Introduction: The incidence of both hospital and community acquired methicillin-resistant Staphylococcus aureus (MRSA) infections is progressively increasing. Previously, we have reported that MRSA-induced sepsis caused more severe vascular leakage and significantly higher plasma levels of nitrite/nitrate compared to Pseudomonas aeruginosa sepsis. The aim of present study was to investigate mechanical aspects underlying MRSA sepsis-related vascular hyperpermeability. We hypothesized that peroxynitrite is primarily responsible for the severe vascular permeability in MRSA sepsis via promoting the angiogenic growth factor angiopoietin-2 (ang-2). We have tested the hypothesis, using our well-established ovine sepsis model and in vitro endothelial cell culture technique.

Method: I. Ovine model. Sheep were instrumented with Swan Ganz, femoral artery, and left atrium catheters to monitor hemodynamics for 24 h. Sepsis was induced by instillation of live MRSA (2.5X10^11CFU) into lungs by bronchoscope under anesthesia. After the injury, sheep were awakened, placed on ventilator, and fluid resuscitated. Urine output was measured via Foley catheter. Groups: 1) control: MRSA instillation, no treatment, n=5; 2) treated: MRSA instillation + peroxynitrite decomposition catalyst (PDS), n=5. Continuous i.v. infusion of PDS was started 6 h post-injury with dose of 0.02 mg/kg/h following bolus dose of 0.1 mg/kg.

II. In vitro study. Human umbilical vein endothelial cells (HUVEC) were challenged for 4 h with 105 CFU of live MRSA and pretreated with or without 5 µM PDC and cell permeability (the amount FITC-Dextran that passed through the confluent HUVEC monolayer) and ang-2 mRNA (RT-PCR) were determined. Cells infected with MRSA were also co-incubated with or without ang-2 (5 µg/mL) and its receptor Tie-2 (5 µg/mL) antibodies and cell permeability was determined (n=4).

Results: Injured animals showed severe hypotension (MAP) and excess fluid retention. Control animals also showed severe hemoconcentration associated with decreased plasma protein and plasma oncotic pressure. However, these pathological changes were attenuated by PDS treatment. MAP (mmHg) was 94±3 at baseline and 66±3 at 24 h in control vs. 93±1 at baseline and 82±10 at 24 h in treated groups. Fluid retention (mL/kg) was 102±32 and 30±8 in control and treated groups at 24 h, respectively. PDC (34±1), and ang-2 (30±1) and Tie-2 receptor (32±1) antibodies reduced HUVEC cell permeability induced by MRSA (73±4) 4 h post co-incubation. PDC reduced ang-2 release from MRSA infected HUVEC cells as well.

Conclusion: It has been shown that interaction of ang-2 and its receptor Tie-2 induces endothelial permeability. Our data strongly indicate that peroxynitrite decomposition catalyst reduces/prevents microvascular hyperpermeability by modulating the ang-2/Tie-2 interaction. Inhibition of reactive nitrogen species should be considered for the future therapy for septic patients.