Updated concepts on the pathophysiology and the clinical management of trauma hemorrhage and coagulopathy / 中华创伤杂志(英文版)

Uncontrolled hemorrhage and subsequent trauma-induced coagulopathy (TIC) are still the principle causes for preventable death after trauma and early detection and aggressive management have been associated with reduced mortality. Despite increasing knowledge about trauma resuscitation, best practice to treat this newly defined entity is still under debate. A synopsis of best current knowledge with reference to the updated European trauma guideline on the management of severe trauma hemorrhage and TIC is presented. The implementation of evidence-based local protocols and algorithms including clinical quality and safety management systems together with parameters to assess key measures of bleeding control and outcome is advocated.

Effect of salvianolic acid B on TNF-α induced cerebral microcirculatory changes in a micro-invasive mouse model / 中华创伤杂志(英文版)

<p><b>PURPOSE</b>To investigate the effects of salvianolic acid B (SAB) on tumor necrosis factor a (TNF-α) induced alterations of cerebral microcirculation with a bone-abrading model.</p><p><b>METHODS</b>The influences of craniotomy model and bone-abrading model on cerebral microcirculation were compared. The bone-abrading method was used to detect the effects of intracerebroventricular application of 40 μg/kg·bw TNF-α on cerebral venular leakage of fluorescein isothiocyanate (FITC)- albulmin and the rolling and adhesion of leukocytes on venules with fluorescence tracer rhodamine 6G. The therapeutical effects of SAB on TNF-α induced microcirculatory alteration were observed, with continuous intravenous injection of 5 mg/kg·h SAB starting at 20 min before or 20 min after TNF-α administration, respectively. The expressions of CD11b/CD18 and CD62L in leukocytes were measured with flow cytometry. Immunohistochemical staining was also used to detect E-selectin and ICAM-1 expression in endothelial cells.</p><p><b>RESULTS</b>Compared with craniotomy method, the bone-abrading method preserved a higher erythrocyte velocity in cerebral venules and more opening capillaries. TNF-α intervention only caused responses of vascular hyperpermeability and leukocyte rolling on venular walls, without leukocyte adhesion and other hemodynamic changes. Pre- or post-SAB treatment attenuated those responses and suppressed the enhanced expressions of CD11b/CD18 and CD62L in leukocytes and E-selectin and ICAM-1 in endothelial cells induced by TNF-α.</p><p><b>CONCLUSIONS</b>The pre- and post-applications of SAB during TNF-α stimulation could suppress adhesive molecular expression and subsequently attenuate the increase of cerebral vascular permeability and leukocyte rolling.</p>

Role of RAGE in lipopolysaccharide-induced cytoskeletal changes in mouse pulmonary microvascular endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate lipopolysaccharide (LPS)-induced changes of cytoskeletal filamentous actin in primary isolated pulmonary microvascular endothelial cells (PMVECs) from wild-type and RAGE knock-out mouse.</p><p><b>METHODS</b>The lungs of wild-type and RAGE knock-out mice were digested with collagenase type I to obtain endothelial cells purified by anti-CD31-coupled magnetic beads. The PMVEC identified by factor VIII labeling were stimulated with LPS at different concentrations and the changes of filamentous actin were observed by confocal microscopy.</p><p><b>RESULTS</b>The cultured primary cells showed typical endothelial cell phenotype as examined with factor VIII labeling. LPS stimulation caused rearrangement of the cytoskeletal filament F-actin in wild-type mouse PMVECs with stress fiber formation, but such changes were not obvious in RAGE knock-out mouse PMVECs.</p><p><b>CONCLUSION</b>Mouse PMVECs of a high purity can be obtained by immune magnetic beads. RAGE is involved in LPS-induced destruction of mouse PMVEC cytoskeletons.</p>

Barrier stabilizing mediators in regulation of microvascular endothelial permeability / 中华创伤杂志(英文版)

Increase of microvascular permeability is one of the most important pathological events in the pathogenesis of trauma and burn injury. Massive leakage of fluid from vascular space leads to lose of blood plasma and decrease of effective circulatory blood volume, resulting in formation of severe tissue edema, hypotension or even shock, especially in severe burn injury. Fluid resuscitation has been the only valid approach to sustain patient's blood volume for a long time, due to the lack of overall and profound understanding of the mechanisms of vascular hyperpermeability response. There is an emerging concept in recent years that some so-called barrier stabilizing mediators play a positive role in preventing the increase of vascular permeability. These mediators may be released in response to proinflammatory mediators and serve to restore endothelial barrier function. Some of these stabilizing mediators are important even in quiescent state because they preserve basal vascular permeability at low levels. This review introduces some of these mediators and reveals their underlying signaling mechanisms during endothelial barrier enhancing process.

