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.

Figure 1

Evaluation of tight junction proteins of HRECs treated with and without Hcy-thiolactone. (A) Immunofluorescence of ZO-1 (green) (B) Immunofluorescence of occludin (green) (C) Immunofluorescence of claudin-5 (red) with nuclear staining DAPI (blue) in cultured HRECs subjected to treatment with Hcy (50 µM) or vehicle (PBS). Calibration bar: 50 μm. (A) and 100 μm. (B and C). (D) Western blot analysis of ZO-1 (198 KDa), (E) Western blot analysis of occludin (59 kD). (F) Western blot analysis of claudin-5 (17–26 KDa). *p < 0.05 and **p < 0.01.

Figure 2

Transendothelial electric resistance (TER) and FITC dextran flux assays of HRECs cells treated with and without Hcy-thiolactone. (A) TER and (B) FITC dextran flux through the confluent monolayer in the Hcy-treated endothelial cells. **p < 0.01.

Figure 3

Evaluation of antigenic potential and cell viability of Hcy-treated HRECs. Cell were treated with different concentrations of Hcy and VEGF was used as a positive control. (A) Tube formation assay of HRECs, white arrows indicate tube formation. (B) Assessment of tube number. (C) Cell Viability (MTT) assay. (D) Apoptosis (Caspase3/7) assay. Error bars indicate SD from the mean, n = 3.

Figure 4

Evaluation of ROS production induced by Hyperhomocysteinemia. (A) DCF staining (green) of the cbs ^+/+and cbs ^−/− retinas. (B) DHE staining for superoxide (red) in the cbs ^+/+ and cbs ^−/− retinas. (C) Quantification of the color intensity of DCF and DHE staining, **p < 0.01 and ***p < 0.001. (D) Cell ROX green staining for ROS in HRECs treated with and without Hcy (green).

Figure 5

Evaluation of the retinal antioxidant capacity of the hyperhomocysteinemic (cbs ^−/−) mice. (A) Retinal frozen sections from the cbs ^+/+ and cbs ^−/− mice immunostained for GSH (green). (B) Retinal frozen sections from the cbs ^+/+ and cbs ^−/− mice stained for H₂S (green). (C) Quantification for the color intensity of GSH, H₂S and NRF2. (D) Retinal frozen sections from the cbs ^+/+ and cbs ^−/− mice immunostained for NRF2 (green). (E) Western blot analysis of GSH reductase expression in the cbs ^−/− retina compared to control retina. (F) RT-PCR evaluation of anti-oxidant gene expression in the cbs ^−/− retina compared to control retina. Calibration bar is 50 μm.

Figure 6

Evaluation of the antioxidant capacity of HRECs treated with and without Hcy-thiolactone. (A) Measurement of GSH activity in HRECs treated with Hcy (20, 50 and 100 μM) *p < 0.05. (B) IF staining of ROS (green) in HRECs treated with 50 μM of Hcy with and without the addition of anti-oxidant NAC, (50 mM). Calibration bar; 100 μm. (C) FITC-dextran assay illustrating fluorescein leakage in HRECs treated with 50 μM of Hcy, with and without the addition of 50 mM of NAC, *p < 0.05.