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Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.018
      Caveolin-1 is an essential structural protein of caveolae, specialized plasma membrane organelles highly abundant in endothelial cells, where they regulate multiple functions including angiogenesis. Caveolin-1 exerts a tonic inhibition of endothelial nitric oxide synthase (eNOS) activity. Accordingly, caveolin-1 gene–disrupted mice have enhanced eNOS activity as well as increased systemic nitric oxide (NO) levels. We hypothesized that excess eNOS activity, secondary to caveolin deficiency, would mediate the decreased angiogenesis observed in caveolin-1 gene–disrupted mice. We tested tumor angiogenesis in mice lacking either one or both proteins, using in vitro, ex vivo, and in vivo assays. We show that endothelial cell migration, tube formation, cell sprouting from aortic rings, tumor growth, and angiogenesis are all significantly impaired in both caveolin-1–null and eNOS-null mice. We further show that these parameters were either partially or fully restored in double knockout mice that lack both caveolin-1 and eNOS. Furthermore, the effects of genetic ablation of eNOS are mimicked by the administration of the NOS inhibitor N-nitro-l-arginine methyl ester hydrochloride (L-NAME), including the reversal of the caveolin-1–null mouse angiogenic phenotype. This study is the first to demonstrate the detrimental effects of unregulated eNOS activity on angiogenesis, and shows that impaired tumor angiogenesis in caveolin-1–null mice is, at least in part, the result of enhanced eNOS activity.
      Caveolae are specialized plasma membrane subdomains that are highly abundant in terminally differentiated cells including endothelial cell (EC).
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      The formation and functions of these organelles require caveolin-1, a protein that structurally supports caveola biogenesis and interacts with multiple signaling molecules, directly regulating their activity. In the endothelium, one such signaling protein is endothelial nitric oxide (eNOS), and the role of caveolin-1 in regulating eNOS activity has been abundantly studied in vitro
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      Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice.
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      Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.
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      The literature documenting the role of caveolin-1 in angiogenesis shows a picture more complex than initially thought (reviewed by Parat
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      Antitumor effects of in vivo caveolin gene delivery are associated with the inhibition of the proangiogenic and vasodilatory effects of nitric oxide.
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      Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis.
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      Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells.
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      Similarly, the number of caveolae in the EC of capillaries regressing on anti-VEGF treatment increases dramatically.
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      Caveolin-1 is critical for the maturation of tumor blood vessels through the regulation of both endothelial tube formation and mural cell recruitment.
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      Caveolin-1-deficient mice have increased tumor microvascular permeability.
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      Endothelial caveolin-1 regulates pathologic angiogenesis in a mouse model of colitis.
      and cells derived from these mice exhibit reduced capillary-like tube formation in vitro
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      Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.
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      Caveolin-1 expression is critical for VEGF-induced ischemic hindlimb collateralization and nitric oxide-mediated angiogenesis.
      . Adenoviral-mediated caveolin-1 overexpression increased in vitro tube formation.
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      • Park D.S.
      • Lisanti M.P.
      Caveolin-1 expression enhances endothelial capillary tubule formation.
      Accordingly, down-regulation of caveolin-1 reduced EC transmigration to serum,
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      Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement.
      capillary like tube formation
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      Caveolin-1 expression enhances endothelial capillary tubule formation.
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      Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo.
      and angiogenesis in the chorioallantoic membrane assay.
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      Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo.
      These discrepant results suggest an ambiguous role for caveolin-1. It has been proposed that an optimal, physiological amount of caveolin-1 promotes normal angiogenesis, whereas excess or lack of caveolin-1 impairs angiogenesis.
      • Sonveaux P.
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      • Gregoire V.
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      • Balligand J.L.
      • Feron O.
      Caveolin-1 expression is critical for VEGF-induced ischemic hindlimb collateralization and nitric oxide-mediated angiogenesis.
      One major mechanism by which caveolin-1 and caveolae regulate angiogenesis involves endothelial production of nitric oxide (NO), a signaling molecule key to the angiogenic process.
      • Lee P.C.
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      • Shears L.L.
      • Watkins S.C.
      • Edington H.D.
      • Billiar T.R.
      Impaired wound healing and angiogenesis in eNOS-deficient mice.
      The regulation by caveolin-1 and caveolae of endothelial nitric oxide production occurs at multiple levels; while in unstimulated conditions, eNOS is maintained in an inactive state by interaction with the scaffolding domain of caveolin-1, activation requires the compartmentalization in caveolae of eNOS as well as upstream and downstream players of the pathway leading to nitric oxide production.
      • Sbaa E.
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      • Feron O.
      The double regulation of endothelial nitric oxide synthase by caveolae and caveolin: a paradox solved through the study of angiogenesis.
      • Solomonson L.P.
      • Flam B.R.
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      • Goodwin B.L.
      • Eichler D.C.
      The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells.
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      • Schmidt H.H.
      Calcium-dependent membrane association sensitizes soluble guanylyl cyclase to nitric oxide.
      In addition, caveola internalization has been shown to further regulate eNOS activation.
      • Maniatis N.A.
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      Novel mechanism of endothelial nitric oxide synthase activation mediated by caveolae internalization in endothelial cells.
      • Chatterjee S.
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      • Shah V.
      Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production.
      Therefore, caveolin-1 is a tonic inhibitor of eNOS but is required for caveola formation, which in turn is necessary for signaling cascades leading to eNOS activation and NO production. The direct inhibitory effect of caveolin-1 on eNOS has been taken advantage of by two in vivo approaches, both successful, to inhibit tumor angiogenesis: overexpression of caveolin-1 in angiogenic EC,
      • Brouet A.
      • DeWever J.
      • Martinive P.
      • Havaux X.
      • Bouzin C.
      • Sonveaux P.
      • Feron O.
      Antitumor effects of in vivo caveolin gene delivery are associated with the inhibition of the proangiogenic and vasodilatory effects of nitric oxide.
      and delivery of a peptide comprising the domain of caveolin-1 responsible for eNOS inhibition.
      • Gratton J.P.
      • Lin M.I.
      • Yu J.
      • Weiss E.D.
      • Jiang Z.L.
      • Fairchild T.A.
      • Iwakiri Y.
      • Groszmann R.
      • Claffey K.P.
      • Cheng Y.C.
      • Sessa W.C.
      Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice.
      In agreement with caveolin-1 being a tonic inhibitor of eNOS, caveolin-1–null mice display excessive NO production as a result of persistent eNOS activation.
      • Drab M.
      • Verkade P.
      • Elger M.
      • Kasper M.
      • Lohn M.
      • Lauterbach B.
      • Menne J.
      • Lindschau C.
      • Mende F.
      • Luft F.C.
      • Schedl A.
      • Haller H.
      • Kurzchalia T.V.
      Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.
      • Razani B.
      • Engelman J.A.
      • Wang X.B.
      • Schubert W.
      • Zhang X.L.
      • Marks C.B.
      • Macaluso F.
      • Russell R.G.
      • Li M.
      • Pestell R.G.
      • Di Vizio D.
      • Hou Jr, H.
      • Kneitz B.
      • Lagaud G.
      • Christ G.J.
      • Edelmann W.
      • Lisanti M.P.
      Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.
      • Zhao Y.Y.
      • Liu Y.
      • Stan R.V.
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      • Schmeisser A.
      • Strasser R.H.
      Disruption of caveolin-1 leads to enhanced nitrosative stress and severe systolic and diastolic heart failure.
      In the present study, we hypothesized that the angiogenic phenotype of caveolin-1 gene–disrupted mice was imputable at least in part to excess basic NO production. To test this hypothesis, we quantified tumor angiogenesis in double knock-out mice lacking both eNOS and caveolin-1, compared with caveolin-1 single knock-out, eNOS single knock-out, and wild-type mice as controls. We validated our findings from this genetic approach by using a pharmacological inhibitor of NO production in wild-type and caveolin-1 gene–disrupted mice.

      Materials and Methods

      Materials

      The following reagents were purchased from Gibco (Invitrogen, Carlsbad, CA): DMEM/F12, fetal bovine serum (FBS), penicillin/streptomycin, and type II collagenase. The following items were obtained from Sigma-Aldrich (St. Louis, MO): hematoxylin, alcoholic eosin, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT), heparin, collagenase, fibronectin, collagen from rat tail, Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME), Drabkin's reagent, hemoglobin, Brij's reagent, and dimethyl sulfoxide (DMSO). Human vascular endothelial growth factor (VEGF) was obtained from R&D Systems (Minneapolis, MN).

      Animals

      Caveolin-1–deficient (stock no. 004585) and eNOS-deficient (stock no. 002684) mice were obtained from Jackson Laboratories (Bar Harbor, ME). The experimental animals of the four genotypes of interest (Cav+/+eNOS+/+, Cav−/−eNOS+/+,Cav+/+eNOS−/−,Cav−/−eNOS−/−) were obtained through continuously renewed heterozygous matings to avoid selection of a background trait and to overcome poor breeding performance of the double knock-out mice. The Cav−/−eNOS−/− mice presented no obvious phenotypic alteration but were obtained at lower than Mendelian ratios. Experiments involving only caveolin-1 and wild-type mice were performed using littermates generated through simple heterozygous matings between Cav+/−eNOS+/+ mice. All animal experiments were approved by the Animal Ethics Committee of the University of Queensland or the Animal Care and Use Committee guidelines of the Cleveland Clinic.

      Isolation and Culture of Mouse Lung Endothelial Cells

      Lungs were harvested in ice-cold DMEM/F12 medium supplemented with FBS (10%), penicillin 50 U/mL, and streptomycin 50 μg/mL. The lungs were cut into small pieces, suspended in medium, and centrifuged at 1200 rpm for 5 minutes at room temperature. The tissue fragments were cut into smaller pieces, suspended in medium containing 0.5 mg/mL of type II collagenase and incubated for 30 minutes at 37°C with frequent agitation. The digest was passed through a 100-μm-mesh sieve and then through a 70-μm-mesh sieve (Becton Dickinson, North Ryde, NSW, Australia). The cells were suspended in medium supplemented with VEGF (2 ng/mL) and heparin (2 μg/mL). Cells from each mouse were seeded onto a fibronectin-coated dish (2 μg/mL in phosphate buffered saline [PBS]). The cells were cultured in a humidified atmosphere of 95% air and 5% CO2. Endothelial origin of the isolated cells was verified by uptake of 1,1′-dioctadecyl-3,3,3′-tetramethyl-indocarcocyanine perchlorate acetylated low-density lipoprotein (Dil-Ac-LDL), CD31 and von Willebrand factor (vWF) immunoreactivity. Cells from passage 2 were used for experiments.

      Characterization of Mouse Lung Endothelial Cells

      To test DiI-Ac-LDL uptake, mouse lung endothelial cells (MLEC) were seeded in eight-well chamber slides (Becton Dickinson). After overnight incubation, the culture medium was replaced with fresh medium containing 10 μg/mL of Dil- Ac-LDL (Invitrogen) and incubated for 6 hours. The cells were rinsed in PBS, fixed in 4% formaldehyde for 20 minutes, rinsed in PBS, and mounted in fluorescent mounting medium containing DAPI (Merck, Kilsyth, Australia). Immunostaining for CD31 and vWF were performed on cells similarly seeded in eight-well chamber slides. After overnight incubation, the cells were rinsed in PBS and fixed in 4% formaldehyde for 20 minutes. The cells were washed in PBS, permeabilized with 0.1% Triton X-100 for 10 minutes, washed in PBS, and blocked with 3% goat serum in PBS for 20 minutes. The cells were then incubated with monoclonal anti-mouse CD31 antibody (Sapphire Bioscience, Waterloo, Australia), or polyclonal anti-rabbit vWF, or rabbit or mouse nonimmune IgGs for 60 minutes. The antibodies were used at a concentration of 1 μg/mL in PBS containing 1.5% goat serum. After washing in PBS, the cells were incubated for 45 minutes with respective secondary antibodies (1:1000) labeled with Alexa Fluor 488 (Invitrogen). Cells were washed in PBS, and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) (see Supplemental Figure S1 at http://ajp.amjpathol.org).

      Cell Proliferation Assay

      The DNA content of cells was measured using the CyQUANT cell proliferation assay following the protocol of the manufacturer (Invitrogen). Briefly, 5 × 103 cells were seeded in 96-well black culture plates with optical bottom (Nalge Nunc International, NY) and grown for 48 hours. The culture medium was removed and the cells were incubated in 100 μl of serum-free medium or medium with 0.5% FBS for 24 hours. A 100-μL of CyQUANT reagent, prepared in culture medium, was added to the wells and incubated for a further 60 minutes. The fluorescence was measured using a microplate reader (FLUOstar Omega, BMG Laboratories, Victoria, Australia) with excitation at 480 nm and emission detection at 520 nm.

      Transmigration Assay

      The transmigration assay was performed using 48-well Boyden chambers as previously described.
      • Santilman V.
      • Baran J.
      • Anand-Apte B.
      • Fox P.L.
      • Parat M.O.
      Caveolin-1 polarization in migrating endothelial cells is directed by substrate topology not chemoattractant gradient.
      Briefly, polycarbonate membranes with 8-μm pores (Neuro Probe, Gaithersburg, MD) were precoated overnight with rat tail collagen (100 μg/mL in 0.2 N acetic acid), air dried, and used to separate the chambers. The bottom wells were prepared with serum-free medium or medium containing 0.5% FBS. After pH equilibration of the chambers in a humidified atmosphere of 95% air and 5% CO2, a suspension of 2 × 104 MLEC in 50 μl of serum-free medium was added to the upper wells. Migration was allowed to proceed for 4 hours. The membranes were washed in PBS, and the cells remaining in the upper surface of the membrane were scraped. The membranes were fixed in 10% buffered formalin for 20 minutes, stained with hematoxylin overnight, rinsed with water, and mounted in Permount mounting medium (Fisher Scientific, PA). Cells on the lower face of the membranes were counted under a microscope by an observer blinded to experimental conditions.

      Tube Formation Assay

      Wells of a 96-well plates were coated with 50 μL of Matrigel (BD Biosciences, North Ryde, NSW, Australia), and the matrigel was allowed to solidify at 37°C for 60 minutes. MLEC suspended in serum-free medium or in medium containing 0.5% FBS were seeded at a density of 2.5 × 104 cells onto the Matrigel. The organization of cells in tubes after 7 hours was documented by phase contrast micrographs. The number of tubes and the average tube length per field were determined.

