P22: Sodium nitrite promotes the viability and proliferation of endothelial cells but inhibits the growth of smooth muscle cells under hypoxia

Abstract

Percutaneous coronary intervention (PCI) is essential following myocardial infarction to restore cardiac perfusion. However, in the long term, the beneficial effects of PCI with stent insertion are limited by restenosis (associated with vascular injury and smooth muscle cell proliferation) and stent thrombosis (associated with inhibition of/delayed re-endothelialisation). Pre-clinical studies suggest that vascular injury and intimal hyperplasia are associated with lack of endogenous nitric oxide (NO) and that nitrite, via its conversion to NO decreases intimal hyperplasia following vascular injury (Alef et al., J. Clin. Invest. 121 (2011) 1646–1656). The aim of this study was to determine the effect of nitrite on the proliferation/viability of EC and VSMC in normoxia, hypoxia and ischaemia/reperfusion (I/R). Human umbilical vein (HUVEC) and coronary artery (HCAEC) endothelial cells and aortic VSMC (HAVSMC) were incubated for 24 h in the absence or presence of nitrite (0.1–10 μM) under normoxic (21% O 2 ) or hypoxic (1–5% O 2 ) conditions. In a separate series of experiments, ECs were subjected to 24 h of 0.1% O 2 followed by 48 h of normoxia to simulate I/R injury. Cell proliferation (cell count) and cell viability (flow cytometry with apoptosis assay-staining for annexin V/propidium iodide) were determined in control cells or cells treated with inhibitors of two potential nitrite reductases: xanthine oxidoreductase and endothelial nitric oxide synthase (eNOS) using allopurinol (100 μM) or l -NMMA (300 μM), respectively. Hypoxia attenuated proliferation of HUVECs with 5% O 2 causing a 21.2 ± 2.8% and 1% O 2 a 24.1 ± 3.1% reduction ( P < 0.001, n = 10) compared to normoxia. Similarly, with HCAECs 5% O 2 caused 30.1 ± 4.1% and 1% a 31.4 ± 3.6% reduction ( P < 0.001, n = 10). This effect on cell number was associated with an increase in the numbers of apoptotic cells by 7.4 ± 1.4% in 5% O 2 and 9.0 ± 1.8% at 1% in HUVECs and 9.5 ± 1.9% and 9.6 ± 1.3% in HCAECs ( P < 0.001, n = 10). Treatment of cells with nitrite caused a concentration-dependent increase in EC number and viability in hypoxia with a peak effect (near complete reversal) occurring at 3 μM in both HUVECs and HCAECs ( P < 0.001, n = 10). This effect of nitrite was attenuated by l -NMMA by ∼85% ( P < 0.001, n = 10) or allopurinol (∼80.0%, P < 0.001, n = 10). Reperfusion with nitrite (3 μM) following 0.1% O 2 for 24 h resulted in a significant concentration dependent improvement in cell growth compared to reperfusion alone at all timepoints ( P < 0.01). In contrast, nitrite had no effect on the proliferation or viability of either cell type under normoxic conditions ( n = 12). Hypoxia also caused a decrease in HAVSMC numbers although this was not associated with an increase in apoptosis ( n = 7). The addition of nitrite caused a concentration-dependent decrease in cell growth with a peak effect occurring at 1 μM of 29 ± 13% ( n = 7) vs normoxia. These studies demonstrate that nitrite protects ECs from the damaging effects of ischaemia or I/R injury but exerts an opposing repressive influence over smooth muscle cell growth. These results suggest that in an environment where improved EC growth but repressed SMC growth is desired, such as in restenosis following stent implantation, raising circulating nitrite levels may be of therapeutic utility. D.A.J. and this work are funded by an NIHR Doctoral Fellowship. Supported by NIHR Doctoral Research fellowship.