P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin

The goal of our study was to investigate the impact of p-cresylsulfate (PCS) on the barrier integrity in human umbilical vein endothelial cell (HUVEC) monolayers and the renal artery of chronic kidney disease (CKD) patients. We measured changes in the transendothelial electrical resistance (TEER) of HUVEC monolayers treated with PCS (0.1–0.2 mM) similar to serum levels of CKD patients. A PCS dose (0.2 mM) significantly decreased TEER over a 48-h period. Both PCS doses (0.1 and 0.2 mM) significantly decreased TEER over a 72-h period. Inter-endothelial gaps were observed in HUVECs following 48 h of PCS treatment by immunofluorescence microscopy. We also determined whether PCS induced the phosphorylation of VE-cadherin at tyrosine 658 (Y658) mediated by the phosphorylation of Src. Phosphorylated VE-cadherin (Y658) and phosphorylated Src levels were significantly higher when the cells were treated with 0.1 and 0.2 mM PCS, respectively, compared to the controls. The endothelial barrier dysfunction in the arterial intima in CKD patients was evaluated by endothelial leakage of immunoglobulin G (IgG). Increased endothelial leakage of IgG was related to the declining kidney function in CKD patients. Increased endothelial permeability induced by uremic toxins, including PCS, suggests that uremic toxins induce endothelial barrier dysfunction in CKD patients and Src-mediated phosphorylation of VE-cadherin is involved in increased endothelial permeability induced by PCS exposure.

Signaling Mechanisms Regulating Vascular Endothelial Barrier Function

During inflammatory conditions, such as sepsis, myocardial infarction and acute respiratory distress syndrome, endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications and ultimately leads to the multi-organ failure and death. Thus, to determine the molecular mechanisms underlying the opening and resolution phase of the gap formation, will provide an insight to better understand the pathology of the cardiovascular and pulmonary inflammatory disorders. In this chapter, we will discuss about how the signaling mechanisms activated by the known inflammatory molecules increase endothelial permeability.

ID: 120: REGULATION OF ENDOTHELIAL BARRIER PERMEABILITY BY PHOSPHOLIPASE D2

Pulmonary edema is a hallmark of several diseases including acute respiratory distress syndrome (ARDS) and is characterized by the disruption of the pulmonary endothelial barrier at its early stage. Maintaining the integrity of the adherens junctions (AJs) by stabilizing VE-Cadherin (VEC) at the cell membrane after injury could potentially be important to minimize endothelial barrier disruption. Since Phospholipase D (PLD) and its catalytic product, phosphatidic acid (PA), has been shown to be critical in membrane trafficking and in recycling of a number of cell surface receptors, we hypothesized that PLD/PA pathway accelerates the rate of VEC recycling to the lamellipodia to reassemble the AJs. We demonstrate, by measuring the trans endothelial resistance of human lung microvascular endothelial cells (HLMVECs), that inhibiting PLD2-dependent PA production increases the endothelium permeability in response to thrombin. Furthermore, immunostaining shows that the uniform redistribution of VEC to the AJs post thrombin insult is compromised when PLD-dependent PA production is inhibited, and resulted in the appearance of eminent intercellular gaps. Also, PLD2 inhibition prevented the HLMVECs from fully spreading after thrombin stimulation while the protrusive activity remained unaffected, suggesting that PLD2 is not required by HLMVECs to send protrusions, but is critical for the adherence of the protrusions. LPS-induced lung injury was more severe in PLD2 knockout mice compared to WT in an in vivo ARDS model. These observations suggest that PLD/PA signaling plays an important role in resealing of endothelial gaps post LPS-induced lung injury and could potentially be therapeutically utilized to enhance post-injury endothelium recovery.

Abstract 179: Rho Kinase Isoform Contribution to Endothelial Contractility

Background: Rho kinase (ROCK) is the major effector protein of RhoA and is known to mediate F-actin stress fibers. ROCK activates myosin motors. Actomyosin-generated contractile forces can be transmitted to cell-cell junctions, resulting in increased junctional tension, junctional disassembly and endothelial hyperpermeability. In apparent contrast, basal ROCK activity is required for maintenance of barrier integrity . We hypothesize that these dual roles of ROCK could be contributed to the two highly homologous expressed isoforms. Methods: Traction force microscopy was utilized to study the endothelium-generated contractile forces after depletion of ROCK1 and/or ROCK2 by SiRNA approach in the absence and presence of the vaso-active agent thrombin. Results: The traction force landscape showed that ROCK is a major contributor to endothelial contraction and permeability. Depletion of ROCK1 and ROCK2 resulted in a blockade of contractile response to thrombin and a marked reduction in endothelial hyperpermeability. Detailed force distribution maps revealed that the absence of ROCK1, but not of ROCK2, resulted in an increase in baseline contractile forces (siROCK1: 88.1 ± 4.1 Pa, siROCK2: 53.6 ± 3.4 Pa, Control: 56.2 ± 6.1 Pa), which was associated with the formation of numerous inter-endothelial gaps upon stimulation with thrombin. Knockdown of ROCK2 stabilized the endothelial barrier and largely prevented traction force enhancements. Exposure to thrombin resulted in all conditions in an increase of traction forces and hyperpermeability. Conclusion: Both ROCK isoforms have a distinct function in endothelial contractility and permeability. ROCK1 is predominantly contributing to barrier maintenance under baseline conditions, whereas ROCK2 mediates the thrombin-induced contractile force enhancements and subsequent barrier dysfunction. Funding: Dutch Heart Foundation 2011T072

Platelets can enhance vascular permeability

Platelets survey blood vessels, searching for endothelial damage and preventing loss of vascular integrity. However, there are circumstances where vascular permeability increases, suggesting that platelets sometimes fail to fulfill their expected function. Human inflammatory arthritis is associated with tissue edema attributed to enhanced permeability of the synovial microvasculature. Murine studies have suggested that such vascular leak facilitates entry of autoantibodies and may thereby promote joint inflammation. Whereas platelets typically help to promote microvascular integrity, we examined the role of platelets in synovial vascular permeability in murine experimental arthritis. Using an in vivo model of autoimmune arthritis, we confirmed the presence of endothelial gaps in inflamed synovium. Surprisingly, permeability in the inflamed joints was abrogated if the platelets were absent. This effect was mediated by platelet serotonin accumulated via the serotonin transporter and could be antagonized using serotonin-specific reuptake inhibitor antidepressants. As opposed to the conventional role of platelets to microvascular leakage, this demonstration that platelets are capable of amplifying and maintaining permeability adds to the rapidly growing list of unexpected functions for platelets.

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca2+]i, nitric oxide, and gap formation in intact venules

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca2+]i, NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca2+]i was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca2+]i, NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca2+]i preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca2+]i in each cell, suggesting that the initial levels of endothelial [Ca2+]i determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca2+ response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 ± 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 ± 0.6 and 1.4 ± 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity ( Lp), indicating that the opening and closing of endothelial gaps contributed to the transient increase in Lp in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.