      Aortic Ring Assay

      Aortas were harvested from mice, and the surrounding tissues were dissected out. Any residual blood clot was removed, and the aortas were sliced into approximately 1 mm-thick rings. The rings were embedded in a drop of Matrigel. Serum-free medium containing VEGF (2 or 20 ng/mL), or control serum-free medium was added to the dishes, to surround the Matrigel. The endothelial-driven cell sprouting from the rings is confirmed by lack of response in the absence of VEGF and the absence of outward cell migration after endothelial denudation of the rings. Radial outgrowth of cells from the rings was monitored by phase contrast microscopy and documented over a period of 7 days. The number of cells aligned in tubes and the number of tube-like structures were calculated using ImageJ software (National Institutes of Health, Bethesda, MD).

      Mouse Corneal Micropocket Assay

      Angiogenesis was tested in vivo using the corneal micropocket assay as previously described.
      • Ebrahem Q.
      • Chaurasia S.S.
      • Vasanji A.
      • Qi J.H.
      • Klenotic P.A.
      • Cutler A.
      • Asosingh K.
      • Erzurum S.
      • Nand-Apte B.
      Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo.
      Hydron pellets containing 50 ng recombinant human VEGF were inserted into corneal micropockets (1 mm from limbus) of Cav−/− (n = 6) and control WT littermates (n = 7). Control buffer-containing pellets were similarly inserted in corneas of Cav+/+ and control WT mice (n = 4 per group). Corneas were examined with a surgical microscope to monitor angiogenic responses to VEGF. Mice were euthanized 7 days after pellet insertion. To document the neovascular response, animals were perfused with India ink to label the vessels. After enucleation and fixation, the corneas were excised, flattened, and photographed. Angiogenic response was analyzed for mean vessel area using image processing software (Image-Pro Plus 6.1; Media Cybernetics).

      In Vivo Tumor Angiogenesis

      B16-F0 melanoma cells were suspended in ice-cold PBS at a density of 5 × 106 cells/mL. Five mice of each genotype were injected subcutaneously onto the right flank with 100 μl of the cell suspension. Food and water intake, body weight, and tumor growth of the animals were monitored throughout the experiment. For pharmacological NO inhibition experiments, the NOS inhibitor L-NAME was provided in the drinking water 48 hours before the injection of the cells at a concentration of 1 mg/mL for 24 hours, followed by 0.5 mg/mL for the remainder of the experiments. On day 13 after the injection of the cells, the animals were euthanized and the tumors were excised and weighed. Tumor tissue was fixed in ice-cold 4% buffered formalin or frozen and stored at −80°C.

      Hematoxylin and Eosin Staining

      Fixed tumors, were washed in PBS, and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, CA). Cryosections (5 μm) (Leica CM1850 cryostat) were stained with hematoxylin and eosin using standard procedures. The number of blood vessels per area (defined by the graticule of the eye piece, Leica 11527001) in each section, as a measure of neovascularization, was counted under a microscope.

      Hemoglobin Measurements

      To determine the hemoglobin (Hb) concentration of the tumor explants, equal amounts of tissue were suspended in distilled water and sonicated using a Sonics Vibra Cell sonicator (Sonics & Materials, CT) for 10 seconds. The suspension was centrifuged at 13,500 rpm at 4°C for 15 minutes. To quantify the hemoglobin content, 50 μL of the tumor suspension was mixed with 950 μL of Drabkin's reagent and incubated in the dark at room temperature for 15 minutes. The absorbance was read at 450 nm in an iMark micro plate reader (Bio-Rad, Hercules, CA). Human hemoglobin was used as a standard. Results were expressed as milligrams of hemoglobin per milligram of tissue.

      Statistical Analyses

      Results are expressed as mean ± SEM. Comparisons between groups were performed by analysis of variance with Tukey's post hoc test using Prism software (GraphPad, San Diego, CA). P < 0.05 was considered significant. Where Barlett's test for equal variances showed the variances differed significantly between groups, the data were log transformed before statistical analysis, as indicated where appropriate.

      Results

      VEGF-Induced Corneal Neovascularization Is Reduced in Caveolin-1–Null Mice

      The migration of activated endothelial cells across the degraded basement membrane toward the tumor site is an essential step in angiogenesis. We subjected endothelial cells isolated from Cav+/+ or Cav−/− mice to chemotactic transmigration, and showed that the ability of the Cav−/− cells to respond to serum was greatly reduced compared with cells from Cav+/+ mice (Figure 1A). When seeded on Matrigel, the Cav−/− cells also had a reduced ability to form capillary-like structures (Figure 1B). The ability of cells from endothelial-intact, freshly isolated vessels to proliferate and to migrate radially toward a gradient of VEGF is tested with the aortic ring assay. The number of outgrowing tube-like structures and the number of cells aligned in tube-like structures were significantly lower in rings explanted from Cav−/− mice (Figure 1C). To test angiogenesis in vivo, hydron micropellets containing VEGF or buffer were implanted into the corneas of Cav+/+ and Cav−/− mice, and neovascularization was assessed after 7 days. The response to VEGF was reduced in the Cav−/− mice compared with their wild-type littermates, and this was confirmed by quantification of the vessel area (Figure 1D).
      Figure thumbnail gr1
      Figure 1Effect of loss of Cav-1 on in vitro, ex vivo, and in vivo angiogenesis. A: EC were tested for their ability to migrate in a modified Boyden chamber assay using serum-free medium or 0.5% serum containing medium. Results are expressed as percentage of the migration of Cav+/+ cells toward no serum medium. B: Tube formation was tested when EC isolated from Cav+/+ and Cav−/− mice were plated on Matrigel. Representative pictures, number of tubes per field, and average tube length are shown and expressed as percentage of Cav+/+ cells. Scale bar = 400 μm. C: Representative micrographs of aortic ring explants placed in Matrigel surrounded by VEGF-containing, serum-free medium, and quantification of the number of cells aligned in tube-like structures and the number of tubes. Scale bar = 400 μm. D: Angiogenic response to VEGF in the corneal micropocket assay. Representative micrographs of angiogenic response to implants releasing buffer or VEGF in Cav+/+ and Cav−/− mice. Scale bar = 600 μm. Bar graph shows quantification of vessel area per sample. *P < 0.05 and ***P < 0.001.

      Decreased Endothelial Cell Migration and in Vitro Tube Formation in Caveolin-1 Gene–Disrupted Mice Are Restored by eNOS Genetic Ablation

      We used a modified Boyden chamber assay to study the role of caveolin-1 and eNOS in endothelial cell transmigration. No significant difference in random migration (toward serum-free medium) was observed among the four genotypes (Figure 2A). Wild-type endothelial cells (Cav+/+eNOS+/+) were capable of chemotactic stimulation (P < 0.001, 0% FBS versus 0.5% FBS). This chemotactic response was lost in caveolin-1–null (Cav−/−eNOS+/+) and in eNOS-null (Cav+/+eNOS−/−) MLEC. However, when both caveolin-1 and eNOS were absent, transmigration was partially restored although this was not statistically significant (Figure 2A). To rule out the possibility that the observed differences in transmigration were the reflection of differential proliferation rates, we measured proliferation of all four genotypes. No significant difference in proliferation was observed, in unstimulated or serum-stimulated conditions, between cells from the four genotypes (Figure 2B), confirming that the observed difference in transmigration was independent of proliferation. Next we examined the effect of caveolin-1 and eNOS genetic ablation on the ability of MLEC to form capillary-like tubes when seeded onto Matrigel. In serum-free and serum-stimulated conditions, tube formation, as measured by tube length or tube number, was significantly impaired in Cav−/−eNOS+/+ and Cav+/+eNOS−/− when compared to wild-type cells (Figure 2, C–E). In contrast, in Cav−/−eNOS−/− MLEC, tube formation was rescued, and increased when compared with either of the single knockouts, (Figure 2, C–E) and this effect was statistically significant in the presence of serum. In summary, the in vitro angiogenic potential of MLEC is impaired by genetic ablation of either caveolin-1 or eNOS, but partially or totally recovered when both proteins are absent.
      Figure thumbnail gr2
      Figure 2Effect of loss of eNOS, Cav-1, or both eNOS and Cav-1 on in vitro angiogenesis. A: EC were tested for their ability to migrate in a modified Boyden chamber assay using serum-free medium or 0.5% serum-containing medium. Results are expressed as percentage of the migration of Cav+/+eNOS+/+ cells toward no serum medium (n = 3). B: EC proliferation was tested using the CyQUANT DNA content assay, in serum-free medium and in medium containing 0.5% FBS (n = 4). C: Micrographs of EC tube formation on matrigel tested in serum-free medium and in medium containing 0.5% FBS. Scale bar = 400 μm. Tube length (D) and tube numbers (E) were calculated from printed micrographs. ns, Not statistically significant. **P < 0.01 and ***P < 0.001.

      Decreased ex Vivo Angiogenesis in Caveolin-1 Gene–Disrupted Mice Is Rescued by eNOS Genetic Ablation

      The migration of cells and the sprouting of tube-like structures from the aortic rings in response to VEGF was dose-dependent in Cav+/+eNOS+/+ explants (Figure 3A). Quantification of the number of cells aligned in tubes (Figure 3B) and of the tube-like structures (Figure 3C) in response to 20 ng/mL VEGF at daily time points, showed a significant decrease of both parameters in Cav−/−eNOS+/+ and Cav+/+eNOS−/− compared to Cav+/+eNOS+/+ aortic rings. In Cav−/−eNOS−/− aortic rings, sprouting was restored in a statistically significant fashion, returning to the levels of outmigration seen with Cav+/+eNOS+/+ rings (Figure 3, A–C). These ex vivo results therefore confirmed our in vitro data and indicate that genetic ablation of eNOS can compensate the angiogenic phenotype observed in caveolin-1–null mice.
      Figure thumbnail gr3
      Figure 3Effect of loss of eNOS, Cav-1, or both eNOS and Cav-1 on ex vitro angiogenesis. A: Representative micrographs of aortic ring explants placed in matrigel surrounded by serum-free medium or medium containing 2 or 20 ng/mL VEGF for 5 days. Scale bar = 400 μm. B: Quantification of the number of cells aligned in tube-like structures sprouting in response to 20 ng/mL of VEGF. C: Quantification of the number of tube-like structures sprouting in response to 20 ng/mL of VEGF. *P < 0.05 and **P < 0.01, Cav−/−eNOS+/+ and Cav+/+eNOS−/− versus Cav+/+eNOS+/+. P < 0.05 and ††P < 0.01, Cav−/−eNOS+/+ and Cav+/+eNOS−/− versus Cav−/−eNOS−/−.

      Impaired Tumor Growth and Angiogenesis in Vivo in Caveolin-1 Gene–Disrupted Mice Are Partially Recovered in Cav−/−eNOS−/− Mice

      To determine whether caveolin-1 and eNOS were similarly controlling in vivo angiogenesis, we performed a tumor angiogenesis assay with mice from each genotype. Tumor growth was markedly reduced in either Cav−/−eNOS+/+ or Cav+/+eNOS−/− mice compared to Cav+/+eNOS+/+ mice (Figure 4, A and B). However, tumor growth was significantly higher in Cav−/−eNOS−/− mice than in Cav−/−eNOS+/+ or Cav+/+eNOS−/− mice (Figure 4, A and B). H&E-stained sections of tumor tissue (Figure 4C) displayed a marked reduction in number of blood-containing vessels in tumors implanted either in Cav−/−eNOS+/+ or in Cav+/+eNOS−/− mice (Figure 4D). Confirming our in vitro and ex vivo experiments, the reduction in blood vessel counts was rescued in tumors from Cav−/−eNOS−/− mice, where the number of vessels was increased compared to Cav−/−eNOS+/+ or Cav+/+eNOS−/− (Figure 4D). Furthermore, the rescue as measured by the hemoglobin content of the tumors was also highly significant (Figure 4E).
      Figure thumbnail gr4
      Figure 4Effect of loss of eNOS, Cav-1, or both eNOS and Cav-1 on tumor growth and angiogenesis. A: Micrographs of representative tumors. Scale bar = 1 cm. B: Tumor weight 13 days after B16 cell implantation; n = 5. C: Representative H&E-stained sections of the tumors. Scale bar = 200 μm. D: Vessel count of the tumor samples in five randomly selected areas, expressed as number of blood vessels per field. E: Hemoglobin content of the tumors. *P < 0.05, **P < 0.01, ***P < 0.001. ns, Not significant.

      Pharmacological NOS Inhibition Recapitulates Effects of eNOS Genetic Ablation on in Vitro and ex Vivo Angiogenesis Alterations Induced by Loss of Caveolin-1

      To generate further evidence that excess NO mediates impaired angiogenesis in Caveolin-1-null mice, we performed experiments using the NOS inhibitor L-NAME. MLEC were exposed to increasing concentrations of L-NAME for 24 hours and cell viability assessed. Concentrations of up to 5 μmol/L L-NAME did not induce any changes in cell viability of MLEC (data not shown) and therefore, this concentration was used for further in vitro and ex vivo experiments. L-NAME did not induce any significant changes in random transmigration (toward serum-free medium) of Cav+/+eNOS+/+ or Cav−/−eNOS+/+ MLEC (Figure 5, A and B). However, L-NAME-treated Cav+/+eNOS+/+ MLEC displayed a dramatic decrease in chemotactic migration (toward 0.5% FBS) compared to untreated Cav+/+eNOS+/+ cells (Figure 4A). This confirms the key role played by nitric oxide production in EC chemotaxis. In contrast, L-NAME increased the chemotactic migration of Cav−/−eNOS+/+ EC (P < 0.001, Cav−/−eNOS+/+− L-NAME versus Cav−/−eNOS+/++ L-NAME) (Figure 5B), in agreement with the data obtained with genetic ablation of eNOS.
      Figure thumbnail gr5
      Figure 5Effect of the NOS inhibitor L-NAME on EC migration and ex vivo angiogenesis from wild-type and caveolin-1–null mice. A: Effect of L-NAME on transmigration of Cav+/+eNOS+/+ MLEC. B: Effect of L-NAME on transmigration of Cav−/−eNOS+/+ MLEC. **P < 0.01; ***P < 0.001. ns, Not significant. C: Representative micrographs of aortic rings showing endothelial cell in response to 20 ng/mL of VEGF at day 7. Scale bar = 400 μm. D: Quantification of the number of cells aligned in tube-like structures sprouting in response to 20 ng/mL of VEGF. E: Quantification of the number of tube-like structures sprouting in response to 20 ng/mL of VEGF. *P < 0.05; **P < 0.01; ***P < 0.001, cav−/−-L-NAME versus cav+/+-L-NAME. P < 0.05, cav−/−+L-NAME versus cav−/−-L-NAME.
      To study the ability of L-NAME to rescue the ex vivo angiogenesis impairment induced by loss of caveolin-1, aortic rings from Cav+/+eNOS+/+ or Cav−/−eNOS+/+ mice were embedded in Matrigel and subjected to serum-free medium containing 20 ng/mL VEGF, in the presence or absence of 5 μmol/L L-NAME for up to 7 days. Representative micrographs of aortic rings at day 7 are shown in Figure 5C. L-NAME impaired the ability of EC from WT rings to respond to VEGF (Figure 5, D and E). However, L-NAME treatment restored EC sprouting in Cav−/−eNOS+/+ aortic rings (Figure 5C). The number of Cav−/−eNOS+/+ aligned cells (Figure 5D) and tube-like structures (Figure 5E) were increased at days 5 to 7 in L-NAME–treated Cav−/−eNOS+/+ rings when compared with untreated Cav−/−eNOS+/+ rings, reaching statistical significance on day 7.

