The Effect And Mechanism Of Pigment Epithelium-derived Factor In Microvascular Permeability In Inflammation

BackgroundSevere burn injury induces a pro-inflammatory status, resulting in a systemic inflammatory response syndrome(SIRS). This inflammatory reaction proceeds from complex phenomenons in which many cellular elements and molecular products are involved. These phenomenons promote significant physiopathologic consequences, especially on cardiovascular homeostasis and endothelial permeability. Despite a better comprehension of the phenomenons underlying this inflammatory process, diagnosis or therapeutic applications are still disappointing.It is now recognised that greater attention should be focused on the progressive subcutaneous and body cavity oedema that typically develops in severe burn patients. The accumulation of parenchymal and interstitial fluids impairs organ function not only by increasing the distance required for oxygen diffusion, but also by compromising microvascular perfusion due to increased interstitial pressure. Tissue oedema has been generally attributed to the widespread increase in vascular permeability in inflammation, which is closely related to microvascular endothelial dysfunction. However, the mechanism by which endothelial dysfunction contributes to hyperpermeability in inflammation has not been clarified.Sepsis is characterised by an elevated level of circulating inflammatory cytokines and dysregulated coagulation, and can adequately mimic inflammatory status. In our previous study, proteomics analysis was applied to compare the changes in serum proteins in post-burn sepsis patients followed by purpura fulminans(PF) to those in healthy individuals. Consistent with previous studies, pigment epithelium-derived factor(PEDF) was found to be significantly increased in septic patients.PEDF is an endogenous protein that is widely expressed throughout the human body and has multiple biological activities. PEDF effectively inhibits vascular endothelial growth factor(VEGF)-driven angiogenesis and vascular permeability by regulating the intracellular proteolysis of VEGF receptors. However, recent reports have raised the possibility that PEDF may act as an important endogenous vasoactive substance. However, the precise mechanisms underlying the effects of PEDF in the vasculature have not been illuminated. The aim of the present study was to investigate the role of PEDF in vascular permeability during inflammation and to identify the molecular mechanism underlying its effects.Materials and Methods1.To determine whether the serum level of PEDF was increased during inflammation, serum samples from septic patients and mice were collected at the indicated times and analysed by enzyme-linked immunosorbent assay(ELISA) and western blotting(WB).2.We evaluated the effects of exogenous PEDF in vivo by using a mouse model of cecal ligation and puncture(CLP)-induced sepsis. We injected exogenous PEDF and PEDF monoclonal antibody(PEDF-mAb), and evaluated the loss of tracheal vessel, cremaster muscle vessel, and dermal vessel integrity by monitoring the extravasation of 100-nm microsphere particles and Evan’s blue(EB).3.In vitro we detected the transendothelial electrical resistance(TER) of human dermal microvascular endothelial cells(HDMECs) under the treatment of different concentrations of PEDF.4.We further analysed the effect of PEDF on paracellular permeability by measuring the leakage of fluorescein isothiocyanate(FITC)-dextran tracer across cultured endothelial cell monolayers.5.To determine whether RhoA contributed to PEDF-induced disruption of cell junctions and hyperpermeability, the effects of C3 transferase(RhoA inhibitor) on PEDF-induced F-actin rearrangement, Zonula occludens-1(ZO-1) redistribution, and FITC-dextran tracer leakage were assessed.6.To identify the receptor that mediates RhoA activation in inflammation, plasma membrane proteins and tissue proteins were extracted, and WB was performed to confirm the differential expression of PEDF receptors-adipose triglyceride lipase(ATGL) and laminin receptor(LR). The interactions between PEDF and the two receptors were investigated by immunoprecipitation(IP).7.To more directly address the role of ATGL in the course of PEDF-induced hyperpermeability during inflammation, we established ATGL-/LR-shRNA-treated HDMECs. Total protein was extracted, and WB was performed to confirm the knockdown effects of ATGL- and LR-shRNA. Besides, RhoA activation, ZO-1 redistribution, and FITC-dextran tracer leakage were assessed.Results1.The results of both the ELISA and WB analysis showed that septic patients had significantly elevated serum PEDF level compared to controls. In post-burn septic mice, the serum PEDF level was significantly elevated from 6 h after establishment of the model and remained elevated up to 24 h post-injury; no significant difference in serum PEDF level was observed at 3 h post-injury. However, in both CLP- and lipopolysaccharide(LPS)-induced septic mice, serum PEDF level was significantly elevated starting 3 h after establishment of the model and remained elevated up to 24 h after