In diabetes, some of the cellular changes are comparable to aging. and reduced replicative capacities. These modifications were pronounced Mouse monoclonal to BMX in microvascular ECs. They developed an irregular and hypertrophic phenotype. Such changes were associated with decreased SIRT (1C7) mRNA expressions. We also found that p300 and SIRT1 regulate each other in such process, as silencing one led to increase of the others manifestation. Furthermore, HG caused reduction in FOXO1s 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 Wortmannin glomerulus and retinal blood vessels along with reduced SIRT1 mRNA expressions in these tissues. Data from this study exhibited that hyperglycemia accelerates aging-like process in the vascular ECs and such process is usually mediated via downregulation of SIRT1, causing reduction of mitochondrial antioxidant enzyme in a p300 and FOXO1 mediated pathway. Introduction Diabetes and its complications account for significant morbidity and mortality throughout the world C. The major factor in the development of chronic diabetic complications is usually vascular EC disorder . The prevailing mechanism leading to EC disorder is usually an increase in reactive oxygen species (ROS) formation . In response to high ambient glucose levels and subsequent oxidative stress, ECs sophisticated large amount of vasoactive factors, growth factors and cytokines , . Such factors lead to increased production of extracellular matrix (ECM) proteins causing structural modifications C. Oddly enough, several such changes seen at the cellular and tissue level in diabetes are comparable to the changes seen in normal aging process C. Oxidative stress causes DNA damage and alters transcriptional machinery both in aging and in diabetes , , , . We have previously shown that glucose induced oxidative stress causes histone acetylation by p300, which regulates several transcripts in diabetes , . p300, a transcriptional coactivator with an intrinsic histone acetyltransferase (HAT) activity, regulates numerous transcription factors , , . Acetylation by p300 and other HATs are balanced by histone deacetylases (HDACs). Silent information regulator 2 proteins or sirtuins (SIRTs) belong to Class III HDACs and regulates epigenetic gene silencing and suppress recombination of rDNA C. In mammals, SIRTs have a range of molecular functions and have emerged as important protein in aging and metabolic regulations , . SIRTs symbolize a small gene family with seven users designated as SIRT1C7, known to be modulated by oxidative stress . Some of the SIRTs activity is usually carried out through deacetylation of the FOXOs, forkhead family O group of transcription factors C. Wortmannin Among the FOXO family, FOXO1 is usually best characterized and plays important functions in cell survival, oxidative stress resistance and cell death C. FOXO1 Wortmannin has a highly conserved DNA binding domain name subjected to posttranslational modifications such as phosphorylation, acetylation and ubiquitination. These modifications can either increase or decrease the transcriptional activity of FOXO1 . FOXO1 acetylation by HAT such as p300, prospects to attenuation of its DNA binding ability and facilitates its phosphorylation by Akt, leading to its export from the nucleus; whereas deacetylation increases FOXO1s transcriptional activity , . The purpose of this study was to investigate whether high glucose causes accelerated aging process in ECs through modification of SIRTs. We further investigated whether the effects of SIRTs are mediated through FOXO1 and if such process is usually regulated by histone acetylase p300. We carried out these studies in numerous ECs as well as in the diabetic animals. Methods Cell Culture Dermal-derived human microvascular EC (HMEC) was obtained from Lonza, Inc. (Walkersville, MD) and produced in EC basal medium 2 (EBM-2, total). Human umbilical vein ECs (HUVECs) were obtained from Lonza and cultured in EC growth medium (EBM total, Walkersville, MD). Bovine retinal microvascular ECs (BRECs) were obtained from VEC Technologies (Rensselaer, NY) and produced in a defined EC growth medium (MCDB-131 total). We have previously explained the culture conditions of these three cells , . No insulin was present in any media. For the long term continuous exposure to glucose, ECs were cultured in 12 well dishes (Corning, Acton, MA) and treated with 5 mM glucose (NG) or 25 mM glucose (HG, D-glucose) or osmotic control (OSM, 25 mM L-glucose). Upon confluence cells were propagated & managed in the same treatment condition until they halted proliferating completely. During each passage subculture cells from each treatment group were stained for SA -gal and collected for RNA.