      NOS Inhibition Decreases Tumor Growth and Angiogenesis in Cav+/+eNOS+/+ Mice but Increases Them in Cav−/−eNOS+/+ Mice

      To test whether NOS inhibition could rescue the deficit in angiogenesis induced by the loss of caveolin-1 in vivo, we performed a tumor angiogenesis assay in wild-type or caveolin-1–null mice exposed to L-NAME. Representative tumors from Cav+/+eNOS+/+ and Cav−/−eNOS+/+ mice treated or not treated with L-NAME are shown in Figure 6A. In response to L-NAME, tumor growth was significantly decreased in Cav+/+eNOS+/+ mice (Figure 6, A and B). In contrast, L-NAME increased tumor growth in Cav−/−eNOS+/+ mice (Figure 6, A and B). H&E-stained sections of tumors from L-NAME–treated Cav+/+eNOS+/+ mice displayed a significant decrease in blood vessels when compared with tumors from control Cav+/+ eNOS+/+ mice (Figure 6C). In contrast, L-NAME dramatically increased the number of blood vessels in tumors from Cav−/−eNOS+/+ mice (Figure 6D). This was apparent from counting the vessels as well as measuring the tumor hemoglobin content (Figure 6, C–E), and was statistically significant. In summary, NOS pharmacological inhibition recapitulated the effect of genetic ablation of eNOS in rescuing the angiogenesis defect induced by the loss of caveolin-1 in vivo as well as in vitro.
      Figure thumbnail gr6
      Figure 6Effect of the NOS inhibitor L-NAME on tumor growth and angiogenesis in Cav+/+eNOS+/+ and Cav−/−eNOS+/+ mice. L-NAME was given in the drinking water 48 hours before B16 cell implantation and for the duration of the assay. A: Representative micrographs of tumors from each group. Scale bar = 1 cm. B: Tumor weight 13 days after inoculation of the B16 cells (n = 5). For statistical analysis data, were log transformed, as Barlett's test showed significantly different variances. C: Representative H&E-stained sections. Scale bar = 200 μm. D: Vessel count from H&E-stained sections (n = 4). E: Quantification of the hemoglobin content in tumor tissue (n = 5). Data were log transformed, as Barlett's test showed significantly different variances. *P < 0.05; **P < 0.01; and ***P < 0.001.

