Pulmonary tissue factor mRNA expression during murine traumatic shock: effect of P-selectin blockade.

Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade and serves as a cell surface receptor and a specific cofactor for plasma factors VII/VIIa. Because there is evidence that TF is regulated by a P-selectin dependent gene, we examined TF mRNA expression in the lungs during murine traumatic shock in the presence and absence of recombinant soluble P-selectin glycoprotein ligand-1 (rsPSGL.Ig) by using ribonuclease protection assays. Moreover, we studied the level of TF mRNA expression in mice with their P-selectin gene deleted (P-selectin -/-). Our data show that TF mRNA was significantly increased (+143%; P < 0.001) in the lungs 2 h after trauma compared with control rats subjected to sham trauma, which exhibited reduced TF mRNA expression (-34%; P < 0.001) after systemic administration of rsPSGL.Ig. The expression of TF mRNA was also significantly decreased (-29%; P < 0.05) in the lungs of P-selectin -/- mice compared with wild-type control C57B16 mice. The present results provide evidence for a P-selectin-dependent mechanism that enhances TF gene expression in traumatic shock. The major support for this mechanism is that either blockade of P-selectin by rsPSGL.Ig or deletion of the P-selectin gene leads to significant decreases in TF mRNA expression in the lung. These results are consistent with the concept that TF interacting with P-selectin may play a significant role in the pathophysiology of trauma.

Pentalyte does not decrease heparinoid release but does decrease circulating thrombotic mediator activity associated with aortic occlusion-reperfusion in rabbits.

Hemorrhage and thrombosis are associated with major vascular and trauma surgery. Release of heparinoids and thrombotic mediators may contribute to these complications and have been described in rabbits after aortic occlusion-reperfusion. We hypothesized that the resuscitative fluid used could reduce heparinoid and thrombotic mediator release after aortic occlusion-reperfusion in rabbits as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). Anesthetized rabbits were administered lactated Ringer's solution (n = 8) or PentaLyte (n = 8) at reperfusion after 30 min of ischemia. Blood was obtained before ischemia and after 30 min of reperfusion for thromboelastography under four conditions: 1) unmodified sample, 2) platelet inhibition, 3) heparinase, and 4) platelet inhibition and heparinase. During reperfusion, unmodified samples demonstrated a significant increase in R and decrease in alpha and G that was not affected by PentaLyte. In the presence of heparinase, no significant fluid-specific thromboelastographic differences were noted. However, thrombotic mediator release (discerned by a decrease in R and an increase in alpha) during reperfusion in samples with platelet inhibition and heparinase was significantly attenuated by PentaLyte. PentaLyte administration does not decrease heparinoid release but does decrease thrombotic mediator release after aortic occlusion-reperfusion.

Tissue factor expression in vital organs during murine traumatic shock: role of transcription factors AP-1 and NF-kappaB.

BACKGROUND: Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. METHODS: Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. RESULTS: TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). CONCLUSIONS: These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

Endothelin-1, endothelin receptors and ecNOS gene transcription in vital organs during traumatic shock in rats.

Endothelin-1 (ET-1) is a vasoconstrictor peptide that may play an important role in the pathophysiology of severe trauma. We examined ET-1 gene expression in vital organs (i.e., heart, lungs, kidneys, liver and small intestine) during murine traumatic shock using ribonuclease protection assays. Our data show that ET-1 mRNA was significantly increased in the lungs two hours after trauma when compared with control anesthetized rats. There was also a significant increase in ET-1 transcripts occurring in the kidneys, heart and liver. During these experimental conditions, we also observed statistically significant increased endothelin type B (ET(B)) receptor mRNA expression in the lung, heart, liver, kidney and small intestine. Expression of endothelial constitutive nitric oxide synthase (ecNOS) gene, which is functionally coupled to ET(B) receptor, also was increased in vital organs during traumatic shock. Endothelin type A (ET(A)) receptor gene expression was slightly decreased in the lung, liver and small intestine. These results suggest that ET-1 and ET(B) mRNA expression are mainly increased in the lung and other vital organs and may play a functional role in the pathophysiology of murine traumatic shock.

Essential role of P-selectin in the initiation of the inflammatory response induced by hemorrhage and reinfusion.

