Stephen M. Vogel

Impairment of Store-Operated Ca2+ Entry in TRPC4−/− Mice Interferes With Increase in Lung Microvascular Permeability

We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4−/−) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4−/− LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 &mgr;mol/L). In TRPC4−/− LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4−/− endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (Kf,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4−/−; La3+ (1 &mgr;mol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4−/−. These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular 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.

Ca2+ Signaling, TRP Channels, and Endothelial Permeability

Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein‐coupled receptors trigger increased endothelial permeability by increasing the intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i activates key signaling pathways that mediate cytoskeletal reorganization (through myosin‐light‐chain‐dependent contraction) and the disassembly of VE‐cadherin at the adherens junctions. The Ca2+‐dependent protein kinase C (PKC) isoform PKCα plays a crucial role in initiating endothelial cell contraction and disassembly of VE‐cadherin junctions. The increase in [Ca2+]i induced by inflammatory agonists such as thrombin and histamine is achieved by the generation of inositol 1,4,5‐trisphosphate (IP3), activation of IP3‐receptors, release of stored intracellular Ca2+, and Ca2+ entry through plasma membrane channels. IP3‐sensitive Ca2+‐store depletion activates plasma membrane cation channels (i.e., store‐operated cation channels [SOCs] or Ca2+ release‐activated channels [CRACs]) to cause Ca2+ influx into endothelial cells. Recent studies have identified members of Drosophila transient receptor potential (TRP) gene family of channels that encode functional SOCs in endothelial cells. These studies also suggest that the canonical TRPC homologue TRPC1 is the predominant isoform expressed in human vascular endothelial cells, and is the essential component of the SOC in this cell type. Further, evidence suggests that the inflammatory cytokine tumor necrosis factor‐α can induce the expression of TRPC1 in human vascular endothelial cells signaling via the nuclear factor‐κB pathway. Increased expression of TRPC1 augments Ca2+ influx via SOCs and potentiates the thrombin‐induced increase in permeability in human vascular endothelial cells. Deletion of the canonical TRPC homologue in mouse, TRPC4, caused impairment in store‐operated Ca2+ current and Ca2+‐store release‐activated Ca2+ influx in aortic and lung endothelial cells. In TRPC4 knockout (TRPC4−/−) mice, acetylcholine‐induced endothelium‐dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4−/− mouse‐lung endothelial cells exhibited lack of actin‐stress fiber formation and cell retraction in response to thrombin activation of protease‐activated receptor‐1 (PAR‐1) in endothelial cells. The increase in lung microvascular permeability in response to PAR‐1 activation was inhibited in TRPC4−/− mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling agonist‐induced increases in endothelial permeability.

Role of TRPM2 Channel in Mediating H2O2-Induced Ca2+ Entry and Endothelial Hyperpermeability

Oxidative stress through the production of oxygen metabolites such as hydrogen peroxide (H2O2) increases vascular endothelial permeability. H2O2 stimulates ADP-ribose formation, which in turn opens transient receptor potential melastatin (TRPM)2 channels. Here, in endothelial cells, we demonstrate transcript and protein expression of TRPM2, a Ca2+-permeable, nonselective cation channel. We further show the importance of TRPM2 expression in signaling of increased endothelial permeability by oxidative stress. Exposure of endothelial cell monolayers to sublytic concentrations of H2O2 induced a cationic current measured by patch-clamp recording and Ca2+ entry detected by intracellular fura-2 fluorescence. H2O2 in a concentration-dependent manner also decreased trans-monolayer transendothelial electrical resistance for 3 hours (with maximal effect seen at 300 &mgr;mol/L H2O2), indicating opening of interendothelial junctions. The cationic current, Ca2+ entry, and transendothelial electrical resistance decrease elicited by H2O2 were inhibited by siRNA depleting TRPM2 or antibody blocking of TRPM2. H2O2 responses were attenuated by overexpression of the dominant-negative splice variant of TRPM2 or inhibition of ADP-ribose formation. Overexpression of the full-length TRPM2 enhanced H2O2-mediated Ca2+ entry, cationic current, and the transendothelial electrical resistance decrease. Thus, TRPM2 mediates H2O2-induced increase in endothelial permeability through the activation of Ca2+ entry via TRPM2. TRPM2 represents a novel therapeutic target directed against oxidant-induced endothelial barrier disruption.

Caveolin-1 Regulates NF-κB Activation and Lung Inflammatory Response to Sepsis Induced by Lipopolysaccharide

Caveolin-1, the principal structural and signaling protein of caveolae, is implicated in NO-mediated cell signaling events, but its precise role in inflammation is not well understood. Using caveolin-1-knockout (Cav-1−/−) mice, we addressed the role of caveolin-1 in the lung inflammatory response to sepsis induced by i.p. injection of LPS. LPS-challenged wild-type (WT) lungs exhibited significant increases in neutrophil sequestration (∼16-fold), lung microvascular permeability Kf,c (∼5.7-fold), and edema formation (∼1.6-fold). Compared with WT, Cav-1−/− lungs showed marked attenuation of LPS-induced neutrophil sequestration (∼11-fold increase) and inhibition of microvascular barrier breakdown and edema formation. Prevention of lung injury in Cav-1−/− mice was associated with decreased mortality in response to LPS challenge. To address the basis of the reduced inflammation and injury in Cav-1−/− lungs, we examined the role of NO because its plasma concentration is known to be increased in Cav-1−/− mice. Cav-1−/− mouse lungs demonstrated a significant increase in endothelial NO synthase (eNOS)-derived NO production relative to WT, which is consistent with the role of caveolin-1 as a negative regulator of eNOS activity. Cav-1−/− lungs concurrently showed suppression of NF-κB activity and decreased transcription of inducible NO synthase and ICAM-1. Coadministration of LPS with the NO synthase inhibitor nitro-l-arginine in Cav-1−/− mice prevented the suppression of NF-κB activity and restored lung polymorphonuclear leukocyte sequestration in response to LPS challenge. Thus, caveolin-1, through its ability to regulate eNOS-derived NO production, is a crucial determinant of NF-κB activation and the lung inflammatory response to LPS.

Activation of Sphingosine Kinase-1 Reverses the Increase in Lung Vascular Permeability Through Sphingosine-1-Phosphate Receptor Signaling in Endothelial Cells

The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1−/− mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1−/− lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1→S1P→S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Cdc42 Regulates the Restoration of Endothelial Barrier Function

Abstract— The mechanisms involved in the restoration of endothelial cell junctions subsequent to barrier disruption remain unclear. It is known that formation of adherens junctions (AJs) affects cytoskeletal actin arrangement and that Rho GTPases regulate the state of actin polymerization. In the present study, we examined the role of the Rho GTPases, Rho, Rac, and Cdc42 in the reannealing of AJs. We studied the response to thrombin, which increases endothelial permeability through disassembly of AJs, followed by recovery of barrier function through junctional reannealing within 2 hours. Cdc42 was activated late, at ≈1 hour after thrombin exposure, concurrent with its translocation from the cytoplasm to the membrane. Activation and translocation of Cdc42 preceded the reformation of AJs. Expression of the dnCdc42 mutant (N17Cdc42) significantly delayed the reformation of the VE-cadherin-containing AJs and restoration of endothelial barrier function. We also studied the lung microcirculation to address the in vivo relevance of Cdc42 signaling in barrier restoration. N17Cdc42 expression in the mouse lung endothelium markedly attenuated the endothelial barrier recovery after the permeability increase induced by activation of the thrombin receptor protease-activated receptor-1. These findings demonstrate the critical function of Cdc42 in restoring AJ-dependent, endothelial cell homotypic adhesion and barrier function. The delayed activation of Cdc42 represents a negative-feedback mechanism that signals AJ reassembly after the increase in endothelial permeability induced by inflammatory mediators such as thrombin.

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.

The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox-sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) had an enhanced inflammatory response and diminished survival relative to that of wild-type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase–mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential–sensitive NADPH oxidase.

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.