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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 May 28;105(22):7738–7743. doi: 10.1073/pnas.0802857105

Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7

Shusheng Wang *, Xiumin Li *, Maribel Parra †, Eric Verdin †, Rhonda Bassel-Duby *, Eric N Olson *,‡
PMCID: PMC2409381  PMID: 18509061

Abstract

VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.

Keywords: angiogenesis, PKD1, MEF2, class II HDAC

The formation of blood vessels through the process of angiogenesis is critical for normal vascular development and numerous vascular disorders (1). In response to angiogenic stimuli, endothelial cells (ECs) proliferate, migrate, and coalesce to form primitive vascular labyrinths that undergo maturation and remodeling, accompanied by recruitment of smooth muscle cells to give rise to mature blood vessels. VEGF plays a key role in angiogenesis by regulating the proliferation, migration, and survival of ECs (2). The sensitivity of ECs to VEGF signaling is exemplified by the lethal vascular abnormalities that result from disruption of even a single VEGF allele in mice (3, 4). Other peptide growth factors, such as fibroblast growth factor (FGF), angiopoietin, transforming growth factors (TGFs), TNF-α, apelin, and insulin-like growth factors (IGFs), also influence endothelial proliferation and function (5, 6). However, most of these factors act on other cell types and complement or coordinate VEGF signaling rather than function as independent regulators of angiogenesis and EC functions.

The binding of VEGF to its receptors induces receptor dimerization and autophosphorylation, which activates several downstream kinases, including protein kinases C and D (PKC and PKD), phosphatidylinositol 3-kinase (PI3K), and MAPK (2). VEGF signaling also leads to the activation of numerous genes, such as those encoding regulator of calcineurin 2 (RCAN2) [formerly Down Syndrome Candidate Region 1 (DCSR1L1)] (7) and the nuclear receptor Nur77 (8), which mediate the biological responses to VEGF. However, how VEGF signals to the nucleus to regulate gene transcription and angiogenesis is far from clear.

Histone acetyltransferases and histone deacetylases (HDACs) are key regulators of chromatin structure and gene expression (9). Mammalian class IIa HDACs (HDAC4, -5, -7, and -9) contain an N-terminal extension that interacts with other transcriptional cofactors and confers responsiveness to extracellular signals (10). Phosphorylation of a series of conserved serine residues in the N-terminal regulatory domain of class IIa HDACs by calcium/calmodulin-dependent kinase (CaMK) and PKD creates docking sites for 14-3-3 chaperone proteins, which drives these HDACs from the nucleus to the cytoplasm and derepresses gene expression (11–14). The MEF2 transcription factor is a key downstream target for repression by class IIa HDACs and for signal-dependent transcription in response to signaling pathways that promote class IIa HDAC phosphorylation (15).

The functions of class IIa HDACs in vivo have been revealed by gene knockout studies. HDAC5 and -9 function as stress-responsive inhibitors of cardiac growth (16, 17), HDAC4 represses chondrocyte growth and differentiation (18), and HDAC7 maintains vascular integrity by repressing matrix metalloprotease 10 (MMP10) expression in ECs (19). A role for HDAC7 in modulating angiogenesis has also been suggested from in vitro assays (19, 20). However, whether VEGF acts through HDAC7 to control angiogenesis and endothelial functions is unknown.

Here, we show that HDAC7 and other class IIa HDACs are phosphorylated and exported from the nucleus to the cytoplasm in response to VEGF signaling. Moreover, blockade of HDAC7 phosphorylation with a signal-resistant HDAC7 mutant represses EC proliferation and migration in response to VEGF. Signaling by VEGF to HDAC7 regulates both MEF2-dependent and independent target genes in ECs. Our results reveal a key role for HDAC7 as a VEGF-dependent molecular switch that governs EC functions.

Results

Effects of Peptide Growth Factors on HDAC7 Phosphorylation in ECs.

