TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

BACKGROUND: Neuroinflammation mediated by microglial pyroptosis is an important pathogenic mechanism of septic encephalopathy (SAE). It has been reported that TRIM45 is associated with tumours and inflammatory diseases. However, the role of TRIM45 in SAE and the relationship between TRIM45 and microglial pyroptosis are unknown. In this study, we found that TRIM45 played an important role in regulating microglial pyroptosis and the molecular mechanism. METHODS: SAE was induced by intraperitoneal injection of LPS in WT and AAV-shTRIM45 mice. BV2 cells were treated with LPS/ATP in vitro. Cognitive function was assessed by the Morris water maze. Nissl staining was used to evaluate histological and structural lesions. ELISA was used to dectect neuroinflammation. qPCR was used to detect the mRNA levels of inflammatory cytokines, NLRP3, and autophagy genes. Western blotting and immunofluorescence analysis were used to analyse the expression of the proteins. Changes in reactive oxygen species (ROS) in cells were observed by flow cytometry. Changes in mitochondrial membrane potential in BV2 cells were detected by JC-1 staining. Peripheral blood mononuclear cells were extracted from blood by density gradient centrifugation and then used for qPCR, western blotting and flow detection. To further explore the mechanism, we used the overexpression plasmids TRIM45 and Atg5 as well as siRNA-TRIM45 and siRNA-Atg5 to analyse the downstream pathway of NLRP3. The protein and mRNA levels of TRIM45 in peripheral blood mononuclear cells from sepsis patients were examined. RESULTS: Knocking down TRIM45 protected against neuronal damage and cognitive impairment in septic mice. TRIM45 knockdown inhibited microglial pyroptosis and the secretion of inflammatory cytokines in vivo and in vitro, which was mediated by NLRP3/Gsdmd-N activation. Overexpression of TRIM45 could activate NLRP3 and downstream proteins. Further examination showed that TRIM45 regulated the activation of NLRP3 by altering Atg5 and regulating autophagic flux. It was also found that overexpression and knockdown of TRIM45 affected the changes in ROS and mitochondrial membrane potential. Thus, knocking down TRIM45 could reduce microglial pyroptosis, the secretion of proinflammatory cytokines, and neuronal damage and improve cognitive function. In addition, the level of TRIM45 protein in septic patients was increased. There was a positive linear correlation between APACHE II score and TRIM45, between SOFA score and TRIM45. Compared to group GCS > 9, level of TRIM45 were increased in group GCS ≤ 8. CONCLUSION: TRIM45 plays a key role in neuroinflammation caused by LPS, and the mechanism may involve TRIM45-mediated exacerbation of microglial pyroptosis via the Atg5/NLRP3 axis.

Advanced glycation end products induce immature angiogenesis in in vivo and ex vivo mouse models.

Proliferative diabetic retinopathy (PDR) is a progressive disease predominantly involving pathological angiogenesis and is characterized by the development of immature, fragile, and easily hemorrhagic new vessels. Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) play important roles in the progression of diabetic retinopathy. Our previous studies demonstrated that AGEs promoted HUVEC angiogenesis by inducing moesin phosphorylation via RhoA/Rho-associated protein kinase (ROCK) pathway. The aim of this study was to further confirm AGE-induced angiogenesis in vivo and the involvement of RAGE, ROCK, and moesin phosphorylation in this process. We performed the study in an AGE-treated mouse model with various angiogenesis assays in multiple in vivo and ex vivo models. The results demonstrated that AGEs promoted significant neovascularization in whole mount retina and mouse aortic ring of adult and postnatal mice and in Matrigel plug as well, which were consistently accompanied by increased moesin phosphorylation. The increase of AGE-evoked neovascularization and moesin phosphorylation were both attenuated by RAGE knockout or ROCK inhibitor Y27632 administration in mice. We also revealed the pathological characteristics of AGE-promoted angiogenesis by demonstrating the decrease of pericyte coverage and the disarranged endothelial alignment in microvessels. In conclusion, this study provides in vivo evidences that AGEs induce immature angiogenesis by binding to RAGE, activating the RhoA/ROCK signal pathway and inducing moesin phosphorylation.NEW & NOTEWORTHY Advanced glycation end product (AGE)-induced formation of neovessels and phosphorylation of moesin in retina and aortic ring required AGE receptors. AGEs increased neovessels and the phosphorylation of moesin in retina and aortic ring via RhoA/ROCK pathway. AGE-induced immature angiogenesis in AGE-treated mouse retina and aortic ring. The AGE-RAGE axis and moesin could be candidate targets for overcoming relative diseases.

RAGE Plays a Role in LPS-Induced NF-κB Activation and Endothelial Hyperpermeability.

Endothelial functional dysregulation and barrier disruption contribute to the initiation and development of sepsis. The receptor for advanced glycation end products (RAGE) has been demonstrated to be involved in the pathogenesis of sepsis. The present study aimed to investigate the role of RAGE in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation in endothelial cells and the consequent endothelial hyperpermeability. LPS-induced upregulation of RAGE protein expression in human umbilical vein endothelial cells (HUVECs) was detected by western blotting. Activation of NF-κB was revealed using western blotting and immunofluorescent staining. LPS-elicited endothelial hyperpermeability was explored by transendothelial electrical resistance (TER) assay and endothelial monolayer permeability assay. The blocking antibody specific to RAGE was used to confirm the role of RAGE in LPS-mediated NF-κB activation and endothelial barrier disruption. We found that LPS upregulated the protein expression of RAGE in a dose- and time-dependent manner in HUVECs. Moreover, LPS triggered a significant phosphorylation and degradation of IκBα, as well as NF-κB p65 nuclear translocation. Moreover, we observed a significant increase in endothelial permeability after LPS treatment. However, the RAGE blocking antibody attenuated LPS-evoked NF-κB activation and endothelial hyperpermeability. Our results suggest that RAGE plays an important role in LPS-induced NF-κB activation and endothelial barrier dysfunction.

Liver X receptor-α and miR-130a-3p regulate expression of sphingosine 1-phosphate receptor 2 in human umbilical vein endothelial cells.

Recent studies have shown that activation of liver X receptors (LXRs) attenuates the development of atherosclerosis, not only by regulating lipid metabolism but also by suppressing inflammatory signaling. Sphingosine 1-phosphate receptor 2 (S1PR2), an important inflammatory gene product, plays a role in the development of various inflammatory diseases. It was proposed that S1PR2 might be regulated by LXR-α. In the present study, the effect of LXR-α on tumor necrosis factor-α (TNF-α)-induced S1PR2 expression in human umbilical vein endothelial cells (HUVECs) was investigated and the underlying mechanism was explored. The results demonstrated that TNF-α led to an increase in S1PR2 expression and triggered a downregulation of LXR-α expression in HUVECs as well. Downregulation of LXR-α with specific small interfering RNA (siRNA) remarkably enhanced the primary as well as TNF-α-induced expression of S1PR2 in HUVECs. Activation of LXR-α by agonist GW3965 inhibited both primary and TNF-α-induced S1PR2 expression. GW3965 also attenuated S1PR2-induced endothelial barrier dysfunction. The data further showed that TNF-α induced a significant decrease in miR-130a-3p expression. Overexpression of miR-130a-3p with mimic product reduced S1PR2 protein expression, and inhibition of miR-130a-3p by specific inhibitor resulted in an increase in S1PR2 protein expression. Furthermore, activation of LXRs with agonist enhanced the expression of miR-130a-3p, and knockdown of LXR-α by siRNA suppressed miR-130a-3p expression. These results suggest that LXR-α might downregulate S1PR2 expression via miR-130a-3p in quiescent HUVECs. Stimulation of TNF-α attenuates the activity of LXR-α and results in enhanced S1PR2 expression.

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

NEW FINDINGS: 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 Ca(2+) 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 Ca(2+) influx was involved in this process. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid in plasma, and its plasma concentration can be adjusted through a complex metabolic process. The alterations in S1P levels and the activation of receptors collaboratively regulate distinct biological effects. This study was performed to investigate comparatively the effect of different concentrations of S1P on endothelial barrier function and to explore the roles of S1P receptors (S1PRs), Rho GTPases and calcium in S1P-induced endothelial responses. Endothelial barrier function was studied using transendothelial electric resistance and a resistance meter in human umbilical vein endothelial cells. Specific agonists or antagonists were applied to control the activation of S1P receptors and the release of calcium from different cellular compartments. The results indicated that at physiological concentrations, S1P preserved endothelial barrier function by binding with S1PR1. The activation of S1PR1 triggered the release of intracellular Ca(2+) from the endoplasmic reticulum through the PI-phospholipase C and inositol trisphosphate pathways. Consequently, the Rho GTPase Rac1 was activated, strengthening the assembly of tight junction proteins and F-actin. However, excessive S1P induced endothelial barrier dysfunction by activating S1PR2 followed by the RhoA/RhoA kinase pathway, causing the disorganization of F-actin and the disassembly of the tight junction protein ZO-1. An influx of extracellular Ca(2+) was involved in this process. These data suggest that physiological and excessive amounts of S1P induce different responses in human umbilical vein endothelial cells; the activation of the 1PR1-PLC-IP3 R-Ca(2+) -Rac1 pathway governs the low-dose S1P-enhanced endothelial barrier integrity, and the activation of S1PR2-calcium influx-RhoA/ROCK dominates the high-dose S1P-induced endothelial monolayer hyperpermeability response.

Gender differences and risk factors for acute kidney injury following cardiac surgery: A single center retrospective cohort study.

Background: We studied AKI incidence and prognosis in cardiac surgery patients under and over 60 years old. Methods: We studied AKI in patients who underwent cardiac surgery at the First Affiliated Hospital of Wenzhou Medical University between Jan 2020 and Dec 2021, using improved global prognostic criteria for diagnosis. Results: After analyzing 781 patients (402 males, 379 females), AKI incidence after surgery was 30.22 %. Adjusting for propensity scores revealed no significant difference in AKI incidence between young males (24.1 %) and females (19.3 %). However, young females had higher AKI stages. Among older patients, AKI incidence was comparable between males (43.4 %) and females (42.2 %), but females had longer intubation times. Independent risk factors for AKI included age, male gender, and BMI, while intraoperative hemoglobin level was protective. Conclusions: No gender gap in AKI frequency for <60 years old and ≥60 years old post-cardiac surgery, yet women display increased AKI severity and extended intubation duration.

The Effect of Transcutaneous Electrical Acupoint Stimulation on High-Risk Patients with PONV Undergoing Laparoscopic Gynecologic Surgery: A Randomized Controlled Trial.

BACKGROUND: Postoperative nausea and vomiting (PONV) is one of the most common complications after general anesthesia. The traditional comprehensive management of PONV usually uses one or two drugs, but this regimen fails to meet the requirements of the latest version of PONV guidelines. The purpose of this study was to evaluate the effect of transcutaneous electrical acupoint stimulation (TEAS) on high-risk PONV patients who are undergoing laparoscopic gynecological surgery. METHODS: In total, 162 high-risk PONV patients were randomly divided into an experimental group (n = 81) and a control group (n = 81). Both groups were injected with 4 mg of dexamethasone and 0.25 mg of palonosetron. In the experimental group, Nei-guan (PC6) and He-gu (LI4) were stimulated by a transcutaneous acupoint electrical stimulation instrument (HANS200E) 30 min before the surgery. The control group also received electrodes but no stimulation. Variance analysis and rank sum test were used to compare the differences between the two groups. RESULTS: The results of the incidence of postoperative nausea, vomiting, NRS score, degree of abdominal distension, and time to first flatus in the experimental group were lower than those in the control group. Nursing satisfaction of the experimental group was higher than that of the control group. CONCLUSIONS: The study demonstrates that TEAS combined with dexamethasone and palonosetron can effectively prevent PONV, reduce postoperative abdominal distension and postoperative pain, and shorten the first postoperative flatus time in high-risk patients with PONV. At the same time, it can improve nursing satisfaction.

SILENCING M 6 A READER YTHDC1 REDUCES INFLAMMATORY RESPONSE IN SEPSIS-INDUCED CARDIOMYOPATHY BY INHIBITING SERPINA3N EXPRESSION.

ABSTRACT: Sepsis-induced cardiomyopathy (SIC) is one of the most common complications of infection-induced sepsis. An imbalance in inflammatory mediators is the main factor leading to SIC . N 6 -methyladenosine (m 6 A) is closely related to the occurrence and development of sepsis. N 6 -methyladenosine reader YTH domain containing 1 (YTHDC1) is an m 6 A N 6 -methyladenosine recognition protein. However, the role of YTHDC1 in SIC remains unclear. Herein, we demonstrated that YTHDC1-shRNA inhibits inflammation, reduces inflammatory mediators, and improves cardiac function in a LPS-induced SIC mouse model. Based on the Gene Expression Omnibus database analysis, serine protease inhibitor A3N is a differential gene of SIC. Furthermore, RNA immunoprecipitation indicated that serine protease inhibitor A3N (SERPINA3N) mRNA can bind to YTHDC1, which regulates the expression of SERPINA3N. Serine protease inhibitor A3N-siRNA reduced LPS-induced inflammation of cardiac myocytes. In conclusion, the m 6 A reader YTHDC1 regulates SERPINA3N mRNA expression to mediate the levels of inflammation in SIC. Such findings add to the relationship between m 6 A reader YTHDC1 and SIC, providing a new research avenue for the therapeutic mechanism of SIC.

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

BACKGROUND: The 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. METHODS: Using 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-moesinT(558A) and pcDNA3/HA-moesinT(558D) were applied in endothelial cells. RESULTS: The 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. CONCLUSION: These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.

Atractyloside induces low contractile reaction of arteriolar smooth muscle through mitochondrial damage.

Atractyloside is the principal naturally occurring active ingredient in ethnomedicines and animal grazing forage. Evidence that atractyloside can induce opening of the mitochondrial permeability transition pore (mPTP) indicates that mitochondrial mechanisms may play an important role in pathophysiological lesions of the heart, liver and kidney after atractyloside poisoning. Therefore, in this study we investigated the association of atractyloside-induced mitochondrial damage in arteriolar smooth muscle cells (ASMCs) with contractile reaction. Atractyloside led to depolarized and swollen or damaged ASMC mitochondria, which might be related to the concentration-dependent induction of mPTP opening. Relative ATP content in ASMCs was significantly reduced by 48%, 63% and 66% of control when cells were treated with 7.5, 10, and 15 µm atractyloside for 10 min, respectively, and ASMCs were hyperpolarized. In addition, the contractile responsiveness of ASMCs was eventually weakened. These results suggest that atractyloside has a toxic effect on vasoreactivity, which is possibly related to mitochondrial damage.

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 Ca(2+)-activated K(+) channels (BK(Ca)) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BK(Ca) to Ca(2+) sparks resulting from its altered beta1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BK(Ca) to Ca(2+) 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 BK(Ca) origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca(2+) 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 BK(Ca) activity to Ca(2+) 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 beta1 subunit of the channel, and single-channel recording substantiated BK(Ca) hypersensitivity at micromolar Ca(2+), which promotes the alpha and beta1 subunit interaction. Treatment of shock animals with the BK(Ca) 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 BK(Ca)-Ca(2+) spark coupling and its therapeutic potential in hemorrhagic shock-associated vascular disorders.

[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.

Identification of Pharmacokinetic Markers for Guanxin Danshen Drop Pills in Rats by Combination of Pharmacokinetics, Systems Pharmacology, and Pharmacodynamic Assays.

This paper reported a feasibility study strategy of identifying pharmacokinetic (PK) markers for a cardiovascular herbal medicine, Guanxin Danshen drop pill (GDDP). First, quantification analysis revealed the constituent composition in the preparation by high-performance liquid chromatography (HPLC). Subsequently, physiochemical property calculation predicted the solubility and intestinal permeability of the constituents in the preparation. Furthermore, HPLC-MS analysis ascertained the absorbable ingredients and their PK properties in rat plasma. The main effective substances from the ingredients absorbed into blood and their cardiovascular effects were also predicted by systems pharmacology study, and were further confirmed by in vivo protective effects on isoprenaline-induced myocardial injury in mice. Finally, the ingredients with high content, representative structure feature, favorable PK properties, high relevant degree to myocardial ischemia (MI) issues, and validated therapeutic effects were considered as the PK markers for the preparation. Ginsenosides Rg1, Rb1, and tanshinone (TS) IIA were identified originally as PK markers for representing PK properties of GDDP. In addition, integrated PK studies were carried out according to previous reports, viz. drug concentration sum method and the AUC weighting method, to understand the in vivo process of GDDP comprehensively. The present study maybe provide a reference approach to identify PK markers for cardiovascular herbal medicines.

Role of Moesin in Advanced Glycation End Products-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells.

Disorders of angiogenesis are related to microangiopathies during the development of diabetic vascular complications, but the effect of advanced glycation end products (AGEs) on angiogenesis and the mechanism has not been completely unveiled. We previous demonstrated that moesin belonging to the ezrin-radixin-moesin (ERM) protein family protein played a critical role in AGE-induced hyper-permeability in human umbilical vein endothelial cells (HUVECs). Here, we investigated the impact of moesin on AGE-induced HUVEC proliferation, migration, and tubulogenesis. Silencing of moesin decreased cell motility and tube formation but not cell proliferation. It also attenuated cellular F-actin reassembly. Further, phosphorylation of threonine at the 558 amino acid residue (Thr 558) in moesin suppressed AGE-induced HUVEC proliferation, migration, and tube formation, while the activating mutation of moesin at Thr 558 enhanced HUVEC angiogenesis. Further, the inhibition of either RhoA activity by adenovirus or ROCK activation with inhibitor Y27632 decreased AGE-induced moesin phosphorylation and subsequently suppressed HUVEC angiogenesis. These results indicate that the Thr 558 phosphorylation in moesin mediates endothelial angiogenesis. AGEs promoted HUVEC angiogenesis by inducing moesin phosphorylation via RhoA/ROCK pathway.

Calcium mobilization is required for peroxynitrite-mediated enhancement of spontaneous transient outward currents in arteriolar smooth muscle cells.

Transiently local release of Ca(2+) from the sarcoplasmic reticulum (SR) activates nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO(-)) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca(2+) mobilization was involved in STOCs alteration by ONOO(-). STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca(2+); by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca(2+) channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO(-) donor, at concentrations of 20-200 microM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca(2+) (200 nM), ryanodine (10 microM), or nifedipine (10 microM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 microM) had little effect on ONOO(-) -induced STOCs activation. These findings suggest that complex Ca(2+)-mobilizing pathways, including external Ca2+ influx through VGCCs activation and subsequent internal Ca(2+) release through RyRs but not IP3Rs, are involved in ONOO(-)mediated STOCs enhancement in ASMCs.

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.

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 100 mmHg and volumetric flow in arterioles in RA group were 2 times more than those in NS treated group within the 2 h 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.

Peroxynitrite induces arteriolar smooth muscle cells membrane hyperpolarization with arteriolar hyporeactivity in rats.

Peroxynitrite (ONOO-) has been recently known to act as a potent cytotoxin during pathogenesis of various diseases. This study aimed to investigate the possible effect of ONOO- on the cremaster muscle arteriolar reactivity in response to noradrenaline and subsequently determined whether membrane hyperpolarization and potassium channel activation were involved in ONOO(-)-induced alteration of arteriolar reactivity. The results demonstrated that 1) ONOO- could decrease arteriolar reactivity in a time- and concentration-dependent manner with no significant alteration of arteriolar diameter; 2) Superfusion with 20 microM ONOO- over 40 minutes showed slight but not significant influence on the resting potential (Em) of arteriolar smooth muscle cells (ASMCs). However, ASMCs subjected to 50 or 100 microM ONOO- administration were significantly hyperpolarized. As control, treatment with 50 microM decomposed ONOO- or Kreb's solution had little effect on the Em of ASMCs; 3) ONOO(-)-induced arteriolar hyporeactivity could be greatly reversed by co-administration of KCl and partially by TEA. The above results indicated that membrane hyperpolarization and potassium channel activation were preferentially responsible for the reduction of cremaster muscle arteriolar reactivity after exposure to ONOO-.

Mobilization of intracellular calcium by peroxynitrite in arteriolar smooth muscle cells from rats.

The present study was designed to investigate the possible effects of peroxynitrite (ONOO(-)) on the intracellular calcium concentration ([Ca(2+)](i)) of mesenteric arteriolar smooth muscle cells (ASMCs), and to reveal the underlying mechanisms by using fluorescence imaging analysis. The results showed that ONOO(-) could exert a concentration- and time-dependent but also a dual effect on [Ca(2+)](i). Bolus administration with a low concentration of ONOO(-) (25 microM) decreased [Ca(2+)](i), whereas higher concentrations (50 or 100 microM) increased [Ca(2+)](i) persistently. Further experiments demonstrated that pretreatment of ASMCs with calcium-free medium completely abolished [Ca(2+)](i) increase by 100 microM ONOO(-). Additionally, nifedipine, an antagonist of selective L-type voltage-gated calcium channels (VGCCs), delayed the [Ca(2+)](i) response to ONOO(-), and ryanodine, an inhibitor of intracellular calcium release from the sarcoplasmic reticulum, effectively antagonized [Ca(2+)](i) increase during the late stage of ONOO(-) exposure. Furthermore, [Ca(2+)](i) alteration by ONOO(-) appeared to be intimately associated with the subsequent membrane potential changes. Although the mechanisms by which ONOO(-) alters [Ca(2+)](i) are complex, we conclude that a series of variables such as external calcium influx, activation of VGCCs, intracellular calcium release, and membrane potential changes are involved. The decrease of [Ca(2+)](i) in ASMCs by a low concentration of ONOO(-) may participate in the pathogenesis of low vasoreactivity in shock, and the increase of [Ca(2+)](i) by high concentrations of ONOO(-) may lead to calcium overload with cellular injury.