Richard D. Minshall

Regulation of Endothelial Junctional Permeability

The endothelium is a semi‐permeable barrier that regulates the flux of liquid and solutes, including plasma proteins, between the blood and surrounding tissue. The permeability of the vascular barrier can be modified in response to specific stimuli acting on endothelial cells. Transport across the endothelium can occur via two different pathways: through the endothelial cell (transcellular) or between adjacent cells, through interendothelial junctions (paracellular). This review focuses on the regulation of the paracellular pathway. The paracellular pathway is composed of adhesive junctions between endothelial cells, both tight junctions and adherens junctions. The actin cytoskeleton is bound to each junction and controls the integrity of each through actin remodeling. These interendothelial junctions can be disassembled or assembled to either increase or decrease paracellular permeability. Mediators, such as thrombin, TNF‐α, and LPS, stimulate their respective receptor on endothelial cells to initiate signaling that increases cytosolic Ca2+ and activates myosin light chain kinase (MLCK), as well as monomeric GTPases RhoA, Rac1, and Cdc42. Ca2+ activation of MLCK and RhoA disrupts junctions, whereas Rac1 and Cdc42 promote junctional assembly. Increased endothelial permeability can be reversed with “barrier stabilizing agents,” such as sphingosine‐1‐phosphate and cyclic adenosine monophosphate (cAMP). This review provides an overview of the mechanisms that regulate paracellular permeability.

Role of Ca2+ signaling in the regulation of endothelial permeability

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.

PKCζ Regulates TNF-α--Induced Activation of NADPH Oxidase in Endothelial Cells

Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKCzeta, has been implicated in the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKCzeta in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47(phox) and membrane-bound gp91(phox) in human pulmonary artery endothelial (HPAE) cells. TNF-alpha failed to activate oxidant generation in lung vascular endothelial cells derived from p47(phox-/-) and gp91(phox-/-) mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-alpha resulted in the phosphorylation of p47(phox) and its association with gp91(phox). Inhibition of PKCzeta by multiple pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation of p47(phox), and its translocation to the membrane. PKCzeta was shown to colocalize with p47(phox), and inhibition of PKCzeta activation prevented the interaction of p47(phox) with gp91(phox) induced by TNF-alpha. Furthermore, inhibition of association of p47(phox) with gp91(phox) prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKCzeta in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.

RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry ROLE IN SIGNALING INCREASED ENDOTHELIAL PERMEABILITY

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+]i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability.

Ca2+ signalling and PKCα activate increased endothelial permeability by disassembly of VE—cadherin junctions

The role of intracellular Ca2+ mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i in both cases was due to release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial 125I‐albumin permeability. Thapsigargin induced activation of PKCα and discontinuities in VE‐cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCα activation and similar alterations in VE‐cadherin junctions, but in association with actin stress fibre formation. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC20), whereas thrombin induced MLC20 phosphorylation consistent with formation of actin stress fibres. Calphostin C pretreatment prevented the disruption of VE‐cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca2+]i elicited by thapsigargin and thrombin may activate a calphostin C‐sensitive PKC pathway that signals VE‐cadherin junctional disassembly and increased endothelial permeability. Results suggest a critical role for Ca2+ signalling and activation of PKCα in mediating the disruption of VE‐cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Size and Dynamics of Caveolae Studied Using Nanoparticles in Living Endothelial Cells

Caveolae are plasma membrane invaginations prominent in all endothelial cells lining blood vessels. Caveolae characteristically bud to form free cytoplasmic vesicles capable of transporting carrier proteins such as albumin through the cell. However, caveolae size distribution and dynamics in living endothelial cells and ability of caveolae to internalize nanoparticles are not well understood. We demonstrate here the design of a dual-color nanoparticle pair to measure noninvasively caveolae size and dynamics. First, we coated nanoparticles with BSA (bovine serum albumin) to address whether albumin promoted their delivery. Albumin has been shown to bind to protein on endothelial cell surface localized in caveolae and activate albumin endocytosis. Imaging of BSA-coated nanoparticles varying from 20 to 100 nm in diameter in endothelial cells demonstrated that caveolae-mediated nanoparticle uptake was dependent on albumin coating of particles. We also showed that caveolae could accommodate up to 100 nm diameter nanoparticles, a size larger than the diameter of typical caveolae, suggesting compliant property of caveolae. Together, our results show the feasibility of tracking multicolored nanoparticles in living endothelial cells and potential usefulness for designing therapeutic nanoparticle cargo to cross the limiting vessel wall endothelial barrier.

Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors : The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells

Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

Novel Mechanism of Endothelial Nitric Oxide Synthase Activation Mediated by Caveolae Internalization in Endothelial Cells

Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor NG-nitro-l-arginine (l-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-&bgr;-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS−/− or caveolin-1−/− lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone.

Tumor Necrosis Factor-α Induces Early-Onset Endothelial Adhesivity by Protein Kinase Cζ–Dependent Activation of Intercellular Adhesion Molecule-1

Abstract— We tested the hypothesis that TNF-&agr; induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the &bgr;2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-&agr; resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-&agr; challenge. We observed that TNF-&agr; induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1–dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-&agr;–induced endothelial adhesivity was protein synthesis–independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKC&zgr;, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-&agr;–induced ICAM-1 gene transcription, we determined the possibility that PKC&zgr; was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-&agr; stimulation of endothelial cells induced PKC&zgr; activation and its association with ICAM-1. Inhibition of PKC&zgr; by pharmacological and genetic approaches prevented the TNF-&agr;–induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-&agr; induces early-onset, protein synthesis–independent expression of endothelial adhesivity by PKC&zgr;-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.

Intercellular adhesion molecule-1-dependent neutrophil adhesion to endothelial cells induces caveolae-mediated pulmonary vascular hyperpermeability.

We investigated the role of caveolae in the mechanism of increased pulmonary vascular permeability and edema formation induced by the activation of polymorphonuclear neutrophils (PMNs). We observed that the increase in lung vascular permeability induced by the activation of PMNs required caveolin-1, the caveolae scaffold protein. The permeability increase induced by PMN activation was blocked in caveolin-1 knockout mice and by suppressing caveolin-1 expression in rats. The response was also dependent on Src phosphorylation of caveolin-1 known to activate caveolae-mediated endocytosis in endothelial cells. To address the role of PMN interaction with endothelial cells, we used an intercellular adhesion molecule (ICAM)-1 blocking monoclonal antibody. Preventing the ICAM-1–mediated PMN binding to endothelial cells abrogated Src phosphorylation of caveolin-1, as well as the increase in endothelial permeability. Direct ICAM-1 activation by crosslinking recapitulated these responses, suggesting that ICAM-1 activates caveolin-1 signaling responsible for caveolae-mediated endothelial hyperpermeability. Our results provide support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of PMNs adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury.