Xuliang Huang

Lung, spleen, and kidney are the major places for inducible nitric oxide synthase expression in endotoxic shock: Role of p38 mitogen-activated protein kinase in signal transduction of inducible nitric oxide synthase expression

Bacterial lipopolysaccharide (LPS) is known to induce endotoxic shock with inducible nitric oxide (NO) synthase (iNOS) expression and NO production. However, the major place for NO production in shock remains unclear. Although there is some literature about p38 mitogen-activated protein kinase (MAPK) in regulating LPS-induced iNOS expression, the results are contradictory. To interpret the precise cell mechanism and the role of p38 MAPK in the expression of iNOS during endotoxic shock, we carried out the following investigations. A severe endotoxic shock model was reproduced in mice 6 h after LPS injection. The plasma NO level was increased in a dose- and time-dependent manner after LPS stimulation and was suppressed by administration of SB203580 [4-(4-fluorophenyl)-2-4-methylsulfonylphenyl-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK. The iNOS expression was increased in many organs, including heart, liver, spleen, lung, gut, and kidney in endotoxic shock. Among them, the highest expression of iNOS mRNA and protein was in the lung, moderate expression was in the spleen and kidney, and the lowest expression was in the heart, gut, and liver. The level of expression in lung was 5.5 times that of iNOS mRNA and was 3.1 times that of iNOS protein than in heart, and 1.6 and 1.8 times that of iNOS mRNA and 1.7 and 1.4 times that of iNOS protein than in spleen and kidney, respectively. The p38 MAPK activity increased after LPS injection, and SB203580 markedly reduced LPS-induced expressions of iNOS protein and mRNA in the lung. The results indicates that lung, spleen, and kidney are the major places for iNOS expression in endotoxic shock and are important therapeutic target organs for attenuating NO production in shock treatment.

ERM protein moesin is phosphorylated by advanced glycation end products and modulates endothelial permeability

Advanced glycation end products (AGEs) accumulated in different pathological conditions have the potent capacity to alter cellular properties that include endothelial structural and functional regulations. The disruption of endothelial barrier integrity may contribute to AGE-induced microangiopathy and macrovasculopathy. Previous studies have shown that AGEs induced the rearrangement of actin and subsequent hyperpermeability in endothelial cells (ECs). However, the mechanisms involved in this AGE-evoked EC malfunction are not well understood. This study directly evaluated the involvement of moesin phosphorylation in AGE-induced alterations and the effects of the RhoA and p38 MAPK pathways on this process. Using immortalized human dermal microvascular ECs (HMVECs), we first confirmed that the ezrin/radixin/moesin (ERM) protein moesin is required in AGE-induced F-actin rearrangement and hyperpermeability responses in ECs by knockdown of moesin protein expression with small interfering RNA. We then detected AGE-induced moesin phosphorylation by Western blot analysis. The mechanisms involved in moesin phosphorylation were analyzed by blocking AGE receptor binding and inhibiting Rho and MAPK pathways. AGE-treated HMVECs exhibited time- and dose-dependent increases in the Thr(558) phosphorylation of moesin. The increased moesin phosphorylation was attenuated by preadministrations of AGE receptor antibody, Rho kinase (ROCK), or p38 inhibitor. Suppression of p38 activation via the expression of dominant negative mutants with Ad.MKK6b or Ad.p38alpha also decreased moesin phosphorylation. The activation of the p38 pathway by transfection of HMVECs with an adenoviral construct of dominant active MKK6b resulted in moesin phosphorylation. These results suggest a critical role of moesin phosphorylation in AGE-induced EC functional and morphological regulations. Activation of the ROCK and p38 pathways is required in moesin phosphorylation.

Hypersensitivity of BKCa to Ca2+ Sparks Underlies Hyporeactivity of Arterial Smooth Muscle in Shock

Large conductance Ca2+-activated K+ channels (BKCa) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BKCa to Ca2+ sparks resulting from its altered &bgr;1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BKCa to Ca2+ sparks that contributes to hypotension and blunted vasoreactivity in acute hemorrhagic shock. In arterial smooth muscle cells under voltage-clamp conditions (0 mV), the amplitude and duration, but not the frequency, of spontaneous transient outward currents of BKCa origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca2+ spark frequency was either unaltered (at 0 mV) or decreased in hyperpolarized resting cells. Examining the relationship between spark and spontaneous transient outward current amplitudes revealed a hypersensitive BKCa activity to Ca2+ spark in hemorrhagic shock, whereas the spark–spontaneous transient outward current coupling fidelity was near unity in both groups. Importantly, we found an acute upregulation of the &bgr;1 subunit of the channel, and single-channel recording substantiated BKCa hypersensitivity at micromolar Ca2+, which promotes the &agr; and &bgr;1 subunit interaction. Treatment of shock animals with the BKCa inhibitors iberiotoxin and charybdotoxin partially restored vascular membrane potential and vasoreactivity to norepinephrine and blood reinfusion. Thus, the results underscore a dynamic regulation of the BKCa–Ca2+ spark coupling and its therapeutic potential in hemorrhagic shock–associated vascular disorders.

New approach to treatment of shock--restitution of vasoreactivity.

Our objective was to observe the therapeutic effect of restituting vasoreactivity agent in severe shock. A hemorrhagic shock (HS) model was reproduced in rat and the response of arterioles of spinotrapezius muscle to norepinephrine (NE) in HS was tested. The diameter, blood velocity, and volumetric flow in arteriole, and the mean arterial pressure (MAP) were measured. The therapeutic effect was observed after the treatment of restituting vasoreactivity agent (glybenclamide—an inhibitor of ATP sensitive potassium channel, and tiron—an oxygen free radical scavenger). The arteriolar vasoreactivity was significantly reduced with 15 fold increase of NE threshold 2 h post HS. After treated with restituting agent(RA), the vascular hyporeactivity of rat was apparently recovered, and the increased level of MAP following injection of dopamine was 1.8 times and 1.9 times more than that in NS-treated and DMSO-treated group respectively. After reinfusion of shed blood, the value of systemic blood pressure maintained more than 100mmHg and volumetric flow in arterioles in RA group were 2 times more than those in NS treated group within the 2h observation periods. The average survival time in RA treated group was also 1.8 times and 1.6 times longer than that in NS-treated and DMSO-treated group respectively. The restituting vasoreactivity agent is able to recover the lower vasoreactivity with excellent anti-shock effect in severe hemorrhagic shock.

RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

BackgroundThe role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.MethodsUsing human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D were applied in endothelial cells.ResultsThe results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.ConclusionThese results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.

Differential activation of receptors and signal pathways upon stimulation by different doses of sphingosine‐1‐phosphate in endothelial cells

What is the central question of this study? Why do different doses of sphingosine‐1‐phosphate (S1P) induce distinct biological effects in endothelial cells? What is the main finding and its importance? S1P at physiological concentrations preserved endothelial barrier function by binding to S1P receptor 1, then triggering Ca2+ release from endoplasmic reticulum through phosphoinositide phospholipase C and inositol triphosphate, and consequently strengthening tight junction and F‐actin assembly through Rac1 activation. Excessive S1P induced endothelial malfunction by activating S1P receptor 2 and RhoA/ROCK pathway, causing F‐actin and tight junction disorganisation. Extracellular Ca2+ influx was involved in this process.

LPS and TNF-α induce expression of sphingosine-1-phosphate receptor-2 in human microvascular endothelial cells

Sphingosine-1-phosphate (S1P) is a bioactive sophospholipid with various S1P receptor (S1PR) expression profiles in cells of different origin. S1PR1, R3 and - to a lesser extent - R2 were the main receptors expressed in most of endothelial cells (ECs). The balances in the expression and activation of S1PR1, R2 and R3 help to maintain the physiological functions of ECs. Reverse transcription-PCR and Western blotting were used to detect the mRNA transcript level and protein expression of S1PR. Endothelial barrier function was measured by transflux of tracer protein through endothelial monolayer. Human dermal microvascular ECs predominantly expressed S1PR1 and S1PR3. Lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) significantly upregulated S1PR2 mRNA and protein levels. The application of S1PR2 antagonist JTE-013 decreased the endothelial monolayer hyper-permeability response induced by LPS and TNF-α. Inflammatory mediators LPS and TNF-α induce S1PR2 expression in endothelium, suggesting that S1PR2 up-regulation may be involved in LPS and TNF-α elicited endothelial barrier dysfunction.

