Qiaobing Huang

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.

Histamine H2 receptor activation exacerbates myocardial ischemia/reperfusion injury by disturbing mitochondrial and endothelial function

There is evidence that H2R blockade improves ischemia/reperfusion (I/R) injury, but the underlying cellular mechanisms remain unclear. Histamine is known to increase vascular permeability and induce apoptosis, and these effects are closely associated with endothelial and mitochondrial dysfunction, respectively. Here, we investigated whether activation of the histamine H2 receptor (H2R) exacerbates myocardial I/R injury by increasing mitochondrial and endothelial permeability. Serum histamine levels were measured in patients with coronary heart disease, while the influence of H2R activation was assessed on mitochondrial and endothelial function in cultured cardiomyocytes or vascular endothelial cells, and myocardial I/R injury in mice. The serum histamine level was more than twofold higher in patients with acute myocardial infarction than in patients with angina or healthy controls. In neonatal rat cardiomyocytes, histamine dose-dependently reduced viability and induced apoptosis. Mitochondrial permeability and the levels of p-ERK1/2, Bax, p-DAPK2, and caspase 3 were increased by H2R agonists. In cultured human umbilical vein endothelial cells (HUVECs), H2R activation increased p-ERK1/2 and p-moesin levels and also enhanced permeability of HUVEC monolayer. All of these effects were abolished by the H2R blocker famotidine or the ERK inhibitor U0126. After I/R injury or permanent ischemia, the infarct size was reduced by famotidine and increased by an H2R agonist in wild-type mice. In H2R KO mice, the infarct size was smaller; myocardial p-ERK1/2, p-DAPK2, and mitochondrial Bax were downregulated. These findings indicate that H2R activation exaggerates myocardial I/R injury by promoting myocardial mitochondrial dysfunction and by increasing cardiac vascular endothelial permeability.

Detrimental effect of fractalkine on myocardial ischaemia and heart failure

AIMS Fractalkine (FKN) is a newly identified membrane-bound chemokine; its role in myocardial ischaemia and heart failure is largely unknown. We attempted to investigate the role of FKN in myocardial ischaemia and ischaemia or pressure overload-induced ventricular remodelling and heart failure. METHODS AND RESULTS FKN-induced changes of heart failure-related genes in cultured rat cardiac cells and the effect of FKN on cultured cardiomyocyte injury during anoxia/reoxygenation (A/R) were examined. The direct influence of FKN neutralization on heart failure and the potential mechanism was also investigated. In mice with failing hearts, myocardial FKN expression was correlated with the lung weight/body weight ratio, left ventricular fractional shortening, and brain natriuretic peptide expression. In cultured rat cells, exposure to FKN increased natriuretic peptide A expression in cardiomyocytes, matrix metalloproteinase-9 expression in fibroblasts, and intercellular adhesion molecule-1 expression in microvascular endothelial cells. FKN also promoted cardiomyocyte damage during A/R and neutralizing FKN antibody treatment improved heart failure induced by myocardial infarction or pressure overload. Neutralizing FKN or its receptor inhibited the activation of mitogen-activated protein kinases (MAPKs) in hypoxic cardiomyocytes or ischaemic myocardium. CONCLUSION FKN promotes myocardial injury and accelerates the progress of heart failure, which is associated with the activation of MAPKs.

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.

Late-phase detection of recent myocardial ischaemia using ultrasound molecular imaging targeted to intercellular adhesion molecule-1

AIMS in this study, we attempted to detect a recent myocardial ischaemic event using ultrasound molecular imaging (UMI) with microbubbles (MB) targeted to intercellular adhesion molecule-1 (ICAM-1) in the late phase of reperfusion. METHODS AND RESULTS we created a myocardial ischaemia-reperfusion model in 60 C57/BL male mice to simulate an angina attack (ischaemia for 15 min, reperfusion for 1-24 h). The degree of myocardial inflammation and levels of ICAM-1 protein were determined by histological and immunohistochemical analyses. UMI with MB targeted to endothelial ICAM-1, as well as routine non-invasive methods including electrocardiography, echocardiography, and plasma troponin I levels, were utilized to evaluate ischaemia over the time course of reperfusion. Levels of ICAM-1 in the vascular endothelium were significantly increased over the time course of reperfusion (8-24 h) of the ischaemic myocardium. The video intensity of ICAM-1 molecular images of the ischaemic anterior wall was almost three times greater than that in the non-ischaemic posterior wall during the late phase (8-24 h) of reperfusion. In contrast, routine methods yielded only weak evidence of ischaemia. CONCLUSION UMI with MB targeted to endothelial ICAM-1 provides reliable evidence of a recent myocardial ischaemic event in the late phase of reperfusion.

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.

Role of Src in Vascular Hyperpermeability Induced by Advanced Glycation End Products.

The disruption of microvascular barrier in response to advanced glycation end products (AGEs) stimulation contributes to vasculopathy associated with diabetes mellitus. Here, to study the role of Src and its association with moesin, VE-cadherin and focal adhesion kinase (FAK) in AGE-induced vascular hyperpermeability, we verified that AGE induced phosphorylation of Src, causing increased permeability in HUVECs. Cells over-expressed Src displayed a higher permeability after AGE treatment, accompanied with more obvious F-actin rearrangement. Activation of Src with pcDNA3/flag-SrcY530F alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-SrcK298M abolished these effects. The pulmonary microvascular endothelial cells (PMVECs) isolated from receptor for AGEs (RAGE)-knockout mice decreased the phosphorylation of Src and attenuated the barrier dysfunction after AGE-treatment. In vivo study showed that the exudation of dextran from mesenteric venules was increased in AGE-treated mouse. This was attenuated in RAGE knockout or PP2-pretreated mice. Up-regulation of Src activity induced the phosphorylation of moesin, as well as activation and dissociation of VE-cadherin, while down-regulation of Src abolished these effects. FAK was also proved to interact with Src in HUVECs stimulated with AGEs. Our studies demonstrated that Src plays a critical role in AGE-induced microvascular hyperpermeability by phosphorylating moesin, VE-cadherin, and FAK respectively.

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells.

AIMS Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs). MAIN METHODS AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry. KEY FINDINGS AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation. SIGNIFICANCE This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.

Olmesartan prevents cardiac rupture in mice with myocardial infarction by modulating growth differentiation factor 15 and p53

Cardiac rupture is a catastrophic complication that occurs after acute myocardial infarction (MI) and, at present, there are no effective pharmacological strategies for preventing this condition. Here we investigated the effect of the angiotensin II receptor blocker olmesartan (Olm) on post‐infarct cardiac rupture and its underlying mechanisms of action.