Resuscitation from hemorrhage induces profound pathophysiologic alterations and activates inflammatory cascades able to initiate neutrophil accumulation in a variety of tissues. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that significant leukocyte-endothelium interactions occur very early in other forms of ischemia/reperfusion (i.e., splanchnic ischemia/reperfusion and traumatic shock) which are largely mediated by increased expression of the adhesion molecule, P-selectin, on the vascular endothelium. Here we postulated that increased endothelial expression of P-selectin in the microvasculature would play an essential role in initiating the inflammatory signaling of hemorrhagic shock. Using intravital microscopy, we found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti-P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-1 immunoglobulin, exhibited markedly attenuated leukocyte-endothelium interaction after hemorrhagic shock. Thus, activation of P-selectin protein on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Moreover, endogenous levels of PSGL-1 mRNA were significantly increased in the lung, liver, and small intestine of wild-type mice subjected to hemorrhagic shock. Since PSGL-1 promotes adhesive interactions largely through P-selectin expressed on the vascular endothelium, this result further supports the crucial role played by P-selectin in the recruitment of leukocytes during hemorrhagic shock.

Effect of recombinant soluble P-selectin glycoprotein ligand-1 on leukocyte-endothelium interaction in vivo. Role in rat traumatic shock.

-Traumatic shock induces profound pathophysiological alterations and initiates inflammatory reactions in many tissues, thus resulting in acute multiple organ damage (eg, intestine, pancreas, and liver). In the rat, Noble-Collip drum trauma increases P-selectin expression on the vascular endothelium as a result of loss of endothelium-derived NO. Here we postulated that blockade of the earliest steps in leukocyte adhesion (ie, leukocyte rolling) via administration of a recombinant soluble form of P-selectin glycoprotein ligand-1 (PSGL-1; the recombinant soluble form is rsPSGL.Ig) would attenuate selectin-mediated events observed in the rat during traumatic shock. Using intravital microscopy of the rat mesenteric microvasculature, we found that intravenous infusion of rsPSGL.Ig significantly inhibited leukocyte-endothelium interaction (ie, leukocyte rolling, adherence, and transmigration) induced by traumatic shock as well as by activation of the microvascular endothelium with 50 micromol/L NG-nitro-L-arginine methyl ester. Immunohistochemical detection of P-selectin on the mesenteric venular endothelial surface demonstrated that rsPSGL.Ig functionally neutralizes effects of P-selectin on the endothelial cell surface rather than attenuating P-selectin expression. Systemic administration of rsPSGL.Ig to traumatized rats prolonged survival time and survival rate, significantly attenuating ileal myeloperoxidase activity and significantly preserving mesenteric endothelial function. Furthermore, PSGL-1 mRNA levels were significantly increased in the blood of traumatized rats and were reduced after systemic administration of rsPSGL.Ig. Thus, soluble recombinant forms of PSGL-1 are able to ameliorate acute shock states by suppressing selectin-mediated leukocyte-endothelium interaction at both the functional and molecular levels.

Occlusion of the pilot tube in a Laser-Shield II endotracheal tube caused by methylene blue crystals: a case report.

The Laser-Shield II (Xomed-Treace, Jacksonville, FL) endotracheal tube is equipped with methylene blue crystals to aid in the prompt detection of tube cuff rupture. As the tube cuff is being inflated with saline, the crystals are supposed to dissolve fully in the saline, forming a solution that is readily visible in the event of cuff rupture. We describe a case of occlusion of the pilot balloon tube caused by undissolved methylene blue crystals, a situation that made it impossible to deflate the Laser-Shield II tube cuff. Our experience may aid in future modification of endotracheal tube design.

P-selectin is up-regulated in vital organs during murine traumatic shock.

Murine traumatic shock is associated with increased adherence of neutrophils to the vascular endothelium resulting in neutrophil infiltration and tissue damage. We examined the effects of trauma on the expression of the adhesion molecule P-selectin in several vital organs (i.e., heart, lungs, liver, kidneys, and small intestine) 2 h after induction of Noble-Collip drum trauma in anesthetized rats. Total RNA was extracted from these organs and P-selectin mRNA was quantified by RNase protection assays. P-selectin mRNA was significantly increased over control nontraumatized, anesthetized rats in all vital organs (P<0.05 or less), with the largest increase occurring in the lung (P<0.01). Immunohistochemical analysis showed increased expression of P-selectin protein in postcapillary venules of all vital organs after trauma. To further investigate the possible mechanisms of increased P-selectin mRNA transcription promoter activity during trauma, we quantified binding of proteins from nuclear extracts to the kappaB site (-218GGGGGTGACCC[-207]) of the P-selectin gene by electrophoretic mobility shift assay. We confirmed the results of NF-kappaB binding by demonstrating increases in p50 and p52, as well as decreases in IkappaB in cytoplasmic and nuclear extracts from the lungs of trauma rats by Western blotting. Increased activity of the transcription factor, nuclear factor kappaB (NF-kappaB), occurred in all vital organs of the trauma rats compared to sham-operated controls. Our findings suggest that severe trauma results in up-regulation of P-selectin at the transcriptional level, which is partly controlled by an NF-kappaB-responsive element in the region of the P-selectin promoter. This increased activation of NF-kappB binding may contribute to the widespread increases in P-selectin expression observed in several vital organs 2 h after trauma, which in turn may play a key role in the pathogenesis of traumatic shock.

