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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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^558Aand pcDNA3/HA-moesinT^558Dwere applied in endothelial cells.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.</p

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

Role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced pulmonary epithelial hyperpermeability

Abstract Background The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI. Methods For in vivo studies, 6–8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein. Results We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS. Conclusions Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process

Endothelial glycocalyx in traumatic brain injury associated coagulopathy: potential mechanisms and impact

Abstract Traumatic brain injury (TBI) remains one of the leading causes of death and disability worldwide; more than 10 million people are hospitalized for TBI every year around the globe. While the primary injury remains unavoidable and not accessible to treatment, the secondary injury which includes oxidative stress, inflammation, excitotoxicity, but also complicating coagulation abnormalities, is potentially avoidable and profoundly affects the therapeutic process and prognosis of TBI patients. The endothelial glycocalyx, the first line of defense against endothelial injury, plays a vital role in maintaining the delicate balance between blood coagulation and anticoagulation. However, this component is highly vulnerable to damage and also difficult to examine. Recent advances in analytical techniques have enabled biochemical, visual, and computational investigation of this vascular component. In this review, we summarize the current knowledge on (i) structure and function of the endothelial glycocalyx, (ii) its potential role in the development of TBI associated coagulopathy, and (iii) the options available at present for detecting and protecting the endothelial glycocalyx

Superimposed traumatic brain injury modulates vasomotor responses in third-order vessels after hemorrhagic shock

Background: Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of death in trauma. Recent studies suggest that TBI may influence physiological responses to acute blood loss. This study was designed to assess to what extent superimposed TBI may modulate physiologic vasomotor responses in third-order blood vessels in the context of HS. Methods: We have combined two established experimental models of pressure-controlled hemorrhagic shock (HS; MAP 50 mmHg/60 min) and TBI (lateral fluid percussion (LFP)) to assess vasomotor responses and microcirculatory changes in third-order vessels by intravital microscopy in a spinotrapezius muscle preparation. 23 male Sprague-Dawley rats (260-320 g) were randomly assigned to experimental groups: i) Sham, ii) HS, iii) TBI + HS, subjected to impact or sham operation, and assessed. Results: HS led to a significant decrease in arteriolar diameters by 20% to baseline (p < 0.01). In TBI + HS this vasoconstriction was less pronounced (5%, non-significant). At completed and at 60 minutes of resuscitation arteriolar diameters had recovered to pre-injury baseline values. Assessment of venular diameters revealed similar results. Arteriolar and venular RBC velocity and blood flow decreased sharply to < 20% of baseline in HS and TBI + HS (p < 0.01). Immediately after and at 60 minutes of resuscitation, an overshoot in arterial RBC velocity (140% of baseline) and blood flow (134.2%) was observed in TBI + HS. Conclusion: Superimposed TBI modulated arteriolar and venular responses to HS in third-order vessels in a spinotrapezius muscle preparation. Further research is necessary to precisely define the role of TBI on the microcirculation in tissues vulnerable to HS

Functional Characterization of S100A8 and S100A9 in Altering Monolayer Permeability of Human Umbilical Endothelial Cells

<div><p>S100A8, S100A9 and S100A8/A9 complexes have been known as important endogenous damage-associated molecular pattern (DAMP) proteins. But the pathophysiological roles of S100A8, S100A9 and S100A8/A9 in cardiovascular diseases are incompletely explained. In this present study, the effects of homo S100A8, S100A9 and their hetero-complex S100A8/A9 on endothelial barrier function were tested respectively in cultured human umbilical venous endothelial cells (HUVECs). The involvement of TLR4 and RAGE were observed by using inhibitor of TLR4 and blocking antibody of RAGE. The clarification of different MAPK subtypes in S100A8/A9-induced endothelial response was implemented by using specific inhibitors. The calcium-dependency was detected in the absence of Ca²⁺ or in the presence of gradient-dose Ca²⁺. The results showed that S100A8, S100A9 and S100A8/A9 could induce F-actin and ZO-1 disorganization in HUVECs and evoked the increases of HUVEC monolayer permeability in a dose- and time-dependent manner. The effects of S100A8, S100A9 and S100A8/A9 on endothelial barrier function depended on the activation of p38 and ERK1/2 signal pathways through receptors TLR4 and RAGE. Most importantly, we revealed the preference of S100A8 on TLR4 and S100A9 on RAGE in HUVECs. The results also showed the calcium dependency in S100A8- and S100A9-evoked endothelial response, indicating that calcium dependency on formation of S100A8 or A9 dimmers might be the prerequisite for this endothelial functional alteration.</p></div

Mdia1 is Crucial for Advanced Glycation End Product-Induced Endothelial Hyperpermeability

Background/Aims: Disruption of endothelial barrier integrity in response to advanced glycation end products (AEGs) stimulation contributes to vasculopathy associated with diabetes mellitus. Mammalian diaphanous-related formin (mDia1) has been reported to bind to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE), which induces a series of cellular processes. This study directly evaluated the participation of mDia1 in AGE-induced hyperpermeability and revealed the precise intracellular signal transductions of this pathological process. Methods: Human umbilical vein endothelial cells (HUVECs) were used in the in vitro studies. Trans-endothelial electric resistance and permeability coefficient for dextran (Pd) were measured to analyze cell permeability. Western blotting, immunofluorescence staining and flow cytometry assay were performed to investigate the underlying mechanism. Dextran flux across the mesentery in mice was monitored to investigate in vivo microvascular permeability. Results: we found that AGEs evoked Nox4 membrane translocation, reactive oxygen species production, phosphorylation of Src and VE-cadherin, dissociation of adherens junctions and eventual endothelial hyperpermeability through RAGE-mDia1 binding. Cells overexpressing mDia1 by recombinant adenovirus infection showed stronger cellular responses induced by AGEs. Down-regulation of mDia1 by infection with an adenovirus encoding siRNA or blockade of RAGE-mDia1 binding by transfection with RAGE mutant plasmids into HUVECs abolished these AGE-induced effects. Furthermore, knockdown of mDia1 using an adenovirus or genetical knockout of RAGE in C57 mice rescued AGE-evoked microvascular hyperpermeability. Conclusion: Our study revealed that mDia1 plays a critical role in AGE-induced microvascular hyperpermeability through binding to RAGE

S100A8, S100A9 and S100A8/A9 induced the phosphorylation and activation of MAPKs.

<p>HUVECs were stimulated with S100A8 (2.0 µg/mL) (<b>A</b>), S100A9 (2.0 µg/mL) (<b>B</b>) and S100A8/A9 (2.0 µg/mL) (<b>C</b>) for 0, 10, 30, 60 and 120 min respectively. Phosphorylation of p38 (p-p38), ERK1/2 (p-ERK1/2) and JNK (p-JNK) were assessed by Western blotting with specific antibodies as described in Materials and Methods. The ration of immunointensity between the phosphorylation of MAPKs (p-p38, p-ERK and p-JNK) and total MAPKs (p38, ERK and JNK) were calculated. The results are expressed in mean ± s.d. from three independent experiments. ^*P<0.05 vs. Control; ^**P<0.01 vs. Control.</p