Glucagon-like peptide-1 inhibits the receptor for advanced glycation endproducts to prevent podocyte apoptosis induced by advanced oxidative protein products.

OBJECTIVE: To investigate whether and how glucagon-like peptide-1 (GLP-1) can protect podocytes from apoptosis induced by advanced oxidative protein products (AOPPs). METHODS: Murine podocytes were stimulated with 200 µg/ml AOPP for 48 h in the presence or absence of GLP-1. Cell viability was assessed using the cell counting kit-8 assay. Podocyte apoptosis was detected by flow cytometry and Hoechst 33258 staining. Superoxide radical production was assayed using lucigenin-enhanced chemiluminescence, and Western blotting was used to measure expression of RAGE, NADPH oxidase subunits p47phox and gp91phox, as well as apoptosis-associated proteins p53, Bax, Bcl-2 and caspase-3. RESULTS: Incubating podocytes with AOPPs reduced cell viability, triggered changes in cell morphology and promoted apoptosis. GLP-1 partially inhibited AOPP-induced apoptosis, O2- overproduction, and AOPP-induced expression of RAGE. GLP-1 inhibited expression of p47phox and gp91phox in AOPP-treated podocytes, and it attenuated AOPP-induced expression of p53, Bax and cleaved caspase-3, whereas it restored expression of Bcl-2. CONCLUSION: GLP-1 partially inhibits AOPP-induced apoptosis in podocytes, perhaps by interfering with the AOPP-RAGE axis, decreasing oxidative stress and inhibiting the downstream p53/Bax/caspase-3 apoptotic pathway. GLP-1 may be a useful anti-apoptotic agent for early intervention in diabetic nephropathy.

Role of moesin, Src, and ROS in advanced glycation end product-induced vascular endothelial dysfunction.

The disruption of endothelial integrity and the occurrence of angiogenesis in response to AGEs contribute greatly to micro- and macrovascular complications associated with DM. Among human dermal, brain, and retinal vascular ECs, activation of ERM, moesin, by phosphorylation of Thr-558 is involved in AGE-induced hyperpermeability and angiogenesis via the Rho and ROCK (Rho/ROCK) and p38 pathways. Src also plays an important role in AGE-induced endothelial barrier dysfunction by phosphorylating moesin, VE-cadherin, and FAK. Furthermore, recent studies have demonstrated that ROS serve as a key mediator of the AGE-induced endothelial response. ROS inhibition would greatly benefit ECs. This review focuses on the role of moesin in microvascular permeability and angiogenesis, and on the involvement of Src and ROS in endothelial barrier disruption.

Updated concepts on the pathophysiology and the clinical management of trauma hemorrhage and coagulopathy.

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.

IRE1α Signaling Pathways Involved in Mammalian Cell Fate Determination.

A diverse array of cellular stresses can lead to accumulation of misfolded or unfolded proteins in endoplasmic reticulum (ER), which subsequently elicits ER stress. Inositol-requiring enzyme 1α (IRE1α) is the most sensitive of the three unfolded protein response (UPR) branches which are triggered to cope with ER stress in mammalian cells. IRE1α signaling is quite context-specific on account of many adaptor and modulator proteins that directly interact with it, including heat shock proteins (HSPs), RING finger protein 13 (RNF13), poly (ADP-ribose) polymerase 16 (PARP16), Bax/Bak, and Bax inhibitor-1 (BI-1). The activated IRE1α triggers different downstream pathways depending on the UPRosome formed by distinct modulator proteins. At the initial phase of ER stress, IRE1α-XBP1 axis functions as an adaptive response. While ER stress sustains or intensifies, signals shift to apoptotic responses. Furthermore, IRE1α signaling can be exploited to the development of a wide range of prevalent human diseases, with cancer the most characterized. Here we provide an overview of recent insights into the complex IRE1α signaling network which makes IRE1α an intriguing cell fate switch. Besides, the functional relevance is presented since IRE1α activation also participates in some other physiological processes beyond protein-folding status.

Effect of salvianolic acid B on TNF-α induced cerebral microcirculatory changes in a micro-invasive mouse model.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Doxycycline hyclate protects lipopolysaccharide-induced endothelial barrier dysfunction by inhibiting the activation of p38 mitogen-activated protein kinase.

Doxycycline hyclate (DOX-h) attenuates inflammatory conditions independent of its antimicrobial effect. This study aimed to observe the effects of DOX-h on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. The endothelial monolayer permeability of human umbilical vein endothelial cells (HUVECs) was monitored by transendothelial electrical resistance (TEER). The phosphorylation of mitogen-activated protein kinases (MAPKs) and the arrangement of F-actin were detected. The results showed that both pretreatment and simultaneous treatment with DOX-h markedly attenuated the LPS-induced reduction in TEER and the disorganization of F-actin on HUVECs in a dose- and time-dependent manner. LPS mediated the phosphorylation of all three MAPKs (p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK)), but DOX-h was only able to inhibit the LPS-induced phosphorylation of p38 and JNK. The data further suggested that DOX-h alleviated LPS-evoked TEER reduction and F-actin redistribution by inhibiting the phosphorylation of p38 and its downstream target, heat shock protein (HSP)27. Thus, DOX-h attenuates LPS-induced endothelial barrier dysfunction via inhibition of the p38 MAPK-HSP27-F-actin pathway.

Barrier stabilizing mediators in regulation of microvascular endothelial permeability.

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.

Role of Moesin Phosphorylation in Retinal Pericyte Migration and Detachment Induced by Advanced Glycation Endproducts.

Proliferative diabetic retinopathy (PDR) involves persistent, uncontrolled formation of premature blood vessels with reduced number of pericytes. Our previous work showed that advanced glycation endproducts (AGEs) induced angiogenesis in human umbilical vein endothelial cells, mouse retina, and aortic ring, which was associated with moesin phosphorylation. Here we investigated whether moesin phosphorylation may contribute to pericyte detachment and the development of PDR. Primary retinal microvascular pericytes (RMPs) were isolated, purified from weanling rats, and identified by cellular markers α-SMA, PDGFR-ß, NG2, and desmin using immunofluorescence microscopy. Effects of AGE-BSA on proliferation and migration of RMPs were examined using CCK-8, wound healing, and transwell assays. Effects on moesin phosphorylation were examined using western blotting. The RMP response to AGE-BSA was also examined when cells expressed the non-phosphorylatable Thr558Ala mutant or phospho-mimicking Thr558Asp mutant of moesin or were treated with ROCK inhibitor Y27632. Colocalization and interaction between CD44, phospho-moesin, and F-actin were observed. Experiments with cultured primary RMPs showed that AGE-BSA inhibited the proliferation, enhanced the migration, and increased moesin phosphorylation in a dose- and time-dependent manner. AGE-BSA also triggered the rearrangement of F-actin and promoted the interaction of CD44 with phospho-moesin in RMPs. These effects were abrogated in cells expressing the non-phosphorylatable moesin mutant and the application of ROCK inhibitor Y27632 attenuated AGE-induced alteration in cultured RMPs by abolishing the phosphorylation of moesin. However, those AGE-induced pathological process occurred in RMPs expressed the phospho-mimicking moesin without AGE-BSA treatment. It is concluded that AGEs could activate ROCK to mediate moesin phosphorylation at Thr558, and resulting phospho-moesin interacts with CD44 to form CD44 cluster, which might stimulate the migration of RMPs and subsequent RMP detachment in microvessel. This pathway may provide new drug targets against immature neovessel formation in PDR.

[Effects of Rho/ROCK signal pathway on AGEs-induced morphological and functional changes in human dermal microvascular endothelial cells.].

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.

Advanced glycation end products induce actin rearrangement and subsequent hyperpermeability of endothelial cells.