Advanced glycation end products induce moesin phosphorylation in murine brain endothelium

Advanced glycation end products (AGEs) have been found to play an important role in the development of diabetes, and AGE levels are correlated with the severity of diabetic complications. We have demonstrated that moesin, a protein linker between actin filaments and the plasma membrane, undergoes phosphorylation of its threonine 558 residue by AGE stimulation in human dermal microvascular endothelial cells through activation of p38 and Rho kinase (ROCK) pathways. In this study, we observed in situ whether AGEs caused phosphorylation of vascular endothelial cells in the brains of AGE-stimulated mice. The animals were injected with AGE-modified mouse serum albumin (AGE-MSA) for 7 consecutive days. Immunohistochemistry was conducted to assess the phosphorylation of moesin in brain vessels. The level of moesin protein phosphorylation was also assessed in cerebral microvessels by western blotting. The effects of p38 and ROCK activation were determined by application of a p38 inhibitor (SB203580) and a ROCK inhibitor (Y27632) at 30 min before each AGE administration. The results showed specific expression of moesin in murine brain vascular endothelial cells. AGE treatment induced a significant increase of threonine 558 phosphorylation in moesin, while inhibition of p38 and ROCK remarkably attenuated the phosphorylation of moesin. The level of moesin protein phosphorylation was also increased in cerebral microvessels, along with an increased permeability of the blood-brain barrier, while inhibition of the p38 and ROCK attenuated these responses. These results demonstrate that AGEs cause the phosphorylation of moesin in murine brain microvascular endothelial cells, with p38 and ROCK being involved in this process.

Effect of sphingosine 1-phosphate on morphological and functional responses in endothelia and venules after scalding injury.

OBJECTIVE The uncontrolled increase of vascular permeability is the major obstacle in treatment of severe burns. Sphingosine 1-phosphate (S1P) has emerged as an important modulator of EC barrier function. This study was designed to explore the effect of S1P on morphological alteration in cultured endothelial cells (ECs) after burned plasma stimulation, and second to investigate the hyper-permeability response in intact vessels after scalding injury. METHODS The distribution of VE-cadherin and F-actin was observed by double staining in primary cultured human umbilical vein endothelial cells (HUVECs) with immunofluorescence and fluorescent probes; respectively. Permeability changes were measured by a fluorescence ratio technique in isolated venules from rat skin. Burned plasma was obtained from a third-degree scald covering 30% of the total body surface area. RESULTS The intervention with burned plasma on injured rats cultured HUVECs caused a significant disruption of intercellular adherens junction labeled by VE-cadherin staining, accompanied by the formation of F-actin stress fibers in the cells. S1P prevented or reversed these burned plasma-induced morphological alterations in cultured endothelial cells. The inhibition of S1P synthesis with N,N-dimethylsphingosine (DMS) mimicked the burned plasma-evoked redistribution of VE-cadherin and reorganization of F-actin. Venules isolated from burned rats demonstrated similar endothelial cytoskeleton changes with cultured cells under the influence of S1P or DMS. Both pre- and post-burn application of S1P attenuated increased permeability in isolated and perfused skin venules after burned plasma stimulation. CONCLUSION Our results indicate that S1P plays a role in maintaining basal vascular barrier function and could be protective in burn injury by enhancing the endothelial barrier function.

Advanced glycation end products induce moesin phosphorylation in murine retinal endothelium

Increase in vascular permeability is the most important pathological event during the development of diabetic retinopathy. Deposition of advanced glycation end products (AGEs) plays a crucial role in the process of diabetes. This study was to investigate the role of moesin and its underlying signal transduction in retinal vascular hyper-permeability induced by AGE-modified mouse serum albumin (AGE-MSA). Female C57BL/6 mice were used to produce an AGE-treated model by intraperitoneal administration of AGE-MSA for seven consecutive days. The inner blood–retinal barrier was quantified by Evans blue leakage assay. Endothelial F-actin cytoskeleton in retinal vasculature was visualized by fluorescence probe staining. The expression and phosphorylation of moesin in retinal vessels were detected by RT–PCR and western blotting. Further studies were performed to explore the effects of Rho kinase (ROCK) and p38 MAPK pathway on the involvement of moesin in AGE-induced retinal vascular hyper-permeability response. Treatment with AGE-MSA significantly increased the permeability of the retinal microvessels and induced the disorganization of F-actin in retinal vascular endothelial cells. The threonine (T558) phosphorylation of moesin in retinal vessels was enhanced remarkably after AGE administration. The phosphorylation of moesin was attenuated by inhibitions of ROCK and p38 MAPK, while this treatment also prevented the dysfunction of inner blood–retinal barrier and the reorganization of F-actin in retinal vascular endothelial cells. These results demonstrate that moesin is involved in AGE-induced retinal vascular endothelial dysfunction and the phosphorylation of moesin is triggered via ROCK and p38 MAPK activation.