorta and aortic sinus were stained with Oil red O. Bone marrow-derived macrophages (mφ) from wildtype or DPP4-KO mice were used in in vitro study. Expression of iNOS, MCP-1, TNF-α and IL-1β (M1 markers), and arginase-1, Mgl2 and Ym1 (M2 markers) mRNA were evaluated by real-time RT-PCR, and mφ polarization was evaluated by flowcytometry. Results: There were no differences on food intake, body weight, lipid profiles and the blood glucose levels by insulin tolerance test between each group, but the blood glucose levels by glucose tolerance test were significantly decreased in DPP4/ApoE-DKO mice. Oil-Red-O staining demonstrated that the size of atherosclerotic lesions was significantly smaller in DPP4/ApoE-DKO mice. In in vitro assay, LPS+IFNγ induced the mRNA expression of M1 markers and there was no difference between wildtype mφ and DPP4-KO mφ. On the other hand, the mRNA expression of M2 markers were significantly increased in DPP4-KO mφ. Flowcytometry revealed that the number of iNOS (M1 marker)-positive cells were increased by LPS+IFNγ, which was comparable between wildtype mφ and DPP4-KO mφ. On the other hand, the number of CD206 (M2 marker)-positive cells was higher in DPP4-KO mφ than that in wildtype mφ. Conclusion: This study suggests that DPP4 deficiency suppresses the progression of atherosclerosis in apoE-KO mice. Moreover, DPP4 deficiency increases the polarization of M2 mφ. These findings may indicate the involvement of DPP4 at least in part on the development of diabetic macrovascular complications. Disclosure Y.Zhou: None. T.Matsumura: Research Support; Shimadzu Corporation, Speaker's Bureau; Eli Lilly Japan K.K., Boehringer Ingelheim Japan, Inc. Y.Yagi: None. T.Yoshinaga: None. T.Senokuchi: None. N.Ishii: None. T.Kondo: None. E.Araki: Research Support; Eli Lilly Japan K.K., Nippon Boehringer Ingelheim, Mitsubishi Tanabe Pharma Corporation, Speaker's Bureau; AstraZeneca, Merck & Co., Inc., Ono Pharmaceutical Co., Ltd., Kowa Company, Ltd., Sumitomo Pharma Co., Ltd., Daiichi Sankyo, Novo Nordisk Pharma Ltd.

Associations of Thyroid Hormone Levels and Macrovascular Complications in Euthyroid Type 2 Diabetic Patients.

PurposeThe purpose of this study is to evaluate whether thyroid hormone in euthyroid patients with type 2 diabetes mellitus (T2DM) is associated with macrovascular complications.Patients and MethodsThe authors examined 311 patients enrolled from February 2019 to December 2019 in Tianjin Medical University Chu Hsien-I Memorial Hospital. A medical record review enabled the collection of demographic and anthropometric information. We classified the patients into two groups based on the echocardiography and vascular ultrasonography results, namely, non-macrovascular complications (n=131) group and macrovascular complications (n=180) group. Odds ratios (OR) and 95% confidence intervals (CI) were calculated, adjusting for potential confounders, the prevalence of macrovascular complications was determined using multivariate logistic regression.ResultsA significant association was observed for diabetic macrovascular complications with normal free triiodothyronine (FT3) (OR=0.534, 95% CI 0.358–0.796, p = 0.002) and free thyroxine (FT4) (OR= 0.844, 95% CI 0.760–0.937, p = 0.001). Nevertheless, there was no evidence of any association between thyroid-stimulating hormone (TSH) and the development of diabetic macrovascular complications. When stratified by the body mass index (BMI), a similar relationship existed with the overall results. The positive association remained in restricted analyses involving only patients with HbA1c abnormalities.ConclusionOverweight or obese T2DM patients are at high risk due to the implicit association between low but clinically normal thyroid hormone levels and elevated risk of macrovascular complications. However, there were no statistically significant associations between TSH and diabetic macrovascular complications.

High glucose upregulates osteopontin expression by FoxO1 activation in macrophages.

Hyperglycemia plays a major role in the development of diabetic macrovascular complications, including atherosclerosis and restenosis, which are responsible for the most of disability and mortality in diabetic patients. Osteopontin (OPN) is an important factor involved in atherogenesis, and hyperglycemia enhances the transcriptional activity of FoxO1 which is closely association with insulin resistance and diabetes. Here, we showed that plasma OPN levels were significantly elevated in type 2 diabetic patients and positively correlated with glycated albumin (GA). The more atherosclerotic lesions were observed in the aorta of diabetic ApoE-/- mice analyzed by Sudan IV staining. High glucose increased both the mRNA and protein expression levels of OPN and inhibited the phosphorylation of FoxO1 in RAW 264.7 cells. Overexpression of WT or constitutively active mutant FoxO1 promoted the expression levels of OPN, while the dominant-negative mutant FoxO1 decreased slightly the expression of OPN. Conversely, knockdown of FoxO1 reduced the expression of OPN. Luciferase reporter assay revealed that high glucose and overexpression of FoxO1 enhanced the activities of the OPN promoter region nt -1918 ~ -713. Furthermore, the interactions between FoxO1 and the OPN promoter were confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results suggest that high glucose upregulates OPN expression via FoxO1 activation, which would play a critical role in the development of diabetic atherogenesis.

Sodium butyrate attenuates diabetes-induced aortic endothelial dysfunction via P300-mediated transcriptional activation of Nrf2

Oxidative stress and inflammation are major contributors to diabetes-induced endothelial dysfunction which is the critical first step to the development of diabetic macrovascular complications. Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in combating diabetes-induced oxidative stress and inflammation. Sodium butyrate (NaB) is an inhibitor of histone deacetylase (HDAC) and an activator of NRF2. However, NaB's effect on diabetes-induced aortic injury was unknown. It was also not known whether or to what extent NRF2 is required for both self-defense and NaB's protection in the diabetic aorta. Additionally, the mechanism by which NaB activates NRF2 was unclear. Therefore, C57BL/6 Nrf2 knockout (KO) and wild type (WT) mice were induced to diabetes by streptozotocin, and were treated in the presence or absence of NaB, for 20 weeks. The KO diabetic mice developed more severe aortic endothelial oxidative stress, inflammation and dysfunction, as compared with the WT diabetic mice. NaB significantly attenuated these effects in the WT, but not the KO, mice. In high glucose-treated aortic endothelial cells, NaB elevated Nrf2 mRNA and protein without facilitating NRF2 nuclear translocation, an effect distinct from that of sulforaphane. NaB inhibited HDAC activity, and increased occupancy of the transcription factor aryl hydrocarbon receptor and the co-activator P300 at the Nrf2 gene promoter. Further, the P300 inhibitor C646 completely abolished NaB's efficacies. Thus, NRF2 is required for both self-defense and NaB's protection against diabetes-induced aortic endothelial dysfunction. Other findings suggest that P300 mediates the transcriptional activation of Nrf2 by NaB.

Melatonin Attenuates Aortic Endothelial Permeability and Arteriosclerosis in Streptozotocin-Induced Diabetic Rats: Possible Role of MLCK- and MLCP-Dependent MLC Phosphorylation.

The development of diabetic macrovascular complications is a multifactorial process, and melatonin may possess cardiovascular protective properties. This study was designed to evaluate whether melatonin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light-chain kinase (MLCK)/myosin light-chain phosphorylation (p-MLC) system via the mitogen-activated protein kinase (MAPK) signaling pathway or by suppressing the myosin phosphatase-targeting subunit phosphorylation (p-MYPT)/p-MLC system in diabetes mellitus (DM). Rats were randomly divided into 4 groups, including control, high-fat diet, DM, and DM + melatonin groups. Melatonin was administered (10 mg/kg/d) by gavage for 12 weeks. The DM significantly increased the serum fasting blood glucose and lipid levels, as well as insulin resistance and endothelial dysfunction, which were attenuated by melatonin therapy to various extents. Importantly, the aortic endothelial permeability was significantly increased in DM rats but was dramatically reversed following treatment with melatonin. Our findings further indicated that hyperglycemia and hyperlipidemia enhanced the expressions of MLCK, p-MYPT, and p-MLC, which were partly associated with decreased membrane type 1 expression, increased extracellular signal-regulated kinase (ERK) phosphorylation, and increased p38 expression. However, these changes in protein expression were also significantly reversed by melatonin. Thus, our results are the first to demonstrate that the endothelial hyperpermeability induced by DM is associated with increased expressions of MLCK, p-MYPT, and p-MLC, which can be attenuated by melatonin at least partly through the ERK/p38 signaling pathway.

Metabolic Signatures of Oxidative Stress in the Red Blood Cells: Editorial Commentary.

