Role of p-selectin in platelet sequestration in pulmonary capillaries during endotoxemia.

BACKGROUND: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. METHODS: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. RESULTS: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. CONCLUSION: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction.

Role of PARP on iNOS pathway during endotoxin-induced acute lung injury.

OBJECTIVE: Excessive nitric oxide (NO) and especially peroxynitrite may cause pulmonary tissue damage, e.g., through lipid peroxidation and/or exhaustion of cellular energy depletion induced by activation of poly (ADP-ribose) polymerase (PARP). Furthermore, PARP seems to aggravate tissue destruction by regulating the expression of respective genes. DESIGN: Prospective animal study. SETTING: University research laboratory. INTERVENTION: We investigated the effect of competitive PARP inhibition by 3-aminobenzamide (3-AB) on the pulmonary iNOS pathway after infusion of lipopolysaccharide (LPS). MEASUREMENTS AND RESULTS: The pretreatment of rabbits with 3-AB attenuated the LPS-induced iNOS mRNA and protein expression analyzed by RT-PCR and Western blot, and plasma nitrite concentrations quantified by Griess reaction (71+/-6%, 93+/-6% vs baseline). Electromobility shift assay showed an enhanced NF-kappaB and attenuated AP-1 activation after 3-AB vs LPS alone. Lipid peroxidation determined as levels of thiobarbituric acid reactive substances in plasma and lung tissue was reduced by 50% in the LPS+3-AB in comparison to LPS alone. Simultaneously, 3-AB was able to inhibit correspondingly the LPS-induced extravasation of gold-labeled albumin and increase of alveolo-arterial oxygen difference. CONCLUSION: PARP regulates the pulmonary NO pathway during endotoxemia via AP-1 and not NF-kappaB. Thus, pharmacological inhibition of PARP might be an effective intervention to prevent endotoxin-induced lung injury, interrupting the vicious circle of NO production and PARP activation.

Platelet-endothelial cell interaction in pulmonary micro-circulation: the role of PARS.

Accumulation of platelets might contribute to acute lung injury during systemic inflammation. The aim of the study was to elucidate the role of the poly (ADP-ribose) synthetase, a nucleotide-polymerizising enzyme, in mediation of platelet-endothelial cell interaction through regulation of adhesion molecules within the pulmonary microcirculation during endotoxemia. We used in vivo fluorescence microscopy to quantify kinetics of fluorescently labeled erythrocytes and platelets in rabbit pulmonary arterioles and venules. Six hours after onset of endotoxin infusion we observed a massive interaction of platelets with the microvascular endothelial cells, whereas under control conditions, no platelet sequestration was measured. An up-regulation of P- and E-selectin was detected in lung tissue following endotoxin infusion by immunohistochemistry and Western blot analysis. Blockade of endothelial P-selectin with fucoidin resulted in a reduction of the endotoxin-induced platelet-endothelial cell interaction. Inhibition of poly (ADP-ribose) synthetase by 3-aminobenzamide inhibited the endotoxin-induced expression of endothelial P- and E-selectin and the subsequent recruitment of platelets. In summary, we provide first in vivo evidence that platelets accumulate in pulmonary microcirculation following endotoxemia. Poly (ADP-ribose) synthetase seems to mediate this platelet-endothelial cell interaction via P- and E-selectin expressed on the surface of microvascular endothelium.

Role of poly(ADP-ribose) synthetase in pulmonary leukocyte recruitment.

During systemic inflammation, recruitment and activation of leukocytes in the pulmonary microcirculation may result in a potentially life-threatening acute lung injury. We elucidated the role of the poly(ADP-ribose) synthetase (PARS), a nucleotide-polymerizing enzyme, in the regulation of leukocyte recruitment within the lung with regard to the localization in the pulmonary microcirculation and in correlation to hemodynamics in the respective vascular segments and expression of intercellular adhesion molecule 1 during endotoxemia. Inhibition of PARS by 3-aminobenzamide reduced the endotoxin-induced leukocyte recruitment within pulmonary arterioles, capillaries, and venules in rabbits as quantified by in vivo fluorescence microscopy. Microhemodynamics and thus shear rates in all pulmonary microvascular segments remained constant. Simultaneously, inhibition of PARS with 3-aminobenzamide suppressed the endotoxin-induced adhesion molecules expression as demonstrated for intercellular adhesion molecule 1 by immunohistochemistry and Western blot analysis. We confirmed this result with the use of PARS knockout mice. The inhibitory effect of 3-aminobenzamide on leukocyte recruitment was associated with a reduction of pulmonary capillary leakage and edema formation. We first provide evidence that PARS activation mediates the leukocyte sequestration in pulmonary microvessels through upregulation of adhesion molecules. As reactive oxygen species released from leukocyte are supposed to cause an upregulation of adhesion molecules we conclude that PARS inhibition contributes to termination of this vicious cycle and inhibits the inflammatory process.