Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

The Children's Hospital of Philadelphia, University of Pennsylvania

Alterado el &oacute;xido n&iacute;trico (NO) bios&iacute;ntesis se piensan para desempe&ntilde;ar un papel en la iniciaci&oacute;n y progresi&oacute;n de la aterosclerosis y pueden contribuir a un mayor riesgo en otras enfermedades cardiovasculares. Se presume que alterados NO biodisponibilidad no puede resultar de un aumento en end&oacute;geno sintasa (NOS) inhibidores, araginine dimethly asim&eacute;trica (ADMA) y N (G)-monometil arginina, que normalmente son metabolizados por dimethyarginine Dimetilamina hidrolasa (DDAH). L&iacute;pidos hidroper&oacute;xidos y sus productos de degradaci&oacute;n se generan durante la inflamaci&oacute;n y el estr&eacute;s oxidativo y han sido implicados en la patogenia de las enfermedades cardiovasculares. Aqu&iacute;, demostramos que el l&iacute;pido hidroper&oacute;xido degradaci&oacute;n producto 4-hidroxi-2-nonenal (4-HNE) causa una disminuci&oacute;n dosis-dependiente ninguna generaci&oacute;n de c&eacute;lulas endoteliales a&oacute;rticas bovinas, acompa&ntilde;ada por una disminuci&oacute;n en la actividad enzim&aacute;tica DDAH. Los efectos inhibitorios del 4-HNE (50 microM) de NO endotelial producci&oacute;n se revirtieron parcialmente con los suplementos de L-Arg (1 mM). Sobreexpresi&oacute;n de DDAH-1 humano junto con suplementos antioxidantes completamente restaurada NO endotelial producci&oacute;n despu&eacute;s de la exposici&oacute;n a 4-HNE (50 microM). Estos resultados demuestran un papel cr&iacute;tico para el methylarginines end&oacute;genos en la patogenesia de la disfunci&oacute;n endotelial. Porque l&iacute;pidos hidroper&oacute;xidos y sus productos de degradaci&oacute;n se saben para estar implicado en la aterosclerosis, modulaci&oacute;n de DDAH y methylarginines puede servir como una nueva diana terap&eacute;utica en el tratamiento de las enfermedades cardiovasculares asociadas con el estr&eacute;s oxidativo.

Papel de DDAH-1 en la inhibici&oacute;n de la peroxidaci&oacute;n de l&iacute;pidos mediada por producto de NO endotelial generaci&oacute;n.

Alt&eacute;r&eacute; l&#39;oxyde nitrique (aucun) biosynth&egrave;se est cens&eacute;e jouer un r&ocirc;le dans l&#39;apparition et la progression de l&#39;ath&eacute;roscl&eacute;rose et peut contribuer &agrave; un risque accru vu dans d&#39;autres maladies cardiovasculaires. On fait l&#39;hypoth&egrave;se que modifi&eacute; non biodisponibilit&eacute; ne peut r&eacute;sulter d&#39;une augmentation endog&egrave;ne synthase (NOS) inhibiteurs, araginine dimethly asym&eacute;trique (ADMA) et N (G)-monom&eacute;thyl arginine, qui sont normalement m&eacute;tabolis&eacute; par dimethyarginine dim&eacute;thylamine hydrolase (DDAH). Les hydroperoxydes lipidiques et leurs produits de d&eacute;gradation sont g&eacute;n&eacute;r&eacute;s au cours de l&#39;inflammation et du stress oxydatif et ont &eacute;t&eacute; impliqu&eacute;s dans la pathogen&egrave;se des maladies cardiovasculaires. Ici, nous montrons que le lipides hydroperoxyde d&eacute;gradation produit 4-hydroxy-2-nonenal (4-HNE) provoque une diminution dose-d&eacute;pendante dans aucune g&eacute;n&eacute;ration de cellules endoth&eacute;liales aortiques bovines, accompagn&eacute;e d&#39;une diminution de l&#39;activit&eacute; enzymatique DDAH. Les effets inhibiteurs du 4-HNE (50 microM) non endoth&eacute;liale production &eacute;taient partiellement renvers&eacute; avec la suppl&eacute;mentation en L-Arg (1 mM). La surexpression de DDAH-1 humain ainsi que la suppl&eacute;mentation en antioxydants enti&egrave;rement restaur&eacute;e endoth&eacute;lial non production apr&egrave;s une exposition au 4-HNE (50 microM). Ces r&eacute;sultats d&eacute;montrent un r&ocirc;le critique pour la m&eacute;thylarginines endog&egrave;nes dans la pathogen&egrave;se de la dysfonction endoth&eacute;liale. Parce que les hydroperoxydes lipidiques et leurs produits de d&eacute;gradation sont connus pour &ecirc;tre impliqu&eacute;s dans l&#39;ath&eacute;roscl&eacute;rose, modulation de DDAH et m&eacute;thylarginines peut-&ecirc;tre constituer une nouvelle cible th&eacute;rapeutique dans le traitement des troubles cardiovasculaires associ&eacute;es au stress oxydatif.

R&ocirc;le de DDAH-1 inhibition d&#39;induite par le produit de la peroxydation de lipides d&#39;endoth&eacute;lial non g&eacute;n&eacute;ration.

&#x6539;&#x53D8;&#x4E00;&#x6C27;&#x5316;&#x6C2E; &#xFF08;&#x65E0;&#xFF09; &#x751F;&#x7269;&#x5408;&#x6210;&#x60F3;&#x53D1;&#x6325;&#x4F5C;&#x7528;&#xFF0C;&#x542F;&#x52A8;&#x548C;&#x52A8;&#x8109;&#x7CA5;&#x6837;&#x786C;&#x5316;&#x7684;&#x8FDB;&#x5C55;&#xFF0C;&#x53EF;&#x80FD;&#x6709;&#x52A9;&#x4E8E;&#x89C1;&#x4E8E;&#x5176;&#x4ED6;&#x5FC3;&#x8840;&#x7BA1;&#x75BE;&#x75C5;&#x7684;&#x98CE;&#x9669;&#x589E;&#x52A0;&#x3002;&#x5B83;&#x5047;&#x8BBE;&#x7684;&#x6539;&#x53D8;&#x65E0;&#x751F;&#x7269;&#x5229;&#x7528;&#x5EA6;&#x53EF;&#x80FD;&#x4F1A;&#x5BFC;&#x81F4;&#x4ECE;&#x589E;&#x52A0;&#x5185;&#x6E90;&#x6027;&#x6C27;&#x5316;&#x6C2E;&#x5408;&#x9176; (NOS) &#x6291;&#x5236;&#x5242;&#x3001; &#x975E;&#x5BF9;&#x79F0;&#x4E8C; araginine (&#x57C3;&#x7279;&#x739B;&#xFF09; &#x548C; N (G)-&#x5355;&#x7532;&#x916F;&#x7CBE;&#x6C28;&#x9178;&#xFF0C;&#x8FD9;&#x901A;&#x5E38;&#x7531; dimethyarginine &#x4E8C;&#x7532;&#x80FA;&#x6C34;&#x89E3;&#x9176; (DDAH) &#x4EE3;&#x8C22;&#x3002;&#x5728;&#x708E;&#x75C7;&#x548C;&#x6C27;&#x5316;&#x5E94;&#x6FC0;&#x8FC7;&#x7A0B;&#x4E2D;&#x751F;&#x6210;&#x7684;&#x8102;&#x8D28;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x548C;&#x53CA;&#x5176;&#x964D;&#x89E3;&#x4EA7;&#x7269;&#x6709;&#x7275;&#x8FDE;&#x7684;&#x5FC3;&#x8840;&#x7BA1;&#x75C5;&#x53D1;&#x75C5;&#x673A;&#x5236;&#x7684;&#x7814;&#x7A76;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x8102;&#x8D28;&#x8FC7;&#x6C27;&#x5316;&#x6C22;&#x964D;&#x89E3;&#x4EA7;&#x54C1; 4-&#x7F9F;&#x57FA;-2-&#x58EC;&#x70EF; (4-HNE) &#x5BFC;&#x81F4;&#x6CA1;&#x6709;&#x4EE3;&#x4ECE;&#x725B;&#x4E3B;&#x52A8;&#x8109;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#xFF0C;&#x4F34;&#x968F;&#x7740; DDAH &#x9176;&#x6D3B;&#x6027;&#x51CF;&#x5C11;&#x5242;&#x91CF;&#x4F9D;&#x8D56;&#x6027;&#x4E0B;&#x964D;&#x3002;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x7684; 4-HNE (50 microM&#xFF09; &#x4E0A;&#x76AE;&#x65E0;&#x751F;&#x4EA7;&#x90E8;&#x5206;&#x88AB;&#x626D;&#x8F6C;&#x4E0E; L-&#x7CBE;&#x6C28;&#x9178;&#x8865;&#x5145; &#xFF08;1 mM&#xFF09;&#x3002;&#x9AD8;&#x6548;&#x8868;&#x8FBE;&#x4EBA; DDAH-1 &#x548C;&#x8865;&#x5145;&#x6297;&#x6C27;&#x5316;&#x8425;&#x517B;&#x7D20;&#x5B8C;&#x5168;&#x6062;&#x590D;&#x5185;&#x76AE; NO 4-HNE (50 microM&#xFF09; &#x63A5;&#x89E6;&#x4E4B;&#x540E;&#x7684;&#x751F;&#x4EA7;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x5185;&#x6E90;&#x6027; methylarginines &#x8840;&#x7BA1;&#x5185;&#x76AE;&#x529F;&#x80FD;&#x969C;&#x788D;&#x7684;&#x53D1;&#x75C5;&#x673A;&#x5236;&#x4E2D;&#x53D1;&#x6325;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x3002;&#x56E0;&#x4E3A;&#x8102;&#x8D28;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x548C;&#x53CA;&#x5176;&#x964D;&#x89E3;&#x4EA7;&#x7269;&#x5DF2;&#x77E5;&#x53C2;&#x4E0E;&#x52A8;&#x8109;&#x7CA5;&#x6837;&#x786C;&#x5316;&#xFF0C;DDAH &#x548C; methylarginines &#x7684;&#x8C03;&#x8282;&#x53EF;&#x4F5C;&#x4E3A;&#x4E0E;&#x6C27;&#x5316;&#x5E94;&#x6FC0;&#x76F8;&#x5173;&#x7684;&#x5FC3;&#x8840;&#x7BA1;&#x75BE;&#x75C5;&#x6CBB;&#x7597;&#x65B0;&#x9776;&#x70B9;&#x3002;

&#x8102;&#x8D28;&#x8FC7;&#x6C27;&#x5316;&#x4EA7;&#x54C1;&#x4ECB;&#x5BFC;&#x6291;&#x5236;&#x5185;&#x76AE; no DDAH-1 &#x7684;&#x4F5C;&#x7528;&#x751F;&#x6210;&#x3002;

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation.

Ge&auml;nderte Stickstoffmonoxid (NO) Biosynthese ist vermutlich eine Rolle bei der Initiierung und Progression der Atherosklerose und kann zu erh&ouml;hten Risiko gesehen in anderen Herz-Kreislauf-Krankheiten beitragen. Es ist die Hypothese, die Nr. ver&auml;ndert Bioverf&uuml;gbarkeit kann resultieren aus einer Zunahme der endogene keinen-Synthase (NOS)-Inhibitoren, asymmetrische Dimethly Araginine (ADMA) und N (G)-Monomethyltetrachlordiphenylmethan Arginin, die normalerweise von Dimethyarginine Dimethylamin Hydrolase (DDAH) verstoffwechselt werden. Lipid-Peroxide und ihre Abbauprodukte w&auml;hrend Entz&uuml;ndung und oxidativem Stress und haben bei der Entstehung von Herz-Kreislauf-Erkrankungen verwickelt worden. Hier zeigen wir, dass die Lipid Cumolhydroperoxid Abbau Produkt 4-hydroxy-2-Nonenal (4-HNE) eine dosisabh&auml;ngige Abnahme der keine Generation von Rindern Aorten Endothelzellen, begleitet von einem R&uuml;ckgang der DDAH Enzym-Aktivit&auml;t verursacht. Die hemmende Wirkung von 4-HNE (50 MicroM) endotheliale No Produktion teilweise aufgehoben wurden L-Arg-Erg&auml;nzung (1 mM). Eine &Uuml;berexpression von menschlichen DDAH-1 zusammen mit antioxidative Nahrungserg&auml;nzung vollst&auml;ndig restauriert und endothelialen NO Produktion nach Exposition gegen&uuml;ber 4-HNE (50 MicroM). Diese Ergebnisse zeigen eine entscheidende Rolle f&uuml;r die endogene Methylarginines in der Pathogenese der endothelialen Dysfunktion. Da Lipid-Peroxide und ihre Abbauprodukte Atherosklerose beteiligt werden bekannterma&szlig;en, kann Modulation der DDAH und Methylarginines als ein neuartiges therapeutisches Ziel bei der Behandlung von Herz-Kreislauf-Erkrankungen, die mit oxidativem Stress verbundenen dienen.

Rolle der DDAH-1 Lipid Peroxidation Produkt-vermittelte Hemmung der endothelialen NO Generierung.

Alterato l&#39;ossido nitrico (NO) biosintesi &egrave; pensata per svolgere un ruolo nell&#39;iniziazione e progressione dell&#39;aterosclerosi e pu&ograve; contribuire all&#39;aumento del rischio visto in altre malattie cardiovascolari. &Egrave; stato ipotizzato che alter&ograve; NO biodisponibilit&agrave; non pu&ograve; derivare da un aumento endogeno sintasi (NOS) inibitori, araginine dimethly asimmetrica (ADMA) e N (G)-monometil arginina, che normalmente vengono metabolizzati da dimethyarginine dimetilammina idrolasi (DDAH). Idroperossidi lipidici e loro prodotti di degradazione vengono generati durante l&#39;infiammazione e stress ossidativo e sono stati implicati nella patogenesi delle malattie cardiovascolari. Qui, ci mostra che il lipide idroperossido degrado prodotto 4-idrossi-2-nonenale (4-HNE) provoca una diminuzione dose-dipendente in nessuna generazione da bovine cellule endoteliali aortiche, accompagnato da una diminuzione di attivit&agrave; dell&#39;enzima DDAH. Gli effetti inibitori di 4-HNE (50 microM) su NO endoteliale produzione erano parzialmente invertita con supplementazione di L-Arg (1 mM). Sovraespressione di umano DDAH-1 insieme a supplementazione antiossidante completamente restaurato NO endoteliale produzione dopo esposizione a 4-HNE (50 microM). Questi risultati dimostrano un ruolo critico per il methylarginines endogeno nella patogenesi della disfunzione endoteliale. Perch&eacute; idroperossidi lipidici e loro prodotti di degradazione sono noti per essere coinvolti nella aterosclerosi, modulazione del DDAH e methylarginines pu&ograve; servire come un romanzo obiettivo terapeutico nel trattamento di disturbi cardiovascolari associati a stress ossidativo.

Ruolo del DDAH-1 nell&#39;inibizione di prodotto-mediata perossidazione lipidica di NO endoteliale generazione.

&#x5909;&#x66F4;&#x3055;&#x308C;&#x305F;&#x4E00;&#x9178;&#x5316;&#x7A92;&#x7D20; (NO) &#x751F;&#x5408;&#x6210;&#x958B;&#x59CB;&#x3068;&#x52D5;&#x8108;&#x786C;&#x5316;&#x306E;&#x9032;&#x884C;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x3066;&#x3057;&#x3001;&#x306E;&#x5FC3;&#x8840;&#x7BA1;&#x75BE;&#x60A3;&#x3067;&#x898B;&#x3089;&#x308C;&#x308B;&#x30EA;&#x30B9;&#x30AF;&#x306E;&#x5897;&#x52A0;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;&#x3053;&#x308C;&#x306F; NO &#x5909;&#x66F4;&#x3053;&#x3068;&#x304C;&#x4EEE;&#x5B9A;&#x3055;&#x308C;&#x308B;&#x30D0;&#x30A4;&#x30AA;&#x30A2;&#x30D9;&#x30A4;&#x30E9;&#x30D3;&#x30EA;&#x30C6;&#x30A3;&#x53EF;&#x80FD;&#x6027;&#x5185;&#x56E0;&#x6027;&#x306E;&#x5897;&#x52A0;&#x304B;&#x3089;&#x306A;&#x3044;&#x5408;&#x6210;&#x9175;&#x7D20; (NOS) &#x963B;&#x5BB3;&#x5264;&#x3001;&#x975E;&#x5BFE;&#x79F0; dimethly araginine &#xFF08;ADMA&#xFF09;&#x3001;&#x304A;&#x3088;&#x3073; N &#xFF08;G&#xFF09;-&#x30E2;&#x30CE;&#x30E1;&#x30C1;&#x30EB; &#x30A2;&#x30EB;&#x30AE;&#x30CB;&#x30F3;&#x306F;&#x3001;&#x901A;&#x5E38; dimethyarginine &#x30B8;&#x30E1;&#x30C1;&#x30EB;&#x30A2;&#x30DF;&#x30F3;&#x52A0;&#x6C34;&#x5206;&#x89E3;&#x9175;&#x7D20; (DDAH) &#x306B;&#x3088;&#x3063;&#x3066;&#x4EE3;&#x8B1D;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x8102;&#x8CEA;&#x904E;&#x9178;&#x5316;&#x7269;&#x304A;&#x3088;&#x3073;&#x305D;&#x306E;&#x5206;&#x89E3;&#x7523;&#x7269;&#x708E;&#x75C7;&#x3084;&#x9178;&#x5316;&#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x6642;&#x306B;&#x751F;&#x6210;&#x3055;&#x308C;&#x3001;&#x5FC3;&#x8840;&#x7BA1;&#x75BE;&#x60A3;&#x306E;&#x75C5;&#x56E0;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x8102;&#x8CEA;&#x30D2;&#x30C9;&#x30ED;&#x30DA;&#x30EB;&#x30AA;&#x30AD;&#x30B7;&#x30C9;&#x52A3;&#x5316;&#x88FD;&#x54C1; 4-&#x30D2;&#x30C9;&#x30ED;&#x30AD;&#x30B7;-2-&#x30CE;&#x30CD;&#x30CA;&#x30FC;&#x30EB; (4-HNE&#xFF09; DDAH &#x9175;&#x7D20;&#x6D3B;&#x6027;&#x306E;&#x4F4E;&#x4E0B;&#x3092;&#x4F34;&#x3063;&#x3066;&#x3001;&#x30A6;&#x30B7;&#x5927;&#x52D5;&#x8108;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x751F;&#x6210;&#x306A;&#x3057;&#x3067;&#x7528;&#x91CF;&#x4F9D;&#x5B58;&#x6027;&#x306E;&#x6E1B;&#x5C11;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6291;&#x5236;&#x52B9;&#x679C;&#x5185;&#x76AE; NO &#x306E; 4-HNE (50 microM&#xFF09; &#x306E;&#x751F;&#x7523;&#x306F;&#x3001;&#x90E8;&#x5206;&#x7684;&#x306B;&#x9006; L Arg &#x88DC;&#x5145; (1 mM)&#x3002;&#x6297;&#x9178;&#x5316;&#x30B5;&#x30D7;&#x30EA;&#x30E1;&#x30F3;&#x30C8;&#x6295;&#x4E0E;&#x3068;&#x5171;&#x306B;&#x4EBA;&#x9593; DDAH 1 &#x306E;&#x904E;&#x5270;&#x767A;&#x73FE;&#x306F;&#x8840;&#x7BA1;&#x5185;&#x76AE; NO &#x3092;&#x5B8C;&#x5168;&#x306B;&#x5FA9;&#x5143;&#x3055;&#x308C;&#x305F; 4-HNE (50 microM) &#x66B4;&#x9732;&#x5F8C;&#x306E;&#x751F;&#x7523;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x5185;&#x56E0;&#x6027;&#x306E; methylarginines &#x5185;&#x76AE;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x306E;&#x75C5;&#x56E0;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x8102;&#x8CEA;&#x904E;&#x9178;&#x5316;&#x7269;&#x304A;&#x3088;&#x3073;&#x305D;&#x306E;&#x5206;&#x89E3;&#x7523;&#x7269;&#x30A2;&#x30C6;&#x30ED;&#x30FC;&#x30E0;&#x6027;&#x52D5;&#x8108;&#x786C;&#x5316;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x306E;&#x3067;&#x3001;DDAH &#x3068; methylarginines &#x306E;&#x5909;&#x8ABF;&#x9178;&#x5316;&#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x5FC3;&#x8840;&#x7BA1;&#x75BE;&#x60A3;&#x306E;&#x6CBB;&#x7642;&#x306B;&#x304A;&#x3051;&#x308B;&#x65B0;&#x898F;&#x306E;&#x6CBB;&#x7642;&#x4E0A;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3068;&#x3057;&#x3066;&#x5F79;&#x7ACB;&#x3064;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;

DDAH 1 &#x306E;&#x30ED;&#x30FC;&#x30EB;&#x88FD;&#x54C1;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x8102;&#x8CEA;&#x904E;&#x9178;&#x5316;&#x6291;&#x5236;&#x5185;&#x76AE; no &#x751F;&#x6210;&#x3002;

&#xBCC0;&#xACBD; &#xB41C; &#xC9C8;&#xC18C; &#xC0B0;&#xD654;&#xBB3C; (NO) &#xC0DD; &#xD569;&#xC131; &#xAC1C;&#xC2DC; &#xBC0F; &#xB3D9;&#xB9E5; &#xACBD;&#xD654; &#xC99D;&#xC758; &#xC9C4;&#xD589;&#xC5D0; &#xC5ED;&#xD560;&#xC744; &#xC0DD;&#xAC01; &#xD558; &#xACE0; &#xB2E4;&#xB978; &#xC2EC;&#xD608; &#xAD00;&#xACC4; &#xC9C8;&#xD658;&#xC5D0; &#xC704;&#xD5D8; &#xC99D;&#xAC00;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83; &#xC544;&#xB2C8;&#xC624; &#xBCC0;&#xACBD; &#xD558;&#xB294; &#xAC00;&#xC124;&#xC744; &#xC138;&#xC6E0;&#xB2E4;&#xB294; bioavailability &#xC99D;&#xAC00; &#xB0B4; &#xC0DD;&#xC5D0; &#xBC1C;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC544;&#xBB34; synthase (NOS) &#xC5B5;&#xC81C;&#xC81C;, &#xBE44;&#xB300;&#xCE6D; dimethly araginine (ADMA)&#xC640; N (G)-&#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; dimethyarginine dimethylamine &#xAC00;&#xC218;&#xBD84;&#xD574; &#xD6A8;&#xC18C; (DDAH)&#xC5D0; &#xC758;&#xD574; &#xB300;&#xC0AC; monomethyl &#xC544;&#xB974;&#xAE30;&#xB2CC;. &#xC9C0;&#xC9C8; hydroperoxides &#xBC0F; &#xC800;&#xD558; &#xC81C;&#xD488; &#xC5FC;&#xC99D;&#xACFC; &#xC0B0;&#xD654; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4; &#xC911;&#xC5D0; &#xC0DD;&#xC131; &#xB418; &#xACE0; &#xC2EC;&#xD608; &#xAD00; &#xC9C8;&#xD658;&#xC758; &#xBCD1; &#xC778;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4;. &#xC5EC;&#xAE30;&#xC5D0;&#xC11C;, &#xC6B0;&#xB9AC;&#xB294; &#xC9C0;&#xC9C8; hydroperoxide &#xC800;&#xD558; &#xC81C;&#xD488; 4-&#xD788; &#xB4DC; &#xB85D; &#xC2DC;-2-nonenal (4-HNE) DDAH &#xD6A8;&#xC18C; &#xD65C;&#xB3D9;&#xC758; &#xAC10;&#xC18C;&#xC640; &#xD568;&#xAED8; &#xC18C; &#xB300;&#xB3D9;&#xB9E5; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xD558;&#xC9C0; &#xBCF5;&#xC6A9;&#xB7C9; &#xC758;&#xC874; &#xAC10;&#xC18C; &#xD558;&#xBA74; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC5B5;&#xC81C; &#xD6A8;&#xACFC; (50 microM) &#xB0B4; &#xD53C; &#xC5C6;&#xC74C;&#xC5D0; 4 HNE&#xC758; &#xC0DD;&#xC0B0; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; L-Arg &#xBCF4;&#xCDA9; (1 mM)&#xC73C;&#xB85C; &#xBC18;&#xC804; &#xD588;&#xB2E4;. &#xD56D; &#xC0B0;&#xD654; &#xBCF4;&#xCDA9; &#xD568;&#xAED8; &#xC778;&#xAC04;&#xC758; DDAH-1 overexpression&#xB294; &#xC644;&#xC804;&#xD788; &#xB0B4; &#xD53C; &#xC5C6;&#xC74C; &#xBCF5;&#xC6D0; &#xB178;&#xCD9C; 4 HNE (50 microM)&#xC5D0; &#xB2E4;&#xC74C;&#xACFC; &#xC0DD;&#xC0B0;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xB0B4; &#xD53C; &#xAE30;&#xB2A5; &#xC7A5;&#xC560;&#xC758; &#xBCD1; &#xC778;&#xC5D0; &#xB0B4; methylarginines&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC9C0;&#xC9C8; hydroperoxides &#xBC0F; &#xC800;&#xD558; &#xC81C;&#xD488; &#xB3D9;&#xB9E5; &#xACBD;&#xD654; &#xC99D;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xAC83;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;, &#xC65C;&#xB0D0;&#xD558;&#xBA74; &#xC0B0;&#xD654; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4;&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC2EC;&#xD608; &#xAD00; &#xC9C8;&#xD658;&#xC758; &#xCE58;&#xB8CC;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xCE58;&#xB8CC; &#xB300;&#xC0C1;&#xC73C;&#xB85C; DDAH&#xC640; methylarginines&#xC758; &#xBCC0;&#xC870; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB0B4; &#xD53C; &#xC5C6;&#xC74C;&#xC758; &#xC9C0;&#xC9C8; &#xACFC;&#xC0B0;&#xD654; &#xC81C;&#xD488; &#xC911;&#xC7AC; &#xC800;&#xD574;&#xC5D0; DDAH-1&#xC758; &#xC5ED;&#xD560; &#xC138;&#xB300;.

Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)-containing enzyme that through hydrolysis of methylated l-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated Km values of 68.7 and 53.6 microM and Vmax values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >or=100 microM were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.

Mechanism of 4-HNE mediated inhibition of hDDAH-1: implications in no regulation.

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and N (G)-monomethyl- l-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH 4) depletion eNOS also generates (*)O 2 (-); however, the effects of methylarginines on eNOS-derived (*)O 2 (-) generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on (*)O 2 (-) production from eNOS under conditions of BH 4 depletion. In the absence of BH 4, ADMA dose-dependently increased NOS-derived (*)O 2 (-) generation, with a maximal increase of 151% at 100 microM ADMA. L-NMMA also dose-dependently increased NOS-derived (*)O 2 (-), but to a lesser extent, demonstrating a 102% increase at 100 microM L-NMMA. Moreover, the native substrate l-arginine also increased eNOS-derived (*)O 2 (-), exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either l-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for l-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and (*)O 2 (-) generation from eNOS. These observations have important implications with regard to the therapeutic use of l-arginine and the methylarginine-NOS inhibitors in the treatment of disease.

Regulation of eNOS-derived superoxide by endogenous methylarginines.

Cells modified with magnetically responsive nanoparticles (MNP) can provide the basis for novel targeted therapeutic strategies. However, improvements are required in the MNP design and cell treatment protocols to provide adequate magnetic properties in balance with acceptable cell viability and function. This study focused on select variables controlling the uptake and cell compatibility of biodegradable polymer-based MNP in cultured endothelial cells.

Formulation and in vitro characterization of composite biodegradable magnetic nanoparticles for magnetically guided cell delivery.

Magnetic guidance is a physical targeting strategy with the potential to improve the safety and efficacy of a variety of therapeutic agents--including small-molecule pharmaceuticals, proteins, gene vectors, and cells--by enabling their site-specific delivery. The application of magnetic targeting for in-stent restenosis can address the need for safer and more efficient treatment strategies. However, its translation to humans may not be possible without revising the traditional magnetic targeting scheme, which is limited by its inability to selectively guide therapeutic agents to deep localized targets. An alternative two-source strategy can be realized through the use of uniform, deep-penetrating magnetic fields in conjunction with vascular stents included as part of the magnetic setup and the platform for targeted delivery to injured arteries. Studies showing the feasibility of this novel targeting strategy in in-stent restenosis models and considerations in the design of carrier formulations for magnetically guided antirestenotic therapy are discussed in this review.

Magnetically targeted delivery of therapeutic agents to injured blood vessels for prevention of in-stent restenosis.

The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolyzable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37 °C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolyzable cross-linkers with structure-specific release kinetics.

Adenoviral vector tethering to metal surfaces via hydrolyzable cross-linkers for the modulation of vector release and transduction.

Gene therapy with viral vectors encoding for NOS enzymes has been recognized as a potential therapeutic approach for the prevention of restenosis. Optimal activity of iNOS is dependent on the intracellular availability of L-Arg and BH4 via prevention of NOS decoupling and subsequent ROS formation. Herein, we investigated the effects of separate and combined L-Arg and BH4 supplementation on the production of NO and ROS in cultured rat arterial smooth muscle and endothelial cells transduced with AdiNOS, and their impact on the antirestenotic effectiveness of AdiNOS delivery to balloon-injured rat carotid arteries.

Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: implications for gene therapy of restenosis.

Scott P. Forbes

Department of Pediatrics,
Division of Cardiology,
Department of Pediatrics, Division of Cardiology

The Children's Hospital of Philadelphia, University of Pennsylvania

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

Scott P. Forbes

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

The Children's Hospital of Philadelphia, University of Pennsylvania

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

Department of Pediatrics,
Division of Cardiology,
Department of Pediatrics, Division of Cardiology