This issue of the journal contains an interesting article—“Metabolic Signatures of Oxidative Stress and their Relationship with Erythrocyte Membrane Surface Roughness among Workers of Manual Material Handling (MMH)”. Oxidative stress has been a longtime research interest that is yet to be assessed by a clinical laboratory for evidence-based practice, because it is perceived to be implicated in virtually every disease or condition. The report surely has backing from the literature. For instance, oxidative stress has known metabolic signatures,[1,2] which is hallmarked by glutathione (GSH), as well as reactive oxygen species.[3] The concept of erythrocyte oxidative stress (EOS) further denotes that the biomarkers vis-a-vis metabolic signatures are detectable in red blood cells. Indeed, cardiovascular complications in diabetes represent a major health problem worldwide, but knowledge of progression and degree of risk in prediabetes is limited. Significant changes in biomarkers of EOS and indicators of related macrovascular events that may underlie the development of diabetic macrovascular complications, have been consistently reported. The biomarkers are speculated to be emerging risk indicators, namely, (1) indices of EOS, including erythrocyte-reduced GSH, malondialdehyde, and associated enzymes; (2) indices of vasculopathy, including plasma D-dimer, homocysteine, and whole blood viscosity; and (3) indices of dyslipidemia, namely, total cholesterol (TC), high-density lipoprotein (HDL), and TC/HDL ratio. Thus, there has been the recognition of a spectrum of metabolic profile[3] that should be narrowed down to a useful panel of tests to improve early identification and intervention.[4] The present article highlights the significant impact of EOS on the surface of the blood cell membranes. While this effect may be known,[5] it is probably yet to be appreciated that apparently healthy individuals may suffer oxidative damage as occupational hazard. The authors have attempted to develop a linear model to demonstrate how individual oxidative stress marker is related to surface roughness of the erythrocyte membrane. It will be interesting to see corroborative reports that will exemplify and validate the speculated model. Further, the authors have, albeit passively, also mentioned a phenomenon that the carriage of oxygen inside the blood and/or body must be assessed by means of biochemical parameters and the nanoscale levels of changes in the surface roughness of the erythrocyte membrane. This may follow the concept of EOS being associated with hypoxia — e.g., oxidative stress can induce hyperviscosity that in turn leads to the sequence of reduced blood flow, less oxygen supply, and tissue hypoxia. The phenomenon may follow the concept of oxidative stress-inducing anemia or exacerbating iron-deficiency anemia as well. It would be interesting to see corroborative reports that will expatiate on this phenomenon.

The effects of gliclazide and other sulfonylureas on low-density lipoprotein oxidation in vitro

Diabetes is associated with increased oxidant stress. This may contribute to the development of diabetic macrovascular complications through increased oxidation of low-density lipoprotein (LDL), which is thought to be a crucial step in the development of atherosclerosis. The sulfonylurea gliclazide has been shown to have free radical—scavenging activity in vitro, but its effects on LDL oxidation, and these effects of other sulfonylureas, are unknown. To investigate this, we studied the effects of in vitro supplementation with gliclazide 1 μmol/L on copper-induced oxidation of LDL isolated from 20 control subjects and 22 type II diabetic patients. The effects of 1 μmol/L vitamin C, a known water-soluble antioxidant, were studied simultaneously. The resistance to oxidation, expressed as the lag time between the addition of copper and commencement of oxidation, was significantly increased by both gliclazide and vitamin C, and the effect was similar for LDL from diabetic and control subjects. The baseline oxidation lag time was 63.4 ± 2.1 minutes, and increased to 108 ± 4.4 minutes with gliclazide and 88.7 ± 5.6 minutes with vitamin C ( P = .0001, baseline v either treatment). The increase in lag time with gliclazide of 70% ± 3% was greater than the 30% ± 5% increase with vitamin C ( P < .0005). In a separate experiment, LDL isolated from eight control and 10 diabetic subjects was supplemented with 1 μmol/L gliclazide, glibenclamide, glipizide, and tolbutamide. For each LDL sample, all drugs were studied simultaneously and the oxidation lag time was compared against that of untreated LDL. Gliclazide increased the lag time from 53.7 ± 2.4 minutes to 108.4 ± 4.5 minutes ( P = .0001). None of the other sulfonylureas had any effect on lag time. These findings demonstrate that gliclazide is an effective inhibitor of in vitro LDL oxidation, and in this respect, it is more potent on a molar basis than vitamin C. This antioxidant property of gliclazide was not shared by the other sulfonylureas studied.