nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

Harvard Medical School

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 μm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and β-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.

Antivascular therapy directed against VEGF or its receptors (VEGFR) has been successful when administered at early stages of tumor vessel growth but is less effective when administered later. Tumor blood vessels are heterogeneous, so vessel subpopulations may differ in their requirements for tumor cell-secreted VEGF and in their susceptibility to anti-VEGF/VEGFR therapy. Human cancers contain several distinct blood vessel types, including mother vessels (MV), glomeruloid microvascular proliferations (GMP), vascular malformations (VM), feeding arteries (FA), and draining veins (DV), all of which can be generated in mice in the absence of tumor cells using expression vectors for VEGF-A(164). In this study, we investigated the sensitivity of each of these vessel types to anti-VEGF therapy with Aflibercept (VEGF Trap), a potent inhibitor of VEGF-A(164). Administering VEGF Trap treatment before or shortly after injection of a recombinant VEGF-A(164)-expressing adenovirus could prevent or regress tumor-free neovasculature, but it was progressively less effective if initiated at later times. Early-forming MVs and GMPs in which the lining endothelial cells expressed high levels of VEGFR-2 were highly susceptible to blockade by VEGF Trap. In contrast, late-forming VMs, FAs, and DVs that expressed low levels of VEGFR-2 were largely resistant. Together, our findings define the susceptibility of different blood vessel subtypes to anti-VEGF therapy, offering a possible explanation for the limited effectiveness of anti-VEGF-A/VEGFR treatment of human cancers, which are typically present for months to years before discovery and are largely populated by late-forming blood vessels.

Tumor-surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy.

&#x51E0;&#x5341;&#x5E74;&#x6765;&#xFF0C;&#x4E8B;&#x540E;&#x4F53;&#x5185;&#x57FA;&#x56E0;&#x6CBB;&#x7597;&#x81EA;&#x4F53;&#x5047;&#x5B9A;&#x4F5C;&#x4E3A;&#x6F5C;&#x5728;&#x7684;&#x66FF;&#x4EE3;&#x529E;&#x6CD5;&#x80A0;&#x5916;&#x884C;&#x653F;&#x91CD;&#x7EC4;&#x86CB;&#x767D;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x5B9E;&#x73B0;&#x6709;&#x6548;&#x7EC6;&#x80DE;&#x690D;&#x5165;&#x72B6;&#x6001;&#x7684;&#x4EE5;&#x524D;&#x68C0;&#x7D22;&#x7684;&#x75C5;&#x4EBA;&#x7EC6;&#x80DE;&#x662F;&#x5177;&#x6709;&#x6311;&#x6218;&#x6027;&#x7684;&#x3002;&#x6700;&#x8FD1;&#xFF0C;&#x6211;&#x4EEC;&#x5DE5;&#x7A0B;&#x5E08;&#x4F53;&#x5185;&#x8840;&#x7BA1;&#x7684;&#x80FD;&#x529B;&#x5DF2;&#x8FDB;&#x5165;&#x7EC4;&#x7EC7;&#x7684;&#x8BF4;&#x660E;&#x4ECB;&#x7ECD;&#x5141;&#x8BB8;&#x901A;&#x8FC7;&#x57FA;&#x56E0;&#x4FEE;&#x6539;&#x751F;&#x6210;&#x8FD9;&#x4E9B;&#x8840;&#x7BA1;&#x7684;&#x8840;&#x7BA1;&#x7EC6;&#x80DE;&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x57FA;&#x56E0;&#x5DE5;&#x7A0B;&#x4EBA;&#x8840;&#x6E90;&#x6027;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x96C6;&#x843D;&#x5F62;&#x6210;&#x7EC6;&#x80DE; (ECFCs) &#x8868;&#x8FBE;&#x4FC3;&#x7EA2;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x7D20; (EPO) &#x56DB;&#x73AF;&#x7D20;&#x8C03;&#x63A7;&#x7CFB;&#x7EDF;&#x7684;&#x63A7;&#x5236;&#x4E0B;&#xFF0C;&#x5E76;&#x5728;&#x514D;&#x75AB;&#x7F3A;&#x9677;&#x5C0F;&#x9F20;&#x4E2D;&#x751F;&#x6210;&#x76AE;&#x4E0B;&#x8840;&#x7BA1;&#x7F51;&#x7EDC;&#x80FD;&#x7CFB;&#x7EDF;&#x6027;&#x7684; EPO &#x91CA;&#x653E;&#x3002;&#x8FD9;&#x4E9B;&#x4E0B;&#x886C;&#x7684;&#x8840;&#x7BA1;&#x7F51;&#x7EDC;&#x5F62;&#x6210;&#x9F20;&#x6807;&#x8109;&#x7BA1;&#x7CFB;&#x7EDF;&#xFF0C;&#x5141;&#x8BB8;&#x91CD;&#x7EC4;&#x4EBA; EPO &#x5230;&#x8840;&#x6DB2;&#x91CC;&#x7684;&#x76F4;&#x63A5;&#x4EA4;&#x4ED8;&#x529F;&#x80FD;&#x543B;&#x5408;&#x3002;&#x7EA2;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x88AB;&#x6FC0;&#x6D3B;&#x540E;&#x7684; EPO &#x8868;&#x8FBE;&#xFF0C;&#x6B63;&#x5E38;&#x548C;&#x8D2B;&#x8840;&#x5C0F;&#x9F20;&#x8BF1;&#x5BFC;&#x662F;&#x5B8C;&#x5168;&#x53EF;&#x9006;&#x7684;&#x8FC7;&#x7A0B;&#x3002;&#x8FD9;&#x79CD;&#x65B9;&#x6CD5;&#x53EF;&#x51CF;&#x8F7B;&#x75C5;&#x4EBA;&#x4ECE;&#x9891;&#x7E41;&#x7684; EPO &#x6CE8;&#x5C04;&#xFF0C;&#x51CF;&#x5C11;&#x8D2B;&#x8840;&#x7684;&#x7BA1;&#x7406;&#x6709;&#x5173;&#x7684;&#x533B;&#x7597;&#x8D39;&#x7528;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x8FD9;&#x4E0B;&#x57FA;&#x4E8E;&#x57FA;&#x56E0;&#x4F20;&#x9012;&#x6218;&#x7565;&#x4F5C;&#x4E3A;&#x4E00;&#x79CD;&#x53EF;&#x884C;&#x7684;&#x66FF;&#x4EE3;&#x6280;&#x672F;&#x65F6;&#x9700;&#x8981;&#x91CD;&#x7EC4;&#x86CB;&#x767D;&#x7684;&#x65E5;&#x5E38;&#x7BA1;&#x7406;&#x3002;

&#x7EA2;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x4F7F;&#x7528;&#x53D7;&#x63A7;&#x4FC3;&#x7EA2;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x7D20;&#x91CA;&#x653E;&#x8F6C;&#x57FA;&#x56E0;&#x4EBA;&#x7C7B;&#x8840;&#x7BA1;&#x7F51;&#x7EDC;&#x7684;&#x8BF1;&#x5BFC;&#x3002;

For decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood-derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

Induction of erythropoiesis using human vascular networks genetically engineered for controlled erythropoietin release.

Pendant des d&#xE9;cennies, autologue th&#xE9;rapie g&#xE9;nique ex vivo a &#xE9;t&#xE9; postul&#xE9;e comme une alternative potentielle &#xE0; l&#x26;#39;administration parent&#xE9;rale de prot&#xE9;ines recombinantes. Toutefois, la r&#xE9;alisation effective greffe cellulaire de cellules de patients pr&#xE9;c&#xE9;demment r&#xE9;cup&#xE9;r&#xE9;s est difficile. R&#xE9;cemment, notre capacit&#xE9; &#xE0; concevoir le syst&#xE8;me vasculaire in vivo a permis l&#x26;#39;introduction d&#x26;#39;instructions dans les tissus en modifiant g&#xE9;n&#xE9;tiquement les cellules vasculaires qui construisent ces vaisseaux sanguins. Dans la pr&#xE9;sente &#xE9;tude, nous avons modifi&#xE9; g&#xE9;n&#xE9;tiquement de l&#x26;#39;homme d&#xE9;riv&#xE9;s du sang endoth&#xE9;liales formant colonie (CPEF cellules) &#xE0; l&#x26;#39;&#xE9;rythropo&#xEF;&#xE9;tine express (OEB) sous le contr&#xF4;le d&#x26;#39;un syst&#xE8;me de t&#xE9;tracycline-r&#xE9;glement&#xE9;, et a g&#xE9;n&#xE9;r&#xE9; des r&#xE9;seaux vasculaires sous-cutan&#xE9;es capables de l&#x26;#39;EPO lib&#xE9;ration syst&#xE9;mique chez des souris immunod&#xE9;ficientes. Ces r&#xE9;seaux ECFC fourr&#xE9;es vasculaires form&#xE9; anastomoses fonctionnelles &#xE0; la vascularisation de la souris, permettant la livraison directe d&#x26;#39;EPO recombinante dans la circulation sanguine. Apr&#xE8;s activation de l&#x26;#39;expression OEB, l&#x26;#39;&#xE9;rythropo&#xEF;&#xE8;se a &#xE9;t&#xE9; induite chez la souris &#xE0; la fois normaux et an&#xE9;miques, un processus qui est compl&#xE8;tement r&#xE9;versible. Cette approche pourrait soulager les patients de fr&#xE9;quentes injections d&#x26;#39;EPO, ce qui r&#xE9;duit les frais m&#xE9;dicaux li&#xE9;s &#xE0; la gestion de l&#x26;#39;an&#xE9;mie. Nous proposons cette ECFC bas&#xE9;e d&#xE9;livrance g&#xE9;nique strat&#xE9;gie comme une technologie alternative viable lorsque l&#x26;#39;administration de routine de prot&#xE9;ines recombinantes est n&#xE9;cessaire.

L&#x26;#39;induction de l&#x26;#39;&#xE9;rythropo&#xEF;&#xE8;se &#xE0; l&#x26;#39;aide de l&#x26;#39;homme des r&#xE9;seaux vasculaires obtenus par g&#xE9;nie g&#xE9;n&#xE9;tique pour la lib&#xE9;ration contr&#xF4;l&#xE9;e &#xE9;rythropo&#xEF;&#xE9;tine

Jahrzehntelang hat autologe ex Vivo Gentherapie als m&ouml;gliche Alternative zur parenteralen Verabreichung von rekombinanten Proteinen postuliert worden. Jedoch ist es schwierig, effektive zellul&auml;re Verteilung der zuvor abgerufenen Patienten Zellen zu erreichen. Vor kurzem hat unsere F&auml;higkeit, Gef&auml;&szlig;system in-vivo-Ingenieur f&uuml;r die Einf&uuml;hrung von Anweisungen in das Gewebe durch genetisch &auml;ndern die vaskul&auml;ren Zellen, die diese Blutgef&auml;&szlig;e zu bauen erlaubt. In der vorliegenden Studie wir genetisch menschliche Blut abgeleitete kolonienbildende Endothelzellen (ECFCs), Erythropoetin (EPO) auszudr&uuml;cken unter der Kontrolle eines Tetracyclin geregelten Systems entwickelt, und subkutane vaskul&auml;re Netzwerken systemische EPO Version f&auml;hig in immungeschw&auml;chte M&auml;use generiert. Diese ECFC ges&auml;umten vaskul&auml;rer Netzwerke gebildet funktionale Anastomoses mit der Maus-Gef&auml;&szlig;system, erm&ouml;glicht direkte &Uuml;bermittlung von rekombinanten menschlichen EPO in den Blutkreislauf. Nach Aktivierung des EPO-Ausdruck wurde Erythropoese im Normal- und an&auml;mischen M&auml;usen, einen Prozess ausgel&ouml;st, die vollst&auml;ndig reversibel war. Dieser Ansatz k&ouml;nnte Patienten aus h&auml;ufigen EPO-Injektionen, Reduzierung der medizinischen Kosten im Zusammenhang mit der Verwaltung von An&auml;mie lindern. Wir schlagen diese ECFC basierende gen-Lieferung-Strategie als tragf&auml;hige alternative Technologie, wenn ordentlichen Verwaltung von rekombinanten Proteinen ben&ouml;tigt wird.

Induktion der Erythropoese mit menschliche vaskul&auml;re Netzwerken gentechnisch f&uuml;r kontrollierte Erythropoetin Release.

Per decenni, autologhe ex vivo gene therapy &egrave; stata postulata come una potenziale alternativa alla somministrazione parenterale di proteine ricombinanti. Tuttavia, il raggiungimento efficacia attecchimento cellulare delle cellule di pazienti precedentemente recuperate &egrave; impegnativo. Recentemente, la nostra capacit&agrave; di vascolarizzazione in vivo l&#39;ingegnere ha permesso l&#39;introduzione delle istruzioni nei tessuti modificando geneticamente le cellule vascolari che costruire questi vasi sanguigni. Nel presente studio, siamo geneticamente ingegnerizzato emoderivati endoteliali formanti cellule umane (ECFCs) per esprimere l&#39;eritropoietina (EPO) sotto il controllo di un sistema regolato da tetraciclina e generato reti vascolari sottocutanei capace di sistemica rilascio EPO in topi immunodeficienti. Queste reti vascolari ECFC-allineati formano anastomosi funzionale con il sistema vascolare del mouse, che consente la consegna diretta di EPO umana ricombinante nel flusso sanguigno. Dopo l&#39;attivazione dell&#39;espressione di EPO, l&#39;eritropoiesi &egrave; stata indotta in topi normali e anemici, un processo che &egrave; stato completamente reversibile. Questo approccio potrebbe alleviare i pazienti da frequenti iniezioni di EPO, riducendo i costi medici associati alla gestione dell&#39;anemia. Vi proponiamo questa strategia basata su ECFC gene-consegna come una tecnologia alternativa valida quando &egrave; necessaria l&#39;ordinaria amministrazione di proteine ricombinanti.

Induzione dell&#39;eritropoiesi utilizzando reti vascolari umani geneticamente ingegnerizzati per il rilascio controllato di eritropoietina.

&#x4F55;&#x5341;&#x5E74;&#x3082;&#x306E;&#x9593;&#x3001;&#x81EA;&#x5BB6;ex vivo&#x3067;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x6CBB;&#x7642;&#x306F;&#x3001;&#x7D44;&#x63DB;&#x3048;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x975E;&#x7D4C;&#x53E3;&#x6295;&#x4E0E;&#x306B;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x4EE3;&#x66FF;&#x7269;&#x3068;&#x3057;&#x3066;&#x4EEE;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x4EE5;&#x524D;&#x306B;&#x53D6;&#x5F97;&#x3057;&#x305F;&#x60A3;&#x8005;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x52B9;&#x679C;&#x7684;&#x306A;&#x7D30;&#x80DE;&#x306E;&#x751F;&#x7740;&#x3092;&#x200B;&#x200B;&#x9054;&#x6210;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x56F0;&#x96E3;&#x3067;&#x3042;&#x308B;&#x3002;&#x6700;&#x8FD1;&#x3067;&#x306F;&#x3001;in vivo&#x3067;&#x8840;&#x7BA1;&#x7CFB;&#x3092;&#x8A2D;&#x8A08;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x6211;&#x3005;&#x306E;&#x80FD;&#x529B;&#x306F;&#x907A;&#x4F1D;&#x7684;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x8840;&#x7BA1;&#x3092;&#x69CB;&#x7BC9;&#x3059;&#x308B;&#x8840;&#x7BA1;&#x7D30;&#x80DE;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x7D44;&#x7E54;&#x3078;&#x306E;&#x547D;&#x4EE4;&#x306E;&#x5C0E;&#x5165;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3057;&#x305F;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x7684;&#x306B;&#x30C6;&#x30C8;&#x30E9;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x8ABF;&#x7BC0;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x5236;&#x5FA1;&#x4E0B;&#x306B;&#x30A8;&#x30EA;&#x30B9;&#x30ED;&#x30DD;&#x30A8;&#x30C1;&#x30F3;&#xFF08;EPO&#xFF09;&#x3092;&#x767A;&#x73FE;&#x3059;&#x308B;&#x30D2;&#x30C8;&#x8840;&#x6DB2;&#x7531;&#x6765;&#x306E;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x30B3;&#x30ED;&#x30CB;&#x30FC;&#x5F62;&#x6210;&#x7D30;&#x80DE;&#xFF08;ECFCs&#xFF09;&#x8A2D;&#x8A08;&#x3001;&#x304A;&#x3088;&#x3073;&#x514D;&#x75AB;&#x4E0D;&#x5168;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5168;&#x8EAB;&#x306E;EPO&#x653E;&#x51FA;&#x304C;&#x53EF;&#x80FD;&#x306A;&#x76AE;&#x4E0B;&#x8840;&#x7BA1;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x3092;&#x751F;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;ECFC&#x88CF;&#x5730;&#x8840;&#x7BA1;&#x7DB2;&#x306F;&#x3001;&#x8840;&#x6D41;&#x3078;&#x306E;&#x7D44;&#x63DB;&#x3048;&#x30D2;&#x30C8;EPO&#x306E;&#x76F4;&#x63A5;&#x914D;&#x4FE1;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3059;&#x308B;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x8840;&#x7BA1;&#x3067;&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x543B;&#x5408;&#x3092;&#x5F62;&#x6210;&#x3057;&#x305F;&#x3002; EPO&#x306E;&#x767A;&#x73FE;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x3057;&#x305F;&#x5F8C;&#x3001;&#x8D64;&#x8840;&#x7403;&#x306F;&#x3001;&#x6B63;&#x5E38;&#x304A;&#x3088;&#x3073;&#x8CA7;&#x8840;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;&#x5B8C;&#x5168;&#x306B;&#x53EF;&#x9006;&#x7684;&#x3067;&#x3042;&#x3063;&#x305F;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x305F;&#x3002;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x306F;&#x3001;&#x8CA7;&#x8840;&#x306E;&#x7BA1;&#x7406;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x533B;&#x7642;&#x8CBB;&#x3092;&#x524A;&#x6E1B;&#x3057;&#x3001;&#x983B;&#x7E41;&#x306B;EPO&#x306E;&#x6CE8;&#x5C04;&#x304B;&#x3089;&#x60A3;&#x8005;&#x3092;&#x548C;&#x3089;&#x3052;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x7D44;&#x63DB;&#x3048;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x65E5;&#x5E38;&#x7684;&#x306A;&#x7BA1;&#x7406;&#x304C;&#x5FC5;&#x8981;&#x306A;&#x5834;&#x5408;&#x306B;&#x6211;&#x3005;&#x306F;&#x3001;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x4EE3;&#x66FF;&#x6280;&#x8853;&#x3068;&#x3057;&#x3066;&#x3053;&#x306E;ECFC&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x6226;&#x7565;&#x3092;&#x63D0;&#x6848;&#x3059;&#x308B;&#x3002;

&#x907A;&#x4F1D;&#x7684;&#x306B;&#x5236;&#x5FA1;&#x3055;&#x308C;&#x305F;&#x30A8;&#x30EA;&#x30B9;&#x30ED;&#x30DD;&#x30A8;&#x30C1;&#x30F3;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x306E;&#x305F;&#x3081;&#x306B;&#x8A2D;&#x8A08;&#x3055;&#x30D2;&#x30C8;&#x8840;&#x7BA1;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x3092;&#x7528;&#x3044;&#x305F;&#x8D64;&#x8840;&#x7403;&#x7523;&#x751F;&#x8A98;&#x5C0E;

&#xC218;&#xC2ED; &#xB144; &#xB3D9;&#xC548;, ex vivo &#xC720;&#xC804;&#xC790; &#xCE58;&#xB8CC; &#xD5CC; &#xC7AC;&#xC870;&#xD569; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBE44;&#xACBD; &#xAD6C; &#xAD00;&#xB9AC;&#xC5D0; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC548;&#xC73C;&#xB85C; postulated &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xD588;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xC804;&#xC5D0; &#xAC80;&#xC0C9; &#xB41C; &#xD658;&#xC790; &#xC138;&#xD3EC;&#xC758; &#xD6A8;&#xACFC;&#xC801;&#xC778; &#xC140;&#xB8F0;&#xB7EC; engraftment&#xB97C; &#xB2EC;&#xC131; &#xD558;&#xB294; &#xB3C4;&#xC804; &#xC774;&#xB2E4;. &#xCD5C;&#xADFC;, &#xD608;&#xAD00; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xD558;&#xB294; &#xC6B0;&#xB9AC;&#xC758; &#xB2A5;&#xB825; &#xC720;&#xC804;&#xC790; &#xAD6C;&#xCD95; &#xC774;&#xB7EC;&#xD55C; &#xD608;&#xAD00; &#xD608;&#xAD00; &#xC140;&#xC744; &#xC218;&#xC815; &#xD558; &#xC5EC; &#xC870;&#xC9C1;&#xC5D0; &#xC9C0;&#xCE68;&#xC758; &#xB3C4;&#xC785;&#xC5D0; &#xB300; &#xD55C; &#xC218; &#xC788;&#xB2E4;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0; &#xC6B0;&#xB9AC; &#xC720;&#xC804;&#xC790; &#xBC0F; &#xC778;&#xAC04; &#xD608;&#xC561; &#xC720;&#xB798; &#xB0B4; &#xD53C; &#xC2DD;&#xBBFC;&#xC9C0;&#xB97C; &#xD615;&#xC131; &#xC138;&#xD3EC; (ECFCs) erythropoietin (EPO)&#xC744; &#xD45C;&#xD604; &#xD558;&#xB294; &#xD56D;&#xC0DD;&#xBB3C;&#xC9C8; &#xADDC;&#xC81C; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xD1B5;&#xC81C; &#xC124;&#xACC4; immunodeficient &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0; &#xD53C;&#xD558; &#xD608;&#xAD00; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xC870;&#xC9C1;&#xC758; EPO &#xCD9C;&#xC2DC;&#xC758; &#xC218;&#xB97C; &#xC0DD;&#xC131;. &#xC774;&#xB7EC;&#xD55C; ECFC &#xC904;&#xC9C0;&#xC5B4; &#xD608;&#xAD00; &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xAE30;&#xB2A5; anastomoses &#xC7AC;&#xC870;&#xD569; &#xC778;&#xAC04; EPO&#xC758; &#xD608; &#xB958;&#xB85C; &#xC9C1;&#xC811; &#xBC30;&#xB2EC; &#xD5C8;&#xC6A9; &#xB9C8;&#xC6B0;&#xC2A4; &#xD608;&#xAD00;&#xC744; &#xD615;&#xC131;. EPO &#xD45C;&#xD604;&#xC758; &#xD65C;&#xC131;&#xD654; &#xD6C4; &#xC801;&#xD608;&#xAD6C;&#xB294; &#xC644;&#xC804;&#xD788; &#xB418;&#xB3CC;&#xB9B4; &#xC218;&#xB294; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC774; &#xACE0; &#xBE48; &#xD608; &#xC950;&#xC5D0;&#xC11C; &#xC720;&#xB3C4; &#xB418;&#xC5C8;&#xB2E4;. &#xC774; &#xC774;&#xB807;&#xAC8C; &#xBE48; &#xD608;&#xC758; &#xAD00;&#xB9AC;&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC758;&#xB8CC; &#xBE44;&#xC6A9;&#xC744; &#xC808;&#xAC10; &#xD558;&#xB294; &#xBE48;&#xBC88;&#xD55C; EPO &#xC8FC;&#xC0AC;&#xC5D0;&#xC11C; &#xD658;&#xC790; &#xC644;&#xD654; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC7AC;&#xC870;&#xD569; &#xD615; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC77C;&#xC0C1;&#xC801;&#xC778; &#xAD00;&#xB9AC; &#xD544;&#xC694;&#xD560; &#xB54C; &#xAC00;&#xB2A5;&#xD55C; &#xB300;&#xCCB4; &#xAE30;&#xC220;&#xB85C;&#xC774; ECFC &#xAE30;&#xBC18; &#xC720;&#xC804;&#xC790; &#xBC30;&#xB2EC; &#xC804;&#xB7B5;&#xC744; &#xC81C;&#xC548; &#xD55C;&#xB2E4;.

&#xC720;&#xB3C4; &#xC81C;&#xC5B4; erythropoietin &#xB9B4;&#xB9AC;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xC804;&#xC790; &#xC870;&#xC791; &#xC778;&#xAC04; &#xD608;&#xAD00; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC801;&#xD608;&#xAD6C;&#xC758;.

Durante d&#xE9;cadas, la terapia g&#xE9;nica in vivo aut&#xF3;logas ex se ha postulado como una alternativa potencial para la administraci&#xF3;n parenteral de prote&#xED;nas recombinantes. Sin embargo, el logro eficaz de injerto celular de las c&#xE9;lulas de pacientes recuperados previamente es un reto. Recientemente, nuestra capacidad para dise&#xF1;ar vascular in vivo ha permitido la introducci&#xF3;n de las instrucciones dentro de los tejidos mediante la modificaci&#xF3;n gen&#xE9;tica de las c&#xE9;lulas vasculares que se basan estos vasos sangu&#xED;neos. En el presente estudio, la ingenier&#xED;a gen&#xE9;tica de la sangre humana derivados del endotelio-c&#xE9;lulas formadoras de colonias (ECFCs) a la eritropoyetina expresa (OEP), bajo el control de un sistema regulado por tetraciclina, y genera redes vasculares subcut&#xE1;neos capaces de liberaci&#xF3;n de EPO sist&#xE9;mica en ratones inmunodeficientes. Estas redes vasculares ECFC forradas en forma anastomosis funcionales de la vasculatura del rat&#xF3;n, lo que permite la entrega directa de la EPO humana recombinante en el torrente sangu&#xED;neo. Despu&#xE9;s de la activaci&#xF3;n de la expresi&#xF3;n de EPO, la eritropoyesis fue inducida en ratones normales y an&#xE9;micas, un proceso que es totalmente reversible. Este enfoque podr&#xED;a aliviar a pacientes de frecuentes inyecciones de EPO, lo que reduce los costos m&#xE9;dicos asociados con el tratamiento de la anemia. Proponemos este ECFC basado en el gen estrategia de distribuci&#xF3;n como una tecnolog&#xED;a alternativa viable cuando la administraci&#xF3;n de rutina de prote&#xED;nas recombinantes que se necesita.

La inducci&#xF3;n de la eritropoyesis mediante redes vasculares humanos gen&#xE9;ticamente modificados para la liberaci&#xF3;n controlada de eritropoyetina

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human β-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium.

Blood-derived endothelial colony-forming cells (ECFCs) have robust vasculogenic potential that can be exploited to bioengineer long-lasting human vascular networks in vivo. However, circulating ECFCs are exceedingly rare in adult peripheral blood. Because the mechanism by which ECFCs are mobilized into circulation is currently unknown, the reliability of peripheral blood as a clinical source of ECFCs remains a concern. Thus, there is a need to find alternative sources of autologous ECFCs. Here we aimed to determine whether ECFCs reside in the vasculature of human white adipose tissue (WAT) and to evaluate if WAT-derived ECFCs have equal clinical potential to blood-derived ECFCs. We isolated the complete endothelial cell (EC) population from intact biopsies of normal human subcutaneous WAT by enzymatic digestion and selection of CD31(+) cells. Subsequently, we extensively compared WAT-derived EC phenotype and functionality to bonafide ECFCs derived from both umbilical cord blood and adult peripheral blood. We demonstrated that human WAT is indeed a dependable source of ECFCs with indistinguishable properties to adult peripheral blood ECFCs, including hierarchical clonogenic ability, large expansion potential, stable endothelial phenotype, and robust in vivo blood vessel-forming capacity. Considering the unreliability and low rate of occurrence of ECFCs in adult blood and that biopsies of WAT can be obtained with minimal intervention in an ambulatory setting, our results indicate WAT as a more practical alternative to obtain large amounts of readily available autologous ECFCs for future vascular cell therapies.

Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential.

Brain pericytes play a critical role in blood vessel stability and blood-brain barrier maturation. Despite this, how brain pericytes function in these different capacities is only beginning to be understood. Here we show that the forkhead transcription factor Foxc1 is expressed by brain pericytes during development and is critical for pericyte regulation of vascular development in the fetal brain. Conditional deletion of Foxc1 from pericytes and vascular smooth muscle cells leads to late-gestation cerebral micro-hemorrhages as well as pericyte and endothelial cell hyperplasia due to increased proliferation of both cell types. Conditional Foxc1 mutants do not have widespread defects in BBB maturation, though focal breakdown of BBB integrity is observed in large, dysplastic vessels. qPCR profiling of brain microvessels isolated from conditional mutants showed alterations in pericyte-expressed proteoglycans while other genes previously implicated in pericyte-endothelial cell interactions were unchanged. Collectively these data point towards an important role for Foxc1 in certain brain pericyte functions (e.g. vessel morphogenesis) but not others (e.g. barriergenesis).

Article	Year
TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration. Angiogenesis\| PubMed ID: 21626280	2011
Tumor-surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy. Cancer research\| PubMed ID: 21937680	2011
Induction of erythropoiesis using human vascular networks genetically engineered for controlled erythropoietin release. Blood\| PubMed ID: 21937702	2011
Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium. Blood\| PubMed ID: 23529929	2013
Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential. Angiogenesis\| PubMed ID: 23636611	2013
Foxc1 is required by pericytes during fetal brain angiogenesis. Biology open\| PubMed ID: 23862012	2013

Shou-Ching Jaminet

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