A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Johns Hopkins University

A molecular basket with four cholate units assembled on a cone-shaped calix[4]arene assumed reversed micelle-like conformation in 5% methanol/carbon tetrachloride. The inwardly facing hydroxyl groups on the cholates concentrated the polar solvent from the mostly nonpolar mixture. Methanolysis of alkyl halides benefited from the concentrated pocket of methanol if the substrate was capable of entering the basket. Substrates that were too large or too hydrophobic to fit within the basket showed no rate acceleration.

Catalyzing methanolysis of alkyl halides in the interior of an amphiphilic molecular basket.

Oligocholate foldamers with different numbers and locations of guanidinium-carboxylate salt bridges were synthesized. The salt bridges were introduced by incorporating arginine and glutamic acid residues into the foldamer sequence. The conformations of these foldamers were studied by fluorescence spectroscopy in homogeneous solution, anionic and nonionic micelles, and lipid bilayers. Environmental effects instead of inherent foldability were found to dominate the folding. As different noncovalent forces become involved in the conformations of the molecules, the best folder in one environment could turn into the worst in another. Preferential solvation was the main driving force for the folding of oligocholates in solution. The molecules behaved very differently in micelles and lipid bilayers, with the most critical factors controlling the folding-unfolding equilibrium being the solvation of ionic groups and the abilities of the surfactants/lipids to compete for the salt bridge. Because of their ability to fold into helices with a nonpolar exterior and a polar interior, the oligocholates could transport large hydrophilic molecules such as carboxyfluorescein across lipid bilayers. Both the conformational properties of the oligocholates and their binding with the guest were important to the transport efficiency.

Environmental effects dominate the folding of oligocholates in solution, surfactant micelles, and lipid membranes.

Hydrophobic interactions normally are not considered a major driving force for self-assembling in a hydrophobic environment. When macrocyclic oligocholates were placed within lipid membranes, however, the macrocycles pulled water molecules from the aqueous phase into their hydrophilic internal cavities. These water molecules had strong tendencies to aggregate in a hydrophobic environment and templated the macrocycles to self-assemble into transmembrane nanopores. This counterintuitive hydrophobic effect resulted in some highly unusual transport behavior. Cholesterol normally increases the hydrophobicity of lipid membranes and makes them less permeable to hydrophilic molecules. The permeability of glucose across the oligocholate-containing membranes, however, increased significantly upon the inclusion of cholesterol. Large hydrophilic molecules tend to have difficulty traversing a hydrophobic barrier. The cyclic cholate tetramer, however, was more effective at permeating maltotriose than glucose.

Water-templated transmembrane nanopores from shape-persistent oligocholate macrocycles.

Macrocyclic oligocholates were found in a previous work (Cho, H.; Widanapathirana, L.; Zhao, Y. J. Am. Chem. Soc.2011, 133, 141-147) to stack on top of one another in lipid membranes to form nanopores. Pore formation was driven by a strong tendency of the water molecules in the interior of the amphiphilic macrocycles to aggregate in a nonpolar environment. In this work, cholate oligomers terminated with guanidinium and carboxylate groups were found to cause efflux of hydrophilic molecules such as glucose, maltotriose, and carboxyfluorescein (CF) from POPC/POPG liposomes. The cholate trimer outperformed other oligomers in the transport. Lipid-mixing assays and dynamic light scattering ruled out fusion as the cause of leakage. The strong dependence on chain length argues against random intermolecular aggregates as the active transporters. The efflux of glucose triggered by these compounds increased significantly when the bilayers contained 30 mol% cholesterol. Hill analysis suggested that the active transporter consisted of four molecules. The oligocholates were proposed to fold into "noncovalent macrocycles" by the guanidinium-carboxylate salt bridge and stack on top of one another to form similar transmembrane pores as their covalent counterparts.

Translocation of hydrophilic molecules across lipid bilayers by salt-bridged oligocholates.

With introverted polar groups and hydrophobic exteriors, cholate-derived amphiphilic molecular baskets were efficient transporters of glucose across lipid membranes.

Cholate-derived amphiphilic molecular baskets as glucose transporters across lipid membranes.

RAD16 &#x4E8C;&#x80BD;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x7EC4;&#x7EC7;&#x5DE5;&#x7A0B;&#x5316;&#x8840;&#x7BA1;&#x6709;&#x5E0C;&#x671B;&#xFF0C;&#x5E76;&#x8868;&#x793A;&#x589E;&#x5F3A;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x4F53;&#x5916;&#x548C;&#x4F53;&#x5185;&#xFF0C;&#x867D;&#x7136;&#x8BE5;&#x673A;&#x5236;&#x4ECD;&#x662F;&#x672A;&#x77E5;&#x3002;&#x6211;&#x4EEC;&#x5047;&#x8BBE; RAD16 II pro &#x8840;&#x7BA1;&#x9020;&#x6210;&#x4E0E; RAD &#x56FE;&#x6848;&#x7684;&#x5FAE;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE; (MVECs) &#x4F4E;&#x4EB2;&#x548C;&#x529B;&#x4F9D;&#x8D56;&#x6574;&#x5408;&#x7D20;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;&#x9F20;&#x6807; MVECs RAD16 &#x7B2C;&#x4E8C;&#x57F9;&#x517B;&#x5177;&#x6709;&#x6216;&#x6CA1;&#x6709;&#x6574;&#x5408;&#x7D20;&#x548C; MAPK/ERK &#x901A;&#x8DEF;&#x6291;&#x5236;&#x5242;&#xFF0C;&#x4E0E;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x54CD;&#x5E94;&#x88AB;&#x91CF;&#x5316;&#x3002;&#x7ED3;&#x679C;&#x8FDB;&#x884C;&#x4E86;&#x9A8C;&#x8BC1;&#x4F7F;&#x7528;&#x9F20;&#x6807;&#x7CD6;&#x5C3F;&#x75C5;&#x521B;&#x9762;&#x6108;&#x5408;&#x4E0E;&#x65B0;&#x751F;&#x8840;&#x7BA1;&#x53D7;&#x635F;&#x6A21;&#x578B;&#x7684;&#x4F53;&#x5185;&#x3002;RAD16 II &#x523A;&#x6FC0;&#x81EA;&#x53D1;&#x7684;&#x6BDB;&#x7EC6;&#x7BA1;&#x5F62;&#x6001;&#xFF0C;&#x5E76;&#x589E;&#x52A0; &beta;(3) &#x6574;&#x5408;&#x7D20;&#x78F7;&#x9178;&#x5316;&#x548C; VEGF &#x8868;&#x8FBE;&#x7684; MVECs&#x3002;&#x8FD9;&#x4E9B;&#x53CD;&#x5E94;&#x88AB;&#x5E9F;&#x9664; &beta;(3) &#x548C; MAPK/ERK &#x901A;&#x8DEF;&#x6291;&#x5236;&#x5242;&#x6216;&#x63A7;&#x5236;&#x80BD;&#x65E0; RAD &#x56FE;&#x6848;&#x4E0A;&#x3002;&#x5168;&#x8C31;&#x6574;&#x5408;&#x7D20;&#x6291;&#x5236;&#x5242; echistatin &#x5B8C;&#x5168;&#x5E9F;&#x9664;&#x4ECB;&#x5BFC; RAD16 &#x4E8C;&#x3001; &#x6BDB;&#x7EC6;&#x7BA1;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x4F53;&#x5916;&#x548C;&#x65B0;&#x751F;&#x8840;&#x7BA1;&#x53CA;&#x4F53;&#x5185;&#x521B;&#x9762; VEGF &#x7684;&#x8868;&#x8FBE;&#x3002;RAD16 &#x7B2C;&#x4E8C; RGD &#x6BCD;&#x9898;&#x7684;&#x52A0;&#x6CD5;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x7ED3;&#x6784;&#x6216;&#x529B;&#x5B66;&#x6027;&#x80FD;&#x4E0D;&#x4F1A;&#x6539;&#x53D8;&#xFF0C;&#x4F46;&#x5BFC;&#x81F4;&#x6BDB;&#x7EC6;&#x8840;&#x7BA1;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x663E;&#x8457;&#x51CF;&#x5C11;&#x3002;&#x603B;&#x4E4B;&#xFF0C;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x4F4E;&#x4EB2;&#x548C;&#x529B;&#x975E;&#x7279;&#x5B9A;&#x5355;&#x5143;&#x683C;&#x548C; RAD &#x56FE;&#x6848;&#x4E4B;&#x95F4;&#x7684;&#x4EA4;&#x4E92;&#x53EF;&#x4EE5;&#x89E6;&#x53D1;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x54CD;&#x5E94;&#x901A;&#x8FC7;&#x78F7;&#x9178;&#x5316; &beta;(3) &#x6574;&#x5408;&#x7D20;&#x4E0E; MAPK/ERK &#x901A;&#x8DEF;&#xFF0C;&#x8868;&#x660E;&#x4F4E;&#x4EB2;&#x548C;&#x529B;&#x5E8F;&#x5217;&#x53EF;&#x4EE5;&#x7528;&#x4E8E; functionalize &#x4E3A;&#x8C03;&#x8282;&#x7EC6;&#x80DE;&#x8FC1;&#x79FB;&#x548C;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#xFF0C;&#x4ECE;&#x800C;&#x6269;&#x5927;&#x5F53;&#x524D;&#x6C60;&#x4E2D;&#x53EF;&#x7528;&#x7684;&#x56FE;&#x6848;&#xFF0C;&#x53EF;&#x4EE5;&#x7528;&#x4E8E;&#x8FD9;&#x7C7B;&#x529F;&#x80FD;&#x5316;&#x7684;&#x751F;&#x7269;&#x76F8;&#x5BB9;&#x6027;&#x6750;&#x6599;&#x3002;RAD &#x6216;&#x7C7B;&#x4F3C;&#x56FE;&#x6848;&#x7EB3;&#x5165;&#x86CB;&#x767D;&#x8D28;&#x5DE5;&#x7A0B;&#x6216;&#x6DF7;&#x5408;&#x80BD;&#x652F;&#x67B6;&#x53EF;&#x80FD;&#x4EE3;&#x8868;&#x8840;&#x7BA1;&#x7EC4;&#x7EC7;&#x5DE5;&#x7A0B;&#x7684;&#x65B0;&#x6218;&#x7565;&#xFF0C;&#x5C06;&#x8FDB;&#x4E00;&#x6B65;&#x52A0;&#x5F3A;&#x8BBE;&#x8BA1;&#x65B0;&#x7684;&#x652F;&#x67B6;&#x6750;&#x6599;&#x7684;&#x673A;&#x4F1A;&#x3002;

&#x6FC0;&#x6D3B;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x548C;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x7684;&#x53EF;&#x6CE8;&#x5C04;&#x80BD;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x7684;&#x89C4;&#x7BA1;&#x3002;

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. The results were validated in vivo using a mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, and increased Î²(3) integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of Î²(3) and MAPK/ERK pathway inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. The addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of Î²(3) integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize biocompatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffold materials.

Regulation of endothelial cell activation and angiogenesis by injectable peptide nanofibers.

RAD16-II peptide nanofibres sont prometteurs pour l&#39;ing&eacute;nierie des tissus vasculaires et ont &eacute;t&eacute; montr&eacute;s pour am&eacute;liorer l&#39;angiogen&egrave;se in vitro et in vivo, bien que le m&eacute;canisme demeure inconnu. Nous avons &eacute;mis l&#39;hypoth&egrave;se que l&#39;effet pro-angiog&eacute;nique de RAD16-II d&eacute;coule de la faible affinit&eacute; des interactions int&eacute;grine-d&eacute;pendante des cellules endoth&eacute;liales microvasculaires (MVECs) avec des motifs de RAD. MVECs de souris ont &eacute;t&eacute; cultiv&eacute;s sur RAD16-II, avec ou sans int&eacute;grine et inhibiteurs de voie MAPK/ERK et angiog&eacute;niques r&eacute;ponses ont &eacute;t&eacute; quantifi&eacute;es. Les r&eacute;sultats ont &eacute;t&eacute; valid&eacute;s in vivo &agrave; l&#39;aide d&#39;un souris diab&eacute;tiques cicatrisation mod&egrave;le avec n&eacute;ovascularisation avec facult&eacute;s affaiblies. RAD16-II stimule morphogen&egrave;se capillaire spontan&eacute;e et augment&eacute; la phosphorylation de l&#39;int&eacute;grine &beta;(3) et expression du VEGF dans MVECs. Ces r&eacute;ponses ont &eacute;t&eacute; abrog&eacute;es en pr&eacute;sence de &beta;(3) et inhibiteurs de voie MAPK/ERK ou sur le peptide de contr&ocirc;le sans motifs RAD. Large spectre int&eacute;grine inhibiteur echistatin abolit compl&egrave;tement la morphogen&egrave;se capillaire induite par le RAD16-II in vitro et n&eacute;ovascularisation et expression du VEGF dans la plaie in vivo. L&#39;ajout du motif RGD &agrave; RAD16-II n&#39;a pas chang&eacute; de nanofibres architecture ou propri&eacute;t&eacute;s m&eacute;caniques, mais a entra&icirc;n&eacute; une diminution significative dans la morphogen&egrave;se capillaire. Globalement, ces r&eacute;sultats sugg&egrave;rent que la faible affinit&eacute; des interactions non sp&eacute;cifiques entre les cellules et les motifs RAD peuvent d&eacute;clencher angiog&eacute;nique r&eacute;ponses par l&#39;interm&eacute;diaire de la phosphorylation de l&#39;int&eacute;grine &beta;(3) et voie de MAPK/ERK, indiquant que les s&eacute;quences de faible affinit&eacute; peuvent &ecirc;tre utilis&eacute;es pour fonctionnaliser mat&eacute;riaux biocompatibles pour la r&eacute;glementation de la migration cellulaire et l&#39;angiogen&egrave;se, &eacute;largissant ainsi la r&eacute;serve actuelle de motifs disponibles qui peut &ecirc;tre utilis&eacute; pour ce fonctionnalisation. Incorporation des motifs RAD ou similaires aux prot&eacute;ines d&#39;ing&eacute;nierie ou hybrides peptide &eacute;chafaudages peuvent repr&eacute;senter une strat&eacute;gie novatrice pour l&#39;ing&eacute;nierie des tissus vasculaires et renforcera encore les possibilit&eacute;s de conception de nouveaux mat&eacute;riaux d&#39;&eacute;chafaudage.

R&eacute;gulation de l&#39;activation des cellules endoth&eacute;liales et angiogen&egrave;se en nanofibres de peptide injectable.

RAD16-II-Peptid-Nanofasern sind vielversprechend f&uuml;r vaskul&auml;re Tissue Engineering und wurden gezeigt, zur Verbesserung von Angiogenesis in vitro und in vivo, obwohl der Mechanismus unbekannt bleibt. Wir die Hypothese, dass der Pro-angiogenen Effekt des RAD16-II aus niedrig-Affinit&auml;t Integrin-abh&auml;ngige Interaktionen mikrovaskul&auml;re Endothelzellen (MVECs) mit RAD Motive ergibt. Maus-MVECs wurden auf RAD16-II kultiviert, mit oder ohne Integrin und MAPK/ERK-Weg-Inhibitoren und angiogenen Antworten wurden quantifiziert. Die Ergebnisse wurden in-vivo unter Verwendung einer Maus Diabetische Wundheilung Modell mit gest&ouml;rter Neovaskularisation validiert. RAD16-II stimuliert spontane Kapillar Morphogenese und erh&ouml;hte &beta;(3)-Integrin-Phosphorylierung und VEGF-Expression in MVECs. Diese Antworten wurden in Anwesenheit von &beta;(3) und MAPK/ERK Stoffwechselweg Inhibitoren oder auf das Steuerelement-Peptid ohne RAD Motive aufgehoben. Weite-Spektrum-Integrin-Inhibitor Echistatin abgeschafft vollst&auml;ndig RAD16-II-vermittelter Kapillar Morphogenese in-vitro- und Neovaskularisation und VEGF-Expression in der Wunde in-vivo. Die Hinzuf&uuml;gung des RGD-Motivs zu RAD16-II Nanofaser-Architektur oder mechanischen Eigenschaften nicht ge&auml;ndert, sondern f&uuml;hrte zu deutlichen R&uuml;ckgang der Kapillare Morphogenese. Diesen Ergebnissen zufolge insgesamt geringer Affinit&auml;t unspezifische Interaktionen zwischen Zellen und RAD Motive ausl&ouml;sen k&ouml;nnen angiogenen Antworten &uuml;ber Phosphorylierung von &beta;(3) Integrin und MAPK/ERK Pathway, darauf hinweist, dass niedrige Affinit&auml;t Sequenzen verwendet werden k&ouml;nnen, zu Stra&szlig;enschlachten biokompatible Materialien f&uuml;r die Regulierung der Zellmigration und Angiogenese, also erweitern den aktuellen Pool der verf&uuml;gbaren Motive, die f&uuml;r solche Funktionalisierung verwendet werden kann. Einbeziehung der RAD oder &auml;hnliche Motive in Protein Engineering oder Hybrid-Peptid-Ger&uuml;ste k&ouml;nnen vertreten, eine neuartige Strategie f&uuml;r vaskul&auml;re Tissue-Engineering und Design-M&ouml;glichkeiten f&uuml;r neue Ger&uuml;st Materialien werden weiter auszubauen.

Verordnung der endothelial Zelle Aktivierung und Angiogenese durch injizierbare Peptid-Nanofasern.

RAD16-II del peptide nanofibre sono promettenti per l&#39;ingegneria dei tessuti vascolari e hanno dimostrate di migliorare l&#39;angiogenesi in vitro e in vivo, anche se il meccanismo resta sconosciuto. Abbiamo ipotizzato che l&#39;effetto pro-angiogenic di RAD16-II risultati da bassa affinit&agrave; interazioni integrina-dipendente delle cellule endoteliali Microvascolari (MVECs) con motivi RAD. MVECs del mouse sono stati coltivati su RAD16-II, con o senza integrina e inibitori della via MAPK/ERK e angiogenic risposte sono state quantificate. I risultati sono stati convalidati in vivo, utilizzando una ferita diabetica mouse guarigione modello con neovascolarizzazione compromessa. RAD16-II stimolato spontaneo morfogenesi capillare e aumentata fosforilazione dell&#39;integrina &beta;(3) e l&#39;espressione di VEGF in MVECs. Queste risposte sono state abrogate in presenza di &beta;(3) e di MAPK/ERK pathway inibitori o sul peptide di controllo senza motivi RAD. Ampio spettro integrina inibitore echistatin completamente abolita RAD16-II-mediata capillare morfogenesi in vitro e neovascolarizzazione ed espressione di VEGF nella ferita in vivo. L&#39;aggiunta del motivo RGD RAD16-II non cambia architettura nanofibre o meccaniche, ma ha provocato una diminuzione significativa nella morfogenesi capillare. Nel complesso, questi risultati suggeriscono che la bassa affinit&agrave; non specifiche interazioni tra cellule e motivi RAD possono innescare angiogenic risposte tramite la fosforilazione dell&#39;integrina &beta;(3) e via MAPK/Erk, indicando che la bassa affinit&agrave; sequenze possono essere utilizzati per funzionalizzare materiali biocompatibili per la regolazione della migrazione cellulare e l&#39;angiogenesi, ampliando cos&igrave; l&#39;attuale piscina di motivi disponibili che possono essere utilizzati per tale funzionalizzazione. Incorporazione di RAD o simili motivi proteine progettate o ibrido del peptide ponteggi possono rappresentare una strategia romanzo per l&#39;ingegneria dei tessuti vascolari e rafforzeranno ulteriormente l&#39;opportunit&agrave; di progettazione per nuovi materiali del ponteggio.

Regolamento di attivazione delle cellule endoteliali e l&#39;angiogenesi di nanofibre iniettabili del peptide.

RAD16 II &#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x306E;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x8840;&#x7BA1;&#x7D44;&#x7E54;&#x30A8;&#x30F3;&#x30B8;&#x30CB;&#x30A2; &#x30EA;&#x30F3;&#x30B0;&#x306E;&#x6709;&#x671B;&#x3067;&#x3001;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x304C; in vitro &#x304A;&#x3088;&#x3073; in vivo &#x8840;&#x7BA1;&#x65B0;&#x751F;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;RAD16 II &#x306E; pro &#x8840;&#x7BA1;&#x65B0;&#x751F;&#x52B9;&#x679C; RAD &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x3068;&#x306E;&#x5FAE;&#x5C0F;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7D30;&#x80DE; (MVECs) &#x306E;&#x4F4E;&#x89AA;&#x548C;&#x6027;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x304B;&#x3089;&#x306E;&#x7D50;&#x679C;&#x3053;&#x3068;&#x3092;&#x4EEE;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x3068; MAPK/ERK &#x7D4C;&#x8DEF;&#x963B;&#x5BB3;&#x5264;&#x6709;&#x7121;&#x30DE;&#x30A6;&#x30B9; MVECs &#x4E0A;&#x306E; RAD16 II &#x57F9;&#x990A;&#x3092;&#x884C;&#x3044;&#x65B0;&#x751F;&#x53CD;&#x5FDC;&#x3092;&#x5B9A;&#x91CF;&#x5316;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#x306F; in vivo &#x30DE;&#x30A6;&#x30B9;&#x7CD6;&#x5C3F;&#x75C5;&#x50B7;&#x6CBB;&#x7652;&#x969C;&#x5BB3;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x3068;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x691C;&#x8A3C;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;RAD16 II &#x306F;&#x3001;&#x81EA;&#x767A;&#x306E;&#x6BDB;&#x7D30;&#x8840;&#x7BA1;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x523A;&#x6FC0;&#x3057;&#x3001;&beta;(3) &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3068; vegf MVECs &#x306E;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x5FDC;&#x7B54;&#x306F;&#x3001;&beta;(3) &#x3068; MAPK/ERK &#x7D4C;&#x8DEF;&#x963B;&#x5BB3;&#x5264;&#x5B58;&#x5728;&#x4E0B;&#x3067;&#x307E;&#x305F;&#x306F;&#x5236;&#x5FA1;&#x30DA;&#x30D7;&#x30C1;&#x30C9; RAD &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306A;&#x3057;&#x306B;&#x5EC3;&#x6B62;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x5E83;&#x57DF;&#x30B9;&#x30DA;&#x30AF;&#x30C8;&#x30EB; &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x963B;&#x5BB3;&#x5264; echistatin &#x306F;&#x3001;&#x5B8C;&#x5168;&#x306B; RAD16 II &#x3092;&#x4ECB;&#x3059;&#x308B;&#x6BDB;&#x7D30;&#x8840;&#x7BA1;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x4F53;&#x5916;&#x3068;&#x65B0;&#x751F;&#x8840;&#x7BA1;&#x3068; vegf in vivo &#x3067;&#x5275;&#x50B7;&#x306E;&#x5EC3;&#x6B62;&#x3002;RGD &#x30E2;&#x30C1;&#x30FC;&#x30D5; RAD16 II &#x3078;&#x306E;&#x6DFB;&#x52A0;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC; &#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x3084;&#x6A5F;&#x68B0;&#x7684;&#x6027;&#x8CEA;&#x5909;&#x5316;&#x3057;&#x306A;&#x304B;&#x3063;&#x305F;&#x304C;, &#x6BDB;&#x7D30;&#x8840;&#x7BA1;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x306E;&#x6709;&#x610F;&#x306A;&#x6E1B;&#x5C11;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x3002;&#x5168;&#x4F53;&#x7684;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x4F4E;&#x89AA;&#x548C;&#x6027;&#x975E;&#x7279;&#x7570;&#x7684;&#x7D30;&#x80DE; RAD &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x76F8;&#x4E92; &beta;(3) &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x304A;&#x3088;&#x3073;&#x7D30;&#x80DE;&#x904A;&#x8D70;&#x3068;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x305D;&#x306E;&#x3088;&#x3046;&#x306A;&#x6A5F;&#x80FD;&#x306E;&#x767A;&#x73FE;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306E;&#x73FE;&#x5728;&#x306E;&#x30D7;&#x30FC;&#x30EB;&#x3092;&#x62E1;&#x5927;&#x3057;&#x3066;&#x306E;&#x898F;&#x5236;&#x306E;&#x751F;&#x4F53;&#x9069;&#x5408;&#x6027;&#x6750;&#x6599;&#x6A5F;&#x80FD;&#x5316;&#x3059;&#x308B;&#x4F4E;&#x89AA;&#x548C;&#x6027;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x3001;MAPK/ERK &#x7D4C;&#x8DEF;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x65B0;&#x751F;&#x53CD;&#x5FDC;&#x3092;&#x8A98;&#x767A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3057;&#x307E;&#x3059;&#x3002;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306B; RAD &#x307E;&#x305F;&#x306F;&#x985E;&#x4F3C;&#x306E;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306E;&#x6DF7;&#x5165;&#x3092;&#x8A2D;&#x8A08;&#x307E;&#x305F;&#x306F;&#x30CF;&#x30A4;&#x30D6;&#x30EA;&#x30C3;&#x30C9; &#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x8DB3;&#x5834;&#x8840;&#x7BA1;&#x7D44;&#x7E54;&#x30A8;&#x30F3;&#x30B8;&#x30CB;&#x30A2; &#x30EA;&#x30F3;&#x30B0;&#x65B0;&#x6226;&#x7565;&#x3092;&#x8868;&#x3059;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3057;&#x3001;&#x30C7;&#x30B6;&#x30A4;&#x30F3;&#x306E;&#x6A5F;&#x4F1A;&#x65B0;&#x3057;&#x3044;&#x8DB3;&#x5834;&#x6750;&#x6599;&#x3092;&#x3055;&#x3089;&#x306B;&#x5F37;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;

&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x6CE8;&#x5C04;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x306E;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x306B;&#x3088;&#x308B;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x306E;&#x5236;&#x5FA1;&#x3002;

