Endothelin-1 production during the acute chest syndrome in sickle cell disease.

To investigate the role of the endothelial-derived vasoactive mediator endothelin (ET-1) in the acute chest syndrome (ACS), we incubated bovine pulmonary artery endothelial cells (BPAEC) with red blood cells (equivalent to a hematocrit of 20%) and/or autologous plasma (1:10 dilution) from two patients during ACS and during routine clinic visits. Cellular RNA was analyzed for ET-1 transcripts by Northern analysis and ET-1 protein levels in BPAEC supernatants and in plasma measured by radioimmunoassay. ET-1 mRNA expression and protein levels increased in BPAEC exposed to plasma obtained during ACS; in contrast, exposure to plasma obtained during routine clinic visits did not alter BPAEC ET-1 mRNA expression or protein levels. Plasma ET-1 level was elevated during ACS, decreased during resolution, and remained slightly elevated during routine clinic visits. Plasma obtained from one patient 4 d prior to hospitalization for vasoocclusive crisis contained the highest ET-1 level and markedly increased BPAEC ET-1 mRNA expression and protein levels. In both patients, BPAEC ET-1 mRNA and protein expression in vitro and plasma ET-1 levels in vivo correlated with stage of disease and occurred in the absence of direct erythrocyte contact in vitro. These observations suggest that ET-1 production contributes to development of ACS.

Distinct effect of hypoxia on endothelial cell proliferation and cycling.

Endothelial cells (EC) occupy a strategic location in the vasculature as a barrier between the intravascular compartment and underlying tissues; as such, they are often exposed to stresses, such as decreases in ambient oxygen, diminished metabolic substrate, or changes in temperature, that could affect their ability to divide and proliferate. The present study characterizes cell counts, cell cycle distribution, and bromodeoxyuridine incorporation in pulmonary artery and aortic EC exposed to acute and/or chronic hypoxia and other cellular stresses. During hypoxia, EC division slows but does not arrest; progression through the G1-to-S transition point and/or progression from S to G2/M is altered with an increased percent of EC in S phase. These changes in EC cell cycle distribution with hypoxia are dependent on the origin of the EC as well as the ambient oxygen concentration; moreover, they are distinct from changes observed with elevated temperature or glucose deprivation. and differ from the quiescent pattern induced by serum deprivation or high-density confluence. These findings demonstrate that hypoxia exerts a distinct effect on the cell cycle distribution and proliferation of EC.

Endothelial cell nitric oxide production in acute chest syndrome.

Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS.