Introduction

Vascular endothelial cells form the lining of blood vessels and separate the underlying tissue from circulating blood. Disruption of the endothelial barrier leads to increased vascular permeability to plasma proteins and inflammatory cells, resulting in edema as seen in acute lung injury and in its more severe form, acute respiratory distress syndrome.¹ Vascular permeability can be transcellular or paracellular. Transcellular permeability involves the formation of transport vesicles, whereas paracellular permeability requires disruption of the adherens junctions between 2 adjacent endothelial cells. Vascular endothelial (VE)-cadherin is an endothelial-specific marker of adherens junctions^2,3 and a determinant of integrity of endothelial junctions. Increased vascular permeability is associated with extravasation of leukocytes into the underlying tissue,⁴ and VE-cadherin has been proposed to play a role in leukocyte transmigration.⁵

Several proinflammatory factors are released into the blood stream during an inflammatory response, among which thrombin, tumor necrosis factor α (TNF-α), interleukin-1β, and histamine are known to disrupt the endothelial barrier. TNF-α is one of the most commonly encountered proinflammatory cytokines in pathological conditions, such as sepsis.⁶ Elevated TNF-α levels are found in the bronchoalveolar lavage (BAL) fluid⁷ and plasma⁸ of patients with acute respiratory distress syndrome. TNF-α increases the permeability of pulmonary microvessel endothelial barrier^9,10 and causes edema in animals.¹¹ However, the mechanisms of TNF-α–induced endothelial barrier dysfunction are not clearly understood. Tyrosine phosphorylation of VE-cadherin,^12,13 production of reactive oxygen species (ROS),^14,15 and activation of the small GTPase Rac¹⁶ have been associated with TNF-α–induced endothelial barrier dysfunction.

Rac is a monomeric GTPase of ≈21 kDa.¹⁷ In endothelial cells, Rac activation downstream of G protein–coupled receptors (GPCRs), such as the thrombin receptor protease-activated receptor-1, induces reannealing of endothelial junctions during endothelial barrier repair phase.¹⁸ However, it was also reported that introduction of a constitutively activated Rac led to endothelial barrier dysfunction.¹⁹ Similar findings were reported in endothelial cells stimulated with vascular endothelial growth factor²⁰ and platelet-activating factor.²¹ In phagocytes, Rac is known for its role in nicotinamide adenine dinucleotide phosphate oxidase (Nox) activation and superoxide production.²² Genetic deletion or silencing of the Rac gene showed that Rac is also essential for Nox-dependent ROS production in endothelial cells.²⁰ Despite these observations, the mechanism by which ROS regulates vascular permeability remains unclear. TNF-α has been shown to induce ROS production and vascular permeability, but the role of Rac in ROS production downstream of the TNF-α pathway has not been established. There are ≈70 Dbl family guanine nucleotide exchange factors (GEFs),²³ yet much fewer Rho family small GTPases (to which Rac belongs), suggesting that Rho GEFs confer specificity of Rho GTPase activation in tissues where the GEFs are expressed. To date, Tiam-1 and Vav-2 are the only GEFs implicated in regulating Rac activation in endothelial cells.^21,24,25

In this study, we examined phosphatidylinositol (3,4,5)-trisphosphate–dependent Rac exchanger 1 (P-Rex1) for its possible involvement in TNF-α–induced endothelial barrier dysfunction. P-Rex1 is a Rac-specific GEF regulated by phosphatidylinositol (3,4,5)-trisphosphate and G protein βγ subunits.^26,27 In neutrophils from P-Rex1–deficient mice, GPCR-induced activation and bactericidal functions are compromised.^28,29 P-Rex1 is primarily expressed in myeloid cells²⁶ and neuronal tissue.³⁰ Its biological functions range from neuron migration and neurite differentiation^30,31 to tumor metastasis.^32–34 In this study, we show that P-Rex1 is a key mediator of TNF-α–induced vascular permeability, which involves Rac activation and ROS production in a phosphoinositide 3-kinase (PI3K)–dependent manner. In addition, we identified a novel role for endothelial P-Rex1 in regulating transendothelial migration of polymorphonuclear leukocytes (PMNs).

Methods

Human lung microvascular endothelial cells (HLMVECs) were obtained from Lonza (Walkersville, MD). These cells were transfected with small interference RNA (siRNA) specific for P-Rex1 or with scrambled siRNA (Sc-siRNA) as negative controls. Experiments with mice were conducted using procedures approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. A detailed, expanded Methods section can be found in the Online Data Supplement Materials.

Results

Endothelial Expression of P-Rex1 and Its Role in the Regulation of Endothelial Barrier Function

P-Rex1 was originally identified in neutrophils and neurons, and its function outside these cells remains unclear.^26,30 We examined P-Rex1 expression in endothelial cells and its potential involvement in endothelial barrier function. Using reverse transcriptase polymerase chain reaction, P-Rex1 transcript was found in 3 different types of endothelial cells tested, including HLMVECs, human pulmonary artery endothelial cells, and human umbilical vein endothelial cells (Online Figure IA). The expression level of P-Rex1 protein in these endothelial cells was comparable with that in bone marrow–derived macrophages (Online Figure IB).

To assess the role of P-Rex1 in endothelial barrier function, HLMVECs were treated with P-Rex1–specific siRNA to reduce P-Rex1 expression. Sc-siRNA was used as a negative control. An ≈80% reduction in P-Rex1 protein level was obtained (Online Figure IC and ID). The siRNA-transfected cells were then subjected to measurement of changes in transendothelial electric resistance (TER) after TNF-α stimulation, which increases vascular permeability. As expected, the control (sc-siRNA–transfected) cells showed a decrease in TER culminating 4 to 5 hours after stimulation, suggesting barrier disruption. In comparison, P-Rex1 siRNA-transfected cells showed much less barrier disruption (Figure 1A). Based on the quantification of barrier disruption against absolute resistance values (Figure 1B), P-Rex1 is a necessary component for TNF-α–induced loss of TER, which reflects endothelial cell barrier dysfunction. TNF-α–induced barrier dysfunction is not a consequence of endothelial cell apoptosis, because the majority of transfected HLMVECs remained healthy after TNF-α treatment in DNA fragmentation assay (Online Figure II).

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Fluorescent imaging analysis was conducted to examine barrier dysfunction of HLMVECs, characterized by intercellular gap formation¹⁴ (Figure 1C). The endothelial adherens junctions were detected with an anti–VE-cadherin antibody (green). After stimulation with TNF-α, sc-siRNA–transfected endothelial cells displayed discontinuities between neighboring cells (marked with arrows). In comparison, HLMVECs receiving P-Rex1 siRNA showed minimal alteration of barrier integrity. The area of interendothelial gaps was quantified (Figure 1D), and the changes were significant (P<0.05). Thus, data from both TER and imaging analysis support a role for P-Rex1 in TNF-α–induced disruption of endothelial barrier.

P-Rex1 Is Essential for TNF-α–Induced Rac Activation

Recent studies have shown that TNF-α, at concentrations that induce opening of interendothelial junctions, activates the small GTPase Rac.¹⁶ However, the GEF responsible for TNF-α–induced Rac activation in endothelial cells remains unidentified. To explore the signal transduction pathway downstream of TNF-α stimulation, we examined possible involvement of P-Rex1 in Rac activation.

TNF-α induced a rapid and transient Rac activation in HLMVECs, which peaked within 1 minute and continued for 2 minutes before it began to decrease (Figure 2A). Based on Rac-GTP pull-down assay, TNF-α induced up to an 8-fold increase in Rac activation compared with unstimulated cells (Figure 2B). In HLMVECs receiving P-Rex1 siRNA, Rac activation was significantly reduced, indicating that P-Rex1 is required for TNF-α–induced Rac activation. In addition, the Rac inhibitor NSC23766 prevented TNF-α–induced endothelial permeability as measured by TER (Online Figure III). We also transfected HLMVECs with a dominant-negative Rac (T17NRac) to exclude the nonspecific effects of NSC23766. The dominant-negative Rac ablated TNF-α–induced barrier dysfunction (Online Figure IV). These results strongly suggest that Rac is required for TNF-α–induced endothelial barrier dysfunction and P-Rex1 is an essential Rac GEF regulating TNF-α–induced Rac activation.

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Several molecules were examined to exclude several possibilities that might have affected the outcome of our experiments. GEF-H1 is not only a Rac GEF but also a Rho GEF³⁵ and has been implicated in TNF-α–induced epithelial barrier integrity.³⁶ To determine whether it plays a role in our experiments, we used siRNA to knock down GEF-H1 in HLMVECs and performed TER. Our results indicate that, unlike P-Rex1, GEF-H1 removal did not reverse barrier dysfunction induced by TNF-α (Online Figure V). We also considered potential involvement of Rho, known to be responsible for endothelial barrier dysfunction.³⁷ To exclude the involvement of Rho downstream of the TNF-α and P-Rex1 pathway, we performed Rho pull-down assay as detailed in the Methods section. The absence of P-Rex1 did not alter Rho activation (Online Figure VI). Therefore, although TNF-α has the ability to activate Rho,¹⁶ it does not require P-Rex1.

TNF-α–Induced ROS Production Is P-Rex1–Dependent

Nox has been implicated in TNF-α–induced endothelial barrier dysfunction.^14,15 However, it is unclear how TNF-α regulates ROS production. In phagocytes, Rac is required for Nox activation, leading to ROS production.^38,39 Therefore, we determined whether P-Rex1 is involved in endothelial ROS production through Rac activation. HLMVECs plated on gelatin-coated glass dishes were treated with either sc-siRNA or P-Rex1 siRNA and then stimulated with TNF-α. ROS production was measured by dihydrorhodamine 123. As shown in Figure 3A and quantified in Figure 3B, siRNA-mediated silencing of P-Rex1 led to a significant reduction in TNF-α–stimulated ROS production. Diphenyleneiodonium, a flavocytochrome inhibitor, diminished TNF-α–induced ROS production, suggesting that inducible activation of the Nox is required (Figure 3C and 3D). Similarly, NSC23766 reduced ROS production, supporting the notion that Rac is involved in TNF-α–induced Nox activation in HLMVECs (Figure 3C and 3D). We next determined whether suppression of ROS production could alter the integrity of the HLMVEC monolayer. Diphenyleneiodonium-treated cells were refractory to TNF-α–induced decrease in TER (Figure 3E and 3F). These results demonstrate a correlation between endothelial P-Rex1 and TNF-α–induced ROS production, leading to a loss of barrier function.

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Silencing P-Rex1 Prevents TNF-α–Induced Src Activation and VE-Cadherin Phosphorylation

We examined the role of P-Rex1 in regulating TNF-α–induced tyrosine phosphorylation of VE-cadherin because this has been reported to be critical to the loss of vascular integrity.¹³ In sc-siRNA–transfected HLMVECs, TNF-α treatment led to phosphorylation of VE-cadherin at 10 minutes and peaked at 20 minutes (Figure 4A and 4B). TNF-α–induced VE-cadherin phosphorylation was diminished in HLMVECs transfected with P-Rex1 siRNA (Figure 4A and 4B), suggesting that VE-cadherin was phosphorylated in a P-Rex1–dependent manner. It was reported that the Src family protein tyrosine kinases undergo ROS-dependent phosphorylation that is required for VE-cadherin phosphorylation.^12,14 To test whether P-Rex1 is required for Src activation downstream of TNF-α stimulation, HLMVECs transfected with sc-siRNA or P-Rex1 siRNA were stimulated with TNF-α for the indicated time points, and phosphorylation of c-Src at Tyr416 was determined. HLMVECs receiving P-Rex1 siRNA displayed >70% reduction in phosphorylation of Tyr416 at 5 and 10 minutes compared with cells transfected with sc-siRNA (Figure 4C and 4D), indicating that P-Rex1 is required for TNF-α–induced Src activation.

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Signaling Mechanism of TNF-α–Induced P-Rex1 Activation

P-Rex1 resides in the cytosol of resting neutrophils and translocates to membrane on cell activation.⁴⁰ We determined whether P-Rex1 is translocated to plasma membrane in TNF-α–stimulated endothelial cells. HLMVECs were stimulated with TNF-α for 0, 1, and 2 minutes, and P-Rex1 in the membrane and cytosolic fractions was determined. P-Rex1 underwent membrane translocation as early as 1 minute and continued to increase at 2 minutes (Figure 5A–5C). We also used an additional approach to test the membrane translocation of P-Rex1. HLMVECs were unstimulated or stimulated with TNF-α for 2 minutes. The cells were then permeabilized, fixed, and incubated with anti–P-Rex1 and an Alexa fluor 488–conjugated secondary antibody. Images acquired by confocal microscopy showed accumulation of P-Rex1 to the membrane periphery after stimulation (Figure 5D). Pretreatment of HLMVECs with the PI3K inhibitor LY294002 prevented membrane translocation of P-Rex1 (Figure 5D), suggesting that it is PI3K-dependent in TNF-α–stimulated HLMVECs.

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Earlier studies have shown that P-Rex1 activation downstream of GPCRs requires both PI3K and the βγ subunits of heterotrimeric G proteins.^26,27 However, TNF-α is not a GPCR agonist and how a non-GPCR activates P-Rex1 remains unclear. To determine whether Gβγ subunits are involved in TNF-α signaling, we pretreated HLMVECs with Gβγ modulator II (Gallein; 3′,4′,5′,6′-tetrahydroxyspiro [isobenzofuran-1(3H),9′-(9H)xanthen]-3-one), which inhibits conformational changes of Gβγ subunits and blocks Gβγ-dependent activation of PI3K and Rac in human leukemia-60 cell line (HL-60) cells.⁴¹ Pretreatment with Gallein did not affect TNF-α–induced membrane translocation of P-Rex1 (Figure 5D, lower panels), whereas it significantly inhibited thrombin-induced calcium mobilization in HLMVECs (Figure 5E and 5F). These findings suggest that Gβγ is not indispensable in TNF-α–induced P-Rex1 activation, but PI3K is necessary. The requirement of PI3K for P-Rex1 activation in TNF-α–stimulated endothelial cells was also confirmed when HLMVECs pretreated with the PI3K inhibitor LY294002 displayed reduced Rac activation by >50% (Online Figure VIIA and VIIB).

P-Rex1 Knockout Mice Display Reduced Lung Vascular Permeability and Edema

To determine an in vivo function of P-Rex1 in acute lung injury, wild-type (WT) and P-Rex1 knockout mice²⁹ were instilled with TNF-α intratracheally. Changes in lung vascular permeability were evaluated based on the accumulation of Evans blue albumin (EBA) after tail vein injection. Significantly less accumulation of EBA was seen in the lungs of mice lacking P-Rex1, compared with WT controls (Figure 6A and 6B). The in vivo role of P-Rex1 in lung edema was also evaluated based on lung wet-to-dry weight ratio after intratracheal instillation of TNF-α. Again, the P-Rex1 knockout mice had significantly less edema compared with WT controls (Figure 6C). These results demonstrate a critical role for P-Rex1 in the dynamic regulation of lung vascular permeability in vivo.

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Lipopolysaccharide-induced sepsis is a clinically relevant model of acute lung injury. We tested a potential role for P-Rex1 in this model where barrier dysfunction contributes to the pathological changes. WT and P-Rex1 knockout mice were intraperitoneally injected with lipopolysaccharide or PBS for 6 hours, which produced septic signs such as decreases in leukocyte count and platelet count (Online Figure XI). The lungs were subjected to K_f,c measurements (Figure 6D) and EBA dye leakage measurement (Figure 6E). Both the K_f,c and EBA data showed that P-Rex1 knockout mice have significantly less lung microvascular capillary filtration and EBA leakage, indicating a role for P-Rex1 in lipopolysaccharide-induced barrier dysfunction during sepsis.

Role of Endothelial P-Rex1 in PMN Transmigration

In acute lung injury, there is marked infiltration of PMNs and macrophages in the lungs. To determine infiltration of phagocytes into BAL fluid, WT and P-Rex1 knockout mice were instilled intratracheally with PBS or 0.5 μg of murine recombinant TNF-α. After 24 hours, mice were anesthetized and BAL fluid was collected. BAL fluid obtained from P-Rex1 knockout mice showed significantly less PMNs and macrophages compared with WT mice (Figure 7A and 7B). This result indicates that P-Rex1 is necessary for transendothelial migration of these leukocytes. In a parallel experiment, WT and P-Rex1 knockout mice were intratracheally injected with murine TNF-α for 24 hours followed by collection of lungs for histological analysis. Hematoxylin and eosin staining showed significantly less cellular infiltration and interstitial tissue thickening in P-Rex1 knockout mouse lungs compared with WT lungs (Online Figure VIII).

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Because the knockout approach results in a loss of P-Rex1 in all tissues, we next determined the relative contribution of P-Rex1 in endothelial cells versus PMNs to the reduced PMN transendothelial migration. Figure 7C shows the effects of eliminating P-Rex1 from endothelial cells on transendothelial migration of WT and P-Rex1^−/− PMNs (a more detailed version of the experiment, with ligand controls included, is shown in Online Figure X). HLMVECs transfected with sc-siRNA or P-Rex1 siRNA were plated on 3-μm membrane pore inserts. PMNs were isolated concurrently from both WT and P-Rex1^−/− mice and applied to the HLMVEC monolayer, which received sc-siRNA (Figure 7C, filled bars) or P-Rex1 siRNA (Figure 7C, open bars) and simulated with TNF-α for 4 hours. Eliminating P-Rex1 from the endothelial cells caused a significant reduction in PMN transmigration, which applies to both WT and P-Rex1^−/− PMNs (Figure 7C). In comparison, removal of P-Rex1 from PMNs does not significantly impact cell migration in this experiment (ns, Figure 7C). Based on these findings, we concluded that endothelial P-Rex1 plays an important role in PMN transendothelial migration.