Effects of Rho/ROCK signal pathway on AGEs-induced morphological and functional changes in human dermal microvascular endothelial cells / 生理学报

The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.

Relationship between the endothelial barrier and vascular permeability after burns and its mechanism / 中华烧伤杂志

Massive burn trauma is characterized by hypovolemic shock induced by the loss of plasma from vessels. The major reasons for this systemic microvascular leakage in burns include an increase in vascular permeability triggered by inflammatory mediators and the increase of vascular hydrostatic pressure caused by vessel dilation. The maintenance of normal vascular permeability depends on the integrity of endothelial barrier function regulated by the interaction of intracellular junctions, cell-matrix adhesion and the cytoskeleton contractile force. This review summarizes some recent discovery in endothelial mechanisms during burn-induced vascular hyperpermeability.

KCNE2 modulates the function of Kv4.3 channel / 南方医科大学学报

<p><b>OBJECTIVE</b>To understand the role of KCNE2 in functional regulation of Kv4.3, the major alpha subunit of transient outward current (I(to)) in human heart.</p><p><b>METHODS</b>The cDNAs of Kv4.3 or Kv4.3 plus KCNE2 were transfected into COS-7 cells and 24-36 h after the transfection, the channel proteins were expressed in the surface membrane of the cells and the channel currents were recorded with patch-clamp technique in whole-cell mode.</p><p><b>RESULTS</b>KCNE2 played an important role in modulating the channel function. The recorded current density was decreased in cells co-expressing KCNE2 and Kv4.3 to 152.96-/+33.71 pA/pF (n=16) as compared with Kv4.3-expressing cells with a mean current density of 375.13-/+112.87 pA/pF (n=11). At the recording voltage of 60 mV, KCNE2 increased the time to peak (TTP) of the current. TTP in only Kv4.3-expressing cells was 4.82-/+0.32 ms (n=11), significantly shorter than the TTP of 20.41-/+2.13 ms (n=16) in cells co-expressing Kv4.3 and KCNE2 (P<0.05). In the presence of KCNE2, the voltage-dependent inactivation of Kv4.3 showed a positive shift. The voltage of half maximum inactivation (V(0.5)) was decreased significantly from -53.62-/+1.24 mV (n=8) in Kv4.3 group to -46.58-/+1.6 mV (n=10) in KCNE2 co-expression group (P<0.05). KCNE2 accelerated the recovery of the channel from inactivation, reducing the recovery time constant (tau) from 193.43-/+17.98 ms to 137.71-/+18.29 ms.</p><p><b>CONCLUSION</b>KCNE2 might serve as an important beta subunit and play a role in the regulation of I(to) function in human heart.</p>

Mechanism of advanced glycation end products-induced hyperpermeability in endothelial cells / 生理学报

The purpose of the present study was to investigate the effects of advanced glycation end products (AGEs) modified protein on the permeability of endothelium monolayers and morphological changes of actin cytoskeleton. The roles of receptor for AGEs (RAGE), oxidant stress and the activation of p38 MAPK pathway in this pathological procedure were elucidated. Human umbilical vein endothelial cells (HUVECs)-derived cell line (ECV304) were incubated with AGEs modified human serum albumin (AGE-HSA) in concentrations of 12.5, 25, 50, and 100 microg/ml respectively, for 2, 4, 8, 12 and 24 h. As control, HSA of the same concentration was administered to cells. Then TRITC-albumin was added to evaluate Pa value that reflects the permeability of endothelial monolayer. Furthermore, to visualize the morphological changes of actin cytoskeleton, the treated cells were incubated with rhodamine-phalloidin to stain F-actin. The results showed that the trans-endothelial membrane flux of albumin was significantly increased in a concentration- and time-dependent manner upon the stimulation of AGE-HSA, accompanying with actin reorganization. The blockage of AGE and RAGE binding with anti-RAGE IgG and the pharmacological inhibition of NADPH oxidase or p38 MAP kinase greatly attenuated the AGE-induced hyperpermeability response, respectively. These results indicate that RAGE, NADPH oxidase and p38 MAPK are possibly involved in the mediation of AGEs-induced barrier dysfunction and actin cytoskeleton reorganization in endothelial cells.

