Toll-like receptor 4 contributes to microvascular inflammation and barrier dysfunction in thermal injury.

Systemic and microvascular inflammation plays a key role in the development of multiple organ failure after infection, sepsis, and traumatic injury. Toll-like receptors (TLRs) regulate host responses to pathogens and sterile, injury-associated inflammatory responses. We investigated whether TLR-4 contributes to microvascular dysfunction during thermal injury in vivo in anesthetized wild-type or TLR-4 (-/-) mice receiving either a 25% total body surface area full-thickness scald burn or sham treatment on the dorsal skin. Using intravital microscopy, we assessed the hemodynamics and leukocyte dynamics in the mesenteric microvasculature as representative of the splanchnic microcirculation at a site remote from the burn wound. The transvascular flux of fluorescein isothiocyanate-albumin across mesenteric venules was measured as an indicator of microvascular permeability. Furthermore, cultured microvascular endothelial cell models were used to evaluate the endothelial-specific mechanisms involved in TLR-4-mediated barrier dysfunction. The results showed significantly elevated microvascular permeability in wild-type mice after burn, whereas this response was markedly attenuated in TLR-4 (-/-) mice. Burn injury also increased leukocyte adhesion in mesenteric venules of wild-type mice, and a blunted leukocyte response was seen in the TLR-4 mice. Treatment of endothelial cell monolayers with burn plasma induced a rapid reduction in the transendothelial electrical resistance measured by electric cell-substrate impedance sensing, indicative of endothelial cell-cell adhesive barrier dysfunction. Reducing expression of TLR-4 with siRNA treatment attenuated this response. Taken together, these data indicate that TLR-4 plays an important role in microvascular leakage and leukocyte adhesion under the inflammatory condition associated with nonseptic thermal injury.

A role for long chain myosin light chain kinase (MLCK-210) in microvascular hyperpermeability during severe burns.

Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunction during trauma. Previous studies suggest the involvement of myosin light chain (MLC) phosphorylation-triggered endothelial contraction in the development of microvascular hyperpermeability. Myosin light chain kinase (MLCK) plays a key role in the control of MLC-phosphorylation status; thus, it is thought to modulate barrier function through its regulation of intracellular contractile machinery. The aim of this study was to further investigate the endothelial mechanism of MLC-dependent barrier injury in burns, focusing on the long isoform of MLCK (MLCK-210) that has recently been identified as the predominant isoform expressed in vascular endothelial cells. An MLCK-210 knockout mouse model was subjected to third-degree scald burn covering 25% total body surface area. The mesenteric microcirculation was observed using intravital microscopy, and the microvascular permeability was assessed by measuring the transvenular flux of fluorescein isothiocyanate-albumin. In a separate experiment, in vivo mesenteric hydraulic conductivity (Lp) was measured using the modified Landis technique. The injury caused a profound microvascular leakage, as indicated by a 2-fold increase in albumin flux and 4-fold increase in Lp at the early stages, which was associated with a high mortality within the 24-h period. Compared with wild-type control, the MLCK-210-deficient mice displayed a significantly improved survival with a greatly attenuated microvascular hyperpermeability response to albumin and fluid. These results provide direct evidence for a role of MLCK-210 in mediating burn-induced microvascular barrier injury and validate MLCK-210 as a potential therapeutic target in the treatment of burn edema.

Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism.

Vascular endothelial growth factor (VEGF)-C plays an important role in lymphangiogenesis; however, functional responses of lymphatic vessels to VEGF-C have not been characterized. We tested the hypothesis that VEGF-C-induced activation of VEGF receptor (VEGFR)-3 increases lymphatic pump output. We examined the in vivo pump activity of rat mesenteric collecting lymphatics using intravital microscopy during basal conditions and during treatment with 1 nM recombinant VEGF-C, the selective VEGFR-3 agonist VEGF-Cys(156)Ser mutation (C156S; 1 nM), or 0.1 nM VEGF-A. Their specific responses were also analyzed during selective inhibition of VEGFR-3 with MAZ-51. Contraction frequency, end-diastolic diameter, end-systolic diameter, stroke volume index, pump flow index, and ejection fraction were evaluated. We also assessed arteriolar diameter and microvascular extravasation of FITC-albumin. The results show that both VEGF-C and VEGF-C156S significantly increased contraction frequency, end-diastolic diameter, stroke volume index, and pump flow index in a time-dependent manner. VEGF-A caused a different response characterized by a significantly increased stroke volume after 30 min of treatment. MAZ-51 (5 muM) caused tonic constriction and decreased contraction frequency. In addition, 0.5 and 5 muM MAZ-51 attenuated VEGF-C- and VEGF-C156S-induced lymphatic pump activation. VEGF-A caused vasodilation of arterioles, whereas VEGF-C and VEGF-C156S did not significantly alter arteriolar diameter. Also, VEGF-A and VEGF-C caused increased microvascular permeability, whereas VEGF-C156S did not. Our results demonstrate that VEGF-C increases lymphatic pumping through VEGFR-3. Furthermore, changes in microvascular hemodynamics are not required for VEGFR-3-mediated changes in lymphatic pump activity.

Validity of arterial and mixed venous oxygen saturation measurements in a canine hemorrhage model after resuscitation with varying concentrations of hemoglobin-based oxygen carrier.

UNLABELLED: In this study, we evaluated the validity of saturation measurements in mixed venous and arterial blood during posthemorrhagic anemia and resuscitation with varying levels of hemoglobin-based oxygen carrier (Hemoglobin glutamer-200 [bovine]; Oxyglobin [Hb-200]). Nineteen anesthetized, splenectomized, mixed-breed dogs were anesthetized (two were excluded from the data because they did not survive the exsanguination, supporting the validity of the model). Their pulmonary arteries were cannulated with the Abbott QVUE Oximetrix 3 catheter. An 18-gauge catheter was placed in the femoral artery, and a reusable Nellcor probe was applied to the tongue. Mixed venous and arterial samples were drawn at baseline, after 40% hemorrhage (to keep arterial pressure at 50 mm Hg), and postresuscitation with 30 mL/kg of 6% hetastarch in lactated Ringer's solution (n = 4), 10 mL/kg of Hb-200, 20 mL/kg of hetastarch (n = 6), 20 mL/kg of Hb-200, and 10 mL/kg of hetastarch (n = 7). Samples were compared with oxygen content from the LEXO2CON-K oxygen analyzer, and oxygen content was calculated for all values from the monitors. Results were compared by using analysis of variance. There was good correlation (0.97 > or = r > or = 0.92) for the measured versus calculated hemoglobin oxygen saturation values at baseline. After resuscitation, the correlation between calculated and measured values of oxygen content was significantly smaller for all tested instruments. The values of oxygen content calculated from the oxygen saturation monitor and from the oximetric pulmonary artery can deviate by as much as 20% from directly measured values. We conclude that the administration of this oxygen therapeutic may interfere with the values of some monitors. IMPLICATIONS: This study evaluated oxygen saturation monitors in a canine model of acute blood loss and resuscitation with a blood substitute and found that these may interfere with the monitors' results in a dose-dependent way.