Orphan nuclear transcription factor TR3/Nur77 regulatesmicrovessel permeability by targeting endothelial nitricoxide synthase and destabilizing endothelial junctionsDezheng Zhaoa,b,1, Liuliang Qinc,1, Pierre-Marie Bourbonc,1, Lauralee Jamesd, Harold F. Dvorakb,c, and Huiyan Zengb,c,d,2

aDivision of Gastroenterology, bCenter for Vascular Biology Research, cDepartment of Pathology, and dDivision of Molecular and Vascular Medicine andDepartment of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215

Edited by Rakesh K. Jain, Harvard Medical School and Massachuestts General Hospital, Boston, MA, and approved June 14, 2011 (received for reviewDecember 14, 2010)

Low-level basal vascular permeability (BVP) provides nutrients tonormal tissues, and increased vascular permeability is characteris-tic of inflammation and cancer. We recently reported that VEGF-A,a potent vascular permeabilizing and angiogenic factor, exertsmuch of its angiogenic activity by up-regulating expression of TR3/Nur77, an orphan nuclear transcription factor, in vascular endo-thelial cells (EC). To determine whether TR3/Nur77 had a moregeneral role in regulating vascular permeability, we found thathistamine, serotonin, and platelet-activating factor, small moleculevascular permeabilizing agents, also increased TR3/Nur77 expres-sion acutely in EC. BVP and the acute vascular hyperpermeability(AVH) induced by these vascular permeabilizing factors weregreatly decreased in Nur77−/− mice, and both BVP and AVH corre-lated with Nur77 expression levels in several different mousestrains. BVP and AVH were enhanced in transgenic mice in whichNur77 was selectively overexpressed in vascular EC, whereas bothwere suppressed in mice overexpressing dominant-negative Nur77.Chronic vascular hyperpermeability (CVH) was induced long be-fore the onset of angiogenesis in a modified, in vivo Matrigelassay that included PT67 cells packaging retroviruses expressingNur77-sense, whereas inclusion of cells packaging viruses ex-pressing Nur77-antisense prevented VEGF-A–induced CVH. TR3/Nur77 modulated vascular permeability by increasing endothelialnitric-oxide synthase expression and bydownregulating several ECjunction proteins that maintain vascular homeostasis. Both func-tions required TR3/Nur77 transcriptional activity. Taking these datatogether, TR3/Nur77 is up-regulated by several vascular permeabi-lizing agents and has critical roles inmediating BVP, AVH, and CVH.

VEGF | TR3 | endothelial NOS | VE-cadherin

TR3 (mouse homolog Nur77, rat homolog NGFI-B) is amember of the class IV subfamily of the orphan nuclear re-

ceptor superfamily of transcription factors (1). TR3/Nur77 playsimportant roles in tumor, lymphocyte, and neural growth andsurvival (2–5). Although Nur77−/− mice develop a normal vascu-lature and lack a developmental phenotype, we recently reportedthe unexpected finding that TR3/Nur77 is strongly up-regulatedby VEGF and has a critical role in pathological angiogenesis (6).VEGF-A was originally described as a potent vascular per-

meabilizing factor (7), and increased vascular permeability is acharacteristic property of “mother” vessels, the first new bloodvessels induced to form in pathological angiogenesis by VEGF-A–secreting tumors, healing wounds, and chronic inflammation(8). Furthermore, endothelial growth factors, such as bFGF andPDGF, which do not affect vascular permeability also did notinduce TR3/Nur77 expression (6). Therefore, we considered thepossibility that TR3/Nur77 might have an important role inregulating vascular permeability in its three different contexts(9): (i) Basal vascular permeability (BVP), the low-level leakageof plasma protein-poor fluid from normal capillaries that pro-vides tissues with nutrients; (ii) Acute vascular hyperpermeability(AVH), the acute, explosive, plasma protein-rich exudate that

proceeds from postcapillary venules in response to short-termexposure to vascular permeabilizing agents, such as VEGF-A;and (iii) Chronic vascular hyperpermeability (CVH), the long-term, plasma protein-rich exudate that extravasates from mothervessels in pathological angiogenesis. We now report that TR3/Nur77 has important roles in regulating vascular permeability inall three of these contexts and that its expression is regulated notonly by VEGF-A but also by the small molecule vascular per-meabilizing agents histamine, platelet-activating factor (PAF),and serotonin. Furthermore, TR3/Nur77, like VEGF, acts at leastin part by regulating the expression of endothelial nitric-oxidesynthase (eNOS) and endothelial cell (EC) junction proteins.

ResultsTR3/Nur77 Is Up-Regulated by Several Vascular Permeabilizing Agentsin Addition to VEGF-A.We hypothesized that TR3/Nur77 might beselectively up-regulated by other agents that increased micro-vascular permeability. Testing this hypothesis, we found that, likeVEGF-A165, three well-known, potent, small-molecule vascularpermeabilizing factors, histamine, PAF and serotonin, stronglyup-regulated TR3 mRNA and protein expression in humanumbilical vein endothelial cells (HUVEC) (Fig. 1 A and B).

BVP and AVH Are Severely Compromised in Nur77−/− Mice in Vivo.The finding that several vascular permeabilizing agents inducedTR3/Nur77 expression suggested that this gene product mightexert a regulatory effect on vascular permeability in vivo. Wetherefore performed the Miles assay in Nur77−/− vs. wild-typemice. The low-level BVP of control skin, or after HBSS injection,was significantly diminished in Nur77−/− mice, as was the AVHresponse induced by VEGF-A, histamine, serotonin, and PAF,even when administered at supraphysiological doses (Fig. 1C).Permeability induced in the peritoneal cavity by intraperitonealinjection of VEGF-A165 was similarly reduced in Nur77−/− mice(Fig. 1C). In addition, protein expression levels of Nur77 (Fig.1D), and both BVP and AVH (Fig. 1E), differed significantly infour different mouse strains, and in the same order: C57BL/6Nur77+/+ > A/J > BALBc >129SV>> C57BL/6 Nur77−/−.

Overexpression of TR3/Nur77 Is Sufficient to Induce CVH. We useda modified Matrigel assay to determine whether TR3/Nur77 wasable to induce CVH in vivo (6). SKMEL-2 melanoma cells

Author contributions: D.Z., L.Q., P.-M.B., and H.Z. designed research; D.Z., L.Q., P.-M.B.,L.J., and H.Z. performed research; D.Z., H.F.D., and H.Z. analyzed data; and D.Z., H.F.D.,and H.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1D.Z., L.Q., and P.-M.B. contributed equally to this work.2To whom correspondence should be addressed. E-mail: hzeng@bidmc.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018438108/-/DCSupplemental.

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transfected to overexpress VEGF-A165, and retrovirus-packagingPT67 cells engineered to express Nur77-related cDNAs, wereincorporated into Matrigel plugs that were implanted sub-cutaneously in nude mice. In this assay, the expectation is thatVEGF-A165 secreted by SKMEL/VEGF cells will induce nearbyvascular EC to divide and therefore to become susceptible toinfection with retroviruses secreted by PT67 packaging cells (6).As expected, inclusion of VEGF-A165-expressing SKMEL/VEGF cells stimulated angiogenesis and the formation of leakymother vessels at 3 d, but not earlier (Fig. 2A, lanes 2 and 3 vs.lane 1; quantification in Fig. S1). Both angiogenesis and micro-vascular permeability were strikingly inhibited when PT67 cellsencoding Nur77-antisense (AS) were included (lane 4). How-ever, when PT67 cells packaging Nur77-sense (S) were also in-cluded (lane 5), permeability developed within 12 h, long beforethe onset of EC division and 60 h before the permeability in-crease induced by SKMEL/VEGF cells.PT67/Nur77-S–expressing cells also rendered vessels hyper-

permeable in a 12-h time-frame, even in the absence of SKMEL/VEGF cells (Fig. 2A, lane 6). Furthermore, whereas the VEGFR-2/KDR inhibitor, SU1498, strikingly inhibited VEGF-A165

–

induced microvascular permeability, it had no effect on the vas-cular permeability induced by PT67 cells packaging retrovirusesexpressing Nur77-S (Fig. S2). Taken together, these data indicatethat Nur77 induces vascular hyperpermeability independentlyand downstream of VEGF-A, and therefore of VEGFR-2/KDR,the VEGF-A165 receptor primarily responsible for both angio-genesis and permeability.

Vascular Permeability in EC-Nur77-S and EC-Nur77-DN Mice. To de-termine whether overexpression of Nur77 in mouse endothelium

was sufficient to regulate microvessel permeability, we generatedFvb mice that selectively overexpressed Nur77 full-length cDNA(Nur77-S), or a dominant-negative mutant (Nur77-DN), in vas-cular EC under the control of a tetracycline-sensitive promoter(Fig. S3 A–C). Expression levels of Nur77-S and Nur77-DNmRNAs and protein in different organs of these mice are shownin Fig. S3 D–F. Following withdrawal of tetracycline, Fvb miceoverexpressing Nur77-S exhibited a striking, highly significant(P < 0.001) increase in BVP and AVH in multiple organs (Fig.2B; quantification in Fig. S4). In addition, vessel size increased asmother vessels formed (P < 0.001) but vascular density was notchanged 2 d after withdrawing tetracycline (Fig. 2C; quantifica-tion in Fig. S5). In Fvb Nur77-DN mice, BVP in skin and AVHin both skin and peritoneum (mesentery) were significantly re-duced from that of wild-type mice (Fig. 2D).

TR3/Nur77 Regulates eNOS Expression Through TranscriptionalActivity. VEGF-A regulates eNOS, the enzyme responsible forsynthesizing nitric oxide (NO), an important mediator of vascularpermeability (10, 11). To determine whether TR3 also regulatedeNOS expression, we transduced HUVEC with Flag-TR3-S, asdescribed (6) (Fig. S6), and found that TR3-overexpressing cellsexpressed greatly increased levels of eNOS, comparable to thoseinduced by stimulating control HUVEC with VEGF-A165 (Fig.3A). Overexpression of TR3 antisense cDNA (TR3-AS) or TR3siRNA (6) (Fig. S6) strikingly inhibited VEGF-A165-inducedeNOS expression (Fig. 3A).Like other orphannuclear receptors,TR3 is comprisedof ligand-

binding (LBD), transactivation (TAD), and DNA-binding (DBD)domains, and both TADandDBDare required for transcriptionalactivity (12). HUVEC transduced with TR3 constructs lacking the

Fig. 1. TR3/Nur77 expression is increased by several different permeability factors and regulates BVP and AVH. (A) HUVEC were stimulated with VEGF-A165

(10 ng/mL), histamine (10 μM), PAF (0.1 μM), or serotonin (10 μM) for 0.5 to 2 h. TR3 mRNA expression was determined by quantitative real-time RT-PCR (mean± SD). (B) Immunoblots demonstrating TR3 protein expression in HUVEC stimulated with histamine (10 μM), PAF (0.1 μM), or serotonin (10 μM) for indicatedtimes. (Right) MAPK protein loading control. (Ci) Representative macroscopic images of Miles vascular permeability assay in flank skin and mesentery ofC57BL/6+/+ (wild-type) mice (Upper) and C57BL/6 Nur77−/− mice (Lower) following intradermal injection of VEGF-A165, histamine, PAF and serotonin, or in-traperitoneal injection of VEGF-A165. Photos were taken 30 min after intravenous injection of 0.2 mL of 0.5% EB dye in saline. (Cii) Quantification of Milesassays in Ci. EB dye was extracted and quantified as microliter per gram (mean ± SD, four mice per group) (6). ***P < 0.001, Tukey–Kramer multiple com-parisons test (flank skin) or unpaired t test (mesentery), comparing Nur77+/+ vs. Nur77−/− mice. (D) Western blot demonstrating expression of Nur77 and eNOSin Nur77+/+ vs. Nur77−/− mice and in several different mouse strains. β-Actin, loading control. (E) Quantification of Miles assays in flank skin of indicated mousestrains following HBSS, VEGF-A165, histamine, or serotonin injection. EB dye was extracted and quantified (mean ± SD, four mice per group). *P < 0.05,**P < 0.01, ***P < 0.001, Tukey–Kramer test.

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LBDdomain (Fig. S6) expressed increased levels of eNOS, similarto those transduced with TR3-S, and eNOS expression was notfurther stimulated by added VEGF-A165 (Fig. 3A). In contrast,HUVEC transduced with TR3 constructs lacking either the TADor DBD domain (Fig. S6) only expressed low levels of eNOS thatwere not increased by VEGF-A165. eNOS protein expression wasgreatly reduced in Nur77−/− mice (Fig. 1D, lane 1 vs. 2). In addi-tion, eNOS expression levels paralleled those of Nur77 (Fig. 1D,lanes 3–5) and both BVP and AVH in several different mousestrains (Fig. 1E).TR3-S cells exhibited significantly greater permeability than

LacZ-transduced cells in EC transwell monolayer assays, aneffect reversed by N-nitro-L-arginine methyl ester (L-NAME)(Fig. 3B). Furthermore, the peritoneal (mesentery) hyperperme-ability of Fvb EC-Nur77-S mice was inhibited >80% by L-NAMEbut not by D-NAME (Fig. 3Ci). In addition, L-NAME (but notD-NAME) significantly inhibited the permeability response ob-

served in vivo in Matrigel plugs in which PT67 cells packagingNur77-S had been incorporated (Fig. 3Cii), and permeability wasgreatly reduced when such Matrigel plugs were implanted ineNOS−/− mice (Fig. 3Ciii).

TR3/Nur77 Suppresses Expression of Adherens and Tight JunctionProteins. EC junction proteins have also been implicated in al-tering vascular permeability (13–15). Like control HUVEC,LacZ-transduced HUVEC expressed increasing levels of themRNAs of both VE-cadherin and claudin 5 as they approachedconfluence, and this effect was strongly inhibited by Flag-fusedTR3-S transduction (Fig. 4A and Fig. S7). We performedWestern blots to determine whether TR3 affected the expressionof HUVEC junction proteins. Expression of the adherens junc-tion-associated proteins VE-cadherin, β-catenin, γ-catenin, andp120 was strikingly down-regulated in TR3-S HUVEC (Fig. 4B,lane 4 vs. lanes 2 and 3). Expression of the tight junction protein

Fig. 3. TR3 regulates eNOS expression through transcriptional activity. (A) HUVEC transduced with indicated TR3 constructs and LacZ (control) were stim-ulated with VEGF-A165 for 0 to 24 h. Cell extracts were immunoblotted with antibodies against eNOS (Left) and β-actin (loading control, Right). (B) Transwellpermeability assay in HUVEC transduced with LacZ or TR3-S ± L-NAME (mean ± SD, n = 4). At the 8.5-h time point, LacZ and TR3-S/L-NAME permeabilitydiffered significantly (P < 0.001) from TR3-S but not from each other (Tukey–Kramer test). (C) Miles permeability assays quantified as in previous figures. (Ci)BVP in peritoneum (mesentery) of EC-Nur77-S mice, greatly increased compared with wild-type mice, was significantly inhibited by L- but not D-NAME. (Cii) L-,but not D-NAME, greatly inhibited vascular permeability in Matrigel assays implanted in Nu/Nu mice in which P67 cells packaging retroviruses expressingNur77-S were incorporated. (Ciii) Nur77-S packaging cells induced expected increased permeability in Matrigel assays in wild-type but not in eNOS−/− mice.***P < 0.001, Mann–Whitney test.

Fig. 2. TR3/Nur77 expression and vascular permeability (A) Macroscopic images illustrate leakage of EB dye at 0.5, 1, and 3 d after implantation of Matrigelplugs with indicated contents of VEGF-A165

–secreting SKMEL/VEGF cells and PT67 cells packaging indicated cDNAs. Dye from 1- and 3-d time points wasextracted and quantified (Fig. S1, eight animals per group). At 1 d, lanes 5 and 6 differed significantly from all other lanes (P < 0.001, Tukey–Kramer test); at3 d, lanes 1 and 4 differed significantly from all other lanes (P < 0.001). (B) BVP, 30 min after intravenous EB dye injection, in selected organs of wild-type(Upper) and EC-Nur77-S mice (Lower), and AVH after VEGF-A165 injection in flank skin. EB dye extravasation was quantified and, in all organs except uterusand brain, EC-Nur77-S was significantly greater than wild-type Fvb mice (P < 0.001 for mesentery, testis, lung, kidney, heart, and skin ± HBSS or VEGF-A165; P <0.01 for liver, Mann–Whitney test). (C) Liver and heart sections immunostained for CD31. Mother vessels that formed in EC-Nur77-S transgenic mice weresignificantly enlarged (P < 0.001), but vascular density vs. control wild-type mice was unchanged (P = 0.15) (unpaired t test). Four mice per group, based onthree different transgenic founders. (D) EB dye leakage induced by HBSS or VEGF-A165 in flank skin and mesentery was significantly greater in wild-type Fvbmice than in EC-Nur77-DN mice 6 d after withholding tetracycline (**P < 0.01, ***P < 0.001, unpaired t test).

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claudin 5 was also substantially down-regulated, and that ofoccludin to a lesser extent; however, JAM-B, ZO-1, and CD31expression levels were not affected. Transduction of TR3-over-expressing HUVEC with Flag-fused VE-cadherin led to increasedexpression of VE-cadherin (Fig. S7), and partial restoration ofβ-catenin expression, but expression levels of the other junctionproteins were unaffected (Fig. 4B, lanes 4–6). L-NAME did notprevent the down-regulation of VE-cadherin, and β- and γ-catenin,but did allow partial restoration of claudin 5 (Fig. 4B, lane 1 vs. 4).We used immunofluorescence to determine the effects of

VEGF-A and TR3 on the cellular distribution of junction pro-teins. VEGF-A caused a rapid loss of junctional VE-cadherin inconfluent HUVEC transduced with LacZ, but this effect waslargely inhibited in HUVEC transduced with TR3-AS or TR3-siRNA (Fig. 4C). A similar loss of VE-cadherin and of severalother junction proteins occurred when HUVEC were transducedwith TR3-S in the absence of added VEGF-A (Fig. 4D). Ex-pression of both VE-cadherin and claudin 5 mRNAs were var-iably increased or decreased in different organs of Nur77−/− andEC-Nur77-S mice (Fig. S8).

Effects of TR3, VE-Cadherin, and Claudin 5 Expression on ECMonolayer Permeability. Macromolecules extravasate across cul-tured EC monolayers, at least in part, by an inter- or paracellularpathway that requires opening of adherens and tight junctions(13–15). The effects of TR3/Nur77 on junction protein expres-sion suggested that TR3 would affect EC monolayer perme-ability. HUVEC transduced with full-length TR3-S exhibited asubstantial increase in monolayer permeability (Fig. 4E). TR3’s

hyperpermeabilizing effect was partially abolished when TR3-SHUVEC were additionally transduced with either VE-cadherinor claudin 5 (Fig. S7), and transduction with both completelyinhibited the effects of TR3 transduction (Fig. 4E). VE-cadherinand claudin 5 had similar, permeability-inhibiting effects inMatrigel assays in vivo. Inclusion of P67 cells packaging retro-viruses expressing Nur77 cDNA rendered microvessels highlypermeable to the Evans blue (EB) dye-albumin complex (Fig.4F, lane 1). However, additional transduction with either VE-cadherin or claudin 5 reduced permeability by ∼50%, and, whenboth VE-cadherin and claudin 5 were transduced, permeabilitywas reduced by 90% (Fig. 4F, lanes 2–4).

TR3/Nur77 Transcriptional Activity Is Required for Regulating VE-Cadherin Expression and Microvascular Permeability. Both thetranscriptional and DNA binding domains are required forVEGF-induced angiogenesis (6) and were essential for regulat-ing eNOS expression (Fig. 3A). HUVEC transduced with Flag-tagged constructs exhibited, as expected, strong expression andnuclear staining (Fig. S9). TR3-S and TR3-ΔLBD–transducedHUVEC exhibited striking reductions in junctional VE-cadherinstaining; however, cells transduced with either TR3-ΔTAD orTR3-ΔDBD exhibited normal, strong junctional VE-cadherinstaining (Fig. 5A). Similar expression data were obtained for VE-cadherin in Western blots of HUVEC transduced with theseconstructs (Fig. 5B).Finally, we performed Matrigel assays to determine whether

transcriptional regulation was also essential for Nur77’s effectson vascular permeability in vivo (Fig. 5C). Inclusion of PT67 cells

Fig. 4. TR3/Nur77 regulates EC junction proteins and permeability. (A) LacZ and TR3-S–transduced HUVEC plated at 30% density were harvested at indicatedtimes for quantitative real-time RT-PCR assay of VE-cadherin and claudin 5 mRNAs, expressed as fold-change vs. the 12-h time point (mean ± SD, n = 2). (B)HUVEC transduced with indicated constructs were cultured for 48 h and immunoblotted for cell junction proteins. (C) Serum-starved HUVEC transduced withLacZ, TR3-AS, or TR3-siRNA were stimulated without (Left) or with 10 ng/mL VEGF-A165 for indicated times and stained with DAPI and antibodies againstVE-cadherin. (D) HUVEC transduced with LacZ or Flag-TR3-S were stained after 48-h culture with DAPI and indicated antibodies. (E) Transwell permeabilityassay on HUVEC that had been transduced with indicated constructs. Data (mean ± SD, n = 4). At the 8.5-h time point, LacZ and TR3-S+VE+Cl5 did not differsignificantly from each other, but both differed significantly (P < 0.01–0.001, Tukey–Kramer test) from the three other conditions. (F) Matrigels containingP67 cells packaging viruses expressing indicated constructs were implanted subuctaneously in nude mice (n = 4). Matrigels were harvested at 24 h, 30 min afterintravenous EB dye injection. Dye was extracted and quantified. Lane 1 differed significantly (P < 0.001) from lanes 2 to 4; lanes 2 and 3 differed significantlyfrom lane 4 (P < 0.01); lane 2 vs. lane 3, NS, not significant (Tukey–Kramer test).

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packaging retroviruses that expressed full-length Nur77-S, orNur77 lacking the LBD domain, caused increased EB dye leak-age, whether or not SKMEL/VEGF-A165 cells were included.However, inclusion of cells packaging retroviruses that expressedNur77 cDNAs lacking either the TAD or DBD domains strikinglyinhibited vascular permeability, even when VEGF-A165-secretingSKMEL cells were present.

TR3 Regulates eNOS and VE-Cadherin mRNA Stability. TR3 regulateseNOS and VE-cadherin expression through transcriptional ac-tivity (Figs. 3 and 5). However, eNOS andVE-cadherin promoterslack TR3 consensus binding elements. Therefore, we hypothe-sized that TR3 might regulate eNOS and VE-cadherin by affect-ing their mRNA stability. To test this possibility, we transducedHUVECwith full-length TR3 or LacZ (control). After culture for24 h, cells were treated with actinomycin D to inhibit mRNAsynthesis. RNA was then collected at various times thereafterand subjected to real-time PCR with eNOS and VE-cadherinprimers. Overexpression of TR3 increased eNOS mRNA stabilitybut decreased VE-cadherin mRNA stability (Fig. 6).

DiscussionOur data indicate that several vascular permeabilizing agents,VEGF-A, as well as the small molecules histamine, serotonin, andPAF, strongly and rapidly induce TR3 mRNA and protein ex-pression in vascular EC. These findings are of interest becausethese agents act through very different receptors: KDR/VEGFR-2in the case of VEGF-A and different G protein-coupled receptorsin the case of histamine, serotonin, and PAF (16). Furthermore,TR3/Nur77 had important roles in regulating vascular perme-ability in all three of its contexts, BVP, AVH, and CVH. More-over, it did so downstream and independent of VEGF-A and,at least in part, by transcriptionally regulating eNOS and ECjunction protein expression (Figs. 3 and 5). However, eNOS andVE-cadherin promoters lack TR3 consensus binding elements,indicating that TR3 regulates eNOS and VE-cadherin expressionindirectly. TR3/Nur77 could act through additional transcriptionfactors that do interact with the eNOS and VE-cadherin pro-moters.However, TR3 acts at least in part by regulating eNOS andVE-cadherin mRNA stability (Fig. 6), perhaps through micro-RNAs or RNA binding proteins. Further work will be required tosort out these mechanisms.

Our data raise several conundrums. The first is that Nur77−/−

mice are viable and develop a normal vasculature (17), whereasdeletion of even one copy of the VEGF gene is lethal (18). Thisdifficulty can be addressed by making the following reasonableassumptions: (i) Pathological angiogenesis and developmentalangiogenesis differ in many respects (9) and TR3/Nur77 has anessential role in the former but not in the latter; (ii) VEGF-A’spermeability-enhancing function, mediated through TR3/Nur77,is dispensable in developmental angiogenesis in that normallydeveloping blood vessels do not exhibit increased permeability;and (iii) VEGF-A’s endothelial cell viability function is mediatedthrough pathways that do not involve TR3/Nur77; closely relatedTR3 family members (Nurr1, Nor-1) may play a compensatoryrole (19, 20).A second conundrum concerns differences in vascular per-

meability in vivo and in vitro. The altered permeability inducedby eNOS or alterations in EC junction proteins in transwellassays takes place over a period of hours (Figs. 3B and 4E). Thisfinding stands in contrast to the AVH induced by VEGF-A165,histamine, serotonin, or PAF in vivo. Single injections of any ofthese permeability factors into skin, peritoneal cavity, and soforth induce a dramatic local increase in vascular permeabilitythat begins within a few minutes and is complete by 20 to 30 min.Furthermore, AVH in vivo is mediated, at least in part, bya transendothelial cell route through vesiculo-vacuolar organ-elles (VVOs), an interconnecting collection of vesicles andvacuoles that spans the cuboidal venular endothelium from lu-men to albumin (21, 22). Cultured endothelial cells, in contrast,are flattened cells that contain few VVOs and that have lost themajority of their cytoplasmic vesicles and caveolae (23). Theythus closely resemble the mother vessel EC associated withtumors and healing wounds (i.e., flattened, vesicle-poor EClacking pericyte coverage) (8, 24, 25). We suggest, therefore, thattranswell assays do not provide a useful model for BVP or AVH,but may closely model the CVH associated with mother vesselpermeability in pathological angiogenesis.The final conundrum also involves the role of TR3/Nur77 in

