Supplemental nitrite increases choroidal neovascularization in mice

Abstract

Low doses of nitrite, close to physiological levels, increase blood flow in normal and ischemic tissues through a nitric oxide (NO) dependent mechanism. Given that nitrite therapy and dietary supplementation with vegetables high in nitrate (e.g. beets) are gaining popularity we decided to determine if low doses of nitrite impact the development of choroidal neovascularization (CNV), a key feature of wet age related macular degeneration (AMD). Sodium nitrite (at 50 mg/L, 150 mg/L, and 300 mg/L), nitrate (1 g/L) or water alone were provided in the drinking water of C57BL/6 J mice aged 2 or 12 months. Mice were allowed to drink ad libitum for 1 week at which time laser-induced choroidal neovascularization (L-CNV) was induced. The mice continued to drink the supplemented water ad libitum for a further 14 days at which point optical coherence tomography (OCT) was performed to determine the volume of the CNV lesion. Blood was drawn to determine nitrite and nitrate levels and eyes taken for histology. CNV volume was 2.86 × 10⁷ μm³ (±0.4 × 10⁷) in young mice on water alone but CNV volume more than doubled to >6.9 × 10⁷ μm³ (±0.8 × 10⁷) in mice receiving 300 mg/L nitrite or 7.34 × 10⁷ μm³ (±1.4 × 10⁷) in 1 g/L nitrate (p < 0.01). A similar trend was observed in older mice. CNV volume was 5.3 × 10⁷ μm³ (±0.5 × 10⁷) in older mice on water alone but CNV volume almost doubled to approximately 9.3 × 10⁷ μm³ (±1.1 × 10⁷) in mice receiving 300 mg/L nitrite or 8.7 × 10⁷ μm³ (±0.9 × 10⁷) 1 g/L nitrate (p < 0.01). Plasma nitrite levels were highest in young mice receiving 150 mg/L in the drinking water with no changes in plasma nitrate observed. In older mice, drinking water nitrite did not significantly change plasma nitrite, but plasma nitrate was increased. Plasma nitrate was elevated in both young and old mice provided with nitrate supplemented drinking water. Our data demonstrate that the CNV lesion is larger in older mice compared to young and that therapeutic levels of oral nitrite increase the volume of CNV lesions in both young and older mice. Therapeutic nitrite or nitrate supplementation should be used with caution in the elderly population prone to CNV.

Keywords: Age-related macular degeneration; Aging; Choroidal neovascularization; Nitrate; Nitrite.