induction-and-analysis-of-epithelial-to-mesenchymal-transition

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

R&D Systems, Inc.

Fusarium moniliforme SW-902, a high-yielding strain for producing D-pantolactone hydrolase was screened. The conditions for enzyme formation of the strain were studied. The suitable carbon source of the medium was glycerol and the suitable nitrogen source was peptone. The optimum temperature and initial pH for enzyme formation were 26 degrees C and pH8.0 respectively. When the organism was cultured in the 60 L-fermentor under the optimum conditions for 45 h, about 7.18 g dry cells/L and 0.92 IU/g dry cells weight were obtained.

[Production of D-pantolactone hydrolase by Fusarium moniliforme SW-902].

&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x63CF;&#x8FF0;&#x4E86;&#x5176;&#x6210;&#x5458;&#x5BF9;&#x4E0D;&#x5BFB;&#x5E38;&#x7684;&#x950C;&#x6307;&#x86CB;&#x767D;&#x8FDB;&#x884C;&#x7F16;&#x7801;&#x7684;&#x5B88;&#x6052;&#x7684; nlz &#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x3002;&#x5728;&#x6591;&#x9A6C;&#x9C7C; neurectoderm&#xFF0C;nlz1 &#x548C;&#x65B0;&#x5206;&#x79BB;&#x7684; nlz2 &#x4E2D;&#x7684;&#x63A8;&#x5B9A;&#x7684;&#x540E;&#x8111;&#x548C;&#x4E2D;&#x8111;/&#x540E;&#x8111;&#x8FB9;&#x754C;&#xFF0C;&#x5176;&#x4E2D; nlz1 &#x7684;&#x8868;&#x8FBE;&#x662F;&#x4F9D;&#x8D56;&#x4E8E; pax2a &#x8868;&#x793A;&#x3002;&#x6B64;&#x5916;&#xFF0C;nlz2 &#x662F;&#x552F;&#x4E00;&#x8868;&#x793A;&#x66F4;&#x6273;&#x624B;&#xFF0C;&#x63A8;&#x5B9A;&#x4E2D;&#x8111;&#x548C;&#x95F4;&#x8111;&#x3002;&#x539F;&#x80A0;&#x9636;&#x6BB5; Nlz &#x86CB;&#x767D;&#x7684;&#x8FC7;&#x5EA6;&#x8868;&#x8FBE;&#x6291;&#x5236;&#x540E;&#x8111;&#x53D1;&#x5C55;&#x3002;&#x5C24;&#x5176;&#x662F;&#xFF0C;ectopically &#x8868;&#x793A;&#x7684; Nlz1 &#x6291;&#x5236;&#x5F62;&#x6210;&#x7684;&#x672A;&#x6765; rhombomeres 2 &#x548C; 3 &#xFF08;r2&#xFF0C;r3&#xFF09;&#xFF0C;&#x800C;&#x76F8;&#x90BB;&#x7684; r1 &#x548C; r4 &#x4E0D;&#x53D7;&#x5F71;&#x54CD;&#x3002;&#x76F8;&#x53CD;&#xFF0C;Nlz1 &#x548C; Nlz2 &#x86CB;&#x767D;&#x7684;&#x540C;&#x65F6;&#x51CF;&#x5C11;&#x51FD;&#x6570;&#x7531;&#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x53CD;&#x4E49; morpholino &#x6539;&#x6027;&#x4F4E;&#x805A;&#x7269;&#x5BFC;&#x81F4;&#x672A;&#x6765; r3 &#x548C; r5&#x3001; r4 &#x76F8;&#x5173;&#x635F;&#x5931;&#x6269;&#x5927;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; nlz &#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x7684;&#x529F;&#x80FD;&#x4E4B;&#x4E00;&#x662F;&#x4EE5;&#x6307;&#x5B9A;&#x6216;&#x7EF4;&#x6301; r4 &#x7684;&#x7279;&#x6027;&#xFF0C;&#x5E76;&#x9650;&#x5236; r3 &#x548C; r5 &#x540E;&#x8111;&#x5F62;&#x6210;&#x8FC7;&#x7A0B;&#x3002;

nlz &#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x662F;&#x5FC5;&#x9700;&#x7684;&#x9635;&#x5217;&#x5728;&#x6591;&#x9A6C;&#x9C7C;&#x4E2D;&#x7684;&#x540E;&#x8111;&#x3002;

This study describes the conserved nlz gene family whose members encode unusual zinc finger proteins. In the zebrafish neurectoderm, both nlz1 and the newly isolated nlz2 are expressed in the presumptive hindbrain and midbrain/hindbrain boundary, where expression of nlz1 is dependent on pax2a. In addition, nlz2 is uniquely expressed more anteriorly, in the presumptive midbrain and diencephalon. Overexpression of Nlz proteins during gastrula stages inhibits hindbrain development. In particular, ectopically expressed Nlz1 inhibits formation of future rhombomeres 2 and 3 (r2, r3), whereas neighboring r1 and r4 are not affected. Conversely, simultaneous reduction of Nlz1 and Nlz2 protein function by expression of antisense morpholino-modified oligomers leads to expansion of future r3 and r5, with associated loss of r4. These data indicate that one function of the nlz gene family is to specify or maintain r4 identity, and to limit r3 and r5 during hindbrain formation.

nlz gene family is required for hindbrain patterning in the zebrafish.