AVH and is the most puzzling. On the one hand, VEGF-A165

and the several other permeability factors tested were unable toincrease permeability when injected into TR3/Nur77−/− mice,even at supraphysiological doses (Fig. 1C); therefore, TR3/Nur77 clearly has an essential role in AVH. However, the ki-netics of AVH is much too rapid to be explained by activation ofa transcription factor that acted to induce expression of down-stream genes or regulate mRNA stability. One possibility is thatthese permeability factors interact with TR3/Nur77 by an addi-tional second mechanism that does not involve transcription.There is precedence for this in that another TR3 function, that ofinducing apoptosis in tumor cells, is independent of transcriptionand occurs when TR3 transmigrates from the nucleus to thecytoplasm (12, 26, 27). Another possibility is that TR3/Nur77,although necessary, is not by itself sufficient for inducing the

Fig. 5. TR3/Nur77 transcriptional activity is required for regulating ECjunction protein expression and permeability. (A) HUVEC transduced withindicated constructs were stained with DAPI and anti–VE-cadherin anti-bodies. (B) Cell extracts from HUVEC transduced as in Awere cultured for 3 dand subjected to immunoblotting. β-Actin, protein loading control. (C)Matrigel plugs, with PT67 cells packaging retroviruses expressing Nur77-S, orindicated mutant constructs, were implanted, without or with SKMEL cellsexpressing VEGF-A165, subcutaneously in nude mice and harvested 3 d later,30 min after intravenous injection of EB dye. Dye was extracted and quan-tified. **P < 0.01, ***P < 0.001; NS, not significant (Tukey–Kramer test).

Fig. 6. TR3 regulates eNOS and VE-cadherin mRNA stability. HUVECtransduced without or with LacZ (control) or TR3 were treated with acti-nomycin D for indicated times. RNA was subjected to real-time RT PCR witheNOS (Left) and VE-cadherin (Right) primers (n = 2 for real-time PCR assay).

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AVH induced by acutely acting permeability factors. Perhapsthese vascular permeabilizing factors act additionally at stepsdownstream of TR3/Nur77, for example to increase eNOS ex-pression, destabilize EC junctions, open VVOs, and so forth. Atpresent we have no evidence to distinguish among these possi-bilities and sorting them out will require additional investigation.

Materials and MethodsSee SI Materials and Methods for further details. Experiments were repeatedat least three times.

Animals. Nur77−/− mice were originally obtained from J. Millbrandt (Wash-ington University School of Medicine, St. Louis, MO) (17). Doubly trans-genic mice overexpressing Nur77-sense (S) or dominant-negative Nur77(Nur77-DN) in vascular endothelial cells under the control of a tetracycline-regulated promoter were derived as described in SI Materials and Methods.Tetracycline, L-NAME and D-NAME were administered in drinking water.

Cell Culture and Monolayer Permeability Assay. For cell culture, 3 × 105 HUVEC(Clonetics) were seeded on polycarbonate membranes in 24-well transwellchambers and transduced with retroviruses (6). Three days later, after cellsachieved confluence, 70 KDa fluoresceinated-dextran (Molecular Probes)was added to the upper chamber. Dye passing through the monolayer fromthe upper to the lower chamber was measured at successive intervals byfluorimetry (28).

Other Assays. TheMiles assay, modified in vivoMatrigel assays, Western blots,and immunohistochemistry were performed as previously described (6).Statistical tests are indicated in the figure legends.

ACKNOWLEDGMENTS. The authors thank Dr. Shou-Ching Shih for quanti-tative real time RT-PCR. This work was supported by National Institutes ofHealth Grant K01 CA098581 (to H.Z.), the American Cancer Society GrantsRSG CSM 109385 and R01CA133235 (to H.Z.), and RSG CSM 113297 (to D.Z.);National Institutes of Health Grants R21DK080970 (to D.Z.) and P01 CA-92644 (to H.F.D.); and by a contract from the National Foundation for CancerResearch (to H.F.D.).

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