RAD16 II &#xD3A9; &#xD2F0; &#xB4DC; nanofibers &#xD608;&#xAD00; &#xC870;&#xC9C1; &#xACF5;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC57D;&#xC18D; &#xC774;&#xBA70; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xC54C; &#xC218; &#xC5C6;&#xB294; &#xB0A8;&#xC544; &#xC788;&#xC9C0;&#xB9CC; &#xC2E0;&#xC0DD; &#xCCB4; &#xC678; &#xADF8;&#xB9AC;&#xACE0; vivo&#xC5D0;&#xC11C; &#xD5A5;&#xC0C1;&#xC744; &#xD45C;&#xC2DC; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; RAD16 II &#xD504;&#xB85C; angiogenic &#xD6A8;&#xACFC; RAD &#xBAA8;&#xD2F0;&#xBE0C; microvascular &#xB0B4; &#xD53C; &#xC138;&#xD3EC; (MVECs)&#xC758; &#xB0AE;&#xC740; &#xC120;&#xD638;&#xB3C4; integrin &#xC885;&#xC18D; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0;&#xC11C; &#xACB0;&#xACFC; &#xAC00;&#xC124;&#xC744; &#xC138;&#xC6E0;&#xB2E4;. Integrin&#xACFC; MAPK/ERK &#xD1B5;&#xB85C; &#xC5B5;&#xC81C;&#xC81C;&#xC758; &#xC720;&#xBB34; &#xB9C8;&#xC6B0;&#xC2A4; MVECs RAD16 II&#xC5D0; &#xACBD;&#xC791; &#xD588;&#xB2E4; &#xADF8;&#xB9AC;&#xACE0; angiogenic &#xC751;&#xB2F5; &#xACC4;&#xB7C9; &#xD588;&#xB2E4;. &#xACB0;&#xACFC; in vivo &#xB9C8;&#xC6B0;&#xC2A4; &#xB2F9;&#xB1E8;&#xBCD1; &#xC0C1;&#xCC98; &#xCE58;&#xC720; &#xC7A5;&#xC560; neovascularization&#xC640; &#xBAA8;&#xB378;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD588;&#xB2E4;. RAD16 II &#xC790;&#xBC1C;&#xC801;&#xC778; &#xBAA8; &#xC138;&#xAD00; morphogenesis &#xC790;&#xADF9; &#xD558; &#xACE0; &#xC99D;&#xAC00; &#xB41C; &beta;(3) integrin &#xC778; &#xC0B0;&#xD654; &#xBC0F; Mvecs&#xC5D0; VEGF &#xC2DD;. &#xC774;&#xB7EC;&#xD55C; &#xC751;&#xB2F5;&#xC744; &beta;(3)&#xC640; MAPK/ERK &#xD1B5;&#xB85C; &#xC5B5;&#xC81C;&#xC81C;&#xC758; &#xC874;&#xC7AC; &#xB610;&#xB294; &#xBC29;&#xC0AC;&#xC120; &#xBAA8;&#xD2F0;&#xBE0C; &#xC5C6;&#xC774; &#xC81C;&#xC5B4; &#xD3A9; &#xD2F0; &#xB4DC;&#xC5D0; &#xD3D0;&#xC9C0; &#xD588;&#xB2E4;. &#xB113;&#xC740; &#xC2A4;&#xD399;&#xD2B8;&#xB7FC; integrin &#xC5B5;&#xC81C;&#xC81C; echistatin RAD16 2 &#xCC28; &#xC911;&#xC7AC; &#xBAA8; &#xC138;&#xAD00; morphogenesis &#xCCB4; &#xC678;&#xC640; neovascularization &#xADF8;&#xB9AC;&#xACE0; vivo&#xC5D0;&#xC11C; &#xC0C1;&#xCC98;&#xC5D0; VEGF &#xC2DD;&#xC5D0; &#xC644;&#xC804; &#xD558; &#xAC8C; &#xD3D0;&#xC9C0; &#xD569;&#xB2C8;&#xB2E4;. RAD16 II&#xC5D0; RGD &#xBAA8;&#xD2F0;&#xBE0C;&#xC758; &#xCD94;&#xAC00; nanofiber &#xC544;&#xD0A4;&#xD14D;&#xCC98; &#xB610;&#xB294; &#xAE30;&#xACC4;&#xC801; &#xD2B9;&#xC131;&#xC744; &#xBCC0;&#xACBD; &#xD558;&#xC9C0; &#xC54A;&#xC740; &#xC788;&#xC9C0;&#xB9CC; &#xBAA8; &#xC138;&#xAD00; morphogenesis&#xC5D0; &#xD070; &#xAC10;&#xC18C; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. &#xC804;&#xBC18;&#xC801;&#xC73C;&#xB85C;, &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xC140;&#xACFC; &#xBC29;&#xC0AC;&#xC120; &#xBAA8;&#xD2F0;&#xD504; &#xAC04;&#xC758; &#xB0AE;&#xC740; &#xC120;&#xD638;&#xB3C4; &#xC544;&#xB2CC; &#xD2B9;&#xC815; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &beta;(3) integrin &#xBC0F; functionalize &#xC140; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158; &#xBC0F; &#xC2E0;&#xC0DD;, &#xB530;&#xB77C;&#xC11C; &#xC774;&#xB7EC;&#xD55C; &#xAE30;&#xB2A5;&#xD654;&#xC5D0; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xC0AC;&#xC6A9; &#xAC00;&#xB2A5;&#xD55C; &#xBAA8;&#xD2F0;&#xD504;&#xC758; &#xD604;&#xC7AC; &#xD480; &#xD655;&#xB300; &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC0DD;&#xCCB4; &#xC7AC;&#xB8CC;&#xB97C; &#xB0AE;&#xC740; &#xC120;&#xD638;&#xB3C4; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC74C;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; MAPK/ERK &#xD1B5;&#xB85C;&#xC758; &#xC778; &#xC0B0;&#xD654;&#xB97C; &#xD1B5;&#xD574; angiogenic &#xC751;&#xB2F5;&#xC744; &#xC2E4;&#xD589;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8;&#xC73C;&#xB85C; &#xBC29;&#xC0AC;&#xC120; &#xB610;&#xB294; &#xC774;&#xC640; &#xC720;&#xC0AC;&#xD55C; &#xBAA8;&#xD2F0;&#xBE0C;&#xC758; &#xD1B5;&#xD569; &#xC124;&#xACC4; &#xB610;&#xB294; &#xD558;&#xC774;&#xBE0C;&#xB9AC;&#xB4DC; &#xD3A9; &#xD2F0; &#xB4DC; &#xAC74;&#xC124; &#xAE30;&#xACC4; &#xC784;&#xB300; &#xD608;&#xAD00; &#xC870;&#xC9C1; &#xACF5;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xC804;&#xB7B5;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xCD94;&#xAC00; &#xBE44; &#xACC4; &#xC2E0;&#xC18C;&#xC7AC;&#xC5D0; &#xB300; &#xD55C; &#xB514;&#xC790;&#xC778;&#xC758; &#xAE30;&#xD68C;&#xB97C; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0AC; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;.

&#xB0B4; &#xD53C; &#xC138;&#xD3EC; &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xC2E0;&#xC0DD; &#xC8FC; &#xC0AC;&#xC6A9; &#xD3A9; &#xD2F0; &#xB4DC; nanofibers&#xC5D0; &#xC758;&#xD574; &#xADDC;&#xC81C;.

