Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells

Abstract

Acute lung exposure to low oxygen results in pulmonary vasoconstriction and redistribution of blood flow. We used human microvascular endothelial cells from lung (HMVEC-L) to study the acute response to oxygen stress. We observed that hypoxia and erythropoietin (EPO) increased erythropoietin receptor (EPOR) gene expression and protein level in HMVEC-L. In addition, EPO dose- and time-dependently stimulated nitric oxide (NO) production. This NO stimulation was evident despite hypoxia induced reduction of endothelial NO synthase (eNOS) gene expression. Western blot of phospho-eNOS (serine1177) and eNOS and was significantly induced by hypoxia but not after EPO treatment. However, iNOS increased at hypoxia and with EPO stimulation compared to normal oxygen tension. In accordance with our previous results of NO induction by EPO at low oxygen tension in human umbilical vein endothelial cells and bone marrow endothelial cells, these results provide further evidence in HMVEC-L for EPO regulation of NO production to modify the effects of hypoxia and cause compensatory vasoconstriction.

Introduction

Erythropoietin (EPO) is a hypoxia-inducible cytokine required for production of mature erythrocytes. EPO acts by binding to and inducing expression of the erythropoietin receptor (EPOR). Beyond erythropoiesis, EPO and EPOR elicit response in numerous tissues, particularly in endothelial cells [1], [2]. The ability for EPO to stimulate cell proliferation and chemotaxis in endothelial cells was first demonstrated in vitro and then in vivo in the chick chorioallantoic membrane and mouse uterine endometrium [3], [4]. The pleiotrophic nature of EPO proliferative and survival response is observed in a variety of non-hematopoietic cells including tumor cells [5], [6]. Moreover, EPO in combination with low oxygen tension can increase the endothelial capacity to produce nitric oxide (NO) by induction of both EPOR and endothelial nitric oxide synthase (eNOS) [7]. EPO activation of NO production in murine erythrocytes has also been reported [8]. EPO stimulated production of NO may compensate for the enhanced NO scavenging in the presence of cell-free hemoglobin followed by haptoglobin binding to facilitate clearance, especially with reduced oxygen availability [9], [10]. Additionally, EPO induced neovascularisation through endothelial progenitor cells recruitment from the bone marrow associated with coronary heart disease and possibly acting in conjunction with stimulated VEGF production by cardiomyocytes [11], [12]. Several in vivo studies demonstrated an EPO protective effect to endothelial cells in diverse models of vascular disease [13], [14].

Endothelial cells exhibited specific responses in adaptation to low oxygen tension in lung [15]. Acute exposure to hypoxia resulted in vasoconstriction of pulmonary arteries, increased pulmonary arterial pressure and redistribution of blood flow from basal to the apical level of lung. In chronic hypoxic lung disease dysfunction of mechanisms regulating vascular tone and remodeling of the pulmonary vasculature contribute to the development of sustained pulmonary hypertension [16]. Chronic hypoxia stimulated polycytemia results in increased blood viscosity and pulmonary hypertension.

NO has a central role in modulation of pulmonary vascular tone. Three isoforms of the enzyme responsible for the NO production are endothelial, inducible (iNOS) and neuronal (nNOS) [17], [18]. All three isoforms are increased in chronic hypoxic pulmonary hypertension [19]. eNOS and nNOS are constitutively expressed NOS isoforms and require calcium for activation, while iNOS is calcium independent but inducible by cytokines. In the normal murine pulmonary circulation nNOS does not modulate tone, whereas eNOS-derived NO is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and both eNOS and iNOS play a role in modulating basal tone chronically [20]. eNOS is the principal isoform expressed in the normal pulmonary vasculature [21].

To clarify the response of lung endothelial cells to EPO stimulation, we examined the levels of EPOR and eNOS/iNOS expression in cultured endothelial cells at normoxia and low oxygen tension. We found that EPO increased expression of EPOR, particularly at low oxygen tension. EPO had little effect on NO production at normoxia. At hypoxia, concomitant with the increase in EPOR expression, EPO increased production of NO up to 4 h. This EPO induction of NO was NOS dependent and evident despite hypoxia induced down regulation of eNOS mRNA and protein expression. The activity of eNOS is regulated primarily by post-translational mechanisms including phosphorylation at multiple sites, but one of the most studied sites is the activation of serine 1177. However, ratio for phosphorylated eNOS at serine 1177 and eNOS, showed increased in there activity at low oxygen but not after EPO stimulation. In contrast, Western blotting showed that iNOS increased at low oxygen tension and even further when combined with EPO treatment. These data suggest that the low level of NO induced at low oxygen tension by EPO stimulation in lung endothelial cells is NOS dependent and concomitant with increase in the low level of iNOS protein expression but decrease in eNOS activity.