To examine whether the long non‐coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is altered in the endothelial cells in response to glucose and the significance of such alteration. We incubated human umbilical vein endothelial cells with media containing various glucose levels. We found an increase in MALAT1 expression peaking after 12 hrs of incubation in high glucose. This increase was associated with parallel increase in serum amyloid antigen 3 (SAA3), an inflammatory ligand and target of MALAT1 and was further accompanied by increase in mRNAs and proteins of inflammatory mediators, tumour necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6). Renal tissue from the diabetic animals showed similar changes. Such cellular alterations were prevented following MALAT1 specific siRNA transfection. Results of this study indicate that LncRNA MALAT1 regulates glucose‐induced up‐regulation of inflammatory mediators IL‐6 and TNF‐α through activation of SAA3. Identification of such novel mechanism may lead to the development of RNA‐based therapeutics targeting MALAT1 for diabetes‐induced micro and macro vascular complications.

OBJECTIVE: Diabetic retinopathy (DR) is a leading cause of blindness. Increased vascular endothelial growth factor (VEGF), promoting angiogenesis and increased permeability, is a key mechanistic abnormality in DR. We investigated microRNA (miRNA) alterations in DR with specific focus on miR-200b, and its downstream target, VEGF. RESEARCH DESIGN AND METHODS: miRNA expression profiling microarray was used to examine the retinas of streptozotocin-induced diabetic rats. Expressions of specific miRNAs were verified with PCR in the rat retina and in glucose-exposed endothelial cells. A target search, based on sequence complementarities, identified specific targets. We analyzed mRNA levels and protein expression in endothelial cells from large vessels and retinal capillaries and in the rat retina, with or without injection of miR-200b mimic or antagomir. Localization of miR-200b and its functional analysis in the rat and human retinas were performed. RESULTS: Alteration of several miRNAs, including downregulation of miR-200b, were observed in the retina in diabetes. Such downregulation was validated in the retina of diabetic rats and in endothelial cells incubated in glucose. In parallel, VEGF (target of miR-200b) mRNA and protein were elevated. In the retina, miR-200b was localized in neuronal, glial, and vascular elements. Transfection of endothelial cells and intravitreal injection of miR-200b mimic prevented diabetes-induced increased VEGF mRNA and protein. Also prevented were glucose-induced increased permeability and angiogenesis. Furthermore, transfection of miR-200b antagonists (antagomir) led to increased VEGF production. Similar alterations were seen in the human retina. CONCLUSIONS: These studies show a novel mechanism involving miR-200b in DR. Identification of such mechanisms may lead to the development of novel miRNA-based therapy.

Oxidative stress is increased in the retina in diabetes, and long-term administration of antioxidants inhibits the development of retinopathy in diabetic rats. The purpose of this study is to determine how diabetes affects the activation of a redox-sensitive nuclear transcriptional factor in the retina, NF-κB, and its inhibition by antioxidants. Alloxan diabetic rats were assigned to receive standard diet or the diet supplemented with multiple antioxidants, including ascorbic acid, Trolox, dl α-tocopherol acetate, N-acetyl cysteine, β-carotene, and selenium for up to 14 months. NF-κB activation, oxidative stress and nitric oxides were measured in the retina at 2, 8 and 14 months of diabetes. Retinal NF-κB was activated by about 60% at two months after induction of diabetes, remained activated for up to 14 months of diabetes, and the duration of diabetes had no effect on the intensity of NF-κB activation. Similarly, oxidative stress and nitric oxides were elevated by over 50% in the retina of rats diabetic for 14 months, and nitrotyrosine levels were elevated by over two folds. Administration of the antioxidants to the rats for the entire duration of diabetes inhibited activation of NF-κB and elevations in oxidative stress, nitric oxides and nitrotyrosine formation without ameliorating the severity of hyperglycemia. These in vivo results were confirmed by in vitro studies showing that high glucose activates NF-κB and elevates NO and lipid peroxides in both retinal endothelial cells and pericytes that can be inhibited by antioxidants. Thus, the results suggest that the activation of retinal NF-κB in diabetes is an early event in the development of retinopathy, and it remains active when the retinal capillary cell death is accelerating, and histopathology is developing. Beneficial effects of antioxidants on the development of diabetic retinopathy might involve inhibition of NF-κB activation and its downstream pathways in the retina.

In diabetes, some of the cellular changes are similar to aging. We hypothesized that hyperglycemia accelerates aging-like changes in the endothelial cells (ECs) and tissues leading to structural and functional damage. We investigated glucose-induced aging in 3 types of ECs using senescence associated β-gal (SA β-gal) staining and cell morphology. Alterations of sirtuins (SIRTs) and their downstream mediator FOXO and oxidative stress were investigated. Relationship of such alteration with histone acetylase (HAT) p300 was examined. Similar examinations were performed in tissues of diabetic animals. ECs in high glucose (HG) showed evidence of early senescence as demonstrated by increased SA β-gal positivity and reduced replicative capacities. These alterations were pronounced in microvascular ECs. They developed an irregular and hypertrophic phenotype. Such changes were associated with decreased SIRT (1–7) mRNA expressions. We also found that p300 and SIRT1 regulate each other in such process, as silencing one led to increase of the others’ expression. Furthermore, HG caused reduction in FOXO1’s DNA binding ability and antioxidant target gene expressions. Chemically induced increased SIRT1 activity and p300 knockdown corrected these abnormalities slowing aging-like changes. Diabetic animals showed increased cellular senescence in renal glomerulus and retinal blood vessels along with reduced SIRT1 mRNA expressions in these tissues. Data from this study demonstrated that hyperglycemia accelerates aging-like process in the vascular ECs and such process is mediated via downregulation of SIRT1, causing reduction of mitochondrial antioxidant enzyme in a p300 and FOXO1 mediated pathway.

Diabetic cardiomyopathy is a cascade of complex events leading to eventual failure of the heart and cardiac fibrosis being considered as one of its major causes. miR‐133a is one of the most abundantly expressed microRNAs in the heart. We investigated the role of miR‐133a during severe hyperglycaemia. And, our aim was to find out what role miR‐133a plays during diabetes‐induced cardiac fibrosis. We saw a drastic decrease in miR‐133a expression in the hearts of streptozotocin‐induced diabetic animals, as measured by RT‐qPCR. This decrease was accompanied by an increase in the transcriptional co‐activator EP300 mRNA and major markers of fibrosis [transforming growth factor‐β1, connective tissue growth factor, fibronectin (FN1) and COL4A1]; in addition, focal cardiac fibrosis assessed by Masson's trichome stain was increased. Interestingly, in diabetic mice with cardiac‐specific miR‐133aa overexpression, cardiac fibrosis was significantly decreased, as observed by RT‐qPCR and immunoblotting of COL4A1, ELISA for FN1 and microscopic examination. Furthermore, Cardiac miR‐133a overexpression prevented ERK1/2 and SMAD‐2 phosphorylation. These findings show that miR‐133a could be a potential therapeutic target for diabetes‐induced cardiac fibrosis and related cardiac dysfunction.