Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed.

Murine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin-positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH.

Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5.

Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor ß receptor I (TGFß-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5-Smad-SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5-mSos-Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFß and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth.

Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis.

BACKGROUND: The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. Intersectin-1s (ITSN-1s) is an important multidomain adaptor protein implicated in the pathophysiology of numerous pulmonary diseases. To date, the role of ITSN-1s in LC has not been studied. METHODS: Human LC cells, human LC tissue and A549 LC cells stable transfected with myc-ITSN-1s construct (A549 + ITSN-1s) were used in correlation with biochemical, molecular biology and morphological studies. In addition scratch assay with time lapse microscopy and in vivo xenograft tumor and mouse metastasis assays were performed. RESULTS: ITSN-1s, a prevalent protein of lung tissue, is significantly downregulated in human LC cells and LC tissue. Restoring ITSN-1s protein level decreases LC cell proliferation and clonogenic potential. In vivo studies indicate that immunodeficient mice injected with A549 + ITSN-1s cells develop less and smaller metastatic tumors compared to mice injected with A549 cells. Our studies also show that restoring ITSN-1s protein level increases the interaction between Cbl E3 ubiquitin ligase and Eps8 resulting in enhanced ubiquitination of the Eps8 oncoprotein. Subsequently, downstream unproductive assembly of the Eps8-mSos1 complex leads to impaired activation of the small GTPase Rac1. Impaired Rac1 activation mediated by ITSN-1s reorganizes the cytoskeleton (increased thick actin bundles and focal adhesion (FA) complexes as well as collapse of the vimentin filament network) in favor of decreased LC cell migration and metastasis. CONCLUSION: ITSN-1s induced Eps8 ubiquitination and impaired Eps8-mSos1 complex formation, leading to impaired activation of Rac1, is a novel signaling mechanism crucial for abolishing the progression and metastatic potential of LC cells.

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

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/Erk1/2(MAPK) 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 (EC(PAH)), 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-EH(ITSN)) with EC proliferative potential and a C-terminal product with dominant negative effects on Ras/Erk1/2. The proliferative potential of GrB-EH(ITSN) is mediated via sustained phosphorylation of p38(MAPK) and Elk-1 transcription factor and abolished by chemical inhibition of p38(MAPK). Moreover, lung tissue of PAH animal models and human specimens and EC(PAH) express lower levels of ITSN-1s compared with controls and the GrB-EH(ITSN) 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/Erk1/2(MAPK) activity ratio, which is critical for EC proliferation. Our findings identify a novel GrB-EH(ITSN)-dependent pathogenic p38(MAPK)/Elk-1 signaling pathway involved in the poorly understood process of PL formation in severe PAH.

In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.

Intersectin-1s (ITSN-1s) is a general endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, via MEK/Erk1/2(MAPK) signaling. To investigate the in vivo effects of ITSN-1s deficiency and the resulting ECs apoptosis on pulmonary vasculature and lung homeostasis, we used an ITSN-1s knocked-down (KD(ITSN)) mouse generated by repeated delivery of a specific siRNA targeting ITSN-1 gene (siRNA(ITSN)). Biochemical and histological analyses as well as electron microscopy (EM) revealed that acute KD(ITSN) [3-days (3d) post-siRNA(ITSN) treatment] inhibited Erk1/2(MAPK) pro-survival signaling, causing significant ECs apoptosis and lung injury; at 10d of KD(ITSN), caspase-3 activation was at peak, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive ECs showed 3.4-fold increase, the mean linear intercept (MLI) showed 48 % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30 %, all compared to controls; pulmonary function was altered. Concomitantly, expression of several growth factors known to activate Erk1/2(MAPK) and suppress Bad pro-apoptotic activity increased. KD(ITSN) altered Smads activity, downstream of the transforming growth factor beta-receptor-1 (TßR1), as shown by subcellular fractionation and immunoblot analyses. Moreover, 24d post-siRNA(ITSN), surviving ECs became hyper-proliferative and apoptotic-resistant against ITSN-1s deficiency, as demonstrated by EM imaging, 5-bromo-deoxyuridine (BrdU) incorporation and Bad-Ser(112/155) phosphorylation, respectively, leading to increased microvessel density and repair of the injured lungs, as well as matrix deposition. In sum, ECs endocytic dysfunction and apoptotic death caused by KD(ITSN) contribute to the initial lung injury and microvascular loss, followed by endothelial phenotypic changes and microvascular remodeling in the remaining murine pulmonary microvascular bed.

Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s.

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.

Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation.

BACKGROUND: The response of lung microvascular endothelial cells (ECs) to lipopolysaccharide (LPS) is central to the pathogenesis of lung injury. It is dual in nature, with one facet that is pro-inflammatory and another that is cyto-protective. In previous work, overexpression of the anti-apoptotic Bcl-XL rescued ECs from apoptosis triggered by siRNA knockdown of intersectin-1s (ITSN-1s), a pro-survival protein crucial for ECs function. Here we further characterized the cyto-protective EC response to LPS and pro-inflammatory dysfunction. METHODS AND RESULTS: Electron microscopy (EM) analyses of LPS-exposed ECs revealed an activated/dysfunctional phenotype, while a biotin assay for caveolae internalization followed by biochemical quantification indicated that LPS causes a 40% inhibition in biotin uptake compared to controls. Quantitative PCR and Western blotting were used to evaluate the mRNA and protein expression, respectively, for several regulatory proteins of intrinsic apoptosis, including ITSN-1s. The decrease in ITSN-1s mRNA and protein expression were countered by Bcl-XL and survivin upregulation, as well as Bim downregulation, events thought to protect ECs from impending apoptosis. Absence of apoptosis was confirmed by TUNEL and lack of cytochrome c (cyt c) efflux from mitochondria. Moreover, LPS exposure caused induction and activation of inducible nitric oxide synthase (iNOS) and a mitochondrial variant (mtNOS), as well as augmented mitochondrial NO production as measured by an oxidation oxyhemoglobin (oxyHb) assay applied on mitochondrial-enriched fractions prepared from LPS-exposed ECs. Interestingly, expression of myc-ITSN-1s rescued caveolae endocytosis and reversed induction of iNOS expression. CONCLUSION: Our results suggest that ITSN-1s deficiency is relevant for the pro-inflammatory ECs dysfunction induced by LPS.

Epsin15 Homology Domains: Role in the Pathogenesis of Pulmonary Arterial Hypertension.

Intersectin-1s (ITSN) deficiency and expression of a biologically active ITSN fragment, result of granzyme B cleavage under inflammatory conditions associated with pulmonary arterial hypertension (PAH), are characteristics of lung tissue of human and animal models of PAH. Recently, we have shown that this ITSN fragment comprising two Epsin15 homology domains (EHITSN) triggers endothelial cell (EC) proliferation and the plexiform arteriopathy in PAH. Limited evidence also indicates that the EH domains of endocytic proteins such as ITSN, upregulate compensatory endocytic pathways in cells with impaired vesicular trafficking. Thus, we sought to investigate whether the EHITSN may be involved in this compensatory mechanism for improving the EC endocytic dysfunction induced by ITSN deficiency and possibly contribute to PAH pathogenesis. We used stably-transfected human pulmonary artery ECs expressing the Myc-EHITSN (ECEH-ITSN) and ITSN knockout heterozygous mice (K0 ITSN+/- ) transduced with the Myc-EHITSN, in conjunction with functional assays: the biotin assay for caveolae internalization and 8 nm gold (Au)- and dinitrophenylated (DNP)-albumin perfusion of murine lung microvasculature. Pulmonary artery ECs of PAH patients (ECPAH), ITSN knockdown ECs (ECKD-ITSN), the monocrotaline (MCT)-induced mouse and rat models of PAH, as well as untreated animals, served as controls. ELISA via streptavidin-HRP or anti-DNP antibody (Ab), applied on ECs and lung lysates indicated greater than 30% increase in biotin internalization in ECEH-ITSN compared to ECCtrl. Despite their endocytic deficiency, ECPAH internalized biotin similar to ECCtrl which is twofold higher compared to ECKD-ITSN. Moreover, the lung microvascular bed of Myc-EHITSN-transduced mice and MCT-treated animals showed greater than twofold increase in DNP-BSA transendothelial transport, all compared to untreated controls. Electron microscopy (EM) revealed the increased occurrence of non-conventional endocytic/transcytotic structures (i.e., caveolae clusters, tubulo-vesicular and enlarged endocytic structures, membranous rings), usually underrepresented. Most of these structures were labeled by Au-BSA, consistent with their involvement in the transendothelial transport. Furthermore, ITSN deficiency and EHITSN expression alter the subcellular localization of the EH-binding protein 1 (EHBP1) and cortical actin organization, altogether supporting the increase occurrence/trafficking of the alternative endocytic structures. Thus, the EHITSN by shifting the physiological vesicular (caveolae) transport toward the alternative endocytic pathways is a significant contributor to the dysfunctional molecular phenotype of ECPAH.