Cette &#xE9;tude d&#xE9;crit la famille des g&#xE8;nes conserv&#xE9;s NLZ dont les membres coder inhabituelles prot&#xE9;ines &#xE0; doigts de zinc. Dans le poisson z&#xE8;bre neurectoderme, &#xE0; la fois nlz1 et les nlz2 nouvellement isol&#xE9;s sont exprim&#xE9;s dans le cerveau post&#xE9;rieur pr&#xE9;somptif et le m&#xE9;senc&#xE9;phale / rhombenc&#xE9;phale fronti&#xE8;re, o&#xF9; l&#x26;#39;expression de nlz1 d&#xE9;pend pax2a. En outre, nlz2 est unique exprim&#xE9; plus en avant, dans le m&#xE9;senc&#xE9;phale et le dienc&#xE9;phale pr&#xE9;somptif. La surexpression de prot&#xE9;ines au cours des &#xE9;tapes NLZ gastrula inhibe le d&#xE9;veloppement du cerveau post&#xE9;rieur. En particulier, exprim&#xE9;s de mani&#xE8;re ectopique Nlz1 inhibe la formation de futurs rhombom&#xE8;res 2 et 3 (R2, R3), alors que voisins R1 et R4 ne sont pas affect&#xE9;s. Inversement, la r&#xE9;duction simultan&#xE9;e de Nlz1 et Nlz2 fonction de la prot&#xE9;ine par l&#x26;#39;expression de antisens morpholino-oligom&#xE8;res modification conduit &#xE0; l&#x26;#39;expansion de r3 futures et R5, avec une perte associ&#xE9;e de la R4. Ces donn&#xE9;es indiquent qu&#x26;#39;une fonction de la famille des g&#xE8;nes NLZ est de pr&#xE9;ciser ou de maintenir l&#x26;#39;identit&#xE9; r4, et de limiter R3 et R5 lors de la formation du cerveau post&#xE9;rieur.

NLZ g&#xE8;ne de la famille est requise pour la mod&#xE9;lisation du cerveau post&#xE9;rieur chez le poisson z&#xE8;bre

NLZ-Gen-Familie ist f&#xFC;r Hinterhirn Musterbildung im Zebrafisch ben&#xF6;tigt.

Questo studio descrive la famiglia genica nlz conservati i cui membri codificano proteine dito di zinco insolito. Nella neurectoderm di zebrafish, sia nlz1 e il nlz2 recente isolati sono espressi nel mesencefalo/hindbrain limite, dove &egrave; dipendente da pax2a espressione di nlz1 e di hindbrain presuntiva. Inoltre, nlz2 &egrave; univocamente espressa pi&ugrave; anteriormente, nella presuntiva mesencefalo e diencefalo. Sovraespressione di proteine Nlz durante fasi di gastrula inibisce lo sviluppo hindbrain. In particolare, ectopically espresso Nlz1 inibisce la formazione di futuri rhombomeres 2 e 3 (r2, r3), considerando che la r4 e r1 vicini non sono interessati. Al contrario, la simultanea riduzione della proteina Nlz1 e Nlz2 funzione mediante espressione dell&#39;antisenso modificati oligonucleotides oligomeri conduce all&#39;espansione del futuro r3 e r5, con associata perdita della r4. Questi dati indicano che una funzione della famiglia genica nlz &egrave; di specificare o mantenere identit&agrave; r4 e limitare r3 e r5 durante la formazione del hindbrain.

famiglia genica nlz &egrave; necessaria per la creazione di serie in the zebrafish hindbrain.

&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x305D;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306F;&#x5909;&#x308F;&#x3063;&#x305F;&#x30B8;&#x30F3;&#x30AF;&#x30D5;&#x30A3;&#x30F3;&#x30AC;&#x30FC;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x30B3;&#x30FC;&#x30C9;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;nlz&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;neurectoderm&#x3067;&#x306F;&#x3001;nlz1&#x3001;&#x65B0;&#x305F;&#x306B;&#x5206;&#x96E2;&#x3055;&#x308C;&#x305F;nlz2&#x4E21;&#x65B9;&#x304C;&#x63A8;&#x5B9A;&#x5F8C;&#x8133;&#x3068;nlz1&#x306E;&#x767A;&#x73FE;&#x304C;pax2a&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x308B;&#x4E2D;&#x8133;/&#x5F8C;&#x8133;&#x5883;&#x754C;&#x3067;&#x767A;&#x73FE;&#x3055;&#x308C;&#x308B;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;nlz2&#x3092;&#x4E00;&#x610F;&#x306B;&#x63A8;&#x5B9A;&#x4E2D;&#x8133;&#x3068;&#x9593;&#x8133;&#x306B;&#x3001;&#x3088;&#x308A;&#x524D;&#x65B9;&#x306B;&#x8868;&#x73FE;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x539F;&#x8178;&#x80DA;&#x6BB5;&#x968E;Nlz&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x904E;&#x5270;&#x767A;&#x73FE;&#x306F;&#x5F8C;&#x8133;&#x306E;&#x767A;&#x9054;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x3002;&#x8FD1;&#x96A3;&#x306E;R1&#x3068;R4&#x304C;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3044;&#x306A;&#x3044;&#x306E;&#x306B;&#x5BFE;&#x3057;&#x3001;&#x7279;&#x306B;&#x3001;&#x7570;&#x6240;&#x7684;&#x306B;&#x767A;&#x73FE;Nlz1&#x306F;&#x3001;&#x5C06;&#x6765;rhombomeres 2&#x304A;&#x3088;&#x3073;3&#xFF08;R2&#x3001;R3&#xFF09;&#x306E;&#x5F62;&#x6210;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x3002;&#x9006;&#x306B;&#x3001;&#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9;&#x30E2;&#x30EB;&#x30D5;&#x30A9;&#x5909;&#x6027;&#x30AA;&#x30EA;&#x30B4;&#x30DE;&#x30FC;&#x306E;&#x767A;&#x73FE;&#x306B;&#x3088;&#x308A;Nlz1&#x3068;Nlz2&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6A5F;&#x80FD;&#x306E;&#x540C;&#x200B;&#x200B;&#x6642;&#x6E1B;&#x5C11;&#x306F;&#x3001;R4&#x306E;&#x95A2;&#x9023;&#x640D;&#x5931;&#x306F;&#x3001;&#x5C06;&#x6765;&#x306E;R3&#x3068;R5&#x306E;&#x62E1;&#x5927;&#x306B;&#x3064;&#x306A;&#x304C;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;nlz&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x4E00;&#x3064;&#x306E;&#x95A2;&#x6570;&#x306F;&#x3001;&#x6307;&#x5B9A;&#x3059;&#x308B;&#x304B;&#x3001;&#x307E;&#x305F;&#x306F;&#x7DAD;&#x6301;&#x3059;&#x308B;R4&#x306E;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x3092;&#x3001;&#x3068;&#x5F8C;&#x8133;&#x306E;&#x5F62;&#x6210;&#x4E2D;&#x306B;R3&#x3068;R5&#x3092;&#x5236;&#x9650;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

Nlz&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306F;&#x3001;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x5F8C;&#x8133;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;

&#xC774; &#xC5F0;&#xAD6C; &#xBCF4;&#xC874;&#xB41C; nlz &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC744; &#xAD6C;&#xC131;&#xC6D0;&#xC774; &#xC778;&#xCF54;&#xB529; &#xD2B9;&#xC774; &#xD55C; &#xC544;&#xC5F0; &#xC190;&#xAC00;&#xB77D; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. Zebrafish neurectoderm nlz1 &#xBC0F; &#xC0C8;&#xB85C; &#xACA9;&#xB9AC; &#xB41C; nlz2 &#xCD94;&#xC815; hindbrain&#xC5D0; midbrain/hindbrain &#xACBD;&#xACC4;, nlz1&#xC758; &#xC2DD; pax2a&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, nlz2 &#xCD94;&#xC815; midbrain diencephalon&#xC5D0; anteriorly &#xB354; &#xD45C;&#xD604; &#xACE0;&#xC720; &#xD558; &#xAC8C; &#xB429;&#xB2C8;&#xB2E4;. Gastrula &#xB2E8;&#xACC4; &#xB3D9;&#xC548; Nlz &#xB2E8;&#xBC31;&#xC9C8;&#xC758; overexpression hindbrain &#xAC1C;&#xBC1C;&#xC744; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;. &#xD2B9;&#xD788;, ectopically &#xD45C;&#xD604;&#xB41C; Nlz1 &#xC774;&#xC6C3; r 1&#xC640; r 4 &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xC9C0; &#xC54A;&#xB294; &#xBC18;&#xBA74; 2, 3 (r2, r3), &#xD5A5;&#xD6C4; rhombomeres&#xC758; &#xD615;&#xC131;&#xC744; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;. &#xBC18;&#xB300;&#xB85C;, Nlz1 &#xBC0F; Nlz2 &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB3D9;&#xC2DC; &#xAC10;&#xC18C; antisense &#xC62C;&#xB9AC;&#xACE0; morpholino &#xC218;&#xC815; &#xB9AC;&#xB4DC; &#xBBF8;&#xB798; r 3&#xC640; r5, r 4&#xC758; &#xAD00;&#xB828; &#xC190;&#xC2E4; &#xD655;&#xB300;&#xC758; &#xC2DD;&#xC5D0; &#xC758;&#xD574; &#xC791;&#xB3D9;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; nlz &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC758; 1 &#xAC1C;&#xC758; &#xAE30;&#xB2A5; &#xC9C0;&#xC815; &#xB610;&#xB294; r4 &#xC815;&#xCCB4;&#xC131;&#xC744; &#xC720;&#xC9C0; &#xD558; &#xACE0; r 3&#xC640; r5 hindbrain &#xD615;&#xC131; &#xB3D9;&#xC548; &#xC81C;&#xD55C; &#xD558;&#xB294; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

nlz &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871; hindbrain &#xD328;&#xD134;&#xD654; &#xB41C; zebrafish&#xC5D0; &#xD544;&#xC694; &#xD558;&#xB2E4;.

Este estudio describe la conserva de la familia, cuyos miembros nlz gen codifica las prote&#xED;nas dedo de zinc inusuales. En el pez cebra neurectoderm, tanto nlz1 y los nlz2 reci&#xE9;n aisladas se expresan en el cerebro posterior presuntiva y el mesenc&#xE9;falo / metenc&#xE9;falo frontera, donde la expresi&#xF3;n de nlz1 depende de pax2a. Adem&#xE1;s, nlz2 se expresa singularmente m&#xE1;s anterior, en el mesenc&#xE9;falo y el dienc&#xE9;falo presuntiva. La sobreexpresi&#xF3;n de las prote&#xED;nas Nlz durante las etapas de g&#xE1;strula inhibe el desarrollo posterior del cerebro. En particular, expres&#xF3; ect&#xF3;pica Nlz1 inhibe la formaci&#xF3;n de los futuros rhombomeres 2 y 3 (r2, r3), mientras que el vecino R1 y R4 no se ven afectados. A la inversa, la reducci&#xF3;n simult&#xE1;nea de Nlz1 y Nlz2 funci&#xF3;n de las prote&#xED;nas mediante la expresi&#xF3;n de antisentido morfolino-modificado olig&#xF3;meros conduce a la expansi&#xF3;n de r3 futuras y R5, con la p&#xE9;rdida asociada de r4. Estos datos indican que una funci&#xF3;n de la familia de genes nlz es especificar o mantener la identidad R4, y para limitar R3 y R5 durante la formaci&#xF3;n del cerebro posterior.

Nlz Familia gen es necesario para modelar cerebro posterior en el pez cebra

Bcl-2 is the founding member of a family of proteins which influences cell survival in response to a variety of stimuli including those from growth factor receptors and integrins. However, how these activities are coordinated through bcl-2 requires further investigation. bcl-2 interacts with paxillin, potentially linking cell survival and cell adhesive pathways. Paxillin is an adapter protein implicated in growth factor and integrin-mediated signal transduction pathways. Previous work in this laboratory demonstrated that loss of bcl-2 affects cell adhesion and migration characteristics of renal epithelial cells, perhaps through disruption of its interaction with paxillin. Here studies were performed to determine the bcl-2 binding motif in paxillin. The amino-terminal portion of paxillin, specifically its LD4 motif, was found to associate with bcl-2. However, the amino-terminal portion of paxillin with the LD4 domain deleted did not associate with bcl-2. The corresponding LD motif in other paxillin family members, Hic-5 and leupaxin, did not associate with bcl-2. Mutations in paxillin's LD4 motif made to mimic Hic-5 and leupaxin LD4-like motifs (E(268) --> R or S(272) --> H) abolished its association with bcl-2. Incubation of embryonic kidneys with paxillin's LD4 motif disrupted ureteric bud branching and morphogenesis, while incubation with the comparable Hic-5 LD motif did not significantly affect morphogenesis. These data suggest that paxillin's association with bcl-2 plays a unique role during kidney development that other paxillin family members may not be able to fulfill.

Paxillin's LD4 motif interacts with bcl-2.

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2.

Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression.

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.

CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

Yixin Tang

Antibody Development Department

R&D Systems, Inc.

Induction and Analysis of Epithelial to Mesenchymal Transition