Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress

Abstract

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs −/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

Faulty homocysteine recycling in diabetic retinopathy

“…Although hyperglycemia is considered as the main instigator of its development, systemic factors including hyperlipidemia and blood pressure are also intimately associated with the development of diabetic retinopathy [35]. Nondiabetic normal population generally has > 15 μM plasma homocysteine, but in diabetic patients, they can go as high as 50-100 μM [10,11]. High homocysteine in diabetic patients is associated with increased macular thickness without macular edema [36], and in patients with retinopathy, high homocysteine is considered to act as a common link through which other systemic factors could exert their deleterious effect on the progression of diabetic retinopathy [6,37].…”

“…Cells from the 7th-8th passage were incubated in the DMEM incubation medium containing reduced serum and growth supplement (2% and 2 μg/ml, respectively) for 48 h in the presence or absence of 100 μM L-Homocysteine thiolactone hydrochloride (Cat No. S784036, Sigma-Aldrich, St Louis, MO) [10], and were analyzed for mitochondrial damage. Incubation of HRECs with homocysteine for 48 h had no effect on their cell phenotype.…”

“…High plasma homocysteine levels are associated with endothelial dysfunction, and in diabetic patients, with many complications including nephropathy, cardiomyopathy and neuropathy [7][8][9]. Studies using genetically manipulated mice that can accumulate homocysteine have suggested a role for homocysteine in diabetic retinopathy; these animals have impaired visual function and damaged blood retinal barrier [10,11]. Homocysteine was also shown to induce mitochondrial dysfunction, and in retinal ganglion cells, it was implicated in the dysregulation of mitochondrial dynamics [12].…”

Homocysteine Induces Inflammation in Retina and Brain

“…Inflammatory markers TNF-α and IL1β were assessed using immunofluorescence in frozen sections (eye and brain prepared from cbs +/+ , cbs +/− , and cbs −/− mice) as per our published method [19]. Briefly, frozen sections were fixed with 4% paraformaldehyde and blocked with Power Block, then incubated with primary antibody for TNF-α (1/200, ab1793) and IL1β (1/250, ab9722) from Abcam Corp. (Cambridge, MA) and biotinylated with GSL I-isolectin B4 (Vector Laboratories, Burlingame, Ca, Cat#: B-1205, 7µL/mL) for 3 h at 37 • C, followed by incubation with an appropriate secondary antibody (Alexafluor and Texas red avidin, Invitrogen).…”

“…We used microglia as the main cell activated in inflammation in addition to HRECs and RPE, which play crucial roles in inner and outer BRB dysfunction in DR and AMD, respectively. Different concentrations of Hcy were also used to evaluate the effect of various levels of HHcy on the induction of inflammation in different cells and in different disease models, as reported in our previous studies [19,20,24].…”