injury. The level of circulating PEDF peaked at 12 h in both post-burn septic and CLP-induced septic mice and peaked at 3 h in LPS-induced septic mice. To verify these results, serum samples collected from septic mice at the peak level of expression were pooled and further analysed by WB, and the results demonstrated similar elevations in serum PEDF among treated mice in comparison to sham controls.2.Extravasation of microsphere particles was not detected in control mice but detected in PEDF mice, a similar disruption in vasculature integrity was observed in the cremaster muscle microvasculature following PEDF injection. In CLP septic mice, exogenous PEDF aggravated the barrier disruption observed in both the tracheal vasculature and cremaster muscle microvasculature. Moreover, blockade of PEDF by using PEDF-mAb prevented the vascular damage previously observed in the trachea and cremaster muscle of CLP septic mice. The decrease in blood fluorescence of FITC-dextran suggested that tracer shifted into the interstitial space after PEDF injection. In addition, blood fluorescence further declined in septic mice injected with PEDF compared to septic mice that were not injected. However, fluorescence was notably increased in septic mice following PEDF-mAb injection, as compared to septic mice, and then recovered to near-normal level. In the EB dye assay, circulating dye that escaped into the skin was increased after local injection of PEDF compared to the control group injected with phosphate buffer solution(PBS). In CLP septic mice, leakage of EB dye was increased after PEDF injection. Notably, septic mice with PEDF-mAb injection exhibited significantly less EB leakage compared to the septic group that was not injected.3. Treatment of HDMECs with PEDF decreased the TER in a dose- and time-dependent manner. Stimulation with PEDF caused an increase in fluorescent leakage under normal or LPS-/ Tumor necrosis factor-α(TNF-α)-induced inflammatory conditions. Staining of HDMECs for F-actin revealed a profound change in cell morphology. In particular, cells in the control group demonstrated confluency with few intercellular gaps and long thin F-actin fibres in the central portion of cells. However, in cells stimulated with PEDF or LPS/TNF-α, the intercellular space was significantly increased and central F-actin bundles were obvious. In cells co-stimulated with PEDF+LPS or PEDF+TNF-α, these stress fibres were thicker and dense wider peripheral F-actin occurred. Moreover, nearly all the cell–cell junctions were disrupted with considerable gaps in the monolayer. ZO-1 fluorescence was concentrated at cell-cell borders in the normal group, while in cells treated with PEDF, LPS or TNF-α, ZO-1 staining was weak and localised in a discontinuous fashion around the cell membrane. Moreover, disassembly of ZO-1 along the cell membrane was more severe in cells co-stimulated with PEDF+LPS or PEDF+TNF-α.4.Although total RhoA expression was unchanged, the activated RhoA level was significantly increased following treatment with PEDF, LPS or TNF-α. Furthermore, cells treated with PEDF+LPS or PEDF+TNF-α demonstrated greater activation of RhoA compared to cells treated with LPS or TNF-α alone. However, Rho-associated coiled-coil protein kinase(ROCK) was not activated following PEDF stimulation. RhoA activity returned to the basal level after treatment with C3 transferase. Correspondingly, the increase in FITC-dextran leakage was also blocked by treatment with C3 transferase. Moreover, C3 transferase abolished the effects of PEDF, including F-actin rearrangement and loss of ZO-1 along cell-cell junctions.5.ATGL expression was significantly increased in both the total cell lysate and in the membrane fraction after stimulation, while LR expression remained unchanged. Treatment with LPS or TNF-α resulted in increased ATGL/PEDF association, whereas the association of LR/PEDF was not altered. Moreover, in conditions of inflammation, the expression of ATGL was increased in five tissues, including the heart, liver, lung, kidney, and intestine, while LR expression remained unchanged.6. RhoA activation induced by PEDF and PEDF+TNF-α was inhibited in ATGL-shRNA-treated HDMECs. FITC-dextran leakage was decreased, and similar effects on ZO-1 distribution were observed. However, in LR-shRNA-treated HDMECs, there were no significant differences in RhoA activity or fluorescent leakage when compared with scrambled control cells, and PEDF treatment still led to the loss of junctional ZO-1 expression.ConclusionIn summary, this study demonstrates that elevated level of serum PEDF induce increased extravasation both in normal and in inflammation in vivo. Exogenous PEDF increases paracellular permeability by targeting the plasma membrane ATGL receptor, leading to F-actin rearrangement and endothelial junctions ZO-1 disruption through activating the RhoA pathway in vitro. Importantly, this damage could be blocked by treatment with PEDF-mAb in vivo or ATGL-shRNA in vitro, which may provide new potential therapeutic strategies for the treatment of hyperpermeability in inflammatory status such as burn shock and sepsis.