Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats

Abstract

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Figures

Figure 1. Vascular injury increases arginase-1 and decreases NO production.

(A) Arginase-1 immunohistochemistry (red) within uninjured control vessels and increased expression within vessels 7 days after injury. Scale bar: 100 μm. (B) Arginase activity is increased 7 days after vascular injury compared with uninjured vessels (*P < 0.05; results are mean ± SEM of 2 separate carotid lysates, each lysate pooled from 2 rats). (C) Ex vivo NOS activity is decreased in vessels 7 days after injury compared with uninjured vessels (*P < 0.05); however, the arginase inhibitor nor-NOHA increases NOS activity in injured vessels (#P < 0.01; 4 vessels per condition, each measured in duplicate). (D) Vascular injury results in decreased S-nitroso­thiol–modified protein concentrations within the vessel wall (*P < 0.05).

Figure 2. Sodium nitrite limits the formation of IH following vascular injury.

(A) Intraperitoneal injection of sodium nitrite decreased the I/M ratio by 60% ± 9% compared with controls (n = 6/group; *P < 0.01). Nebulized sodium nitrite decreased IH by 77% ± 7% compared with controls (n = 8/group). Oral supplementation of sodium nitrite resulted in a 47% ± 5% reduction versus controls (n = 6/group). *P < 0.01. (B and C) H&E staining of representative carotid arteries. (B) Control injured artery; original magnification, ×40. (C) Artery pretreated with oral nitrite supplementation prior to injury; original magnification, ×40. Scale bar: 50 μm. (D) Serum nitrite levels were increased 3.5- to 4-fold in the rats following delivery by all 3 strategies (n = 6/group; *P < 0.01).

Figure 3. Sodium nitrite increases NO generation.

(A) NO generation from injured carotid arteries ex vivo demonstrated a significant increase with the addition of sodium nitrite (9.1 ± 0.44 pmol NO/min/mg protein) compared with vehicle-exposed vessels (0.9 ± 0.2 pmol NO/min/mg protein; *P < 0.01) even in the presence of the NOS inhibitor L-NAME. n = 4 arteries per group, each measured in triplicate. (B) NO generation from cultured SMCs demonstrated a significant increase with the addition of sodium nitrite (7.2 ± 1.4 pmol NO/min/mg protein) compared with vehicle (0.87 ± 0.14 pmol NO/min/mg protein; *P < 0.01). The results are the mean ± SEM for 3 independent experiments, with experiments performed in triplicate for each condition. (C) S-nitrosothiol–modified protein concentration in carotid arteries 4 weeks after injury demonstrated decreased S-nitrosothiol concentration compared with uninjured vessels (**P < 0.05). Oral sodium nitrite supplementation during days 15–28 increased S-nitrosothiol content in injured vessels (#P < 0.05 compared with non-nitrite injured vessels). n = 4 vessels per group. (D) Immunohistochemistry for S-nitrosocysteine (red) demonstrated minimal expression within the injured vessels of control rats versus the injured vessels of rats supplemented with nitrite. Scale bar: 50 μm.

Figure 4. Sodium nitrite inhibited SMC proliferation and was dependent upon p21Waf1/Cip1.

(A) Proliferation detected from 3H-thymidine in cultured SMCs demonstrated inhibition with sodium nitrite in a dose-dependent fashion (*P < 0.01). The results are the mean ± SEM of 4 independent experiments, with experiments performed in triplicate for each condition. (B) Western blot analysis demonstrated increased expression of p21 in the presence of the NO donor DETA-NONOate (50 μM) and sodium nitrite (0–100 μM). Blot is representative of 3 independent experiments. (C) Sodium nitrite inhibited wild-type and rapidly proliferating p53-knockout mouse SMC proliferation (*P < 0.05). However, sodium nitrite did not inhibit p21-knockout mouse SMC proliferation. The results are the mean ± SEM of 3 independent experiments, with experiments performed in triplicate for each condition.

Figure 5. Sodium nitrite–induced NO generation, inhibition of SMC proliferation, and p21 induction are dependent on XOR.

Allopurinol (100 μM) inhibited nitrite-induced (250 μM) NO generation within ex vivo carotid arteries (A; n = 4 independent arteries, each condition measured in triplicate; *P < 0.01 compared with vehicle, #P < 0.05 compared with nitrite-treated, injured vessels) and cultured SMCs (B; each condition measured in triplicate in 3 independent experiments; *P < 0.01 compared with vehicle, control SMCs, #P < 0.05 compared with nitrite-treated, control SMCs). (C) Allopurinol prevented nitrite-induced inhibition of SMC proliferation (*P < 0.01 compared with non-nitrite controls; #P < 0.05 compared with nitrite-treated SMCs). The results are the mean ± SEM of 3 independent experiments, with experiments performed in triplicate for each condition. (D) Western blot analysis demonstrated increased p21 protein levels following nitrite treatment, an effect that was inhibited by the addition of allopurinol (representative blot of 3 independent experiments). (E) Protection against IH formation by oral sodium nitrite pretreatment was inhibited in the presence of allopurinol (100 μM/kg/d; 48 hours prior to injury and 24 hours after injury) (n = 6/group; *P < 0.01 compared with non-nitrite-treated rats; #P < 0.01 compared with nitrite-treated rats). Brief allopurinol treatment alone had no effect on IH in non-nitrite-treated, injured vessels.

Figure 6. Arterial injury increased XOR expression and activity.

(A) XOR immunohistochemistry (red) within uninjured control vessels and injured vessels. Scale bar: 50 μm. (B) Western blot analysis demonstrated increased XOR within injured versus uninjured vessels (representative results of 4 independent experiments). (C) XOR activity is increased within injured versus uninjured control vessels (*P < 0.05; n = 7–8 vessels per group). (D) A NOx– diet resulted in significantly greater I/M ratios compared with rats kept on regular chow (n = 6/group, *P < 0.01). (E) A tungsten-rich diet, which inhibits XOR activity, resulted in significantly increased I/M compared with standard chow (n = 6/group, *P < 0.01).

Figure 7. Sodium nitrite reversed established IH following vascular injury.

(A) IH continues to progress from 2 to 4 weeks after injury (#P < 0.05). Sodium nitrite treatment on days 15–28 after vascular injury decreased I/M ratios by 66% ± 6% compared with untreated injured controls as measured 4 weeks after injury (*P < 0.01, n = 6/group). (B) Rats kept on a NOx– diet, which had an exaggerated injury response, were also rescued by sodium nitrite supplementation in drinking water from days 15 to 28 after injury. Sodium nitrite resulted in a 72% ± 8% reduction in I/M ratios compared with rats kept on a NOx– diet without nitrite supplementation (*P < 0.01, n = 6/group). (C) Immunohistochemistry for p21 (red) demonstrated minimal expression within injured vessels versus nitrite-treated injured vessels. Scale bar: 50 μm.

Figure 8. Schematic representation of vascular injury–induced arginase and XOR and generation of NOS-independent NO.

Under normal conditions, eNOS utilizes l-arginine to produce vasoregulatory NO. Following vascular injury, an increase in arginase competes for l-arginine as a substrate and decreases NOS-generated NO production. As part of an adaptive response, XOR reduces circulating nitrite (NO2–) to NO to limit injury. Supplementing nitrite can augment this response to serve as a therapeutic.