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 ...

Rush University

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.

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

Intersectine-1 s (ITSN-1 s), une homologie de Src cinq 3 (SH3) contenant du domaine prot&eacute;ique, est critique requise pour les cav&eacute;oles et endocytose par la clathrine (CME), en raison de ses interactions avec la dynamine (dyn). Les cinq domaines de SH3A-E, SH3A est unique en raison de sa forte affinit&eacute; pour dyn et inhibition puissante du CME. Toutefois, le m&eacute;canisme mol&eacute;culaire par lequel SH3A s&#39;int&egrave;gre dans la fonction globale de ITSN-1 s de r&eacute;glementer le processus d&#39;endocytose n&#39;est pas compris. En utilisant des approches biochimiques et fonctionnelles ainsi que la microscopie &eacute;lectronique &agrave; haute r&eacute;solution, nous montrons que SH3A exog&egrave;ne exprim&eacute;e dans les cellules endoth&eacute;liales pulmonaires humaines caus&eacute;es des structures intervenant dans l&#39;endocytose anormales, grappes de cav&eacute;oles d&eacute;form&eacute;e, fr&eacute;quentes anneaux coloration denses autour de l&#39;inhibition de caveolar cous et 60 % de l&#39;internalisation des cav&eacute;oles. De plus, des &eacute;tudes in vitro a r&eacute;v&eacute;l&eacute; que SH3A, semblable &agrave; pleine longueur ITSN-1 s stimule dyn2 oligom&eacute;risation et guanosine triphosphatase (GTP) activit&eacute; ase, effets non d&eacute;tect&eacute;s lorsque d&#39;autres domaines SH3 de ITSN-1 ont &eacute;t&eacute; utilis&eacute;s comme t&eacute;moins. &Eacute;tonnamment, en pr&eacute;sence de SH3A, dyn2-dyn2 interactions sont stabilis&eacute;es et malgr&eacute; l&#39;hydrolyse de la GTP continu, oligom&egrave;res dyn2 ne peut pas d&eacute;monter. SH3A peut contenir jusqu&#39;&agrave; cav&eacute;oles lib&eacute;ration de la membrane plasmique et de la formation de v&eacute;sicules de transport gratuit, en prolongeant la dur&eacute;e de vie de dyn2 assembl&eacute;s. Au total, nos r&eacute;sultats indiquent que l&#39;ITSN-1 s, par l&#39;interm&eacute;diaire de son SH3A a la capacit&eacute; unique de r&eacute;glementer dyn2 montage-d&eacute;montage et fonction au cours de l&#39;endocytose.

R&egrave;glement de la dynamine-2 Montage-d&eacute;montage et fonctionnent par l&#39;interm&eacute;diaire du domaine de SH3A d&#39;intersectine-1 s.

Intersectin-1s (ITSN-1s), un cinque Src homology 3 (SH3) dominio-contenente della proteina, &egrave; critico richiesto per caveolae ed endocytosis clathrin-mediata (CME), a causa delle sue interazioni con dynamin (dyn). I cinque domini SH3A-E, SH3A &egrave; unico a causa della sua elevata affinit&agrave; per dyn e potente inibizione della CME. Tuttavia, il meccanismo molecolare con cui SH3A si integra nella funzione generale di ITSN-1s per regolare il processo endocitico non &egrave; capito. Utilizzando approcci biochimici e funzionali, cos&igrave; come la microscopia elettronica ad alta risoluzione, mostriamo che SH3A esogenicamente espressa nelle cellule endoteliali umane polmone causate strutture endocitico anormale, caveolae distorta dei cluster, frequenti colorazione-densi anelli intorno il caveolar collo e 60% inibizione di interiorizzazione caveolae. Ulteriori studi in vitro hanno rivelato che SH3A, simili a full-length ITSN-1s stimola dyn2 oligomerizzazione e guanosina trifosfato (GTP) ase attivit&agrave;, effetti non rilevati quando altri domini SH3 di ITSN-1s sono stati utilizzati come controlli. Sorprendentemente, in presenza di SH3A, dyn2-dyn2 interazioni sono stabilizzate e nonostante continuo idrolisi di GTP, oligomeri dyn2 non possono smontare. SH3A pu&ograve; contenere fino caveolae rilascio dalla membrana plasmatica e formazione di vescicole di trasporto gratis, prolungando la durata del dyn2 assemblati. Complessivamente, i nostri risultati indicano che ITSN-1s, tramite il suo SH3A ha la capacit&agrave; unica di regolare dyn2 montaggio-smontaggio e funzione durante endocitosi.

Regolamento di dynamin-2 Montaggio-smontaggio e funzione attraverso il dominio di SH3A di intersectin-1s.

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.

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

La r&eacute;ponse des cellules endoth&eacute;liales microvasculaires pulmonaires (ECs) &agrave; lipopolysaccharide (LPS) est au c&oelig;ur de la pathogen&egrave;se des maladies pulmonaires. Il est double, avec une facette qui est pro-inflammatoire et une autre qui est cyto-protecteur. Dans un travail ant&eacute;rieur, la surexpression de l&#39;anti-apoptotique Bcl-XL a sauv&eacute; ECs l&#39;apoptose d&eacute;clench&eacute;e par les siARN knockdown d&#39;intersectine-1 (ITSN-1 s), une prot&eacute;ine apoptotiques cruciale pour la fonction de l&#39;ECs. Ici, nous avons caract&eacute;ris&eacute; davantage la r&eacute;ponse d&#39;EC cyto-protecteur de LPS et dysfonction pro-inflammatoires.

Le dysfonctionnement endoth&eacute;lial pro-inflammatoires est associ&eacute; &agrave; intersectine-1 s down-regulation.

La risposta delle cellule endoteliali Microvascolari (ECs) di polmone al lipopolisaccaride (LPS) &egrave; centrale nella patogenesi delle lesioni polmonari. &Egrave; doppio nella natura, con una sfaccettatura che &egrave; pro-infiammatoria e un altro che &egrave; cyto-protettivo. Nel precedente lavoro, iperespressione del anti-apoptotic Bcl-XL salvato ECs da apoptosi innescata da siRNA atterramento di intersectin-1s (ITSN-1s), una proteina pro-survival cruciale per la funzione di ECs. Qui abbiamo caratterizzato ulteriormente la risposta CE cyto-protettivo per LPS e disfunzione pro-infiammatoria.

Disfunzione delle cellule endoteliali pro-infiammatorie &egrave; associata con intersectin-1s down-regulation.

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.

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

Intersectine-1 s (ITSN-1 s), une prot&eacute;ine contenant SH3 cinq domaines (A-E), r&eacute;gule, par l&#39;interm&eacute;diaire de la SH3A, la fonction de dynamine-2 (dyn2) sur le site de l&#39;endocytose. Expression ITSN-1 s a &eacute;t&eacute; modul&eacute;e dans l&#39;endoth&eacute;lium pulmonaire de souris par livraison de liposomes d&#39;un plasmide codant myc cDNA-SH3A ou un sp&eacute;cifique siARN ciblant le g&egrave;ne ITSN-1. La vascularisation pulmonaire de SH3A-transduites and ITSN-1 s-deficient mice a &eacute;t&eacute; perfus&eacute;e avec or albumine (UA-BSA) &agrave; analyser au microscope &eacute;lectronique la morphologiques interm&eacute;diaires et les voies impliqu&eacute;es dans le transport transendoth&eacute;liale ou avec dinitrophenylated (DNP)-BSA &agrave; quantifier, par ELISA, son transport. Aigu&euml; modulation de l&#39;expression de l&#39;ITSN-1 s a diminu&eacute; le nombre des cav&eacute;oles, affaiblies &agrave; leur transport et ouvert les jonctions inter-endoth&eacute;liales, tandis que cytoprotectrices compensatoire non-conventionnelles endocytose/transcytotiques structures. Inhibition chronique de ITSN-1 s a encore augment&eacute; la pr&eacute;sence de produits interm&eacute;diaires non conventionnels et partiellement restaur&eacute; l&#39;int&eacute;grit&eacute; jonctionnelle. Ces r&eacute;sultats indiquent qu&#39;expression ITSN-1 s est requise pour la fonction cav&eacute;oles et transport transendoth&eacute;liale efficace. De plus, nos r&eacute;sultats d&eacute;montrent que l&#39;ECs sont hautement adapt&eacute;s de remplir leur fonction de transport tout en conservant l&#39;hom&eacute;ostasie du poumon.

Alt&eacute;ration fonction cav&eacute;oles et upregulation de fili&egrave;res alternatives d&#39;endocytose induite par exp&eacute;rimentale modulation de l&#39;expression d&#39;intersectine-1 s dans l&#39;endoth&eacute;lium pulmonaire de souris.

Intersectin-1s (ITSN-1s), una proteina contenente cinque SH3 domini (A-E), regola tramite il SH3A la funzione di dynamin-2 (dyn2) presso il sito endocitico. Espressione ITSN-1s &egrave; stato modulato in endotelio polmonare del mouse dalla consegna di liposomi di un cDNA del plasmide codifica myc-SH3A o a specifici siRNA gene ITSN-1 di targeting. Il sistema vascolare polmonare di transduced SH3A e ITSN-1s-deficient mice &egrave; stato perfuso con albumina d&#39;oro (Au-BSA) per analizzare da microscopia elettronica il morfologici intermedi e vie coinvolte nel trasporto transendoteliale o dinitrophenylated (DNP)-BSA quantificare da ELISA suo trasporto. Modulazione acuta dell&#39;espressione ITSN-1s &egrave; diminuito il numero delle caveolae, ha alterato il loro trasporto e aperto le giunzioni interendothelial, mentre upregulating compensativi non convenzionali strutture endocitico/transcytotic. Inibizione cronica di ITSN-1s ulteriormente incrementato la presenza di prodotti intermedi non convenzionali e parzialmente restaurato l&#39;integrit&agrave; giunzionale. Questi risultati indicano che espressione ITSN-1s &egrave; richiesto per la funzione caveolae e trasporto efficiente transendoteliale. Inoltre, i nostri risultati dimostrano che la ECs sono estremamente adattati a svolgere la loro funzione di trasporto, mantenendo l&#39;omeostasi del polmone.

Alterata funzione caveolae e sovraregolazione di alternativi percorsi endocitico indotta da sperimentale modulazione dell&#39;espressione di intersectin-1s nell&#39;endotelio polmonare del mouse.

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 48Â % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30Â %, 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 (TÎ²R1), 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.

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

Intersectine-1 s (ITSN-1 s) est une prot&eacute;ine intervenant dans l&#39;endocytose g&eacute;n&eacute;rale impliqu&eacute;e dans la r&eacute;gulation de la perm&eacute;abilit&eacute; vasculaire pulmonaire et endoth&eacute;liale cellules survie (ECs), par l&#39;interm&eacute;diaire de signalisation MEK/Erk1/2(MAPK). Afin d&#39;&eacute;tudier les effets in vivo de l&#39;ITSN-1 s et l&#39;apoptose ECs qui en r&eacute;sulte sur le syst&egrave;me vasculaire pulmonaire et l&#39;hom&eacute;ostasie du poumon, nous avons utilis&eacute; une ITSN-1 s knocked-down (KD(ITSN)) souris g&eacute;n&eacute;r&eacute;e par une distribution r&eacute;p&eacute;t&eacute;e d&#39;un g&egrave;ne de l&#39;ITSN-1 ciblage sp&eacute;cifique siARN (siRNA(ITSN)). Les analyses histologiques et biochimiques ainsi que la microscopie &eacute;lectronique (EM) a r&eacute;v&eacute;l&eacute; que KD(ITSN) aigu&euml; [traitement de post-siRNA(ITSN) (3d) 3 jours] inhib&eacute;e Erk1/2(MAPK) signalisation apoptotiques, porter pr&eacute;judice significatif ECs pulmonaires et l&#39;apoptose ; &agrave; 10D de KD(ITSN), activation de la caspase-3 &eacute;tait au sommet, terminal deoxynucleotidyl transferase dUTP nick-fin &eacute;tiquetage (TUNEL)-ECs positives a montr&eacute; 3.4-fold d&#39;augmentation, l&#39;interception moyenne lin&eacute;aire (MLI) augmenter de 48 % et la densit&eacute; des microvaisseaux pulmonaire telle que r&eacute;v&eacute;l&eacute;e par aquaporin-1 coloration (AQP-1) ont diminu&eacute; de 30 %, tous par rapport aux t&eacute;moins ; la fonction pulmonaire est alt&eacute;r&eacute;e. En m&ecirc;me temps, l&#39;expression de plusieurs facteurs de croissance connu pour activer Erk1/2(MAPK) et de supprimer l&#39;activit&eacute; pro-apoptotique Bad augment&eacute;e. KD(ITSN) a modifi&eacute; l&#39;activit&eacute; SMAD, en aval de la transformant le facteur de croissance b&ecirc;ta-r&eacute;cepteur-1 (T&beta;R1), comme le montrent des analyses immunoblot et de fractionnement subcellulaires. En outre, post-siRNA(ITSN) 24D, survivant ECs devenue hyper prolif&eacute;ration et apoptose r&eacute;sistant contre la carence de l&#39;ITSN-1 s, comme en t&eacute;moigne l&#39;imagerie EM, 5-bromo-deoxyuridine (BrdU) incorporation et la phosphorylation de Bad-Ser(112/155), respectivement, conduisant &agrave; la densit&eacute; des microvaisseaux accrue et de la r&eacute;paration des poumons bless&eacute;s, et le d&eacute;p&ocirc;t de matrice. En somme, ECs dysfonction endocytose et la mort apoptotique par KD(ITSN) contribuent l&eacute;sion pulmonaire initiale et une perte microvasculaire, suivie par endoth&eacute;liales changements ph&eacute;notypiques et microvasculaire transformant dans le lit de microvasculaire pulmonaire murin restant.

Knockdown in vivo des intersectine-1 modifie le ph&eacute;notype cellulaire endoth&eacute;liale et provoque un remodelage microvasculaire chez les poumons de souris.

Intersectin-1s (ITSN-1s) &egrave; una generale endocitico proteine coinvolte nella regolazione della permeabilit&agrave; vascolare polmonare ed endoteliali cellule sopravvivenza (ECs), via MEK/Erk1/2(MAPK) di segnalazione. Per studiare gli effetti in vivo di deficit ITSN-1s e il apoptosis ECs risultante sul sistema vascolare polmonare e omeostasi del polmone, abbiamo utilizzato un ITSN-1s abbattuto (KD(ITSN)) mouse generato da ripetute consegna di un gene targeting ITSN-1 di specifici siRNA (siRNA(ITSN)). Analisi biochimiche e istologiche, cos&igrave; come la microscopia elettronica (EM) ha rivelato che KD(ITSN) acuta [trattamento post-siRNA(ITSN) (3d) 3 giorni] inibito Erk1/2(MAPK) pro-survival segnalazione, significativo ECs apoptosis del polmone e del pregiudizio; a 10d di KD(ITSN), attivazione di caspase-3 era al culmine, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positivo ECs ha mostrato 3.4-fold aumento, l&#39;intercetta lineare medio (MLI) ha mostrato 48% potenziati e densit&agrave; del microvessel polmonare come rivelato da aquaporina-1 colorazione (AQP-1) &egrave; diminuito del 30%, tutti rispetto ai controlli; funzione polmonare &egrave; stato alterato. Simultaneamente, l&#39;espressione di diversi fattori di crescita noti per attivare Erk1/2(MAPK) e sopprimere le attivit&agrave; di pro-apoptotic male aumenta. KD(itsn) alterata attivit&agrave; SMAD, a valle del transforming growth factor beta-receptor-1 (T&beta;R1), come mostrato dalle analisi sottocellulari di frazionamento e immunoblot. Inoltre, 24d post-siRNA(ITSN), sopravvivendo ECs divenne iper-proliferativa e apoptotic-resistente contro deficit ITSN-1s, come dimostrato dall&#39;imaging EM, 5-bromo-deoxyuridine (BrdU) incorporazione e Bad-Ser(112/155) fosforilazione, rispettivamente, portando a microvessel maggiore densit&agrave; e riparazione dei polmoni feriti, come pure la deposizione di matrice. In somma, ECs endocitico disfunzione e morte apoptotic causata da KD(ITSN) contribuire al danno polmonare iniziale e perdita microvascolare, seguita dai cambiamenti fenotipici endoteliale e microvascolare rimodellamento nel restante murino letto microvascolare polmonare.

Cristina Bardita

Department of Pharmacology

Rush University

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

Cristina Bardita

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology

Rush University

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

Department of Pharmacology