We have also taken an ex vivo approach to determine the effect of P-Rex1 in PMN transmigration into the lung tissue. Lungs from WT and P-Rex1 knockout mice were perfused to remove blood cells and then exposed to murine TNF-α. Freshly isolated PMNs from WT and P-Rex1^−/− mice were radiolabeled with ¹¹¹Indium oxine and perfused through WT and P-Rex1^−/− lungs, or vice versa. As shown in Figure 7D, the P-Rex1^−/− lungs showed significantly less radioactivity accumulation than WT lungs, suggesting that absence of P-Rex1 in lung tissue could significantly reduce PMN transmigration. In contrast, no significant difference in radioactivity accumulation was observed in WT lungs perfused with either WT PMNs or P-Rex1^−/− PMNs (Figure 7D). These findings suggest that endothelial P-Rex1 is highly important in PMN transmigration into the lung tissue.

P-Rex1 Is Important for TNF-α–Induced ICAM-1 Expression in Endothelial Cells

In the above experiments, we observed that TNF-α treatment of HLMVECs is necessary for PMN transmigration. Several proteins are induced upon TNF-α stimulation of endothelial cells, among which intercellular adhesion molecule-1 (ICAM-1) is required for efficient PMN transendothelial migration. We examined TNF-α–induced ICAM-1 expression in HLMVECs transfected with either sc-siRNA or P-Rex1 siRNA and found that cells receiving P-Rex1 siRNA displayed significantly less ICAM-1 expression compared with cells treated with sc-siRNA (Online Figure IX). A significantly lower ICAM-1 induction may contribute to the decreased PMN transmigration across the P-Rex1–deficient HLMVEC monolayer.

Discussion

The present study examines P-Rex1 expression in endothelial cells and its role in mediating TNF-α–induced increase in vascular endothelial permeability. Many new findings were made. (1) P-Rex1 is highly expressed in vascular endothelial cells and plays important roles in these cells. (2) Our results show for the first time that P-Rex1 can be activated by a non-GPCR, in this case the TNF receptor, in endothelial cells. (3) This study reaffirms a role for Rac in TNF-α–induced vascular endothelial dysfunction, which has been an unsettled issue. (4) P-Rex1 activation leads to ROS production in endothelial cells. (5) Endothelial P-Rex1 is important for PMN transmigration into the lung tissue. These findings are summarized schematically in a working model (Figure 8).

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P-Rex1 Expression and Functions in Lung Vascular Endothelial Cells

In this model, P-Rex1 is a major Rho GEF downstream of TNF-α receptor in endothelial cells. Before this study, P-Rex1 is mainly known for its functions in the brain and in PMNs, where it was first discovered.²⁶ Thus, the finding of P-Rex1 in various endothelial cells suggests that this Dbl family Rho GEF is more broadly expressed than previously thought. Our data demonstrate that P-Rex1 is expressed in vascular endothelial cells, and it mediates TNF-α–induced vascular permeability as well as PMN infiltration into the lung tissue. These functions of P-Rex1 require Rac activation, which leads to Nox-dependent ROS production, c-Src activation, and VE-cadherin phosphorylation in HLMVECs. Our in vitro results are corroborated by data from P-Rex1 knockout mice, which are refractory to TNF-α–induced increase in vascular permeability in the lungs as demonstrated by reduced edema. Collectively, these results demonstrate that endothelial P-Rex1 is critical to TNF-α signaling that leads to increased vascular endothelial permeability.

