Histone deacetylases induce angiogenesis by negative ... - Nature

© 2001 Nature Publishing Group http://medicine.nature.com

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Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes MYOUNG SOOK KIM1, HO JEONG KWON3, YOU MIE LEE1, JIN HYEN BAEK1, JAE-EUN JANG1, SAE-WON LEE1, EUN-JOUNG MOON1, HAE-SUN KIM1, SEOK-KI LEE1, HAE YOUNG CHUNG2 CHUL WOO KIM4 & KYU-WON KIM5 1 3

Department of Molecular Biology, 2Department of Pharmacy, Pusan National University, Pusan, Korea Department of Bioscience and Biotechnology, Sejong University, Seoul, Korea4Department of Pathology, College of Medicine and 5Angiogenesis Research Laboratory, College of Pharmacy, Seoul National University, Seoul, Korea. Correspondence should be addressed to K.-W. K.; e-mail: [email protected]

Low oxygen tension influences tumor progression by enhancing angiogenesis; and histone deacetylases (HDAC) are implicated in alteration of chromatin assembly and tumorigenesis. Here we show induction of HDAC under hypoxia and elucidate a role for HDAC in the regulation of hypoxia-induced angiogenesis. Overexpressed wild-type HDAC1 downregulated expression of p53 and von Hippel–Lindau tumor suppressor genes and stimulated angiogenesis of human endothelial cells. A specific HDAC inhibitor, trichostatin A (TSA), upregulated p53 and von Hippel–Lindau expression and downregulated hypoxia-inducible factor-1α and vascular endothelial growth factor. TSA also blocked angiogenesis in vitro and in vivo. TSA specifically inhibited hypoxia-induced angiogenesis in the Lewis lung carcinoma model. These results indicate that hypoxia enhances HDAC function and that HDAC is closely involved in angiogenesis through suppression of hypoxia-responsive tumor suppressor genes.

The acetylation and deacetylation of histones have significant roles in regulation of gene transcription in many cell types1. Until now, studies on HDAC have been focused on its role as a corepressor in the chromatin remodeling process because HDAC associates with specific transcription factors and contributes to repression of these genes2,3 . Specific HDAC inhibitors have been used to elucidate HDAC function and suggested as a therapy for some types of cancer4. Deregulation of HDAC recruitment to specific promoters of genes involved in cell-cycle progression and differentiation seems to be one of the mechanisms by which HDAC contributes to tumorigenesis5. However, more precise mechanisms underlying involvement of HDAC in tumorigenesis have not yet been defined. Hypoxia, a common feature of malignant tumors, can be detected in central regions of solid tumors as well as in embryonic development6,7,8. Hypoxia regulates many transcription factors including hypoxia-inducible factor (HIF)-1α, which controls hypoxia-inducible angiogenic factors such as vascular endothelial growth factor9 (VEGF). VEGF functions as a survival factor in endothelial10,11 and tumor cells12 via VEGF receptors that are upregulated by hypoxia12,13,14. Thus, enhancement of angiogenesis by hypoxia is a prerequisite for progressive growth and metastasis of solid tumors. Here we investigated HDAC regulation under hypoxia and the effect of TSA on angiogenesis. We found that HDAC1 is hypoxiainducible and that TSA has a potent anti-angiogenic effect, which is more evident in hypoxia-induced angiogenesis. In addition, TSA induce expression of p53 and von Hippel-Lindau (VHL) under hypoxic conditions, whereas it reduced the expression of HIF-1α and VEGF. Correspondingly, expression of p53 and VHL were decreased by HDAC1 overexpression, but expression of HIF1α and VEGF were induced. Moreover, we found that HDAC1 stimulated angiogenesis in vitro and in vivo, suggesting a signifiNATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001

cant role for HDAC in hypoxia-induced angiogenesis. Modulation of HDAC activity in hypoxic condition We determined HDAC activity of HepG2 human hepatoblastoma cells in conditions of hypoxia, hypoglycemia, 0% serum or 0% serum combined with hypoxia. All conditions significantly increased HDAC activity compared to the control (Fig. 1a). Among them, cells exposed to hypoxia or serum-deprived hypoxia for 16 hours showed a relatively higher increase. We also compared HDAC activity in different cell lines: bovine aortic endothelial cells (BAECs), human Chang liver cells, human umbilical vein endothelial cells (HUVECs) and HepG2 cells. The HDAC activity was similar in each cell line under normoxia and significantly increased with hypoxia. Compared to other cell lines, HepG2 cells showed the most sensitivity to hypoxia (Fig. 1b). When the changes of HDAC activity in HepG2 cells were evaluated over time, the hypoxia-induced activity responded in a time-dependent manner, reaching a maximum at 16 hours (Fig. 1c). At the same time, mRNA and protein expression of HDAC1 was upregulated by 4 and 16 hours of hypoxia (Fig. 1d), and expression of HDAC2 and HDAC3 was also induced by hypoxia (data not shown). These results indicate that increased HDAC in hypoxia might be involved in oxygen-regulated gene expression and hypoxia-induced angiogenesis. Effect of TSA on the expression of p53, VHL, HIF-1α and VEGF Hypoxia-induced HDAC activity was effectively blocked by TSA as verified in HDAC assays (Fig. 2a) and acid-urea-triton (AUT) gel analysis15, which revealed hyperacetylated histones (Fig. 2b, lane 3). Also, acetylation of histone species decreased under hypoxia compared with normoxia (Fig. 2b, lanes 1 and 2). Normoxic HDAC activity was also significantly inhibited by TSA (Fig. 2a). 437


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Fig. 1 Activity and expression of HDAC are regulated by oxygen tension. a, HDAC activity was determined in HepG2 cells after exposure to hypoxia ( ), hypoglycemia ( ), serum deprivation (0% serum) ( ) or serum-deprived hypoxic condition (0% serum + hypoxia) (), control (). *, P < 0.05 compared to control cells maintained in complete growth medium under normal conditions. b, HDAC activities of BAECs, Chang liver cells, HUVECs and HepG2 cells (, normoxia; , hypoxia). c, Time-dependent increase of HDAC activity of hypoxic () compared to normoxic () HepG2 cells. *, P < 0.05 compared to normoxia. Independent experiments repeated three times (mean ± s.d. of triplicate). d, mRNA and protein expression of HDAC1 by northern- (upper two) and western- (lower two, α-HDAC1) blot analyses after exposure of HepG2 cells to normoxic or hypoxic conditions. Ethidium bromide staining of 18s and 28s ribosomal transcripts and expression of α-tubulin were used as internal controls.

The tumor suppressor gene products p53 and VHL have inhibitory effects on angiogenesis16,17,18. Mutation or loss of function of either of them induces highly vascularized malignant tumors19,20. When HepG2 cells were grown in a neutralizing pH-buffered culture medium21, transcription and translation of p53 and VHL were significantly reduced by hypoxia (Fig. 2c and d). The decreases of VHL and p53 were most evident at 4 and 16 hours, respectively. As reported22, however, a prolonged hypoxia (more than 48 h) induced the expression of p53 (data not shown). TSA at noncytotoxic concentrations (100 ng/ml) significantly increased p53 and VHL under hypoxic conditions (Fig. 2c and d). VHL and p53 promote degradation of HIF-1α by ubiquitin-mediated proteolysis and suppress HIF-1α–stimulated transcription16,23,24. Expression of VEGF is negatively regulated by p53 and VHL (refs. 25,26,27) and is frequently overexpressed along with HIF-1α in human cancers28. TSA substantially reduced the hypoxic increase of expression and DNA-binding activity of HIF-1α (Fig. 2e and f). Similarly, TSA reduced VEGF expression in hypoxic conditions (Fig. 2g). Together, these data suggest that TSA inhibits hypoxia-induced angiogenesis through reactivation of p53 and VHL and concurrent suppression of HIF-1α and VEGF. TSA functions as a potent angiogenic inhibitor Treatment with TSA substantially reduced new vessel formation in chick embryos without any signs of thrombosis and hemorrhage and with negligible egg lethality (Fig. 3a). The anti-angiogenic activity of TSA (0.5 µg/egg) was 59.21 ± 2.40% (n = 33) (Fig. 3b). We also evaluated the angiogenic inhibition of TSA by using the mouse Matrigel plug assay29, an established in vivo angiogenesis model (Fig. 3c). Control plugs in which Matrigel was injected with heparin alone showed few vessels, but basic fibroblast growth factor (bFGF) (100 ng/ml) enhanced abundant vessel development inside the plugs. However, TSA strongly inhibited the bFGF-induced angiogenesis. To quantify the functional vasculature, we measured the hemoglobin contents of the Matrigel plugs (Fig. 3d); TSA (10 µg/ml) significantly reduced the hemoglobin levels to 1.13 ± 0.36 g/dl (n = 6). To explore mechanisms of action of TSA, we performed in vitro angiogenesis assays in BAECs. TSA strongly inhibited the hypoxia438

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stimulated networks as shown by foreshortened and severely broken tubes (data not shown). Moreover, TSA effectively suppressed hypoxia-stimulated DNA synthesis (Fig. 3e) and significantly inhibited hypoxia-enhanced migratory and invasive activity of BAECs (refs. 30,31; Fig. 3f and g). We performed an assay for ECM-cell attachment using collagen type IV, vitronectin, fibronectin or collagen type I. Attachment of BAECs to these ECM beds was significantly increased by hypoxia with marked stimulation of fibronectin, whereas TSA lowered cell adherence in all ECM components tested (Fig. 3h). Regulation of hypoxia-responsive genes by HDAC1 To evaluate whether HDAC modulates genes involved in hypoxiainduced angiogenesis, we transfected HepG2 cells with vector expressing wild-type HDAC1 (wt-HDAC1) or mutant HDAC1 (mt-HDAC1) which has a point mutation of H141A on wt-HDAC1. In contrast to the suppressed levels of p53 and VHL, expression of HIF-1α and VEGF was significantly upregulated by overexpression of wt-HDAC1 (Fig. 4a, lane 3). Compared to wt-HDAC1, however, mt-HDAC1 did not induce a significant change in expression of p53, VHL, HIF-1α and VEGF (Fig. 4a, lane 4). The mt-HDAC1 inactivated both the deacetylation and transcriptional repression activities of HDAC1 and mimicked the effect of HDAC inhibitors32. Thus, HDAC activity is critically involved in the regulation of these factors. The increase of HIF-1α levels by HDAC1 was also confirmed in wtHDAC1–transfected human embryonic kidney (HEK) 293 cells (Fig. 4b, lane 2), whereas VHL coexpressed cells significantly reduced the increased expression level of HIF-1α by HDAC1 (Fig. 4b, lane 3). Angiogenic stimulation by HDAC1 overexpression We transiently transfected HepG2 cells with wt-HDAC1 and mtHDAC1 vectors in low serum medium (M199, 1% FBS) before collecting conditioned medium (CM). We then directly applied CM to HUVECs to examine angiogenesis in vitro. Morphological differentiation of HUVECs on Matrigel-coated culture plates was stimulated by complete medium (M199 containing 20% FBS, 3 ng/ml bFGF and 5 U/ml heparin), whereas incubation of HUVECs with CM of non-transfectants formed incomplete or degraded microvessels (data not shown). Similarly, CM of mock transfectants NATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001



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did not induce tube-like structures. However, CM collected from wt-HDAC1 transfectants enhanced the formation of capillaries, which developed elongated and complex networks (Fig. 5a). We did not detect stimulation of tube formation in CM of mt-HDAC1 transfectants. To investigate whether VEGF mediates HDAC1-stimulated tube formation, we incubated CM collected from wt-HDAC1 transfectants with neutralizing antibody against VEGF (10 µg/ml) at 4 °C for 16 hours. The antibody almost completely blocked the tube formation stimulated by CM collected from wt-HDAC1–overexpressed cells (Fig. 5a, wt-HDAC1+Ab), indicating that VEGF might be an important molecule in HDAC-stimulated angiogenesis. In contrast, the control goat IgG had no inhibitory effect on the stimulation of tube formation in vitro (data not shown). Similarly, proliferation (Fig. 5b), migration (Fig. 5c) and viability (Fig. 5d) of HUVECs were significantly increased by wt-HDAC1–overexpressed CM, but not by CM of mt-HDAC1 transfectants or CM previously incubated with anti-VEGF antibody. The concentrated CM of wt-HDAC1–overexpressed HepG2 cells augmented neovascularization compared with mock transfectants or Matrigel alone (Fig. 5e). We observed intact red blood cells inside the new vessels, indicating functional vasculature. However, as shown in vitro (Fig. 5a–d), the CM of mt-HDAC1

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transfectants or CM incubated with neutralizing antibody against VEGF (50 µg/ml) could not induce new vessels in vivo (Fig. 5e). The control goat IgG did not inhibit the angiogenic stimulation of the CM of wt-HDAC1 transfectants (data not shown). Quantification of hemoglobin in Matrigel revealed the angiogenic response of wt-HDAC1 (Fig. 5f). The hemoglobin contents of control was 1.14 ± 0.41 g/dl (n = 9), which was similar to that of non-transfectants (1.11 ± 0.16 g/dl, n = 9) or mock transfectants (0.95 ± 0.18 g/dl, n = 9). However, the hemoglobin contents of wt-HDAC1–overexpressed CM considerably increased to 5.68 ± 1.05 g/dl (n = 11). Hemoglobin contents of mtHDAC1 transfectants were 0.73 ± 0.18 g/dl (n = 9) and those of anti-VEGF antibody–treated CM were 1.41 ± 0.39 g/dl (n = 10). Together, these results indicate that HDAC is involved in angiogenic stimulation. Upregulation of HDAC in hypoxic regions of tumors To delineate hypoxia and expression of hypoxia-induced HDAC in tumor cells, we performed in vivo tumor-formation experiments with Lewis lung carcinoma cells. Seven days after inoculation of tumor cells, we treated tumor-bearing mice with TSA (1 mg/kg body weight) or control vehicle. Tumors grew rapidly to more than 170 mm3 in size 10 days after inoculation; however, we observed a sig-

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Fig. 2 Effect of TSA on the regulation of p53, VHL, HIF-1α and VEGF expression. a, HDAC activity of HepG2 cells was determined in the presence or absence of TSA. normoxia; , normoxia + TSA; , hypoxia; hypoxia + TSA. *, P < 0.05 compared to normoxia, **, P < 0.05 to hypoxia. Independent experiments were repeated twice (mean ± s.d. of triplicate). b, AUT gel electrophoresis. Lane 1, normoxia; lane 2, hypoxia; lane 3, hypoxia + TSA. c, p53 expressions determined by northern- (upper two) and western-blot (α-p53, lower two) analyses. d, Transcription levels of VHL were examined by RT-PCR analysis (topmost). Anti-VHL western blotting from NATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001

immunoprecipitates of HepG2 cell lysates (100 µg) with 2 µg of anti-VHL antibody (α-VHL, third from top). β-actin and α-tubulin served as loading controls. Incubation times and conditions are indicated. e, Immunoblotting with anti-HIF-1α antibody. f, Electromobility shift assay was performed with oligonucleotides of the VEGF-specific HIF-1α-binding sequences41. Competitor, unlabeled oligonucleotides added in extracts of the hypoxic HepG2 cells as indicated. g, Northern- (top two) and western-blot (bottom two) analyses against VEGF (α-VEGF) were performed on total RNA and cell lysate isolated from HepG2 cells exposed to 16 h of hypoxia. 439

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Fig. 3 Anti-angiogenic activity of TSA. a and b, In a CAM assay, angiogenic inhibition of TSA was photographed in a microscope in a (left, control; middle, 1 µg retinoic acid; right, 0.5 µg TSA) or quantitatively evaluated in b ( , TSA at indicated concentrations; , empty coverslip as control; , retinoic acid). *, P < 0.05 compared to control. Independent experiments were repeated 3 times, and each value indicates mean ± s.e. of more than 20 eggs. c and d, Matrigel plug assay. In c: left, control; middle, bFGF; right, bFGF + TSA. E, endothelial cells; M, Matrigel; SM, smooth muscle; V, vacuole. Samples were stained with Masson-trichrome; scale bars, 40 µm. In d, , control (Matrigel containing heparin alone); , bFGF + heparin (10 U/ml); , TSA at indicated concentrations. Each value represents mean ± s.e. *, P < 0.05 compared to control, **, P < 0.05 compared to bFGF-induced hemoglobin quantity. e–h, Proliferation (e, 24 h), migration (f, 8 h), invasion (g, 18 h) and attachment (h, 30 min) of BAECs showed similar patterns with that of tube formation. , normoxia; , normoxia + TSA; , hypoxia; , hypoxia + TSA. In h, Col IV, type IV collagen (5 µg/ml); VN, vitronectin (1 µg/ml); FN, fibronectin (1 µg/ml); Col I, type I collagen (5 µg/ml). *, P < 0.05 compared to normoxia and **, P < 0.05 to hypoxia. Independent experiments were repeated two or three times, and each value indicates mean ± s.d. of triplicate.

nificant decrease of tumor size (∼40%) at day 10 of TSA treatment (data not shown). To examine tumor masses during active tumor growth for signs of angiogenic regression, we killed the mice at 10 days after treatment. We visualized hypoxic regions with the hypoxia marker pi-

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monidazole near the center or in sporadic regions of tumor mass, and HIF-1α colocalized with hypoxic regions (Fig. 6a). HDAC1 was overexpressed in the hypoxic regions about 100–150 µm away from the vessels. VEGF was also upregulated in regions containing high levels of HIF-1α and HDAC1. These results show that hypoxia enhances the expression of HDAC1 as well as HIF-1α and VEGF in vivo. Inhibition of hypoxia-induced angiogenesis in vivo by TSA Because TSA inhibited the proliferation and migration of endothelial cells especially in hypoxic conditions in vitro (Fig. 3), we investigated whether TSA could specifically inhibit the hypoxia-induced angiogenesis in tumors in vivo. In control tumors, we found many microvessels in HIF-1α–expressing regions (Fig. 6b, TSA–, arrows), in which we saw invading vessels containing erythrocytes (Fig. 6b,

Fig. 4 Regulation of hypoxia-responsible genes by HDAC overexpression. a, HepG2 cells were transiently transfected with mock (lane 2), wt-HDAC1 (lane 3) or mt-HDAC1 expression vectors (lane 4), total cell lysate was isolated and immunoblot analysis was performed with each specific antibody. Lane 1 shows non-transfectants treated with the same amount of transfection reagents without plasmids. b, HEK 293 cells were transiently transfected with wt-HDAC1 and/or pCMV-VHL expression vectors using the calcium phosphate method. Total cell lysates were isolated and immunoblot analysis was performed with anti-HIF-1α antibody. Lane 1, empty vector-transfected control cells; lane 2, wt-HDAC1-transfected cells; lane 3, wt-HDAC1 and CMV-VHL-cotransfected cells. NATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001


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Fig. 5 Angiogenic stimulation of wt-HDAC1 overexpression. HepG2 cells were transiently transfected with empty, wt-HDAC1 or mt-HDAC1 vectors and the CM was collected. Neutralizing anti-VEGF (10 µg/ml) or control goat IgG antibody was incubated with the wt-HDAC1-overexpressed CM before application on HUVECs. a, Stimulated tube formation of HUVECs at 28 h. Left, wt-HDAC1; middle, mt-HDAC1; right, wt-HDAC1 + anti-VEGF antibody. Scale bars, 40 µm. b–d, Proliferation (b, 48 h), migration (c, 4 h) and viability (d, 48 h) of HUVECs observed in CM from transfected HepG2 , mt-HDAC1; , wt-HDAC1 + anti-VEGF cells. , mock; , wt-HDAC1; antibody. *, P < 0.05 compared with CM of mock transfectants, **, P < 0.05 compared with CM of wt-HDAC1 transfectants. Independent experiments

were repeated two or three times, and each value indicates mean ± s.d. of triplicate. e, Anti-VEGF (50 µg/ml) was incubated with concentrated CM (100×)of wt-HDAC1 before performing Matrigel plug assay. All Matrigels were mixed with heparin. Samples were stained with Masson-trichrome. Left, wt-HDAC1; middle, mt-HDAC1; right, wt-HDAC1 + anti-VEGF antibody. V, vacuole; R, red blood cells. f, Hemoglobin contents were determined to quantify the intact vessels. , control; , non-transfected ; , mock transfectant; , wt-HDAC1; , mt-HDAC1; , wt-HDAC1 + anti-VEGF antibody. Each value represents mean ± s.e. *, P < 0.05 compared with CM of mock transfectants, **, P < 0.05 compared with CM of wt-HDAC1 transfectants. Independent experiment was repeated 4 times; scale bars, 40 µm.

TSA–, arrowheads). HDAC1 and VEGF were also simultaneously expressed in HIF-1α–expressing regions and we detected invading vessels in these regions as well (Fig. 6b, TSA–). In TSA-treated tumors, we found hypoxic regions apart from large vessels and microvessels (Fig. 6a and b, TSA+). However, most large vessels and microvessels in these tumors were regressed in hypoxic regions detected by pimonidazole and HIF-1α (Fig. 6b, TSA+). In contrast to control tumors, we found no invading microvessels in hypoxic regions where HDAC and VEGF were also expressed. Although we observed hypoxic regions and induction of HIF-1α, HDAC and VEGF in TSA-treated tumors, microvessel formation was markedly reduced by TSA. PECAM staining also

showed that endothelial cells were not found in hypoxic regions of TSA-treated tumor (data not shown). This indicates that induction of new blood vessels was largely inhibited in the hypoxic regions of TSA-treated tumors. Although TSA did not diminish the hypoxia in the central regions of tumor, it might decrease the migration, proliferation and/or invasion of endothelial cells into the hypoxic regions in vivo. Although it was difficult to compare the relative expression levels of VEGF and HIF-1α between the control and TSA-treated tumors by immunohistochemical staining, we observed weaker staining of VEGF and HIF1α in TSA-treated tumors (Fig. 6). Together, these data indicate that TSA might inhibit hypoxia-induced angiogenesis in vivo.

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Fig. 6 Induction of HDAC in hypoxic tumor regions and inhibition of hypoxiainduced angiogenesis by TSA in Lewis lung carcinoma model. a, Tumor sections treated with saline (left panels) or TSA (right panels) and immunostained by pimonidazole (top panels) or antibodies against HIF-1α (second two from top), HDAC1 (third two from top) and VEGF (bottom two). Arrows indicate large vessels containing erythrocytes. b, Tumor sections treated with saline (left panels) or TSA (right panels) immunostained by antibodies against HIF-1α (top panels), HDAC1 (middle) and VEGF (bottom). Arrows indicate microvessels inside or around the immunoreative regions and arrowheads indicate invading vessels. Scale bars, 100µm. NATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001

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Discussion Histone deacetylation is involved in transcriptional silencing and a variety of genes are upregulated as the level of acetylated histones increases. However, it is unclear whether gene transcription is selectively influenced by changes in the acetylation status of chromatin. To date, HDAC regulation by external signals is not well understood except for the recent findings that human HDAC3 and a mouse HDAC gene, Hd1, are inducible by phytohemagglutinin and IL-2, respectively33,34. The increase of HDAC activity under hypoxia (Fig. 1) indicates that HDAC might be involved in oxygen-regulated gene expression and hypoxia-induced angiogenesis. The Methyl-CpG-binding protein MeCP2 has been shown to interact with HDAC to repress its transcription35, and promoter activity of p53 and VHL are inactivated by CpG methylation36,37,38. In addition, methylation-silenced genes can be reactivated by TSA (refs. 39,40). Thus, lowered levels of p53 and VHL in hypoxia and the increase by TSA (Fig. 2c and d) indicate that HDAC modulation can be closely involved in regulation of tumor suppressor genes, and methylation of tumor suppressor genes by HDAC might occur more frequently under hypoxic conditions. We will conduct further investigations of these matters. Loss of p53 function amplifies HIF-1α–dependent responses to hypoxia16, and VHL targets HIF-1α for oxygen-dependent proteolysis24. These reports indicate that p53 and VHL negatively regulate HIF-1α. Experiments shown here using the HDAC inhibitor TSA indicate that HDAC influences HIF-1α and VEGF, perhaps by direct regulation of p53 and VHL (Fig. 2c-g). However, we cannot exclude the possibility that HDAC may be able to directly control the transcriptional regulation of HIF-1α and VEGF as well as their functions. Given that TSA blocked angiogenesis of endothelial cells more effectively under hypoxic conditions than in normoxia (Fig. 3), HDAC inhibition might be a signal for cells not to stimulate angiogenesis. The increase of HIF-1α and VEGF in HDACoverexpressed HepG2 cells and HEK293 cells strongly support the idea that HDAC overexpression could be an activating signal for angiogenesis (Fig. 4). When HUVECs were incubated with the CM of wt-HDAC1–transfected HepG2 cells or when mice were injected with concentrated CM in Matrigel mixture, both in vitro and in vivo angiogenesis were significantly enhanced (Fig. 5). More importantly, neutralizing antibody against VEGF blocked wt-HDAC1–stimulated angiogenesis (Fig. 5), and no response occurred in mt-HDAC1–overexpressed CM. Therefore, the stimulation of angiogenesis by wt-HDAC1 is likely to be mediated through at least VEGF. In the adopted Lewis lung carcinoma model, hypoxic regions were found at considerable distances (about 100–150µm) from the large vessels as judged by the spatial relationship with large or microvessels. In hypoxic regions of the control tumor tissues, HIF1α, HDAC and VEGF were highly expressed (Fig. 6a). Aggressive invasion of erythrocytes inside hypoxic regions was also observed in the control tumor (Fig. 6b, TSA–), indicating that hypoxia stimulates migration and invasion of endothelial cells in the control tumor tissues. These results confirm that HDAC is over-expressed within hypoxic tumor, and hypoxia-induced angiogenic molecules behave together with HDAC at similar regions in vivo. Interestingly, TSA reduced the vessels inside hypoxic regions of tumor. Even though there were also normoxic areas containing larger vessels, few blood vessels were developed near or around the hypoxic areas (Fig. 6b, TSA+). This indicates that TSA selectively inhibits the hypoxia-induced angiogenesis in vivo. 442

TSA treatment decreased the relative levels of HIF-1α and VEGF (Fig. 6), though the quantitative comparison using immunohistochemistry was difficult. Until now, little was known about the role of HDAC in hypoxiainduced angiogenesis and its response to environmental stresses. The anti-angiogenic effects of TSA and the angiogenic stimulation by HDAC overexpression shown here indicate an important role for HDAC in the regulation of angiogenesis. We suggest that specific HDAC inhibition has potential therapeutic value for cancer therapy, and that HDAC can be used as a novel target in developing angiogenic inhibitors. Methods Primers and antibodies. Antibody against HDAC1 was from Santa Cruz Biotechnology (Santa Cruz, California). Antibody against HIF-1α was from Novus (Littleton, Colorado) and Labvision (Fremont, California). Antibodies against VHL and PECAM-1 were from Pharmingen (San Diego, California). Antibodies against p53 and VEGF were from Upstate (Lake Placid, New York). Neutralizing antibody against VEGF was from R&D (Minneapolis, Minnesota). Specific primers and thermocycler parameters for VHL were as follows: 94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min, 5′-AGAGATGCAGGGACACACGAT-3′ and 5′-TCACAACTGTAGAATGTCAATCC-3′. The double-stranded oligonucleotides for the VEGF-specific HIF-1α–binding sequence (5′-TGCATACGTGGGCTCCAACAG-3′) was synthesized as described41. Cells. BAECs (passage 8–12), the 786-0 (VHL-negative) HEK 293 cells and Lewis lung carcinoma cells were cultured in DMEM, and HepG2 cells were cultured in MEM, supplemented with 10% FBS and antibiotics. Primary HUVECs (passage 5–8) were grown on 0.3% gelatin-coated plates in M199 supplemented with 20% FBS, 3 ng/ml bFGF and 5 U/ml heparin. For the hypoxic condition, cells were incubated at CO2 level of 5% with 1% O2 balanced with N2. Mice. 7-wk-old, specific-pathogen–free male C57BL/6J mice were provided with autoclaved tap water and lab chow ad libitum and were kept at 23 ± 0.5 °C and 55 ± 10% humidity in a 12-h light–dark cycle. Plasmid construction. mt-HDAC1–expressing vector was constructed as described32. The deacetylase activity was reduced 85% compared with that of wt-HDAC1. pCMV-VHL expression vector was constructed by subcloning the full-length VHL cDNA into the pCMV–Tag3B vector at EcoRI/XhoI site. Preparation of [3H]-labeled histones and HDAC assay. HepG2 cells were incubated with 0.5 mCi/ml [3H]acetate (100 mCi/mmol; New England Biolabs, Beverly, Massachusetts) with TSA (100 ng/ml; Sigma) for 1 h. The labeled histone fraction as substrate for the HDAC assay was extracted, and total HDAC extraction was performed as described15. For HDAC assay under normoxic or hypoxic conditions, HepG2 cells were incubated in RPMI 1640 glucose-free medium supplemented with 10% FBS for hypoglycemia and in MEM with 0% FBS for 0% serum condition, respectively. AUT gel electrophoresis. HepG2 cells were treated with TSA for 6 h. Cellular histones were isolated and analyzed by gel electrophoresis using AUT gel as described15. The equal amount of histone (10 ug/lane) was loaded after quantification by BCA methods. Gels were stained with Coomassie Brilliant Blue R-250. Chorioallantoic membrane (CAM) assay and mouse Matrigel plug assay. CAM assay was conducted by using 4.5-day-old chick embryos as described42. The mouse Matrigel plug assay in vivo was performed as described29. Tube formation assay. 250 µl of Matrigel (10 mg/ml) (Becton Dickinson, Franklin Lakes, New Jersey) was polymerized for 30 min at 37 °C. BAECs (5 × 105 cells) were seeded on the surface of Matirgel. Then, TSA was added and incubated for 24 h under normoxic or hypoxic condition. HUVECs were seeded at a density of 1 × 105 cells/well on Matrigel and grown in the CM collected from HDAC1 transfectants of HepG2 cells for 28 h. Morphologic changes of cells were photographed at ×40 magnification in ImagePro Plus software (Media Cybernetics, Silver Spring, Maryland). NATURE MEDICINE • VOLUME 7 • NUMBER 4 • APRIL 2001


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Chemomigration, chemoinvasion and cell attachment assay. 24-well transwell chambers of 8.0-µm–pore polycarbonate filter inserts were used, and cell migration and invasion were determined by counting whole cell numbers at single filter with optical microscopy at ×40 magnification. A cell attachment assay was performed as previously described43. Transient transfection and CM preparation. Transfection of HepG2 cells was performed using LipofectAMINE Plus Reagent kit (Life Technologies, Gaithersburg, Maryland) according to the supplier’s protocol. The CM was filtered through 0.45-µm–pore membrane (Millipore, Bedford, Massachusetts) and directly applied onto HUVECs. To conduct the Matrigel plug assay, the medium of transfected HepG2 cells was changed with serum-free medium, and cells were further incubated for 24 h. The medium was concentrated using a ultrafiltration kit (5 kD cut-off, Millipore). The concentrated CM was mixed with Matrigel and injected into mice. Non-transfectants were treated with the same amounts of transfection reagents without plasmids. β-galactosidase expression vector (1 µg) was cotransfected to normalize transfection efficiency. Completion of transient transfection was confirmed by performing RT-PCR and Western blotting, respectively, in addition to β-galactosidase assay (data not shown). Tumor formation and TSA treatment. Anesthetized C57BL/6J mice were implanted with Lewis lung carcinomas (∼1 × 106 cells/0.1 ml media) at the flank of mouse. When tumors were about 20 mm3 in size at 7 d after implant, TSA (1 mg/kg body weight) or saline (containing 10% DMSO) was treated daily intraperitoneally. After 10 d of treatment, mice were injected with pimonidazole (60 mg/kg body weight) for the detection of tumor hypoxia44. After 3 h mice were killed and tumor masses were removed. Immunohistochemistry. Tumors were fixed in 10% buffered formalin solution (pH 7.2) for 16 h, frozen in OCT compound and serially sectioned to 15 µm at –20 °C. Immunohistochemistry was performed as described45.

Acknowledgments We thank S.L. Schreiber for the gift of pBJ5-wt-HDAC1–expressing vector; J.A. Raleigh for hypoxia marker, pimonidazole and its associated antibody; and B.P. Yu for critical reading of the manuscript. Financial support was from the National Research Laboratory Fund (2000-N-NL-01-C-015), the Ministry of Science and Technology, Korea (to K-W.K.) and the Korea Science and Engineering Foundation (to H.J.K.).

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