RAD16-II p&eacute;ptido nanofibras son prometedoras para la ingenier&iacute;a del tejido vascular y se proyectaron para realzar la angiog&eacute;nesis in vitro e in vivo, aunque se desconoce el mecanismo. Presumimos que el efecto pro-angiog&eacute;nicos de RAD16-II resultado de interacciones de integrina-dependiente de baja afinidad de las c&eacute;lulas endoteliales microvasculares (MVECs) con motivos RAD. Rat&oacute;n MVECs fueron cultivadas en RAD16-II con o sin integrina e inhibidores de la v&iacute;a MAPK/ERK y angiog&eacute;nicos respuestas fueron cuantificadas. Los resultados fueron validados en vivo usando un rat&oacute;n diab&eacute;tico cicatrizaci&oacute;n modelo con neovascularizaci&oacute;n deteriorada. RAD16-II estimulado morfog&eacute;nesis capilar espont&aacute;nea y aumento de la fosforilaci&oacute;n de integrina &beta;(3) y expresi&oacute;n de VEGF en MVECs. Estas respuestas fueron derogadas en presencia de &beta;(3) y los inhibidores de la v&iacute;a MAPK/ERK o en el p&eacute;ptido de control sin motivos RAD. Inhibidor de amplio espectro integrina echistatin hab&iacute;a suprimido totalmente RAD16-II-mediada capilar morfog&eacute;nesis in vitro y neovascularizaci&oacute;n y expresi&oacute;n de VEGF en la herida en vivo. La adici&oacute;n del motivo RGD RAD16-II no cambiaron nanofibras arquitectura o propiedades mec&aacute;nicas, pero produjo una disminuci&oacute;n significativa en la morfog&eacute;nesis capilar. En general, estos resultados sugieren que la baja afinidad inespec&iacute;fica interacciones entre c&eacute;lulas y RAD motivos pueden desatar angiog&eacute;nicos respuestas mediante la fosforilaci&oacute;n de la integrina &beta;(3) y V&iacute;a MAPK/ERK, indicando que pueden utilizarse secuencias de baja afinidad para funcionalizar materiales biocompatibles para la regulaci&oacute;n de la migraci&oacute;n celular y la angiog&eacute;nesis, ampliando la actual piscina de motivos disponibles que pueden utilizarse para tal funcionalizaci&oacute;n. Incorporaci&oacute;n de motivos RAD o similares en la prote&iacute;na dise&ntilde;ado o h&iacute;bridos p&eacute;ptido andamios pueden representar una nueva estrategia para ingenier&iacute;a de tejidos vasculares y mejorar&aacute;n a&uacute;n m&aacute;s las posibilidades de dise&ntilde;o de nuevos materiales de andamio.

Regulaci&oacute;n de la activaci&oacute;n de la c&eacute;lula endotelial y la angiog&eacute;nesis por nanofibras de p&eacute;ptido inyectable.

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

Diabetes alters intracellular calcium transients in cardiac endothelial cells.

Low-amplitude electric field (EF) is an important component of wound-healing response and can promote vascular tissue repair; however, the mechanisms of action on endothelium remain unclear. We hypothesized that physiological amplitude EF regulates angiogenic response of microvascular endothelial cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. A custom set-up allowed non-thermal application of EF of high (7.5 GHz) and low (60 Hz) frequency. Cell responses following up to 24 h of EF exposure, including proliferation and apoptosis, capillary morphogenesis, vascular endothelial growth factor (VEGF) expression and MAPK pathways activation were quantified. A db/db mouse model of diabetic wound healing was used for in vivo validation. High-frequency EF enhanced capillary morphogenesis, VEGF release, MEK-cRaf complex formation, MEK and ERK phosphorylation, whereas no MAPK/JNK and MAPK/p38 pathways activation was observed. The endothelial response to EF did not require VEGF binding to VEGFR2 receptor. EF-induced MEK phosphorylation was reversed in the presence of MEK and Ca(2+) inhibitors, reduced by endothelial nitric oxide synthase inhibition, and did not depend on PI3K pathway activation. The results provide evidence for a novel intracellular mechanism for EF regulation of endothelial angiogenic response via frequency-sensitive MAPK/ERK pathway activation, with important implications for EF-based therapies for vascular tissue regeneration.

Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field.

Controlled translocation of molecules and ions across lipid membranes is the basis of numerous biological functions. Because synthetic systems can help researchers understand the more complex biological ones, many chemists have developed synthetic mimics of biological transporters. Both systems need to deal with similar fundamental challenges. In addition to providing mechanistic insights into transport mechanisms, synthetic transporters are useful in a number of applications including separation, sensing, drug delivery, and catalysis. In this Account, we present several classes of membrane transporters constructed in our laboratory from a facially amphiphilic building block, cholic acid. Our "molecular baskets" can selectively shuttle glucose across lipid membranes without transporting smaller sodium ions. We have also built oligocholate foldamers that transiently fold into helices with internal hydrophilic binding pockets to transport polar guests. Lastly, we describe amphiphilic macrocycles, which form transmembrane nanopores in lipid bilayers through the strong associative interactions of encapsulated water molecules. In addition to presenting the different transport properties of these oligocholate transporters, we illustrate how fundamental studies of molecular behavior in solution facilitate the creation of new and useful membrane transporters, despite the large difference between the two environments. We highlight the strong conformational effect of transporters. Because the conformation of a molecule often alters its size and shape, and the distribution of functional groups, conformational control can be used rationally to tune the property of a transporter. Finally, we emphasize that, whenever water is the solvent, its unique properties--small size, strong solvation for ionic functionalities, and an extraordinary cohesive energy density (i.e., total intermolecular interactions per unit volume)--tend to become critical factors to be considered. Purposeful exploitation of these solvent properties may be essential to the success of the supramolecular process involved--this is also the reason for the "learning through water play" in the title of this Account.