Role of Rho kinase in reorganization of the vascular endothelial cytoskeleton induced by rat burn serum / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the changes in endothelial cytoskeletal reorganization and the role of Rho in the signal transduction pathway.</p><p><b>METHODS</b>ECV304 cells were cultured and randomly divided into following groups: i.e. sham (with normal rat serum treatment), burn (with burn rat serum treatment), Y (with 30 micromol/L Rho kinase inhibitor Y-27632 treatment), burn plus Y (pretreatment of cells with burn serum before treated with 30 micromol/L Y-27632), Y plus burn (pretreatment of cells with Y-27632 for 1 hour before treated with burn serum), LPA (with normal rat serum and 13 micromol/L LPA), and LPA plus Y (pretreatment of cells with LPA before treated with Y-27632) groups. The indices were examined at 6, 7 and 8 posttreatment hours (PTH) in all groups except in Y group. The endothelial morphology was observed with HE staining. Endothelial cytoskeleton was observed by dual-fluorescence labeling of filamenta (F) with Rhodamine-phalloidin and monomer (G) with oregon green labeled DNAase. The actin content in the cells in all groups was measured with flow cytometry.</p><p><b>RESULTS</b>In sham and control group, the cells were in fusiform or polygonal shape, with satisfactory growth; filamentous actin (F-actin) was mainly distributed in the peripheral site of the cytoplasm and formed peripheral filamental band. The cells became confluent to form a single layer with reticular structure. Globular actin (G-actin) was concentrated in the nucleus and per nucleus. In burn group, after 6 hours of burn serum treatment, the ability of cells to adhere to vessel wall was weakened, and a striking reorganization of the actin cytoskeleton and the formation of the stress fibers were found. Furthermore, the fluorescent intensity of the peripheral filament bands was weakened, and dispersed actin monomers were seen in the cytoplasm. This reaction was enhanced along with elapse of stimulation time. In burn plus Y or Y plus burn group, the cells grew and adhered well to the wall of culture vessel. The distribution of the filamentous actin was the same as the sham group, while the stress fiber decreased in amount obviously. The structure of globular actin was condensed with little G-actin in the cytoplasm. The changes in actin cytoskeleton in LPA group was similar to that in burn group. The effects of LPA on actin reorganization could also be reversed by Y-27632. The content of F-actin in burn group at 6 PTH (0.63 +/- 0.07) was lower than that in sham group (0.75 +/- 0.08), while the content of G-actin in burn group (1.28 +/- 0.27) was higher than that in sham group (1.16 +/- 0.16, P > 0.05).</p><p><b>CONCLUSION</b>Burn serum induces vascular endothelial actin cytoskeleton reorganization in endothelial cells via the Rho-dependent signal pathway. Similar to the effect of LPA, this effect could be reversed by Y-27632.</p>

Effects of lipopolysaccharide on actin reorganization and actin pools in endothelial cells / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304.</p><p><b>METHODS</b>F-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry.</p><p><b>RESULTS</b>F-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool.</p><p><b>CONCLUSIONS</b>LPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.</p>

Effect of protein kinase on endothelial cytoskeleton induced by septic shock / 中华外科杂志

<p><b>OBJECTIVE</b>To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock.</p><p><b>METHODS</b>Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with LPS or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 was used to pretreat the endothelial cells before the administration of LPS or PKG activator 8-Br-cGMP.</p><p><b>RESULTS</b>Exposure to LPS for 5, 10, 30 and 60 minutes led to a rapid time-dependent increase in endothelial PKG activity (P < 0.01 compared to the blank) and the polar distribution of intracellular filamentous actin and preincubation with KT5823 abolished these effects. 8-Br-cGMP was similar to LPS.</p><p><b>CONCLUSIONS</b>The results suggested that LPS can mediate PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably induce the endothelial hyperpermeability after septic shock.</p>