Septic shock is a clinical entity that is increasing in importance because of its mounting prevalence and associated high mortality rate. A understanding of the pathophysiologic mechanisms through which septic shock manifests itself is a requirement for effective therapeutic intervention. Endothelial cells, especially those of the microcirculation (MEC), are a prime target for circulating bacterial lipopolysaccharide (LPS). LPS-mediated alterations in MEC function can include perturbations in vascular hemodynamics, permeability, coagulation, leukocyte interactions and immune function. Prostaglandins (PG) have been implicated in each of these changes in MEC function. The objective of these studies is to elucidate the relationship between LPS and the regulation of endogenous PG formation within MECs. These studies will be carried out in a transformed line of human microvessel-derived ECs. The initial aim of the proposal is to fully characterize this transformed cell line and to compare its response to primary cultures of MECs with regard to LPS responsiveness and other parameters.
A second aim i s to use the transformed MECs to investigate whether they possess an LPS receptor protein. Equilibrium and kinetic receptor binding, (125)I-LPS competitive binding, receptor occupancy and receptor affinity studies will be used to characterize the nature of the receptor.
A third aim i s to investigate the inter-relationships between LPS-mediated PG production and the induction of cytokines and vasodilator systems within the cells. Experiments will be made to assess the time course of LPS-mediated intracellular mRNA production for PGHS-1, PGHS-2, cytoplasmic PLA(2) and secretory PLA(2) as well as for IL-1alpha, IL-1beta, and several nitric oxide enzymes. Attempts to mimic the LPS effect on mediator gene expression will be made by the administration of IL-1, TNF, PAF and other factors. Stimulation and ablation studies will be carried out to determine the inter-dependence of the expression of one mediator gene system upon the expression of other mediator systems. The effect of PGE(2) upon mediator gene expression will be determined. In summary, a novel model of MECs will be used to assess the relationships between LPS and the regulation of PG production as well as possible modulation of the PG system by other compounds stimulated by LPS. A knowledge of the mechanisms by which LPS alters MEC function can be used to improve therapeutic approaches to the treatment of pediatric and geriatric septic shock, burn- and surgery-associated septicemia, antibiotic resistant infectious diseases and immune deficiency-related sepsis.
