| Objective:This study was performed to determine the effects of advanced glycation end products (AGEs) modified protein on actin cytoskeleton morphology and permeability of endothelium monolayer and to detect the underlying signaling mechanisms involved in these responses. Methods:AGE-modified human serum albumin (AGE-HSA) was prepared by incubation of 150 mM human serum albumin with 250 mM of D-glucose for 6 weeks. Endothelial cells (ECs ) grown on gelatin-coated glass bottom microwell Petri dishes or Transwell-clear polyester filters were allowed to reach confluence and subjected to AGE-HSA treatments. In separate groups, ECs were treated with soluble anti-AGE receptor (RAGE) IgG, Apocynin, PD98059, SB203580 or SP600125 respectively before AGE-HSA administration. ECs were also pre-infected with recombinant adenovirus of constitutive dominant negative forms of MEK1, MKK6b or p38α and then subjects to AGE-HSA stimulation. The morphological changes of ECs were monitored by observing fluorescence of F-actin stained with Rhodamine-phalloidin orFITC- phalloidin and G-actin with Oregon Green-Dnase I, respectively. Endothelialpermeability was assessed by measuring the flux TRITC-albumin across ECsmonolayer.Results:1. AGE-HSA induced actin cytoskeleton morphologic changes of ECsIn ECV304, continuous F-actin was mainly distributed in endothelial cellular cortical area under normal condition, forming a typical peripheral actin rim (PAR) and few stress fiber could be seen in the cells, whereas G-actin mostly located on perinuclear and nucleus regions with evenly staining and smooth distinct borderline in control cells. Exposure of ECs to AGE-HSA caused a shift in F-actin distribution from web-like structure to polymerized stress fiber. Cells subjected to higher-concentration and longer-time AGE-HSA exposure showed more and more stress fiber accumulation. Also the edge of G-actin became coarse and illegible and merged with each other, even formed tufts of flock extending all around nuclear area.In ECs pretreated with anti-RAGE IgG, Apocynin, PD98059 or SB203580, more F-actin at intercellular junction area and fewer stress fiber were observed, and these inhibitors also abolished AGE-HSA induced redistribution of G-actin. All the inhibitors themselves had no effect on actin morphology.In primary HUVEC, F-actin showed similar changes to those seen in ECV304 when exposed to AGE-HSA. Further more, the stress fiber formation and peripheral actin rim disruption were coincident with increased permeability in ECV304 monolayer pre-infected with recombinant virus of constitutive active forms, MEK1(E) or MKK6b(E). Infections of primary HUVEC with constitutive negative forms, MEK1(A) or MKK6b(A), abolished AGE-HSA elicited stress fiber formation, while SP600125 and dominant negative form of MKK7 (MKK7(A)) failed to inhibit the effects of AGEs. 2. AGE-HSA caused hyperpermeability in ECV-304 monolayerStimulation ECs with AGE-HSA induced a significant elevation of permeability coefficient for TRITC-albumin (Pa) in a dose- and time-dependent pattern. 12.5, 25, 50, 100 |ug/ml of AGE-HSA increased Pa to 107.68 ± 3.76 %, 123.24 ± 2.55 %,134.06 ± 7.70 %, 155.48 ± 5.30 % of control, respectively. The permeability response occurred within 2 h after 50 ug/ml AGE-HSA administrationa and Pa increased continually for the rest of the observation period. Pa value in 24h is 157.82 ± 4. 81 % of control. Incubation with HSA alone had no effect on ECs permeability. We can see that the more greatly the actin morphology changed the higher the relative Pa was.Pretreatment of ECs monolayer with soluble anti-RAGE IgG for 1 h attenuated AGE-HSA-induced elevations in Pa in a concentration-dependent manner. Anti-RAGE IgG (50 and 100 ^ig/ml) reduced Pa from 134.06 ± 7. 70% tol 13.34 ± 5.04 % and 105.74 ± 3.06 % of control, respectively in the presence of 50 ug/ml AGE-HSA, while it did not affect baseline permeability in the absence of AGE-HSA.In the presences of Apocynin (100, 500 umol/L), a specific NADPH oxidase inhibitor, AGEs treatment yielded Pa of 112.10 ± 2.85% and 107.29 ± 2.84% compared with 134.06 ± 7. 70% without Apocynin. Incubation with Apocynin alone had no effect on ECs permeability.Pretreatment of ECV-304 with PD98059 (50,100 |amol/L), specific inhibitors of ERK, and SB203580 (10, 25 umol/L), specific inhibitors of p38 MAPK could both abolished AGE-HSA-induced hyperpermeability, as Pa were reduced from 134.06 ± 7. 70% to 117.09 ± 2. 74% and 110.02 ± 0. 92% or 120.34 ± 9. 79% and 107.25 ± 7. 58%, respectively. The effects of upstream kinases of ERK and p38 (MEK1 and MKK6b) were detected by infecting ECs with recombinant virus of dominant negative forms of MEK1 (MEK1(A)), MKK6b (MKK6b(A)) 24 h prior to AGE-HSA treatment. Consistently, the results showed potent inhibitory effects upon the stimulation of AGE. This hyperpermeability response was also attenuated by infecting the cells with recombinant virus of dominant negative form of p38a (p38a(A)). Further more, the infection of recombinant virus of constitutive active form of MEK1 (MEK1(E)) or MKK6b (MKK6b(E)) could as well increase Pa to 127.63 ± 10. 19% andl29.60 ± 3. 56% of control respectively, while the effect elicited by MKK6b (E) was eliminated by co-infection with p38a(A). SP600125 and dominant negative form of MKK7 (MKK7(A)) failed to inhibit the effects of AGEs. Conclusion:1. AGEs-HSA could induce actin cytoskeleton reorganization and significantly increase the permeamblilility of endothelial monolayer in time- and dose- dependent manners.2. Anti RAEG-IgG could attenuate the cytoskeleton reorganization and hyperpermeability induced by AGEs which suggests AGE-RAGE interaction plays a central role in mediating the signal into cells.3. NADPH oxidase inhibitor could abolish the effects induced by AGEs, which support our hypothesis that oxidative stress takes part in this pathological process.4. ERK and p38 MAPK are involved in the signaling mechanisms in AGE-induced dysfunction of endothelial cells, but not JNK MAPK. Additional, p38a is the main participating isoform.
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