This study aimed to determine the effects of advanced glycation end products (AGEs) on endothelial cytoskeleton morphology and permeability, and to detect the underlying signaling mechanisms involved in these responses. Cultured endothelial cells (ECs) were exposed to AGE-modified human serum albumin (AGE-HSA), and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of TRITC-albumin across the EC monolayers. To explore the signaling pathways behind AGE-induced EC alteration, ECs were treated with either soluble anti-AGE receptor (RAGE) IgG, or the MAPK inhibitors PD98059 and SB203580 before AGE-HSA administration. To further elucidate possible involvement of the ERK and p38 pathways in AGE-induced EC changes, adenovirus-carried recombinant constitutive dominant-negative forms of upstream ERK and p38 kinases, namely MEK1(A) and MKK6b(A), were pre-infected into ECs 24 h prior to AGE-HSA exposure. AGE-HSA induced actin cytoskeleton rearrangement, as well as EC hyperpermeability, in a dose and time-dependent manner. The effects were attenuated in cells pretreated with anti-RAGE IgG, PD98059 or SB203580, respectively. EC pre-infection with MEK1(A) and MKK6b(A) also alleviated the effect of AGEs. Furthermore, adenovirus-mediated administration of activated forms of either MEK1 or MKK6b alone induced rearrangement of F-actin and hyperpermeability. The results indicate that ERK and p38 MAPK play important roles in the mediation of AGE-induced EC barrier dysfunction associated with morphological changes of the F-actin.

[Mechanism of advanced glycation end products-induced hyperpermeability in endothelial cells].

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 RAGE in lipopolysaccharide-induced cytoskeletal changes in mouse pulmonary microvascular endothelial cells].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Mouse PMVECs of a high purity can be obtained by immune magnetic beads. RAGE is involved in LPS-induced destruction of mouse PMVEC cytoskeletons.

Role of Rho kinase and actin filament in the increased vascular permeability of skin venules in rats after scalding.

OBJECTIVE: To investigate the role of the Small GTPase Rho and endothelial cytoskeleton in the increased vascular permeability of rat skin after scalding. METHODS: Rats were subjected to scalding local ventral skin and a venule was isolated from scalded skin and cannulated by micropipette. The venular permeability was measured with a fluorescence ratio technique and expressed with the permeability coefficient to albumin (P(a)). The venular F-actin filaments were observed by staining with rhodamine phalloidin and laser confocal scanning microscopy. A specific Rho kinase inhibitor Y-27632 was added into vessel bathing solution or preincubated with vessels to evaluate the role of Rho kinase in regulating of vascular barrier function. RESULTS: Scalding increased P(a) value of skin venule about threefold compared to normal skin venules (P<0.01) and was maintained for 120 min. Inhibition of Rho kinase with Y-27632 (30 micromol/l in low-concentration group; 60 micromol/l in high-concentration group) significantly attenuated the hyperpermeability responses to scalding in a dose dependent fashion. A prominent peripheral actin rim (PAR) existed at the outer area of endothelial cells and apparently delineated the cell-to-cell borders. In the control group, the PARs were arranged smoothly and fairly continuously. However, occasionally PARs did show focal interruption with focal fluorescein isothiocyanate (FITC)-albumin leakage. In the burned group, PARs were less organized and accompanied by a large amount of FITC-albumin leakage. Inhibition of Rho kinase with Y-27632 dramatically reduced P(a) value with recovery of actin filament arrangement in venule after scalding. CONCLUSION: Burn leads to dermal venular permeability increase with endothelial cytoskeleton depolymerization and disruption. Rho signal transduction pathway is involved in these responses.

p38 MAPK Signal is Necessary for TNF-alpha Gene Expression in RAW Cells.

The luciferase reporter gene system driven by the TNF-alpha promoter was constructed for the studies of the effect of p38 MAPK signal transduction pathway on the gene expression of TNF-alpha. In the experiments of co-transfection in RAW cells it was found that there was a significant relevance between the activation of p38 by LPS and the induction of TNF-alpha reporter gene transcription. The induction of TNF-alpha expression was not shown after transfection of p38 only co-transfection of p38 with the active mutant of its upstream kinase, MKK6b, however, did induce the high expression of luciferase. Further studies showed that the induction of TNF-alpha promoter activity by MKK6b(E) and LPS was similar. In addition, the dominant negative form of p38 or p38 inhibitor gave an inhibitory effect on the TNF-alpha promoter activity induced by MKK6b(E) or LPS. All these results suggest that a possible mechanism for TNF-alpha production in endotoxic shock is the increase in gene transactivity induced by LPS in monocytes/macrophages, and that p38 MAPK signal pathway participates in the regulation of TNF-alpha gene expression induced by LPS.

Effects of lipopolysaccharide on actin reorganization and actin pools in endothelial cells.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Myosin light-chain kinase contributes to short-term endothelial cell cytoskeletal alteration induced by serum from burned rats.

OBJECTIVES: To investigate the time-dependent effects of serum from burned rats on cytoskeletal filamentous actin (F-actin) reorganization by visualizing their distribution in human umbilical vein endothelial cell line ECV-304 and evaluate the role of myosin light-chain kinase (MLCK) in this process. METHODS: The serum-starved ECV-304 cells were incubated with the serum from burned rats for 30 min, 1, 2, 4, and 6 h, respectively, and 30 min before or after the incubation, the cells were treated with 5 micromol/L ML-7 for 30 min. F-actin was stained with rhodamine-phalloidin and observed under fluorescence microscope. RESULTS: Under normal condition, F-actin was distributed mainly in the cortical area of the endothelial cells. After stimulation with the burn serum, stress fiber formation could be clearly seen in the endothelial cells, exhibiting a time-dependent enhancement in a time course ranging from 30 min to 6 h. Such an effect could be significantly inhibited by a 30-min pretreatment of the cells with MLCK-specific inhibitor ML-7. Inhibition of MLCK also reversed actin reorganization in the endothelial cells pretreated with the burn serum. CONCLUSION: Serum from burned rats induces characteristic morphological changes in the endothelial cell actin cytoskeleton mainly due to the MLCK activation, an effect that can be reversed by the inhibition of MLCK.

[Effects of aging and hypoxia on the proliferation of cultured rat pulmonary arterial smooth muscle cells].

OBJECTIVE: To study the effects of aging and hypoxia on the proliferative behavior of cultured pulmonary arterial smooth muscle cells (PASMCs). METHODS: PASMCs isolated from aged (18-24 months) and young (3-4 months) rats were divided, according to the different treatments the cells were subjected to, into young and aged normoxic groups (groups A and B) and young and aged hypoxic groups (groups C and D) respectively. MTT cell proliferation assay, 3H-TdR incorporation assay, flow cytometriy and immunocytochemical analysis were respectively employed to observe the proliferative behavior. RESULTS: Compared with the cells from young rats, PASMCs from aged rats had a higher proliferation rate, more 3H-TdR incorporation, increased mitotic cell ratio, reduced amount of the total protein, and elevated content of proliferating cell nuclear antigen (PCNA). In comparison with normoxic condition, hypoxia elicited higher proliferation rate of the cells with inhibition of 3H-TdR incorporation that was subsequently increased. Higher percentage of mitotic cells, less total protein amount and increased PCNA were also observed in response to hypoxia. CONCLUSIONS: Aging and hypoxia may directly induce PASMC proliferation, and in aging PASMCs, the proliferation is the most obvious in response to hypoxic stimulation.

[Effect of protein kinase on endothelial cytoskeleton induced by septic shock].

OBJECTIVE: To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Relationship between the endothelial barrier and vascular permeability after burns and its mechanism].

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.