P-Rex1 Activation by a Non-GPCR

Our model places P-Rex1 downstream of the TNF-α receptor, whereas published reports depict P-Rex1 as a Rac-specific GEF activated by GPCRs.²⁶ In endothelial cells, P-Rex1 can be activated by a GPCR.⁴² Our finding that P-Rex1 is activated by TNF-α is totally unexpected because TNF-α is not known to couple to G proteins. We observed rapid membrane translocation of P-Rex1 in endothelial cells, which is characteristic of its activation.⁴⁰ It is also evident that TNF-α stimulates P-Rex1–dependent Rac activation in HLMVECs. Because the time course of Rac activation and P-Rex1 membrane translocation is consistent with that of GPCR signaling, we examined the requirement for P-Rex1 activation in TNF-α–stimulated cells. A reported feature of P-Rex1 is its dependence on phosphatidylinositol (3,4,5)-trisphosphate and Gβγ for activation.^26,27 Our results show that TNF-α–induced Rac activation is PI3K-dependent. However, we observed no effect for the Gβγ inhibitor Gallein to affect TNF-α–induced P-Rex1 membrane translocation, thus challenging the conventional view that Gβγ is required for P-Rex1 activation. It is notable that TNF-α signaling has not been associated with activation or transactivation of heterotrimeric G proteins, although TNF-α is known for its activation of PI3K,⁴³ suggesting that TNF-α–induced phosphatidylinositol (3,4,5)-trisphosphate production might be sufficient to trigger P-Rex1 activation in HLMVECs.

A Role for Rac in Endothelial Barrier Dysfunction

In endothelial cells stimulated with GPCR agonists such as thrombin, a reversible endothelial barrier disruption occurs. Although there are multiple pathways for disrupting the endothelial barrier, Rac has been associated with reannealing of junctions in response to GPCR activation.¹⁸ Contrary to this view, there is evidence supporting a role of Rac in endothelial barrier dysfunction. For instance, van Wetering et al¹⁹ reported that expression of a constitutively active Rac in human umbilical vein endothelial cells could cause changes leading to increased vascular permeability. Rac has been implicated in TNF-α and vascular endothelial growth factor–induced increase in vascular permeability.^16,20 As shown in our model, GTP-bound Rac is required for TNF-α–induced ROS production, and the Rac inhibitor NSC23766 abrogated TNF-α–induced vascular endothelial permeability in HLMVEC. Similarly, dominant-negative RacT17N, when expressed in endothelial cells, prevented TNF-α–induced endothelial barrier dysfunction. Our data support a role for Rac in TNF-α–induced endothelial barrier dysfunction, which is mediated through P-Rex1 activation and ROS production. These findings connect a Rho GEF to previously reported functions of ROS in the regulation of vascular permeability.^{15,19,44–46}

Endothelial P-Rex1 and PMN Transmigration

Disruption of endothelial barrier is a triggering factor for infiltration of PMNs into tissues.^4,47 We tested whether TNF-α–induced disruption of endothelial barrier aggravates PMN infiltration, and if so, whether blocking P-Rex1 expression in endothelial cell prevents PMN transmigration. We found that, in the absence of P-Rex1, PMN transmigration was significantly reduced. Much fewer PMNs and macrophages were present in BAL fluid of P-Rex1 knockout mice compared with WT mice after instillation of murine recombinant TNF-α into the airways. Silencing endothelial P-Rex1 expression resulted in significantly less PMN transmigration compared with sc-siRNA–transfected endothelium. This phenomenon was also confirmed ex vivo by PMN sequestration studies where radiolabeled WT or P-Rex1 knockout PMNs were perfused into WT and P-Rex1 knockout mice, respectively, and vice versa. It seems that the crosstalk between PMN and endothelial cell is key to PMN transendothelial migration.⁴ Our Western blot data showed that there is significantly less ICAM-1 expression in the absence of P-Rex1. As depicted in the model, P-Rex1 seems to play a role in the regulation of TNF-α–induced ICAM-1 expression, which involves nuclear factor-κB activation. The mechanism underlying P-Rex1 regulation of nuclear factor-κB activation is yet to be delineated. An increase in ICAM-1 expression may in turn affect PMN transendothelial migration.

A Potential Target for Therapeutic Intervention

Our findings strongly implicate P-Rex1 in regulating TNF-α–induced vascular permeability and lung edema. This function is mediated through the activation of Rac and generation of ROS, thus promoting endothelial barrier disruption and transendothelial PMN migration. These results demonstrate that downregulation of P-Rex1 may affect multiple proinflammatory pathways, and P-Rex1 may be a new therapeutic target in controlling lung vascular injury and PMN-mediated lung inflammation.

Acknowledgments

We thank Dr Richard Minshall and Dr Chinnaswamy Tiruppathi for guidance to the graduate student and for sharing their knowledge in endothelial biology. We also thank Dr Marcus Thelen for the gift of anti–P-Rex1 antibodies.

Footnotes