Perturbation of endothelial junction proteins by Staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin.

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell-cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus alpha-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus alpha-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, alpha-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked alpha-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after alpha-toxin application. Although no RhoA activation was noted after endothelial alpha-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.

Adrenomedullin reduces Staphylococcus aureus alpha-toxin-induced rat ileum microcirculatory damage.

OBJECTIVE: Increased microvascular permeability and perfusion mismatch are hallmarks of sepsis or septic shock. The intestinal mucosa is very sensitive to tissue hypoxia. Intestinal mucosa dysfunction may allow translocation of bacteria and their products, thereby perpetuating sepsis and inflammation. Staphylococcus aureus alpha-toxin is a major pathogenicity determinant of this bacterium, provoking cardiovascular collapse. Current evidence suggests that the endogenous peptide adrenomedullin stabilizes circulatory homeostasis in systemic inflammatory response. Using alpha-toxin as a well-defined strong initiator of an inflammatory reaction, we tested the hypothesis that exogenously applied adrenomedullin stabilizes gut microcirculation. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated, perfused ileum from male Sprague-Dawley rats and human umbilical vein endothelial cells. INTERVENTIONS: Administration of S. aureus alpha-toxin before or after infusion of adrenomedullin. MEASUREMENTS AND MAIN RESULTS: Injection of a bolus of 1 microg of alpha-toxin in the superior mesenteric artery in a constant-flow, blood-perfused preparation of rat ileum increased perfusion pressure and relative hemoglobin concentration and decreased mucosal hemoglobin oxygen saturation. Continuous infusion of adrenomedullin (0.1 micromol/L) significantly reduced these alpha-toxin-related effects. Severe microvascular hyperpermeability observed in alpha-toxin-exposed ileum was abolished by adrenomedullin pretreatment. In addition, adrenomedullin blocked alpha-toxin-induced endothelial myosin light chain phosphorylation, endothelial cell contraction, and subsequent loss of endothelial barrier function in vitro. Treatment of alpha-toxin (infusion of 0.05 microg/mL)-exposed ileum with adrenomedullin (0.1 micromol/L) started 10 mins after onset of toxin application also significantly reduced superior mesenteric artery pressure and permeability increase. CONCLUSIONS: In summary, these data suggest that exogenous adrenomedullin protects ileum by reducing alpha-toxin-induced microcirculatory disturbances and by stabilizing endothelial barrier function.

Porphyromonas gingivalis strain-dependent activation of human endothelial cells.

Porphyromonas gingivalis is an important bacterium involved in periodontal diseases. Colonization by periodontopathogens has been associated with severe local inflammatory reactions in the connective tissue. In this study we characterized P. gingivalis-mediated infection and activation of human umbilical vein endothelial cells by using two strains of different virulence capacities, strains ATCC 53977 and DSMZ 20709. Both strains were able to adhere to and infect endothelial cells with an infection rate of 0.48% for ATCC 53977 and 0.007% for DSMZ 20709. The triggering of two signal transduction pathways in P. gingivalis-infected endothelial cells was demonstrated for both strains, with a rapid increase of p38 mitogen-activated protein kinase phosphorylation and a more delayed degradation of IkappaBalpha, followed by nuclear translocation of NF-kappaB. In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1). Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 DeltafimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT). Thus, two P. gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation.