Advanced Glycation End Products Induce Moesin Phosphorylation In Endothelia In Murine Brain And Other Organs

Objective:Our previous study has demonstrated that ERM protein moesin is phosphorylated by AGE-induced signaling events and plays an important role in modulating the endothelial cytoskeleton arrangement and barrier function in human microvascular endothelial cells. We have already detected the increase of murine retinal microvascular permeability in an AGE-stimulated mouse model. The purpose of this present study is to observe the alteration of blood brain barrier function after AGE-stimulation and to explore the effects of threonine phosphorylation of moesin in this response. This AGE-induced moesin phosphorylation was also detected in kidney, heart, and liver. The involvements of ROCK and MAPK in AGE-induced moesin phosphorylation in microvascular endothelial cells were also clarified.Methods:1. Preparation of AGE-MSAAGE-MSA was prepared according to the reported protocoll. Briefly,3.5 mg/ml mouse serum albumin was incubated in PBS (pH 7.4) with 100 mmol/L of D-glucose for 10 weeks at 37℃in a sterile environment. Control albumin was incubated in the same condition without glucose. The solutions were then extensively dialyzed against PBS and concentrated with Millipore 15 ml ultrafiltration column. Endotoxin was removed by using a Pierce Endotoxin Removing Gel. AGE-specific fluorescence was determined by using ratio spectrofluorometry. The AGE-MSA contained 80.50 U/mg protein AGEs, while native albumin contained only 0.79U/mg protein AGEs.2. Animals and anesthesiaFemale C57/BL6 mice used in this experiment were obtained from Laboratory Animal Center of Southern China. The research protocols were approved by the Animal Care Committee of Southern Medical University of China and performed in adherence to National Institute of Health guidelines. The mice were anesthetized with an intramuscular injection of 13.3% ethyl carbamate plus 0.5% chloralose (0.65 ml/kg) before all operational manipulations.3. AGE-MSA treatment of miceTwenty four Mice of 10-12 weeks old were randomly assigned to four groups of equal size. According to the protocol of Moore TC et al (Moore et al.2003), control and AGE-treated groups were injected intraperitoneally (i.p.) with either native mouse serum albumin (MSA,10 mg/kg) or AGE-MSA (10 mg/kg) daily for seven consecutive days. SB203580 (1 mg/kg) or Y-27632 (5 mg/kg) were injected, respectively, i.p.30 min before every AGE-MSA administration in p38 or ROCK inhibition group.4. Immiinohistochemical analysis of moesin expression in brainAfter anesthesia, the animals were perfused with saline from abdominal vein through left ventricle for 2 min and then with 10% neutral buffere formalin for 1 min for primary fixation. The brains were removed and cerebral pia mater discarded and then kept in 10% neutral buffered formalin at least 24 hours at room temperature. The brain, kidney, heart, and liver were embedded in paraffin and were cut on a sliding microtome at 20 mm followed by immunohistochemistry. All immunostaining were carried out by standard protocol using anti-moesin or anti-phospho-moesin antibodies as primary antibodies and biotin free horseradish peroxidase (HRP) antibody as secondary antibody. HRP polymer was labeled with 3,3'-diaminobenzidine (EnVision plus detection system, Dako Ltd, Ely, UK).5. Quantification and observation of blood-brain barrier functionTo clarify the functional impact of AGE-MSA stimulation on blood-brain-barrier permeability, Evans blue (EB) dye extravasation with fluorescein was applied according to Uuama O's protocols. Briefly, mice were anesthetized and EB was injected through tail vein over 10 seconds at a dosage of 45 mg/kg and the uptake and distribution of the dye was confirmed by immediate visibly blue in mice. After the dye had circulated for 60 minutes, the chest cavity was opened, and saline was perfused into the body through left ventricle with 110 mmHg pressure while right atrium was cut open to allow all the blood flushing out. Then 1% paraformaldehyde diluted in 0.05 mol/L citric acid buffer in 37℃with pH 3.5 was affused through left ventricle to discard the EB left in the vessels. The brain was taken out and half of the brain was weighted and then homogenated with 2 ml 50% trichloroacetic acid (TCA) in 4℃and 15000 rpm for 20 min. The supernatant was diluted in 100% ethanol with 1:3 in volume and the fluorescence was detected using fluorescence spectrophotometer. The amount of extravasated EB was determined using a standard curve established with EB from 31.25 ng/ml-2000 ng/ml. To directly observe the leakage of brain vessel, the other half of brain was used for fast frozen coronal sections in the thickness of 8μm. The EB was observed using fluorescence microscope under the excitation of 450-470 nm.6. Western blot analysisBrain microvascular tissues were isolated and collected according to Roux's report. After 2 min of saline perfusion, the anesthetized animals were sacrificed by decollation. The brain was taken out and cerebral pia mater and white matter was discarded upper the ice. The remained brain tissue was cut for 10 times in 6 ml pH7.2 PBS with 0.1 g/L of typeⅡcollagenase and then incubated in 37℃shaker. The tissue was filtrated and the filtrate was collected and centrifuged in 1500 rpm for 10min. The sediment was extracted by lysing and sonicating in lysis buffer with anti-protease and anti-phosphotase cocktail. Samples were subjected to SDS-PAGE, and proteins were transferred to PVDF membranes. Blots were blocked and incubated with 1:1000 dilution of primary antibody of interest overnight at 4℃on a rocker. After three washes for 5 min each with TPBS, the blots were incubated with a 1:1000 dilution of HRP-conjugated species-specific respective secondary antibody (Dako Ltd, Ely, UK) for 1 h at room temperature. After three washes for 5 min each with TPBS, protein bands were visualized by chemiluminescence.Results:1. Effect of AGE-MSA in expression of moesin and threonine 558 phosphorylated moesin in murine brain and other organsCellular expression of moesin was immunohistochemically identified with total moesin antibody or 558 threonine phosphorylated moesin antibody. The immunohistochemistry of brain and other organ tissues showed that moesin was mainly and strongly expressed in microvascular endothelial cells, with some weakly expression in brain micro glial cells and cardiomyocytes. The phosphorylated moesin was almost absent in control organs. The administration of AGE-MS A did not alter the total moesin level, while it significantly strengthened the expression of moesin phosphorylated in 558 threonine residue. The pretreatment of p38MAPK inhibition SB203580 or ROCK inhibition Y27632 had no influence on total moesin expression in vascular endothelia, while they remarkably weakened the AGE-MSA induced moesin phosphorylation. The immunofluorescent assays of those murine organs revealed similar results. The strong and specific expression of moesin in brain endothelial cells was quite striking. AGE-MSA induced moesin phosphorylation was obviously detected with fluorescence secondary antibody and the inhibition of p38MAPK or ROCK pathway attenuated the phosphorylation of moesin in 558 threonine residue.2. Effect of AGE-MSA in protein expression of moesin and threonine 558 phosphorylated moesin in murine brain microvascularSince the amount of protein samples were limited from isolated murine brain microvascular, the expression of moesin and phosphorylated moesin were detected in separated operation by western blotting. The results demonstrated that the expression of total moesin were not altered either by stimulation of AGE-MSA or by pre-treating the mice with SB203580 and Y27632 before AGE-MSA administrations. The expression of 558 threonine phosphorylated moesin in AGE-MSA treated mouse were significantly increased (p<0.05 compared with control).The western blot data also showed that the phosphorylating activation of p38MAPK or ROCK was significantly weakened by the pre-administration of SB203580 or Y27632 in AGE-MSA treated mice. This down-regulation of p38MAPK or ROCK was accompanied by significant attenuation of moesin phosphorylation induced by AGE-MSA stimulation (p<0.05 compare with AGE-MSA treated alone). These results indicated that both p38MAPK and ROCK are involved in this AGE-induced moesin threonine phosphorylation.3. The changes of blood-brain-barrier functionthe cerebral microvascular endothelial barrier function. The amount of Evans blue (EB) dye extravasation increased significantly in AGE-MSA treated-mice (p<0.05,). The fluorescent leakage images from frozen sections also demonstrated that Evans blue dye leaked from the vessels and spread in the brain tissue much more obviously in AGE-MSA treated group than in control. The usages of SB203580 or Y27632 before AGE-MSA stimulation significantly attenuated these AGE-induced increase in brain microvascular permeability both in leakage assay and frozen sections.Conclusion:AGEs treatment induced a significant increase of threonine 558 phosphorylation of moesin in the observed tissue. The intervention of ROCK and p38 activation remarkably attenuated the phosphorylation of moesin in 558 threonine residue. Consistent with these in situ data, the protein level of moesin phosphorylation was also increased in cerebral microvascular, accompanying with the permeability augmentation of brain-blood barrier, whilst the inhibition of ROCK and p38 pathways lessened these responses. These results demonstrated that AGEs did cause the moesin phophosrylation in murine brain microvascular endothelial cells. p38 MAPK and ROCK are involved in this phophosrylating process.