Regulation of P-selectin expression in human endothelial cells by nitric oxide.

P-selectin translocation to the surface of endothelial cells is increased after exposure to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), resulting in increased endothelial adhesiveness. L-NAME (3 mM) was added to human cultured iliac vein endothelial cells for 1, 2, 4, and 6 h, and P-selectin mRNA expression was quantified by a ribonuclease protection assay. In parallel experiments, the NO donor, SPM-5185 (10 microM), was added to human iliac venous endothelial cells, and P-selectin mRNA expression quantified. P-selectin protein synthesis was quantified by Western blot analysis. L-NAME caused increased expression of P-selection RNA at 2-4 h, whereas D-NAME, the stereoisomer lacking NO synthase-inhibitory activity, had no effect. The stimulatory effect of L-NAME was reversed by addition of 3 mM L-arginine. SPM-5185 decreased P-selectin mRNA over the same time period (P < 0.02). The increased P-selectin mRNA expression induced by L-NAME was paralleled by an increase in P-selectin protein synthesis. The effects of SPM-5185 and L-arginine were also paralleled by decreases in P-selectin protein synthesis and in decreased adherence of human neutrophils to human iliac venous endothelial cells. The peak effect of inhibition of NO synthesis or addition of exogenous NO occurred at 2-4 h. These results suggest a regulatory effect of NO on endothelial P-selectin expression that modulates early leukocyte-endothelial cell interactions to preserve vascular homeostasis.

An association between lung lymph endothelin concentration and survival during endotoxemia in awake sheep.

Endotoxin stimulates synthesis of endothelin which can cause pulmonary and systemic vasoconstriction and bronchoconstriction. Prolonged endotoxemia in sheep results in dramatic increases in pulmonary and systemic vascular resistances in nonsurvivors compared with survivors. Experiments were conducted in 12 conscious sheep (seven survivors, five nonsurvivors) to determine if synthesis of endothelin might contribute to the pathophysiology in nonsurvivors. Endotoxin was infused at 10 ng/min/kg for 12 h followed by a 4 h recovery in survivors. Lung lymph endothelin concentration peaked at 38.7 +/- 5.8 pg/mL during the endotoxin infusion in survivors compared with a peak of 128.7 +/- 33.0 pg/mL (p < .05) in nonsurvivors. Cardiac output was lower, and systemic and pulmonary vascular resistances and the Aa gradient in PO2 were significantly greater in nonsurvivors compared with survivors. These variables are those likely to be affected by increased circulating endothelin concentrations which suggests that endothelin contributes to early mortality during prolonged endotoxemia.

Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle.

We examined the effect of several nitric oxide (NO) donors, authentic NO gas, and L-arginine in isolated cat and rat papillary muscles. We did not observe significant inotropic effects in response to any NO donor (ie, SPM-5185, C87-3754, and S-nitroso-N-acetylpenicillamine [SNAP]) from 1 nmol/L to 100 mumol/L. Similarly, authentic NO, at concentrations far in excess of those that maximally dilate the coronary vasculature (ie, 500 nmol/L), also failed to exert a detectable inotropic effect in these preparations. However, in the presence of 5 mumol/L norepinephrine, 500 nmol/L NO exerted a 12 +/- 3% decrease in isolated rat papillary muscle contractility (P < .05). Addition of L-arginine up to 25 mmol/L exerted no inotropic effects in isolated rat papillary muscles. However, at 50 mmol/L, L-arginine decreased contractile force by 21 +/- 4% (P < .01). On further examination, the negative inotropic effect of 50 mmol/L L-arginine appeared to be nonspecific, since the inactive stereoisomer, D-arginine, at 50 mmol/L exerted the same effect. Further studies in isolated adult rat cardiac myocytes elicited similar results, in that 50 mmol/L of L- and D-arginine equally decreased contraction amplitude and the underlying cytosolic calcium transient. Moreover, 500 nmol/L of the NO donor SPM-5185 only modestly decreased contraction amplitude or intracellular calcium in isolated rat cardiac myocytes. These results indicate that administration of physiological concentrations of exogenous NO does not acutely depress the inotropic state of the rat or cat heart to a physiologically significant extent.(ABSTRACT TRUNCATED AT 250 WORDS)