Jiajun Tang

Protective effects of ulinastatin on pancreatic and renal damage in rats following early scald injury.

Organ protection is a routine therapy in severe burn/scald injuries, and damage following early scald injury was not been fully elucidated. Our aim was to verify the beneficial effects of ulinastatin on pancreatic and renal damage associated with scald injury. Lewis rats were subjected to 30% total body surface area (TBSA) scald injury, and were randomly divided into a burn control (S group) and an ulinastatin-treated group (U group). Pancreatic malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined. Serum amylase, serum creatinine (Scr) and blood urea nitrogen (BUN) were identified and the kidneys were examined histologically with immunohistochemistry (IHC) as well for the MHC class I chain-related antigen A (MICA) and Bcl-2 at 0, 1, 6, 12, 18, 24, 48 and 72 h after the injury. Ulinastatin decreased MDA levels and ameliorated the down-regulation of SOD activity. MICA was up-regulated after the scald, and this up-regulation was greatly diminished by ulinastatin. Bcl-2 was up-regulated after the scald, especially in the U group. From 24 to 72 h, in comparison with the U group, higher levels of BUN, Scr and serum amylase were observed in the S group which were all lower than the common upper limits. Our results demonstrated that pancreatic and renal damage associated with autoimmunity and oxidant attack occurred after severe scald. Ulinastatin exhibits significant protective effects on these effects.

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.

Guanine-nucleotide exchange factor H1 mediates lipopolysaccharide-induced interleukin 6 and tumor necrosis factor α expression in endothelial cells via activation of nuclear factor κB.

ABSTRACT The development of sepsis is multifactorial. Tissue damage and organ dysfunction may be caused not only by the microorganisms but also by the inflammatory mediators released in response to the infection. Interleukin 6 (IL-6) and tumor necrosis factor &agr; (TNF-&agr;) levels in serum are well known to be upregulated in humans with sepsis and can be used to predict outcome. Using human umbilical vein endothelial cells, we analyzed the role of guanine-nucleotide exchange factor H1 (GEF-H1) on lipopolysaccharide (LPS)–dependent IL-6/TNF-&agr; expression in endothelial cells. Lipopolysaccharide upregulated IL-6 secretion in a dose- and time-dependent manner. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced nuclear factor &kgr;B (NF-&kgr;B) p65 phosphorylation, IL-6/TNF-&agr; messenger RNA (mRNA), and IL-6/TNF-&agr; protein productions. Guanine-nucleotide exchange factor H1 protein expression remained on a high level among 1 to 9 h in response to LPS challenge of endothelial cells. Inhibition of GEF-H1 by specific small interfering RNA or inactivation of Rho-associated kinase with Y-27632 not only significantly reduced LPS-induced p38 and extracellular signal–regulated kinase 1/2 (ERK1/2) activities but also blocked LPS-induced NF-&kgr;B translocation and activation, thereby inhibiting IL-6/TNF-&agr; mRNA and protein productions. Furthermore, SB203580 (p38 inhibitor) but not PD98059 (ERK1/2 inhibitor) blocked LPS-induced NF-&kgr;B activation; however, both inhibitors significantly suppressed IL-6/TNF-&agr; mRNA and protein expression. In summary, our data suggest that LPS rapidly upregulates GEF-H1 expression. Activated Rho-associated kinase by GEF-H1 subsequently activates p38 and ERK1/2, thereby increasing IL-6/TNF-&agr; expression in endothelial cells. P38 and ERK1/2 regulate LPS-induced IL-6/TNF-&agr; expression through an NF-&kgr;B–dependent manner and an NF-&kgr;B–independent manner, respectively.

Specific inhibition of AQP1 water channels in human pulmonary microvascular endothelial cells by small interfering RNAs.

Background: Aquaporin (AQP)-1 is expressed in most microvasculature endothelial cells forming water channels that play major roles in a variety of physiologic processes. Our aim was to investigate the regulatory functions of AQP1 on trancellular and paracellular permeability. Methods: We designed, synthesized, and used small interfering RNAs (siRNAs) selective for AQP1 and investigated their effectiveness in altering AQP1-mediated permeability in human pulmonary microvascular endothelial cells. Results: Twenty-four hours after transfection of ECs with siRNAs targeting two different regions of the AQP1 transcript, AQP1 protein was inhibited by 47.8% to 74.6%. siRNAs containing the same percent of base pairs as the AQP1-siRNAs but in random sequence (i.e., scrambled siRNAs) had no effect. Suppression of AQP1 expression in ECs resulted in decreases in epithelial Na+ channel (ENaC) and Na-K ATPase of ECs, and the suppression ENaC &agr;, &bgr;, &ggr;, and Na-K ATPase were 43.1% to 48.2%,70.0% to 76.0%, 52.6% to 55.0%, and 72.7% to 79.3%, respectively. The reduced AQP1expression also resulted in decreased cell-cell junction protein level of VE-cadherin, which was suppressed by 36.5% to 59.5% but had no effect on occludin protein. Tube formation assay and tranwell assay showed AQP1 siRNAs induced high permeability of human pulmonary microvascular endothelial cells. Rho-kinase (ROCK) I and ROCK II were increased by 46.0% to 50.0% and 59% to 81%, respectively, AQP1 siRNA treatment accelerated the formation of F-actin bundles, demonstrating the activation of Rho/ROCK signaling pathway, and decreased mitochondrial membrane potential after AQP1 siRNA treatment, showing an important event of apoptosis process. Conclusions: The data demonstrate that AQP1 is a critical participate in regulating endothelial permeability and barrier function and provide direct evidence of the contribution of AQP1 to blood vessel formation.

Guanine nucleotide exchange factor-H1 signaling is involved in lipopolysaccharide-induced endothelial barrier dysfunction

BACKGROUND Gram-negative bacterial lipopolysaccharide (LPS) leads to the pathologic increase of vascular leakage under septic conditions. However, the mechanisms behind LPS-induced vascular hyperpermeability remain incompletely understood. In this study, we tested hypothesis that guanine nucleotide exchange factor-H1 (GEF-H1) signaling might be a key pathway involved in endothelial cells (ECs) barrier dysfunction. METHODS The roles of GEF-H1 signaling pathway in LPS-induced ECs barrier dysfunction were accessed by Evans blue dye-labeled albumin (EB-albumin) leak across the human umbilical vein EC (HUVEC) monolayers and Western blot assays. Furthermore, the effect of GEF-H1 signaling on LPS-induced alteration of cytoskeletal proteins and disruption of cell-cell junctions were analyzed by immunofluorescent analysis and Western blot assays, respectively. RESULTS We found that LPS could rapidly activated GEF-H1/RhoA/Rho-associated protein kinase (ROCK) signaling pathway in ECs. The LPS-mediated increase in EB-albumin flux across human HUVECs monolayers could be prevented by GEF-H1 depletion or ROCK inactivation. ECs permeability is controlled by actin filaments and cell-cell contact protein complexes. Actin stress fiber formation and/or cell-cell contact proteins loss cause vascular barrier disruption. Here, GEF-H1 knockdown or ROCK inactivation both not only significantly inhibited LPS-induced actin stress fiber formation, phosphorylation of myosin light chain, and myosin-associated phosphatase type 1, but also suppressed LPS-induced loss of occludin, claudin-1, and vascular endothelial (VE)-cadherin in ECs, which suggested that LPS-induced stress fiber formation and cell-cell junctions disruption were closely associated with GEF-H1/RhoA/ROCK signaling activation. CONCLUSION Our findings indicate that GEF-H1/RhoA/ROCK pathway in ECs plays an important role in LPS-mediated alteration of cell morphology and disruption of cell-cell junctions, consequently regulate LPS-induced vascular permeability dysfunction.

Association of platelet counts decline and mortality in severely burnt patients.

Although the dramatic fluctuation of platelet counts (PCs) has always been observed in severely burnt patients, the underlying relationship between the decline of PCs and mortality has never been well defined. In this study, we evaluated the prognostic implications of PC decline in severely burnt patients. We performed a 3-year retrospective chart of adult patients with a burn index of 50 or greater admitted to 2 burn centers. A total of 97 survivors and 51 nonsurvivors were included in this study. The trends of PCs in 2 groups within first 10 days after admission are seen to constitute 2 stages, with a slump to a nadir during the first 3 days and then a variable recovery. With respect to 30-day mortality, the areas under the receiver operating characteristic curve for the percentage decline of PCs (PPC) during the first 3 days and Acute Physiology And Chronic Health Evaluation II score were 0.784 and 0.871, respectively (P = .0549). Furthermore, an optimal cutoff of PPC during the first 3 days, which discriminated survivors with nonsurvivors, was 65.53% using the maximum Youden index (0.4853). Although the sensitivity and specificity of PPC during the first 3 days were not as high as the Acute Physiology And Chronic Health Evaluation II score in the present study, Kaplan-Meier estimation and multiple logistic regression models both indicated that a PPC of 65% or greater during the first 3 days was significantly associated with 30-day death (odds ratio, 1.054; 95% confidence interval, 1.006-1.104; P = .028). In summary, an initial slump of PCs, especially a PPC of 65% or greater during the first 3 days, provides prognostic significance for 30-day mortality in severely burnt patients.

The p38/mitogen-activated protein kinase pathway is implicated in lipopolysaccharide-induced microtubule depolymerization via up-regulation of microtubule-associated protein 4 phosphorylation in human vascular endothelium

BACKGROUND Microtubules (MTs) play an important role in lipopolysaccharide (LPS)-induced overexpression of inflammatory cytokines and vascular barrier dysfunction; however, the mechanisms behind MT dynamics changes in the vascular endothelium under septic conditions are still not well understood. METHODS Human umbilical vein endothelial cells (HUVECs) stimulated with LPS were pretreated with or without the specific p38/mitogen-activated protein kinase (MAPK) inhibitor, SB203580. p38/MAPK cascade-induced signaling events and proteins expression were investigated by Western blotting assay. The interaction between p38/MAPK and microtubule-associated protein 4 (MAP4) was examined by immunoprecipitation. Furthermore, the effects of agonists on LPS-induced MT disruption and alteration of acetylated alpha-tubulin (Acet-tubulin) were analyzed by double-immunofluorescent assay and Western blotting analysis. RESULTS In the present study, our results indicated that LPS induced MT depolymerization, but the effects of LPS could be reversed in endothelial cells pretreated with taxol. Furthermore, phosphor-p38 and MAP4 interacted to form a complex after exposure to LPS. LPS-induced MAP4 phosphorylation was greatly suppressed by SB203580, suggesting that activation of p38/MAPK signaling affected MAP4 phosphorylation linked to MT acetylation after stimulation with LPS. CONCLUSION The present study demonstrated that the p38/MAPK signaling pathway might disrupt MT dynamics via phosphorylation of MAP4 in vascular endothelial cells challenged by LPS. Our findings provide novel insights into the pathogenic mechanism of MT disassembly and consider new targets for therapeutic intervention under sepsis or septic shock conditions.

Lipopolysaccharide Induces Human Pulmonary Micro-Vascular Endothelial Apoptosis via the YAP Signaling Pathway

Gram-negative bacterial lipopolysaccharide (LPS) induces a pathologic increase in lung vascular leakage under septic conditions. LPS-induced human pulmonary micro-vascular endothelial cell (HPMEC) apoptosis launches and aggravates micro-vascular hyper-permeability and acute lung injury (ALI). Previous studies show that the activation of intrinsic apoptotic pathway is vital for LPS-induced EC apoptosis. Yes-associated protein (YAP) has been reported to positively regulate intrinsic apoptotic pathway in tumor cells apoptosis. However, the potential role of YAP protein in LPS-induced HPMEC apoptosis has not been determined. In this study, we found that LPS-induced activation and nuclear accumulation of YAP accelerated HPMECs apoptosis. LPS-induced YAP translocation from cytoplasm to nucleus by the increased phosphorylation on Y357 resulted in the interaction between YAP and transcription factor P73. Furthermore, inhibition of YAP by small interfering RNA (siRNA) not only suppressed the LPS-induced HPMEC apoptosis but also regulated P73-mediated up-regulation of BAX and down-regulation of BCL-2. Taken together, our results demonstrated that activation of the YAP/P73/(BAX and BCL-2)/caspase-3 signaling pathway played a critical role in LPS-induced HPMEC apoptosis. Inhibition of the YAP might be a potential therapeutic strategy for lung injury under sepsis.

Stromal-derived factor-1 up-regulated the expression of MIC on mouse keratinocyte stem cells.

Although previous studies have demonstrated that stromal-derived factor-1 (SDF-1) played a key role in chronic graft dysfunction (CGD), the precise mechanisms underlying this process are not clear. In this study, SDF-1 was injected into keratinocyte stem cells (KSCs) which were isolated and purified from neonatal C57BL/6 (H-2b) mice. Adenylyl cyclase (AC) activity of KSCs was measured and expressions of the human major histocompatibility complex (MHC) class I chain-related antigens A and B (MICA, MICB) detected by immunofluorescence. Cultured KSCs were negative for IA/IE MHC class II molecules by immunofluorescence, indicating the absence of any contamination with Langerhans cells and certifying the purity of KSCs. Over a 7-day culture period, SDF-1 up-regulated AC activity to 2.783 +/- 0.799, which was higher than that of the control group (1.290 +/- 0.476; P < .01). Immunostaining showed that KSCs expressed increased amounts of MICA protein (0.790 +/- 0.134 versus 0.200 +/- 0.022; P < .01) and MICB protein (0.610 +/- 0.832 versus 0.230 +/- 0.016; P < .01). Mixed lymphocyte reaction assays showed that KSCs cultured with SDF-1 injection for 7 days stimulated allogeneic T-cell proliferation. The data indicated that SDF-1 may accelerate the ultimate rejection of allogeneic keratinocytes by enhancing MIC through the AC signal pathway.