p22^phox-containing microparticles from plasma of septic patients are related to reactive oxygen species production and apoptosis of vascular cells in culture

Reactive oxygen species (ROS) are related to vascular dysfunction in several pathologies and may be related to the pathophysiology of sepsis and multiple organ dysfunction.

To investigate their role and sources we evaluated the effects of plasma of septic patients (SPP) on reactive ROS production and apoptosis of rabbit endothelial (REC) and aortic smooth muscle (RASM) cells in culture through NAD(P)H (0.3 mM)-driven lucigenin (5 μM) luminescence and annexin V assay respectively.

SPP alone showed intrinsic luminescence when compared to plasma from healthy controls (HCP) (51.4 ± 9.5 cpm/min/mg protein vs 7.0 ± 1.2, n = 5, P < 0.05). After incubation with REC and RASM cells SPP induced a two-fold increase in ROS production (n = 5, P < 0.05) when compared to incubation with HCP. This enhancement of ROS production by REC and RASM cells were paralleled by increase in apoptosis rates induced by SPP. The fraction responsible for ROS production and apoptosis induction was determined through filtration and ultracentrifugation to be over 50 kD and to contain microparticles (MP), similar to those from activated platelets. MP showed similar luminescence (60.6 ± 4.4 vs 32.9 ± 3.1, n = 11), blocked by DPI (60%), a flavoenzyme inhibitor, and by SOD (80%). Addition of MP from SPP to REC and RASM also induced increased luminescence when compared to the effect of MP from HCP; These signals were also inhibitable by SOD and DPI. Incubation of REC and RASM with MP from SPP doubled apoptosis rates when compared to incubation with MP from HCP. Apoptosis was also inhibited by addition of SOD or DPI. After lipid extraction, western-blot analysis of the MP was positive for the p22^phox and gp91 subunits of the NAD(P)H oxidase.

Thus, SPP has intrinsic and enhances REC and RASM ROS production, due to NAD(P)H oxidase activity. It causes apoptosis of REC and RASM, also inhibited by SOD and DPI. These effects may be attributable to MP containing the p22^phox and gp91 subunits of NAD(P)H oxidase. ROS derived from microparticles may represent a new signaling pathway involved in the pathophysiology of severe sepsis.