Conformationally controlled oligocholate membrane transporters: learning through water play.

Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR.

NRF2 plays a protective role in diabetic retinopathy in mice.

Objective: The effect of chronic hyperglycemic exposure on endothelial cell (EC) phenotype, impaired wound neovascularization, and healing is not completely understood. The hypotheses are: 1) chronic exposure to diabetic conditions in vivo impairs the angiogenic potential of ECs and 2) this deficiency can be improved by an extracellular microenvironment of angiogenic peptide nanofibers. Approach: Angiogenic potential of microvascular ECs isolated from diabetic (db/db) and wild type (wt) mice was assessed by quantifying migration, proliferation, apoptosis, capillary morphogenesis, and vascular endothelial growth factor (VEGF) expression for cell cultures on Matrigel (Millipore, Billerica, MA) or nanofibers under normoglycemic conditions. The in vivo effects of nanofiber treatment on wound vascularization were determined using two mouse models of diabetic wound healing. Results: Diabetic ECs showed significant impairments in migration, VEGF expression, and capillary morphogenesis. The nanofiber microenvironment restored capillary morphogenesis and VEGF expression and significantly increased proliferation and decreased cell apoptosis of diabetic cells versus wt controls. In diabetic wounds, nanofibers significantly enhanced EC infiltration, neovascularization, and VEGF protein levels, as compared to saline treatment; this effect was observed even in MMP9 knockout mice with endothelial progenitor cell (EPC) deficiency. Innovation: The results suggest a novel approach for correcting diabetes-induced endothelial deficiencies via cell interactions with a nanofiber-based provisional matrix in the absence of external angiogenic stimuli. Conclusion: Impaired endothelial angiogenic potential can be restored by angiogenic cell stimulation in the nanofiber microenvironment; this suggests that nanofiber technology for diabetic wound healing and treatment of other diabetes-induced vascular deficiencies is promising.

Nanofiber Microenvironment Effectively Restores Angiogenic Potential of Diabetic Endothelial Cells.

Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I/R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type retinal ganglion cells, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species. The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited reactive oxygen species increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2 knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I/R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. Oxidative stress is thought to be an important mediator of retinal ganglion cell death in ischemia-reperfusion injury. We found that the transcription factor NF-E2-related factor 2 (Nrf2), a major regulator of oxidative stress, is an important endogenous neuroprotective molecule in retinal ganglion cells in ischemia-reperfusion, exerting a cell-autonomous protective effect.  The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) reduces neurodegeneration following ischemia-reperfusion in an Nrf2-dependent fashion. This suggests that Nrf2-activating drugs including triterpenoids could be a therapeutic strategy for retinal neuroprotection.

Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.

Retinal ischemia results in neuronal degeneration and contributes to the pathogenesis of multiple blinding diseases. Recently, the fumaric acid ester dimethyl fumarate (DMF) has been FDA-approved for the treatment of multiple sclerosis, based on its neuroprotective and anti-inflammatory effects. Its potential role as a neuroprotective agent for retinal diseases has received little attention. In addition, DMF's mode of action remains elusive, although studies have suggested nuclear factor erythroid 2-related factor 2 (Nrf2) activation as an important mechanism. Here we investigated the neuroprotective role of monomethyl fumarate (MMF), the biologically active metabolite of DMF, in retinal ischemia-reperfusion (I/R) injury, and examined the role of Nrf2 in mediating MMF action.

Monomethyl fumarate promotes Nrf2-dependent neuroprotection in retinal ischemia-reperfusion.

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM-derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC-based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double-deficiency (EPC+diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: 1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; 2) these impairments can be rescued by SCF administration. Full-thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin-induced diabetes in the presence or absence of tail-vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 post-wounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb-EPC) counts when compared with WT. Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both non-diabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC-deficiency and impaired wound neovascularization in both diabetic and non-diabetic MMP9 KO mice. Overall, the results demonstrate that BM-derived EPCs play a significant role during wound neovascularization and that the SCF-based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds. This article is protected by copyright. All rights reserved.

Diabetic Wound Healing In A MMP9-/- Mouse Model.

Stem cell-based therapy is emerging as a promising approach for chronic diabetic wounds, but strategies for optimizing both cellular differentiation and delivery remain as major obstacles. Here, we study bioengineered vascularized constructs as a therapeutic modality for diabetic wound healing. We developed a wound model in immunodeficient rodent and treated it with engineered vascularized constructs from endothelial progenitors or early vascular cells-derived from human induced pluripotent stem cells (hiPSCs) reprogrammed either from healthy donor or type-1 diabetic patient. We found that all vascularized constructs expedited wound closure and reperfusion, with endothelial progenitor constructs having the earliest maximum closure rate followed closely by healthy and diabetic hiPSC-derivative constructs. This was accompanied by rapid granulation layer formation and regression in all vascularized construct groups. Macrophage infiltration into the hydrogel matrix occurred during early stages of healing, seeming to facilitate rapid neovascularization of the wound that could then better persist in the vascularized constructs. Blood perfusion of the human vasculature could be detected after three days, indicating rapid integration with the host vasculature. Overall, we propose a potential therapeutic strategy using allograft or autologous vascularized constructs to treat type-1 diabetic wounds. This approach highlights the unprecedented prospects of designing patient-specific stem cell therapy.

Hongkwan Cho

Department of Ophthalmology,
School of Medicine,
Department of Ophthalmology, School of Medicine

Johns Hopkins University

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Hongkwan Cho

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

Johns Hopkins University

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Department of Ophthalmology,
School of Medicine,
Department of Ophthalmology, School of Medicine