Sanda Predescu

Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.

Cholesterol-dependent syntaxin-4 and SNAP-23 clustering regulates caveolar fusion with the endothelial plasma membrane

We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.

Nitric Oxide Stimulates Macrophage Inflammatory Protein-2 Expression in Sepsis

NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-κB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IκB and activation of NF-κB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-l-arginine. Despite having relatively modest effects on NF-κB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-l-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.

Transport of nitrated albumin across continuous vascular endothelium

Because modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that may function as a mechanism of nitrogen monoxide clearance from microcirculation, we investigated biochemicaly and morphologically the cell surface binding and the transendothelial transport of NOA. An electron microscopic study was carried out with mouse lungs and hearts perfused in situ with NOA and NOA-Au complexes. The results indicate that NOA-Au can bind to the endothelial cell surface, and its binding can be blocked by albumin plus nitrotyrosine (NO-tyrosine) or abolished by excess NOA. We detected NOA-Au into perivascular spaces as early as 30 sec after the beginning of its perfusion. NOA, unlike native albumin, leaves the vascular lumina via both endothelial caveolae and open junctions. By cross-linking and ligand blotting analysis, we showed that NOA interacted with the same albumin binding proteins of 16–18, 30–32, 60, and 74 kDa as native albumin. ELISA performed on tissue homogenates obtained from the same specimens showed that NOA transport was 2- to 4-fold greater than native albumin. The augmented transendothelial transport of NOA reflects its transcytosis as well as its exit from the microcirculation via open junctions. The increased transport of NOA may serve as an important mechanism that protects a vascular bed against the damaging effects of nitrogen monoxide and peroxynitrite.

Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-XL overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization.

Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PHITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PHITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.

Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities

Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoAs interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities.

Gα12 Interaction with αSNAP Induces VE-cadherin Localization at Endothelial Junctions and Regulates Barrier Function

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified αSNAP as an interactive partner of Gα12 using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of αSNAP with Gα12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of Gα12 (1–37 amino acids) was necessary and sufficient for its interaction with αSNAP. Gα13 with its N-terminal extension replaced by that of Gα12 acquired the ability to bind to αSNAP, whereas Gα12 with its N terminus replaced by that of Gα13 lost this ability. Using four point mutants of αSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of αSNAP was involved in its interaction with Gα12. Co-transfection of human umbilical vein endothelial cells with Gα12 and αSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of αSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with Gα12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the α subunit of G12 and αSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.

A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.

Background: Plexiform lesions comprising proliferative endothelial cells are hallmarks of pulmonary arterial hypertension. Results: Granzyme B cleaves intersectin-1s and generates a fragment with endothelial cell proliferative potential via phosphorylation of p38MAPK and Elk-1 transcription factor. Conclusion: Granzyme B cleavage of intersectin-1s and subsequent p38MAPK/Elk-1 activation are critical for endothelial cell proliferation. Significance: The novel pathogenic p38MAPK/Elk-1 signaling may explain the formation of plexiform lesions. Plexiform lesions (PLs), the hallmark of plexogenic pulmonary arterial hypertension (PAH), contain phenotypically altered, proliferative endothelial cells (ECs). The molecular mechanism that contributes to EC proliferation and formation of PLs is poorly understood. We now show that a decrease in intersectin-1s (ITSN-1s) expression due to granzyme B (GrB) cleavage during inflammation associated with PAH and the high p38/Erk1/2MAPK activity ratio caused by the GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype. We used human pulmonary artery ECs of PAH subjects (ECPAH), paraffin-embedded and frozen human lung tissue, and animal models of PAH in conjunction with microscopy imaging, biochemical, and molecular biology approaches to demonstrate that GrB cleaves ITSN-1s, a prosurvival protein of lung ECs, and generates two biologically active fragments, an N-terminal fragment (GrB-EHITSN) with EC proliferative potential and a C-terminal product with dominant negative effects on Ras/Erk1/2. The proliferative potential of GrB-EHITSN is mediated via sustained phosphorylation of p38MAPK and Elk-1 transcription factor and abolished by chemical inhibition of p38MAPK. Moreover, lung tissue of PAH animal models and human specimens and ECPAH express lower levels of ITSN-1s compared with controls and the GrB-EHITSN cleavage product. Moreover, GrB immunoreactivity is associated with PLs in PAH lungs. The concurrent expression of the two cleavage products results in a high p38/Erk1/2MAPK activity ratio, which is critical for EC proliferation. Our findings identify a novel GrB-EHITSN-dependent pathogenic p38MAPK/Elk-1 signaling pathway involved in the poorly understood process of PL formation in severe PAH.

Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium

Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis.