Growing evidence that high glucose may be a causative agent of the thickened vascular basement membranes characterize diabetic microangiopathy prompted this investigation underlying mechanisms. When exposed to 30 mM glucose, 70% 52 primary cultures human endothelial cells, each derived from single umbilical vein, showed increased levels fibronectin (median 181% control, range 104–549%) and collagen IV mRNA (175% 101–807%). The response two transcripts was concordant in 77% studied (P =...

To investigate whether high glucose (HG) induces mitochondrial dysfunction and promotes apoptosis in retinal Mu ¨ller cells.METHODS.Rat cells (rMC-1) grown normal (N) or HG (30 mM glucose) medium for 7 days were subjected to MitoTracker Red staining identify the network.Digital images of mitochondria captured live under confocal microscopy analyzed morphology changes based on form factor (FF) aspect ratio (AR) values.Mitochondrial metabolic function was assessed by measuring oxygen...

Impairment of retinal vascular homeostasis is associated with the development and progression diabetic retinopathy involving gap junction intercellular communication (GJIC) activity. The principal protein communication, connexin, was investigated to determine effects high glucose concentrations on expression endothelial-specific connexins (Cx37, Cx40, Cx43), connexin phosphorylation pattern, GJIC Rat microvascular endothelial (RME) cells grown in (30 mmol/l)-glucose medium for 9 days had...

OBJECTIVE To investigate early events leading to microvascular cell loss in diabetic retinopathy. RESEARCH DESIGN AND METHODS FOXO1 was tested vivo by DNA binding activity and nuclear translocation cells retinal trypsin digests. In studies were undertaken STZ-induced rats Zucker fatty using the tumor necrosis factor (TNF)-specific blocker, pegsunercept, or inhibiting with RNAi. Microvascular apoptosis, formation of pericyte ghosts, acellular capillaries measured. Upstream downstream effects...

PURPOSE. To examine whether diabetes-induced connexin 43 downregulation promotes retinal vascular lesions characteristic of diabetic retinopathy (DR). METHODS. Two animal models, streptozotocin-induced mice and Cx43 heterozygous knockout (Cx43(+/-)) mice, were studied to directly assess diabetes reduces the expression Cx43, which, in turn, contributes cell loss by apoptosis. Retinal protein levels assessed nondiabetic control Cx43(+/-) Western blot analysis, localization distribution cells...

<h3>Background:</h3> Neuronal abnormalities are associated with the pathogenesis of diabetic retinopathy. However, mechanisms for neuronal cell death in retinopathy remain unclear. <h3>Aim:</h3> To determine whether altered expression Bax, caspase-9 and -3 is degenerative neurons <h3>Methods:</h3> Immunohistochemistry was performed on cryosections obtained from five pairs normal age-matched human retinas. Diabetic eyes had no proliferative histories photocoagulation or ocular surgery. In...

Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, prominent lesion of diabetic retinopathy. High glucose (HG) has been reported induce mitochondrial fragmentation and endothelial cells, contributing apoptosis. In this study, the effects HG on morphology, membrane potential, metabolic changes whether they could contribute HG-induced apoptosis pericytes were investigated.Bovine (BRPs) grown normal or medium for 7 days. Both sets cells double stained with...

To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression cell monolayer permeability.Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, (HG; 30 N medium transfected with Cx43 siRNA, or scrambled siRNA. determine Cx43, occludin, protein expression, Western blot (WB) analysis immunostaining performed. Gap intercellular communication (GJIC) was determined using scrape load dye...

Basement membrane thickening is the most prominent and characteristic feature of early diabetic microangiopathy. Unknown not only causative process but also whether reflects increased synthesis specific components. Because collagen type IV uniquely present in basement membranes represents their predominant structural element, we studied its expression retinas obtained postmortem from five patients with 8 +/- 3 yr diabetes six nondiabetic controls. The transcript proved to be rare adult human...

Purpose: The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) affect vascular permeability in diabetic retinopathy.Materials Methods: To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days confluency normal (N, 5 mM) or high glucose (HG, 30 medium on transwell inserts subjected an vitro assay. Inserts...

This study aims to determine whether high glucose-induced inhibition of connexin 43 (Cx43) expression and reduced gap junction intercellular communication (GJIC) promote microvascular endothelial cell loss.To downregulate Cx43 protein in rat cells (RMECs), the were grown (30 mM) glucose medium for 7 days, or transfected with antisense-Cx43 (AS-Cx43) oligonucleotides. Western blot analysis confirmed significant expression. Scrape load dye transfer (SLDT) assay showed reduction GJIC activity...

In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased permeability, a prominent lesion of early diabetic retinopathy. this study we determined whether altered expression activity lysyl oxidase (LOX), cross-linking enzyme, may compromise functional integrity under high-glucose (HG) conditions.Rat endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed LOX...

To determine whether fenofibric acid (FA) reduces high glucose (HG)-induced basement membrane component overexpression and hyperpermeability in human retinal pigment epithelial (RPE) cells.Retinal cells (ARPE-19) were cultured for 18 days normal (5 mM) or HG (25 medium studied the effects of FA on fibronectin (FN) collagen IV (Coll IV) expression. During last 3 experiment, 100 μM was added to grown plus IL-1β (HG + IL-1β) mimic, at least part, inflammatory aspect diabetic milieu. Real-time...

To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression whether altered mtCx43 channel activity is involved promoting apoptosis retinal endothelial cells.MtCx43 was determined using immunostaining, green fluorescent protein (GFP)-tagged followed by confocal imaging, Western blot analysis isolated from rat cells (RRECs). assess HG expression, RRECs were grown normal (5 mM) or (30 medium for 7 days,...

Purpose.: To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal Müller cells, promotes cell pericyte loss. Methods.: Retinal cells (rMC-1) cocultures of rMC-1 pericytes were grown normal (N) or HG (30 mM glucose) medium. Additionally, transfected with Cx43 small interfering RNA (siRNA) as pericytes, plasmid HG. Expression was determined by Western blotting immunostaining GJIC assessed...

Diabetic retinopathy is a prevalent microvascular complication characterized by apoptotic vascular cell loss in the retina. Previous studies have shown that high glucose (HG)-induced mitochondrial fragmentation plays critical role promoting retinal apoptosis. Here, we investigated whether downregulation of fission genes, Fis1 and Drp1, which are overexpressed HG condition, prevents fragmentation, preserves function, protects endothelial cells from Rat (RRECs) were grown normal (5 mM glucose)...