Alk5/Runx1 signaling mediated by extracellular vesicles promotes vascular repair in acute respiratory distress syndrome.

BACKGROUND: Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. RESULTS: The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73+CD105+Cd34-CD45-). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TßRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. CONCLUSIONS: The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.

Mouse Lung Fibroblast Resistance to Fas-Mediated Apoptosis Is Dependent on the Baculoviral Inhibitor of Apoptosis Protein 4 and the Cellular FLICE-Inhibitory Protein.

A characteristic feature of idiopathic pulmonary fibrosis (IPF) is accumulation of apoptotic resistant fibroblasts/myofibroblasts in the fibroblastic foci. As caveolin (Cav)-null mice develop pulmonary fibrosis (PF), we hypothesized that the participating fibroblasts display an apoptosis-resistant phenotype. To test this hypothesis and identify the molecular mechanisms involved we isolated lung fibroblasts from Cav-null mice and examined the expression of several inhibitors of apoptosis (IAPs), of c-FLIP, of Bcl-2 proteins and of the death receptor CD95/Fas. We found significant increase in XIAP and c-FLIP constitutive protein expression with no alteration of Bcl-2 and lower levels of CD95/Fas. The isolated fibroblasts were then treated with the CD95/Fas ligand (FasL) to induce apoptosis. While the morphological and biochemical alterations induced by FasL were similar in wild-type (wt) and Cav-null mouse lung fibroblasts, the time course and the extent of the alterations were greater in the Cav-null fibroblasts. Several salient features of Cav-null fibroblasts response such as loss of membrane potential, fragmentation of the mitochondrial continuum concurrent with caspase-8 activation, and subsequent Bid cleavage, prior to caspase-3 activation were detected. Furthermore, M30 antigen formation, phosphatidylserine expression and DNA fragmentation were caspase-3 dependent. SiRNA-mediated silencing of XIAP and c-FLIP, individually or combined, enhanced the sensitivity of lung fibroblasts to FasL-induced apoptosis. Pharmacological inhibition of Bcl-2 had no effect. Together our findings support a mechanism in which CD95/Fas engagement activates caspase-8, inducing mitochondrial apoptosis through Bid cleavage. XIAP and c-FLIP fine tune this process in a cell-type specific manner.

New insights into the functions of intersectin-1s.

Intersectin-1s (ITSN) is a ubiquitously expressed multifunctional protein known as a scaffold and regulator of the general endocytic machinery as well as a critical integrator of cellular signaling pathways. We showed recently that ITSN deficiency triggers a transforming growth factor ß (TGFß)/Alk5 signaling switch, from the canonical Smad 2/3 to the Erk1/2 MAPK pathway; moreover, endocytic impairment induced by ITSN deficiency enhances Alk5 ubiquitination and degradation and elicits TGFß-paracrine effects mediated by circulating microparticles, leading to endothelial cell survival and increased proliferation. The studies expand our understanding of how ITSN facilitates cross-regulation of signaling pathways and provide insights into the involvement of ITSN deficiency in human disease.

Long-term silencing of intersectin-1s in mouse lungs by repeated delivery of a specific siRNA via cationic liposomes. Evaluation of knockdown effects by electron microscopy.

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition(1). Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce(2). Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration(3)Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study(4). Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Intersectin-1s: an important regulator of cellular and molecular pathways in lung injury.

Abstract Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe syndromes resulting from the diffuse damage of the pulmonary parenchyma. ALI and ARDS are induced by a plethora of local or systemic insults, leading to the activation of multiple pathways responsible for injury, resolution, and repair or scarring of the lungs. Despite the large efforts aimed at exploring the roles of different pathways in humans and animal models and the great strides made in understanding the pathogenesis of ALI/ARDS, the only viable treatment options are still dependent on ventilator and cardiovascular support. Investigation of the pathophysiological mechanisms responsible for initiation and resolution or advancement toward lung scarring in ALI/ARDS animal models led to a better understanding of the disease's complexity and helped in elucidating the links between ALI and systemic multiorgan failure. Although animal models of ALI/ARDS have pointed out a variety of new ideas for study, there are still limited data regarding the initiating factors, the critical steps in the progression of the disease, and the central mechanisms dictating its resolution or progression to lung scarring. Recent studies link deficiency of intersectin-1s (ITSN-1s), a prosurvival protein of lung endothelial cells, to endothelial barrier dysfunction and pulmonary edema as well as to the repair/recovery from ALI. This review discusses the effects of ITSN-1s deficiency on pulmonary endothelium and its significance in the pathology of ALI/ARDS.

Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.

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.