Effects of sickle erythrocytes on metabolism and gene regulation in cultured human endothelial cells under flow conditions
McIntire, Larry V.
Doctor of Philosophy
Sickle cell anemia is characterized by chronic hemolysis and episodic vasoocclusive crises. There are many factors contributing to vascular occlusion in sickle cell patients, with the enhanced, abnormal adhesion of sickle erythrocytes to endothelial cells being investigated most thoroughly. The vascular endothelium lining on blood vessel walls is in a unique position of direct exposure to these abnormal red blood cells. It is possible that interactions with sickle erythrocytes may modulate endothelial cells to create an environment sensitive to crisis-triggering factors and in favor of adhesion events. However, quantitative information on the metabolic and gene regulatory effects of these interactions on endothelial cells under flow is lacking. In this research, we developed an experimental system that simulates the physiological vascular flow environment to subject cultured human endothelial cell (EC) monolayers to sickle cell perfusion with a well-defined wall shear stress level of 1dyne/cm2 for times up to 24 hours. Effects of sickle erythrocytes on EC production of vasotone mediators (prostacyclin and endothelin-1) and on EC expression of cell adhesion molecules (ICAM-1 and VCAM-1) were examined at the transcription and/or protein levels. The production of prostacyclin and endothelin-1 both increased in ECs after exposure to sickle cell perfusion, in comparison to perfusion with normal red cells. The altered levels of prostacyclin and endothelin-1 may disturb the delicate balance of vasoactive materials, leading to vasotone instability in sickle cell patients. Gene expression and cell surface expression of ICAM-1 in ECs were profoundly elevated by perfusion with sickle erythrocytes. The elevation in membrane-bound ICAM-1 levels may increase the interactions between blood cells and ECs, especially leukocyte endothelium adhesion. This may lead to the entrapment of red cells in regions of low oxygen tension in the microcirculation and trigger the polymerization of sickle hemoglobin. VCAM-1 mRNA, but not membrane-bound VCAM-1, was also significantly increased by perfusion with sickle erythrocytes. The presence of VCAM-1 on ECs may enhance the adhesion of erythrocytes via binding to integrin VLA-4( a 4 b 1) expressed on sickle cell membranes, but not on normal red cell membranes. The release of soluble ICAM-1 and soluble VCAM-1 both increased in ECs after exposure to sickle cell perfusion, and may serve as an indicator of injury and/or activation of ECs. The presence of IL-1 b in the perfusion system, as a model of inflammatory cytokine effects, synergistically increased the production of prostacyclin, ICAM-1 and VCAM-1 in endothelial cells. This is consistent with the close association of inflammation and vasoocclusive crises observed in sickle cell anemia patients.
Biomedical engineering; Chemical engineering