Recombinant human erythropoietin inhibits iNOS activity and reverts vascular dysfunction in splanchnic artery occlusion shock

Abstract

1. We investigated the effects of recombinant human erythropoietin (rh-EPO) in splanchnic artery occlusion (SAO) shock. Sham operated animals were used as controls. Survival rate, mean arterial blood pressure (MAP), serum Tumor Necrosis Factor (TNF-alpha), plasma nitrite/nitrate concentrations, red blood cell (RBC) count, blood haemoglobin (Hb), the responsiveness of aortic rings to phenylephrine (PE, 1 nM-10 microM) and the activity of inducible nitric oxide synthase (iNOS) were studied. 2. SAO shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham shocked rats survived more than 4 h), enhanced serum TNF-alpha concentrations, increased plasma nitrite/nitrate levels (60+/-9.5 microM; sham shocked rats= 2+/-0.4 microM), decreased MAP, unchanged RBC count and blood Hb and enhanced iNOS activity in the aorta. Moreover aortic rings from shocked rats showed a marked hyporeactivity to PE. 3. Rh-EPO (25, 50 and 100 U 100 g(-1), 5 min following the onset of reperfusion) increased survival rate (70% at 4 h of reperfusion with the highest dose), reduced plasma nitrite/nitrate concentrations (10.3+/-3.3 microM), increased MAP, did not change RBC count and blood Hb, and inhibited iNOS activity in thoracic aortae. Furthermore rh-EPO, either in vivo or in vitro (10 U for 1 h in the organ bath), restored to control values the hyporeactivity to PE. Finally rh-EPO inhibited the activity of iNOS in peritoneal macrophages activated with endotoxin. 4. Our data suggest that rh-EPO protects against SAO shock by inhibiting iNOS activity.

Figure 1

Effects of vehicle (1 ml kg⁻¹ i.v., 5 min after the onset of reperfusion) or rh-EPO (100 U 100 g⁻¹ i.v., 5 min following the onset of reperfusion) on serum TNF-α in rats subected to splanchnic ischaemia-reperfusion injury (SAO). Each point represents the mean±s.e.mean of seven experiments.

Figure 2

Contractile response to cumulative doses of phenylephrine (PE) in endothelium denuded aortic rings from sham-operated rats and rats subjected to splanchnic ischaemia-reperfusion injury (SAO) treated with vehicle (1 ml kg⁻¹ i.v., 5 min after the onset of reperfusion) or rh-EPO (100 U 100 g⁻¹ i.v. 5 min following the onset of reperfusion). Each point represents the mean±s.e.mean of seven experiments. ^*P<0.01 vs SAO+vehicle.

Figure 3

Contractile response to cumulative concentrations of phenylephrine in aortic rings without endothelium from untreated sham occluded or splanchnic artery occlusion (SAO) shocked rats. The rings were incubated for 1 h with rh-EPO (10 U), vehicle (10 μl) or MEG (100 μM). Each point represents the mean±s.e.mean of six experiments. ^*P<0.01 vs vehicle.

Figure 4

Effect of rh-EPO on iNOS activity in aortae collected 80 min following the onset of reperfusion in splanchnic artery occlusion (SAO) shocked rats. Each point represents the mean±s.e.mean from 3–5 rats. ^*P<0.05 vs SAO+vehicle: #P<0.01 vs SAO+vehicle. Rh-EPO (i.v.) or MEG (i.p.) were inected 5 min following the onset of reperfusion.

Figure 5

Effect of rh-EPO on nitrite accumulation in the supernatant of the cultured macrophages activated with endotoxin (LPS). Data are expressed as means±s.e.mean of eight wells from 3–4 independent experiments. Macrophages were stimulated for 24 h with LPS and rh-EPO was applied 6 h after LPS. The black column depicts the effect of rh-EPO incubated simultaneously together with LPS without any previous stimulation. ^*P<0.05 vs LPS+vehicle; #P<0.001 vs LPS+vehicle.