      Discussion

      Our results demonstrate reduced angiogenesis elicited by VEGF in the corneal micropocket assay in caveolin-1 gene–disrupted mice. By in vitro, ex vivo, and in vivo experiments, our results further confirm previous studies showing that the individual loss of eNOS
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      impairs the angiogenic response elicited by B16 melanoma cells inoculated in vivo. In addition, we show for the first time that reduced tumor angiogenesis in caveolin-1–null mice is due to unabated endothelial nitric oxide production, as genetic disruption of eNOS in the caveolin-1–null background partially or totally restores angiogenesis quantitatively. Therefore, when combined, the lack of caveolin-1 and the lack of eNOS do not act in an additive or synergistic fashion to decrease angiogenesis but instead counteract each other, and their combination rescues the decrease seen with each individual loss.
      Decreased pathological neovascularization in caveolin-1–null mice has not been unanimously reported. Decreased angiogenesis was shown using bFGF-containing matrigel plugs,
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      Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.
      VEGF-expressing adenoviral vector,
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      Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.
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      Caveolin-1 is critical for the maturation of tumor blood vessels through the regulation of both endothelial tube formation and mural cell recruitment.
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      Caveolin-1-deficient mice have increased tumor microvascular permeability.
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      Our experiments show that ablation of endothelial NO production can increase angiogenesis in caveolin-1–null mice. This may seem counterintuitive, as endothelial NO is clearly pro-angiogenic: NO has been shown to promote increased vascular permeability, endothelial cell migration and proliferation,
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      Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.
      or interleukin-1 receptor–associated kinase.
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      • Garcia-Cardena G.
      • Madri J.A.
      • Sessa W.C.
      Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.
      It is well known that superoxide can be produced by eNOS itself in certain conditions.
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      • Wever R.
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      • van Faassen E.E.
      Origin of superoxide production by endothelial nitric oxide synthase.
      Interestingly, increased superoxide levels have been detected in cardiac and vascular tissues from caveolin-1–null mice but returned to normal after prolonged L-NAME treatment.
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      Nitric oxide synthases are crucially involved in the development of the severe cardiomyopathy of caveolin-1 knockout mice.
      If increased endothelial NOS activity due to caveolin-1 ablation causes decreased endothelial cell migration, tube formation, and angiogenesis, as indicated by our results, then overexpression of eNOS would be expected to have the same consequences. This is not observed, as transgenic eNOS mice have been generated
      • van Haperen R.
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      and display an increased, rather than decreased, neovascularization in response to hind limb ischemia.
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      This indicates that neovascularization is not simply proportional to the activity of eNOS or the amount of NO produced. Other factors may include the circumstances in which eNOS is able to produce superoxide, and the subcellular localization of eNOS, which in the absence of caveolae cannot be compartmentalized with upstream and downstream partners of the pathways leading to NO production. Targeting of eNOS to different cellular compartments has been shown to regulate the mechanism and degree of its activation
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      Functional relevance of Golgi- and plasma membrane-localized endothelial NO synthase in reconstituted endothelial cells.
      ; and, given than NO is short lived, the subcellular localization of its production is known to affect its actions, including protein S-nitrosylation.
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      Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking.
      Another way of interpreting our results is that ablation of caveolin-1 rescues the altered angiogenesis seen in the absence of eNOS. This could be hypothesized to be the result of hyperactivation, in the absence of caveolin-1 and caveolae, of pro-angiogenic proteins other than eNOS. Alternatively, antiangiogenic receptors that need caveolae localization or interaction with caveolin-1 may be inactive when caveolin-1 is ablated. Prostacyclin synthase produces the potent vasodilator prostacyclin in response to VEGF
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      Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.
      and promotes angiogenesis.
      • Hiraoka K.
      • Koike H.
      • Yamamoto S.
      • Tomita N.
      • Yokoyama C.
      • Tanabe T.
      • Aikou T.
      • Ogihara T.
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      • Koike H.
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      • Matsumoto K.
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      • Yokoyama C.
      • Tanabe T.
      • Ogihara T.
      • Kaneda Y.
      Enhanced angiogenesis and improvement of neuropathy by cotransfection of human hepatocyte growth factor and prostacyclin synthase gene.
      Prostacyclin synthase is located in caveolae where it interacts with caveolin-1.
      • Spisni E.
      • Griffoni C.
      • Santi S.
      • Riccio M.
      • Marulli R.
      • Bartolini G.
      • Toni M.
      • Ullrich V.
      • Tomasi V.
      Colocalization prostacyclin (PGI2) synthase–caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis.
      The angiopoietin receptor TEK/Tie2 exerts tight control on angiogenesis (maintaining them quiescent on angiopoetin-1 binding, but promoting endothelial cell activation on angiopoietin-2 binding) and has also been suggested to localize in endothelial cell caveolae.
      • Yoon M.J.
      • Cho C.H.
      • Lee C.S.
      • Jang I.H.
      • Ryu S.H.
      • Koh G.Y.
      Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells.
      CD36 is a critical anti-angiogenic receptor of endothelial cells
      • Simantov R.
      • Silverstein R.L.
      CD36: a critical anti-angiogenic receptor.
      which co-fractionates with caveolae and co-immunoprecipitates with caveolin-1.
      • Uittenbogaard A.
      • Shaul P.W.
      • Yuhanna I.S.
      • Blair A.
      • Smart E.J.
      High density lipoprotein prevents oxidized low density lipoprotein- induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae.
      This suggests that caveolin-1, and caveolae, may regulate angiogenesis by multiple pathways, including NO-mediated and NO-independent mechanisms (Figure 7).
      Figure thumbnail gr7
      Figure 7Proposed regulation of angiogenesis by caveolin-1 and eNOS. A: When both proteins are expressed, caveolin-1 regulation of eNOS compartmentalization and activation promotes optimal endothelial NO production in response to proangiogenic signals. B: In the absence of caveolin-1, excessive endothelial nitric oxide production, together with increased superoxide production, results in impaired angiogenesis. C: In eNOS knockout mice, insufficient endothelial nitric oxide production prevents optimal neovascularization. D: In mice lacking both eNOS and caveolin-1, endothelial nitric oxide production is reduced, but other angiogenic pathways are activated in the absence of caveolin-1, resulting in eNOS-independent angiogenesis.

      Acknowledgments

      We gratefully acknowledge the help of JoAnne Baran and Mila Ellard in the maintenance of the animal colony.

      Supplementary data

      • Supplemental Figure S1

        Characterization of endothelial cells prepared from each genotype. Cells were immunolabeled to detect von Willebrand factor (vWf) or CD31, or labeled with Dil-Ac-LDL as described. The total cell population is shown with either phase-contrast images (vWF and CD31 immunolabels) or DAPI nuclear staining (DiI-Ac-LDL labeling). Scale bar = 200 μm.

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