Publications by authors named "Sanda Predescu"

35 Publications

Up-Regulation of the Long Noncoding RNA X-Inactive-Specific Transcript and the Imbalance Sex/Ratio of Pulmonary Arterial Hypertension.

Am J Pathol 2021 Apr 6. Epub 2021 Apr 6.

Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Rush University, Chicago, Illinois. Electronic address:

Pulmonary arterial hypertension (PAH) is a sex-biased disease. Recent studies demonstrated that the increased expression and activity of the long-noncoding RNA X-inactive-specific transcript (Xist), essential for X-chromosome inactivation and dosage compensation of X-linked genes, may explain the imbalance sex/ratio of PAH. The present studies, using a murine model of plexiform PAH, the intersectin-1s (ITSN) heterozygous knockout (KO) mouse transduced with an ITSN fragment (EH) possessing endothelial cell proliferative activity, in conjunction with molecular, cell biology, biochemical, morphologic, and functional approaches, demonstrate significant sex-centered differences with regard to EH-induced alterations in pulmonary artery remodeling, lung hemodynamics, and p38/Elk1/c-Fos signaling, altogether leading to a more severe female lung PAH phenotype. Moreover, the long-noncoding RNA-Xist is up-regulated in the lungs of female EH-KO mice compared with female wild-type mice, leading to sex-specific modulation of the X-linked gene Elk1 and its target, two molecular events also characteristic to female human PAH lung. More important, cyclin A1 expression in the S and G/M phases of the cell cycle of synchronized pulmonary artery endothelial cells of female PAH patients is greater versus controls, suggesting functional hyperproliferation. Thus, Xist up-regulation leading to females' pulmonary artery endothelial cell sexual dimorphic behavior may provide a better understanding of the origin of sex bias in PAH. Notably, the EH-KO mouse is a unique experimental animal model of PAH that recapitulates most of the sexually dimorphic characteristics of human disease.
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http://dx.doi.org/10.1016/j.ajpath.2021.03.009DOI Listing
April 2021

Sex differences in the proliferation of pulmonary artery endothelial cells: implications for plexiform arteriopathy.

J Cell Sci 2020 05 14;133(9). Epub 2020 May 14.

Department of Internal Medicine, Pulmonary, Critical Care and Sleep Medicine, Rush University Medical Center, Chicago, IL 60612, USA

The sex-biased disease pulmonary arterial hypertension (PAH) is characterized by the proliferation and overgrowth of dysfunctional pulmonary artery endothelial cells (PAECs). During inflammation associated with PAH, granzyme B cleaves intersectin-1 to produce N-terminal (EH) and C-terminal (SH3A-E) protein fragments. In a murine model of PAH, EH triggers plexiform arteriopathy via p38-ELK1-c-Fos signaling. The SH3A-E fragment also influences signaling, having dominant-negative effects on ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively). Using PAECs engineered to express tagged versions of EH and SH3A-E, we demonstrate that the two ITSN fragments increase both p38-ELK1 activation and the ratio of p38 to ERK1 and ERK2 activity, leading to PAEC proliferation, with female cells being more responsive than male cells. Furthermore, expression of EH substantially upregulates the expression and activity of the long non-coding RNA in female PAECs, which in turn upregulates the X-linked gene and represses expression of (). These events are recapitulated by the PAECs of female idiopathic PAH patients, and may account for their proliferative phenotype. Thus, upregulation of could be an important factor in explaining sexual dimorphism in the proliferative response of PAECs and the imbalanced sex ratio of PAH.
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http://dx.doi.org/10.1242/jcs.237776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240306PMC
May 2020

Mesenchymal stem cells-derived extracellular vesicles in acute respiratory distress syndrome: a review of current literature and potential future treatment options.

Clin Transl Med 2019 Sep 12;8(1):25. Epub 2019 Sep 12.

Department of Internal Medicine, Pulmonary, Critical Care and Sleep Medicine, Rush University Medical Center, 1750 W Harrison St. 1535 JS, Chicago, IL, 60612, USA.

Acute respiratory distress syndrome (ARDS) is a life-threatening inflammatory lung condition associated with significant morbidity and mortality. Unfortunately, the current treatment for this disease is mainly supportive. Mesenchymal stem cells (MSCs) due to their immunomodulatory properties are increasingly being studied for the treatment of ARDS and have shown promise in multiple animal studies. The therapeutic effects of MSCs are exerted in part in a paracrine manner by releasing extracellular vesicles (EVs), rather than local engraftment. MSC-derived EVs are emerging as potential alternatives to MSC therapy in ARDS. In this review, we will introduce EVs and briefly discuss current data on EVs and MSCs in ARDS. We will discuss current literature on the role of MSC-derived EVs in pathogenesis and treatment of ARDS and their potential as a treatment strategy in the future.
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http://dx.doi.org/10.1186/s40169-019-0242-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739436PMC
September 2019

Plexiform Arteriopathy in Rodent Models of Pulmonary Arterial Hypertension.

Am J Pathol 2019 06 26;189(6):1133-1144. Epub 2019 Mar 26.

Division of Pulmonary Critical Care and Sleep Medicine, Department of Internal Medicine, Rush Medical College, Chicago, Illinois. Electronic address:

As time progresses, our understanding of disease pathology is propelled forward by technological advancements. Much of the advancements that aid in understanding disease mechanics are based on animal studies. Unfortunately, animal models often fail to recapitulate the entirety of the human disease. This is especially true with animal models used to study pulmonary arterial hypertension (PAH), a disease with two distinct phases. The first phase is defined by nonspecific medial and adventitial thickening of the pulmonary artery and is commonly reproduced in animal models, including the classic models (ie, hypoxia-induced pulmonary hypertension and monocrotaline lung injury model). However, many animal models, including the classic models, fail to capture the progressive, or second, phase of PAH. This is a stage defined by plexogenic arteriopathy, resulting in obliteration and occlusion of the small- to mid-sized pulmonary vessels. Each of these two phases results in severe pulmonary hypertension that directly leads to right ventricular hypertrophy, decompensated right-sided heart failure, and death. Fortunately, newly developed animal models have begun to address the second, more severe, side of PAH and aid in our ability to develop new therapeutics. Moreover, p38 mitogen-activated protein kinase activation emerges as a central molecular mediator of plexiform lesions in both experimental models and human disease. Therefore, this review will focus on plexiform arteriopathy in experimental animal models of PAH.
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http://dx.doi.org/10.1016/j.ajpath.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584781PMC
June 2019

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

Front Physiol 2018 2;9:1393. Epub 2018 Oct 2.

Division of Pulmonary Medicine, Critical Care and Sleep Medicine, Department of Internal Medicine, Rush Medical College, Rush University, Chicago, IL, United States.

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 (EH) 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 EH 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-EH (EC) and ITSN knockout heterozygous mice (K0 ) transduced with the Myc-EH, 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 (EC), ITSN knockdown ECs (EC), 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 EC compared to EC. Despite their endocytic deficiency, EC internalized biotin similar to EC which is twofold higher compared to EC. Moreover, the lung microvascular bed of Myc-EH-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 EH 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 EH by shifting the physiological vesicular (caveolae) transport toward the alternative endocytic pathways is a significant contributor to the dysfunctional molecular phenotype of EC.
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http://dx.doi.org/10.3389/fphys.2018.01393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176378PMC
October 2018

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

Clin Transl Med 2018 Jun 22;7(1):19. Epub 2018 Jun 22.

Department of Internal Medicine, Pulmonary, Critical Care and Sleep Medicine, Rush University Medical Center, 1750W Harrison St. 1535 JS, Chicago, IL, 60612, USA.

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 (CD73CD105Cd34CD45). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TβRI)/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.
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http://dx.doi.org/10.1186/s40169-018-0197-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013417PMC
June 2018

Intersectin-1s deficiency in pulmonary pathogenesis.

Respir Res 2017 09 6;18(1):168. Epub 2017 Sep 6.

Department of Pharmacology and Division of Pulmonary and Critical Care Medicine, Rush University Medical Center and Rush Medical College, 1750 W. Harrison Street, 1535 Jelke, Chicago, IL, 60612, USA.

Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFβRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EH and SH3A-E fragments. The EH fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EH plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.
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http://dx.doi.org/10.1186/s12931-017-0652-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585975PMC
September 2017

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

Front Physiol 2017 14;8:128. Epub 2017 Mar 14.

Department of Internal Medicine, Division of Pulmonary and Critical Care, Rush University, Medical College Chicago, IL, USA.

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.
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http://dx.doi.org/10.3389/fphys.2017.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348516PMC
March 2017

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

Am J Pathol 2017 Mar 6;187(3):528-542. Epub 2017 Jan 6.

Department of Pharmacology, Rush University Medical Center, Chicago, Illinois. Electronic address:

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 (EH), 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 EH 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 EH 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 p38-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.
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http://dx.doi.org/10.1016/j.ajpath.2016.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389368PMC
March 2017

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

Mol Cancer 2016 09 14;15(1):59. Epub 2016 Sep 14.

Department of Pharmacology and Division of Pulmonary and Critical Care Medicine, Rush University Medical Center and Rush Medical College, 1750 W. Harrison Street, 1535 Jelke, Chicago, IL, 60612, USA.

