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Volume 15 Supplement 3

Sepsis 2011

Interplay between angiopoietin-2, vascular endothelial growth factor and peroxynitrite is an important determinant of vascular hyperpermeability during methicillin-resistant Staphylococcus aureus sepsis

Introduction

We have reported that nitric oxide (NO) production and microvascular hyperpermeability were significantly higher in septic sheep with methicillin-resistant Staphylococcus aureus (MRSA) than with Pseudomonas aeruginosa. We hypothesize that peroxynitrite, a byproduct of NO, causes vascular hyperpermeability in MRSA sepsis via promoting vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2). The hypothesis was tested, using both a well-established ovine sepsis model and cultured human umbilical endothelial cells (HUVECs).

Methods

Female ewes were chronically instrumented with multiple catheters and live MRSA (USA300, 1011 CFU) was instilled into the both lungs by bronchoscope under deep isoflurane anesthesia. The sheep were then randomly allocated to control and treated (nonspecific NOS inhibitor L-NAME, 25 mg/kg, i.v., every 12 hours) groups and monitored for 24 hours for cardiopulmonary hemodynamics. The cells were challenged with 105 CFU of live MRSA or 50 μM peroxynitrite and co-incubated with or without L-NAME, peroxynitrite scavenger FeTMPyP, Tie-2 and Ang-2 antibody, and VEGF and its antibody. At different times after the treatment, the permeability was measured by quantifying the amount of FITC-Dextran that passed through the confluent HUVEC monolayer (n = 4). Ang-2 mRNA was determined by RT-PCR in those cells with or without treatment as well (n = 4). Statistical analysis: one-way ANOVA (Bonferroni).

Results

In vivo, L-NAME significantly reduced MRSA-induced fluid accumulation and requirement, as well as expression of VEGF. HUVEC permeability was time-dependently increased following MRSA co-incubation, reaching a plateau at 2 and 4 hours. These permeability changes (73 ± 4 RFUs) were significantly (P < 0.001) inhibited by 1 mM L-NAME (28 ± 1), 5 μM FeTMPy (34 ± 2), 5 μg/ml Tie-2 antibody (32 ± 2), and 5 μg/ml Ang-2 antibody (30 ± 1). In HUVECs, the Ang-2 mRNA was time-dependently (picks at 30 minutes) and dose-dependently increased by peroxynitrite (highest at 50 μM). Treatment of HUVECs with 5 μM VEGF augmented the MRSA-induced Ang-2 mRNA increases. The latter was reversed with FeTMPyP and 5 μM VEGF antibody.

Conclusion

Ang-2 and VEGF, Tie-2 receptor, NO and its byproduct peroxynitrite play an important role in MRSA-induced vascular hyper-permeability. The results strongly suggest that peroxynitrite increases vascular hyper-permeability by promoting Ang-2 release through stimulating the VEGF expression during MRSA-induced Gram-positive sepsis.

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Correspondence to P Enkhbaatar.

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Enkhbaatar, P., Zhu, Y., Traber, L. et al. Interplay between angiopoietin-2, vascular endothelial growth factor and peroxynitrite is an important determinant of vascular hyperpermeability during methicillin-resistant Staphylococcus aureus sepsis. Crit Care 15, P39 (2011). https://doi.org/10.1186/cc10408

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Keywords

  • Nitric Oxide
  • Vascular Endothelial Growth Factor
  • Peroxynitrite
  • Vascular Hyperpermeability
  • Vascular Endothelial Growth Factor Antibody