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.
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http://dx.doi.org/10.1186/s12943-016-0543-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024437PMC
September 2016

New insights into the functions of intersectin-1s.

Commun Integr Biol 2015 May-Jun;8(3):e1034400. Epub 2015 Jun 24.

Departments of Pharmacology and Critical Care Medicine; Rush University Medical Center ; Chicago, IL USA.

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 β (TGFβ)/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 TGFβ-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.
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http://dx.doi.org/10.1080/19420889.2015.1034400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594419PMC
October 2015

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells.

Am J Physiol Lung Cell Mol Physiol 2015 Aug 19;309(4):L425-34. Epub 2015 Jun 19.

Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Department of Medicine, Indiana University, Indianapolis, Indiana; The Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana.

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.
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http://dx.doi.org/10.1152/ajplung.00376.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538232PMC
August 2015

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

J Cell Sci 2015 Apr 26;128(8):1528-41. Epub 2015 Feb 26.

Department of Pharmacology, Rush University, Chicago, IL 60612, USA Pulmonary and Critical Care Medicine, Rush University Medical Center, Chicago, IL 60612, USA

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 β receptor I (TGFβ-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 TGFβ and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth.
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http://dx.doi.org/10.1242/jcs.163030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406123PMC
April 2015

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

Pulm Circ 2013 Sep 5;3(3):478-98. Epub 2013 Dec 5.

1 Department of Pharmacology, Rush University, Chicago, Illinois, USA.

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.
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http://dx.doi.org/10.1086/674439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070809PMC
September 2013

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

PLoS One 2013 27;8(9):e75846. Epub 2013 Sep 27.

Department of Pharmacology and Medicine, Division of Pulmonary and Critical Care, Rush University, Chicago, Illinois, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075846PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785431PMC
July 2014

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

J Biol Chem 2013 Sep 26;288(36):25701-25716. Epub 2013 Jul 26.

From the Departments of Pharmacology and Medicine, Vascular Biology, and Pulmonary and Critical Care Medicine, Rush University Medical Center, Chicago, Illinois 60612,. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M113.502674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764778PMC
September 2013

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.

J Vis Exp 2013 Jun 21(76). Epub 2013 Jun 21.

Department of Pharmacology, Rush University.

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.
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http://dx.doi.org/10.3791/50316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728359PMC
June 2013

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

Am J Physiol Lung Cell Mol Physiol 2013 Aug 14;305(4):L291-300. Epub 2013 Jun 14.

Dept. of Pharmacology, The Univ. of Illinois, College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612.

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 RhoA's 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.
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http://dx.doi.org/10.1152/ajplung.00094.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891015PMC
August 2013

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

Apoptosis 2013 Jan;18(1):57-76

Department of Pharmacology, Rush University, 1735 W. Harrison St., Chicago, IL 60612, USA.

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 (TβR1), 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.
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http://dx.doi.org/10.1007/s10495-012-0762-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543613PMC
January 2013

Impaired caveolae function and upregulation of alternative endocytic pathways induced by experimental modulation of intersectin-1s expression in mouse lung endothelium.

Biochem Res Int 2012 26;2012:672705. Epub 2012 Feb 26.

Department of Pharmacology, Rush University, 1735 W Harrison, Chicago, IL 60612, USA.

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.
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http://dx.doi.org/10.1155/2012/672705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299393PMC
August 2012

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

Respir Res 2011 Apr 12;12:46. Epub 2011 Apr 12.

Pulmonary and Critical Care Medicine, Rush University Medical Center, 1750 W, Harrison Street, 297 Jelke, Chicago, IL 60612, USA.

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.
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http://dx.doi.org/10.1186/1465-9921-12-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096597PMC
April 2011

Stress chaperone GRP-78 functions in mineralized matrix formation.

J Biol Chem 2011 Mar 14;286(11):8729-39. Epub 2011 Jan 14.

Department of Oral Biology, University of Illinois, Chicago, Illinois 60612, USA.

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.
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http://dx.doi.org/10.1074/jbc.M110.179341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059005PMC
March 2011

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

J Cell Mol Med 2011 Nov;15(11):2364-76

Department of Pharmacology, Rush University Medical Center, Medical College, Vascular Biology Section, Chicago, IL 60612, USA.

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.
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http://dx.doi.org/10.1111/j.1582-4934.2010.01226.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072443PMC
November 2011

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

J Biol Chem 2009 Sep 21;284(38):25953-61. Epub 2009 Jul 21.

Department of Pharmacology, Rush University Medical Center, Chicago, Illinois 60612, USA.

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-PH(ITSN-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-PH(ITSN-2L)-transfected cells compared with control and 1 microM 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.
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http://dx.doi.org/10.1074/jbc.M109.035071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757996PMC
September 2009

Tiam1 and Rac1 are required for platelet-activating factor-induced endothelial junctional disassembly and increase in vascular permeability.

J Biol Chem 2009 Feb 17;284(8):5381-94. Epub 2008 Dec 17.

Department of Pharmacology, Rush University Medical Center, Chicago, Illinois 60612, USA.

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.
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http://dx.doi.org/10.1074/jbc.M808958200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643508PMC
February 2009

Molecular determinants of endothelial transcytosis and their role in endothelial permeability.

Am J Physiol Lung Cell Mol Physiol 2007 Oct 20;293(4):L823-42. Epub 2007 Jul 20.

Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois, College of Medicine, Chicago, Illinois 60612, USA.

Caveolae transcytosis with its diverse mechanisms-fluid phase, adsorptive, and receptor-mediated-plays an important role in the continuous exchange of molecules across the endothelium. We will discuss key features of endothelial transcytosis and caveolae that have been studied recently and have increased our understanding of caveolae function in transcytosis at the molecular level. During transcytosis, caveolae "pinch off" from the plasma membrane to form discrete vesicular carriers that shuttle to the opposite front of endothelial cells, fuse with the plasma membrane, and discharge their cargo into the perivascular space. Endothelial transcytosis exhibits distinct properties, the most important being rapid and efficient coupling of endocytosis to exocytosis on opposite plasma membrane. We address herein the membrane fusion-fission reactions that underlie transcytosis. Caveolae move across the endothelial cells with their cargo predominantly in the fluid phase through an active process that bypasses the lysosomes. Endothelial transcytosis is a constitutive process of vesicular transport. Recent studies show that transcytosis can be upregulated in response to pathological stimuli. Transcytosis via caveolae is an important route for the regulation of endothelial barrier function and may participate in different vascular diseases.
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http://dx.doi.org/10.1152/ajplung.00436.2006DOI Listing
October 2007

Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells.

J Biol Chem 2007 Jun 3;282(23):17166-78. Epub 2007 Apr 3.

Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.

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-X(L) 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.
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http://dx.doi.org/10.1074/jbc.M608996200DOI Listing
June 2007

A novel lysophospholipid- and pH-sensitive receptor, GPR4, in brain endothelial cells regulates monocyte transmigration.

Endothelium 2007 Jan-Feb;14(1):25-34

Department of Pharmacology, Rush University Medical Center, Chicago, Illinois 60612, USA.

Abundant evidence documents the highly proinflammatory actions of lysophosphatidylcholine (LPC). Further, LPC, found in high amounts in oxidized low-density lipoprotein (LDL), is implicated as an atherogenic factor. In endothelial cells, LPC impairs endothelial barrier function through GPR4, a novel receptor hypothesized to be sensitive to LPC and protons. The authors investigated the stimulation by LPC or low pH of GPR4 in human brain microvascular endothelial cells (HBMECs) and whether the activated GPR4 regulates in vitro monocyte transmigration. The results indicated that HBMECs stimulated by LPC (5 microM), but not low pH, showed a twofold increase in monocyte transmigration. Using retroviruses containing siRNA to GPR4, a > 60% reduction of GPR4 expression resulted in blockade of the LPC-stimulated transmigration. The inhibited response was restored by co-expression with an small interference RNA (siRNA)-resistant, but functional, GPR4 mutant construct. To investigate potential signaling mechanisms, the siRNA-mediated knockdown of GPR4 also prevented LPC-induced RhoA activation. C3 transferase, a Rho inhibitor, prevented approximately approximately 65% of the LPC-stimulated transmigration. LPC also increased MLC phosphorylation by 5 min, which was inhibited by the Rho kinase inhibitor, Y-27632 (10 microM) or ML-7 (myosin light chain kinase (MLCK) inhibitor). The findings indicate that the proinflammatory and atherogenic LPC stimulated endothelial GPR4, which promoted monocyte transmigration through a RhoA-dependent pathway.
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http://dx.doi.org/10.1080/10623320601177288DOI Listing
June 2007

siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway.

Am J Physiol Lung Cell Mol Physiol 2006 Feb 23;290(2):L405-13. Epub 2005 Sep 23.

Dept. of Pharmacology, Univ. of Illinois College of Medicine, 835 So. Wolcott Ave. M/C868 Chicago, IL 60612, USA.

Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1-/- mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by approximately 90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability.
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http://dx.doi.org/10.1152/ajplung.00292.2005DOI Listing
February 2006

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

J Biol Chem 2005 Nov 23;280(44):37130-8. Epub 2005 Aug 23.

Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.

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.
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http://dx.doi.org/10.1074/jbc.M505659200DOI Listing
November 2005