To begin to explore the signaling pathways that regulate the activity of class IIa HDACs in ECs, we compared the expression of HDAC4, -5, -7, and -9 in human umbilical vein ECs (HUVECs) and human aortic ECs (HAECs). HDAC4 and -7 mRNAs are abundant in these two cell types, whereas HDAC5 and -9 are expressed at relatively low levels [supporting information (SI) Fig. S1]. Because HDAC7 is EC-restricted (19), we tested a variety of growth factors known to modulate EC functions for their effects on HDAC7 phosphorylation in ECs. Three different phospho-HDAC7 antibodies, which specifically recognize the conserved phosphoserines in HDAC7, were used in the assay. These sites correspond to serines 178, 344, and 479 in mouse HDAC7 (Fig. 1A). The specificity of these antibodies has been documented (21). HAEC cells were infected with adenovirus-expressing mouse HDAC7 for 48 h, starved for 24 h with low serum medium, and treated with different growth factors. As shown in Fig. 1B, VEGF-A robustly induced HDAC7 phosphorylation at all three serine residues. Endothelin-1, TGF-β, IGF, and FGF-2 also modestly induced HDAC7 phosphorylation. The three serine residues in HDAC7 showed similar patterns of phosphorylation. Apelin, another angiogenic factor (5), failed to induce HDAC7 phosphorylation, possibly due to the lack of its receptor in the HAEC cell line (5).

Fig. 1.

Fig. 1.

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Regulation of HDAC7 phosphorylation by VEGF. (A) Schematic diagram of HDAC7 showing the three signal-responsive serines in the N-terminal regulatory domain. NLS, nuclear localization sequence. (B–D) HAEC cells were infected with adenovirus expressing FLAG-HDAC7 for 48 h, transferred to EBM-2 medium with 0.1%FBS for 24 h, and then treated with growth factors. Antibodies recognizing HDAC7 phosphorylated at Ser-178, Ser-344, and Ser-479 were used to detect the phosphorylation state of HDAC7. Total amount of overexpressed HDAC7 was monitored by antibody against FLAG tag. The cells were treated with: (B) 20 ng/ml IGF-1; 2.5 ng/ml TGFβ; 10 ng/ml VEGF-A; 10−8 M endothelin-1; 1 ng/ml FGF-2; 10 ng/ml Apelin or no treatment (control) for 30 min, or (C) 10 ng/ml VEGF-A for the indicated times. A portion of the cell lysate was immunoprecipitated with monoclonal anti-FLAG conjugated to agarose beads (IP) and immunoblotted with an antibody against endogenous 14–3-3 protein (IB).

Mutation of the three serine residues to alanine in HDAC7 (HDAC7-S/A) abolished inducible and basal phosphorylation (Fig. 2D, lane 4). These results indicate that HDAC7 is phosphorylated in response to multiple growth factors in ECs, whereas VEGF-A stands out as the most robust inducer of HDAC7 phosphorylation.

Fig. 2.

Fig. 2.

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PKD is necessary and required for HDAC7 phosphorylation by VEGF. (A and B) HAEC cells were infected with adenovirus expressing FLAG-HDAC7 for 48 h, transferred to EBM2 medium with 0.1% FBS for 24 h, treated with kinase inhibitors, and then treated with 20 ng/ml of VEGF-A for 10 min. Antibodies recognizing HDAC7 phosphorylated at Ser-178, Ser-344, and Ser-479 were used to detect the phosphorylation state of HDAC7. The total amount of overexpressed HDAC7 was monitored by antibody against FLAG tag. The kinase inhibitors used were: (A) 30 μM BAPTA/AM or 5 μM KN93; (B) 500 nM Gö6983, 1.3 μM Gö6976; DMSO is the control for the drug vehicle; (C) HAEC cells were transfected with control siRNA or siRNA against PKD1 and infected with adenovirus expressing FLAG-HDAC7, transferred to EBM-2 medium with 0.1% FBS, and treated with VEGF-A as above. Antibody recognizing HDAC7 phosphorylated at Ser-479 was used to detect the phosphorylation state of HDAC7. The efficiency of si-PKD1 was examined by Western blot analysis with PKD1 antibody. The total amount of overexpressed HDAC7 was monitored by antibody against FLAG tag. Loading control is indicated by antibody against GAPDH. (D) HAEC cells were infected with or without adenoviruses expressing constitutively active PKD1 (Myc-tagged) and FLAG-HDAC7 or FLAG-HDAC7-S/A, transferred to EBM-2 with 0.1% FBS, and treated with VEGF-A. Western blot analysis was performed by using antibodies recognizing FLAG or phosphorylated HDAC7 at Ser-178, Ser-344, and Ser-479. For coimmunoprecipitation, the protein lysates were immunoprecipitaed with anti-FLAG or anti-Myc antibodies, and immunoblotted with antibodies against Myc, PKD or FLAG.

VEGF rapidly induced HDAC7 phosphorylation with the strongest response observed 5–10 min after VEGF treatment (Fig. 1C). Phosphorylation of HDAC7 was paralleled by binding to 14-3-3, as detected by coimmunoprecipitation. Phosphorylation of HDAC7 in response to VEGF-A occurred in a dose-dependent manner with a maximum at 20 ng/ml (data not shown). We conclude that VEGF-A dynamically regulates HDAC7 phosphorylation in ECs.

The PKC/PKD Pathway Is Necessary and Sufficient for HDAC7 Phosphorylation by VEGF.

To identify the kinases that phosphorylate HDAC7 in response to VEGF, HAEC cells were infected with HDAC7 virus and pretreated with kinase inhibitors before VEGF-A treatment. The general serine/threonine kinase inhibitor staurosporine completely blocked HDAC7 phosphorylation (data not shown). 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (a Ca2+ chelator), KN93 (a CaMK inhibitor), KN62 (a CaMKII-specific inhibitor), and autocamtide inhibitory peptide II-2 (AIP II-2) did not affect VEGF-induced HDAC7 phosphorylation (Fig. 2A and data not shown), indicating that Ca2+ and CaMKs are not responsible for VEGF-induced HDAC7 phosphorylation. In contrast, the PKC inhibitor Gö6983 and the Ca2+-dependent PKC and PKD inhibitor Gö6976 (22) inhibited VEGF-induced HDAC7 phosphorylation (Fig. 2B). Because the BAPTA/AM studies suggested that calcium is not required for VEGF-induced HDAC7 phosphorylation, these findings suggest that atypical PKC, a known regulator of PKD, is required for VEGF-induced HDAC7 phosphorylation, consistent with a recent report that VEGF can induce PKC dependent PKD phosphorylation (23).

To directly test whether PKD is required for VEGF-induced HDAC7 phosphorylation, a siRNA oligonucleotide targeted to PKD1 or a control siRNA pool was transfected into HAEC cells. The knockdown efficiency of si-PKD1 was confirmed by Western blot analysis against endogenous PKD1 (Fig. 2C). Phosphorylation of HDAC7 in response to VEGF was repressed by knocking down PKD1 in ECs (Fig. 2C), whereas HDAC7 protein levels were unaffected.

To further test whether PKD is sufficient to induce HDAC7 phosphorylation, HDAC cells were infected with adenovirus-expressing Myc-tagged constitutively active PKD (PKD-CA), adenovirus-encoding FLAG-tagged HDAC7 or the signal-resistant HDAC7 mutant HDAC7-S/A, and HDAC7 phosphorylation was examined. As shown in Fig. 2D, PKD-CA induced HDAC7 phosphorylation to comparable levels as VEGF treatment. In contrast, HDAC7-S/A was completely resistant to phosphorylation by PKD-CA and VEGF. Coimmunoprecipitation assays were performed to test whether PKD directly associated with HDAC7 in ECs. HDAC7 interacted weakly with endogenous PKD, without a detectable increase upon VEGF treatment. Interestingly, the HDAC7-S/A mutant protein interacted with PKD much more strongly than did the wild-type HDAC7 protein (Fig. 2D). We interpret these findings to indicate that PKD transiently associates with and phosphorylates HDAC7, whereas the nonphosphorylated HDAC7 mutant forms a stable complex with PKD.

VEGF Induces HDAC7 Nuclear Export in ECs.

Phosphorylation of class IIa HDACs creates binding sites for the 14-3-3 chaperone protein, which drives their export from the nucleus to the cytoplasm (11–14, 16, 17, 24). As shown in Fig. 3A, HDAC7 is exclusively localized in the nucleus in serum-starved HAEC cells in the absence of VEGF. HDAC7 nuclear export was observed beginning 30 min after VEGF treatment. The percentage of cells with cytoplasmic HDAC7 continued to increase thereafter and reached a maximum of ≈70% after 4 h of VEGF treatment. HDAC7 then gradually relocalized to the nucleus and was primarily nuclear 24 h after VEGF treatment. Thus, ECs respond to VEGF signaling by a wave of nucleocytoplasmic HDAC7 shuttling, reflecting the dynamic effects of VEGF on ECs.

Fig. 3.

Fig. 3.

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VEGF induces HDAC7 nuclear export. HAEC cells infected with adenoviruses expressing (A) FLAG-HDAC7 and treated with VEGF-A (10 ng/ml) and kinase inhibitors as indicated; (B) FLAG-HDAC7 or FLAG-HDAC7-S/A in the presence or absence of Myc-PKD1-CA were treated with VEGF-A (10 ng/ml) as indicated. Immunocytochemistry was performed by using antibody against FLAG (red). Nuclei were stained with DAPI (blue). (C) Quantitative analysis of the percentage of cytoplasmic HDAC7 after treatment with VEGF-A for the indicated times (0–24 hr) without or with prior treatment of indicated inhibitor. KN93 (CaMK inhibitor, 5 μM); Gö6976 (PKD inhibitor, 1.3 μM).

Consistent with the requirement of the PKC/PKD pathway for HDAC7 phosphorylation, Gö6976 inhibited HDAC7 nuclear export in response to VEGF, whereas KN93 failed to block VEGF-induced HDAC7 nuclear export (Fig. 3A). PKD-CA also drove HDAC7 into the cytoplasm independent of VEGF treatment (Fig. 3B). In contrast, HDAC7-S/A was localized to the nucleus regardless of VEGF treatment and was insensitive to PKD-CA. These results demonstrate that serines 178, 344, and 479 in HDAC7 are critical for VEGF-induced HDAC7 nucleo-cytoplasmic shuttling.

VEGF Induces Phosphorylation of HDAC4, -5, and -9.

We further tested the response of other class IIa HDACs to VEGF. An antibody against phosphoserines 259 and 498 in HDAC5 also recognizes the corresponding phosphoserines in HDAC4 and MEF2-interacting transcription repressor (MITR) (a splice variant of HDAC9) (13). The specificity of the antibody was demonstrated by the lack of signal with HDAC4-S/A and MITR-S/A mutants (Fig. S2).

Serum-starved HAEC cells showed a basal level of phosphorylation of HDAC4 and -5 and MITR, which was strongly enhanced by VEGF (Fig. S2). Phosphorylation of HDAC4 and -5 and MITR in response to VEGF was nearly abolished by the PKC/PKD inhibitor Gö6976 but not the CaMK inhibitor KN93. Moreover, PKD-CA alone potently induced HDAC4 and -5 and MITR phosphorylation.

HDAC4 and -5 and MITR were primarily located in the nucleus in unstimulated HAEC cells (Fig. S3 A–C). HDAC4 and -5 and MITR became significantly more cytosolic after 4 h of VEGF treatment. PKD-CA was sufficient to induce HDAC4 and -5 and MITR nuclear export independent of VEGF treatment. Mutation to alanine of the key serine residues in HDAC4 (serines 246, 467, and 632), HDAC5 (serines 259 and 498), and MITR (serines 218 and 448) rendered them irresponsive to VEGF and PKD, demonstrating that these conserved residues in HDAC4 and -5 and MITR are required for VEGF-induced nuclear export in ECs.

HDAC7 Is Required for VEGF-Induced EC Proliferation and Migration.

The PKC/PKD pathway regulates EC proliferation in response to VEGF (23). To study the functional significance of VEGF-induced HDAC7 phosphorylation, we overexpressed HDAC7 and HDAC7-S/A in ECs and examined cell proliferation in response to VEGF. The expression level of HDAC7 and HDAC7-S/A was quantified by Western blot analysis (Fig. 4A). As shown in Fig. 4B, overexpression of HDAC7-S/A, but not HDAC7, blunted cell proliferation in response to VEGF as assayed by [3H]-thymidine incorporation. These results demonstrate that the VEGF-responsive phosphorylation sites in HDAC7 are critical for VEGF-induced endothelial proliferation.

Fig. 4.

Fig. 4.

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Regulation of EC proliferation and migration by HDAC7. HAEC cells were infected with adenovirus expressing (A) FLAG-HDAC7 or FLAG-HDAC7-S/A as detected in a Western blot using FLAG antibody. (B) HAEC cells infected with adenovirus expressing lacZ (Ad-LacZ), FLAG-HDAC7 (Ad-HDAC7), or FLAG-HDAC7-S/A (Ad-HDAC7-S/A) and subjected to cell proliferation and (C) migration assays in response to VEGF treatment. (P value <0.007 between Ad-LacZ (or Ad-HDAC7) and Ad-HDAC7-S/A). Of note, there are slightly less cells in Ad-HDAC7-S/A infected samples compared with the other two controls. The results in B and C are representative of three independent experiments.

The impact of HDAC7 on cell migration was examined by a scratch-wound assay in which EC migration depends on VEGF. Overexpression of HDAC7 in HAEC cells did not significantly affect EC migration relative to a LacZ control (Fig. 4C). However, overexpression of HDAC7-S/A significantly repressed VEGF-induced EC migration. Twenty-four hours after wounding, compared with the LacZ control, overexpression of HDAC7 and HDAC7-S/A in ECs resulted in 20% and 85% decreases in the number of cells migrating to the scratched area in response to VEGF, respectively. These results suggest that signaling from VEGF to HDAC7 is required for VEGF-induced EC migration. Importantly, ECs expressing HDAC7-S/A showed no signs of toxicity. Failure of HDAC7 to be phosphorylated by VEGF imposes a blockade to cell migration, suggesting that key genes required for migration are irreversibly repressed by the signal-resistant HDAC7 mutant.

HDAC7 Regulates VEGF-Induced Gene Expression in ECs.

RCAN2 and Nur77 are among the early VEGF response genes implicated in angiogenesis (7, 25). To test whether these genes are controlled by HDAC7 downstream of VEGF signaling, and whether the interaction between HDAC7 and MEF2, a well defined target for repression by HDAC7, confers VEGF responsiveness to these genes, HAEC cells were infected with virus expressing HDAC7, HDAC7-S/A, or HDAC7-S/A-ΔMEF, which lacks the MEF2-binding domain. LacZ was used as a control. Real-time PCR was performed to examine the level of candidate gene expression after 1 h of VEGF treatment. GAPDH and cyclophilin A, which were not significantly affected by VEGF treatment, were used as controls.

RCAN2 and Nur77 were induced ≈8- and 40-fold, respectively, upon 1 h of VEGF treatment (Fig. 5). Although overexpression of wild-type HDAC7 did not significantly affect the expression of RCAN2 and Nur77, presumably because it is phosphorylated and rendered inactive by VEGF, overexpression of HDAC7-S/A blunted VEGF-induced RCAN2 and Nur77 expression. These results indicate that phosphorylation of HDAC7 is required for expression of RCAN2 and Nur77 in response to VEGF. HDAC7-S/A-ΔMEF failed to effectively block expression of Nur77 but was as effective as HDAC7-S/A in blocking VEGF-induced expression of RCAN2, suggesting that VEGF-induced Nur77 expression depends on MEF2, whereas RCAN2 expression is independent of MEF2. Consistent with these findings, the Nur77 gene is a known MEF2 target (26). These results indicate that HDAC regulates VEGF-induced gene expression in both a MEF2-dependent and independent manner.

Fig. 5.

Fig. 5.

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Inhibition of VEGF-dependent gene expression by HDAC7. (A and B) HAEC cells infected with adenovirus expressing lacZ (Ad-LacZ), FLAG-HDAC7 (Ad-HDAC7), FLAG-HDAC7-S/A (Ad-HDAC7-S/A), or HDAC7-S/A-ΔMEF (Ad-HDAC7-S/A-ΔMEF) were treated with 10 ng/ml VEGF for 1 h. Real-time quantative RT-PCR was performed to examine the expression level of (A) RCAN2 and (B) Nur77. (C–E) HAEC cells transfected with control siRNA pool or siRNA oligos targeted to HDAC7 (si-HDAC7) were treated with 10 ng/ml VEGF for 1 h. Expression of (C) HDAC7, (D) RCAN2, and (E) Nur77 was determined by real-time RT-PCR. The results are representative of three independent experiments. The P value shown was calculated by Student's t test.

We further tested the effect of HDAC7 knockdown on VEGF-induced gene expression. siRNA was used to knockdown HDAC7 in HAEC cells (Fig. 5C), causing expression of RCAN2 and Nur77 to increase by 2-fold in the absence of VEGF treatment (Fig. 5 D and E). Moreover, knockdown of HDAC7 decreased the magnitude of induction of RCAN2 and Nur77 expression in response to VEGF compared with cells transfected with control siRNA (Fig. 5 D and E). These data indicate that the presence of HDAC7 is required for repressing VEGF-regulated RCAN2 and Nur77 expression. The reduced effect of siHDAC7 on gene expression might reflect partial HDAC7 knockdown efficiency and redundant roles of other class IIa HDACs in regulating RCAN2 and Nur77 expression by VEGF. In addition, the reduction of HDAC7 might up-regulate other repressors, such as HADC9 (27), which might compromise the VEGF effect on gene expression.

Discussion

The results of this study demonstrate that VEGF activates the PKD signaling pathway, which modulates HDAC7 phosphorylation and nuclear export and in turn the migration and proliferation of ECs by regulating VEGF-responsive gene expression (Fig. 6).

Fig. 6.

Fig. 6.

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A model for the control of EC gene expression by VEGF signaling to HDAC7. In this model, VEGF induces phosphorylation and nuclear export of HDAC7 via a PKC/PKD1 pathway. HDAC7 controls VEGF-induced EC proliferation and migration by regulating VEGF responsive gene expression.

Regulation of VEGF Signaling by HDAC7 in ECs.

How VEGF signaling is dynamically regulated in the nucleus is a subject of great interest. Our results show that VEGF-A rapidly induces HDAC7 phosphorylation and nuclear export, which promotes EC proliferation and migration by relieving HDAC7-dependent repression of VEGF responsive genes. Maximal HDAC7 phosphorylation occurs ≈5–10 min after VEGF-A stimulation, and HDAC7 nuclear export is maximized ≈4 h later. Both HDAC7 phosphorylation and nuclear export then decrease with time, with HDAC7 becoming relocalized to the nucleus after 24 h of VEGF treatment. These findings indicate that HDAC7 acts as a molecular switch to dynamically regulate VEGF signaling. The ability of HDAC7 to mediate VEGF signaling is consistent with recent studies showing that HDAC7 is critical for angiogenesis (19, 20), a process in which VEGF plays a key role. We also found that other peptide growth factors, which complement or coordinate the actions of VEGF in regulating endothelial functions, also induce HDAC7 phosphorylation, albeit to a reduced level relative to VEGF. This suggests that HDAC7 acts as a focal point for multiple signaling pathways. Consistent with this notion, the T cell receptor has been shown to regulate apoptosis in thymocytes via phosphorylation and nuclear export of HDAC7 (28, 29).

Activation of the PKC/PKD pathway is necessary and sufficient to mediate VEGF-A-induced HDAC7 phosphorylation and nucleocytoplasmic shuttling, consistent with a prior report that VEGF can induce PKC-dependent PKD phosphorylation in ECs (23). PKD1, also known as protein kinase Cμ (PKCμ), is the founding member of a the PKD family, which also includes PKD2 and PKD3/PKCν (30). PKD1 has been identified as class IIa HDAC export kinase in cardiomyocytes and thymocytes (29, 31, 32). PKD has been implicated in diverse processes, such as, vesicular transport, metastasis, immune responses, apoptosis, and cell proliferation (30). Our data and those of others showed that PKD mediates VEGF-induced endothelial proliferation and migration (23, 33), indicating an important role for PKD in EC function.

Role of Class II HDACs in VEGF-Induced Angiogenesis.

Our results indicate that HDAC7 controls EC functions by repressing the expression of MEF2-dependent and independent genes. MEF2 likely plays a role in the regulation of VEGF-induced EC proliferation and migration downstream of the PKC/PKD pathway. Prior studies have implicated MEF2 in the control of EC survival downstream of the MAPK pathway (34, 35).

MMP10, a gene regulated by HDAC7 in ECs, was repressed by HDAC7-S/A but not significantly regulated by 1 h of VEGF treatment (data not shown). Similarly, calcineurin-binding protein (calsarcin-1) and growth arrest-specific 1 (GAS1), which have been shown to be up-regulated in HUVEC cells after siRNA-mediated knockdown of HDAC7 (19), were not significantly regulated by 1 h of VEGF treatment (data not shown). These findings suggest that the regulation of these genes by HDAC7 may reflect a late effect of VEGF on HDAC7. Alternatively, HDAC7 might also regulate VEGF-independent gene expression in ECs.

Although we have focused on the regulation of HDAC7 phosphorylation by VEGF because of the specificity and abundant expression of this HDAC in ECs, our results indicate that all class IIa HDACs are phosphorylated in response to VEGF. Thus, it is likely that the responses of ECs to VEGF represent the combined effects of phosphorylation of all class IIa HDACs. However, in vivo functional studies by gene knockouts show that only HDAC7 is required for maintaining endothelial function, whereas the absence of the other HDACs does not evoke a vascular phenotype under normal conditions (16–19). It is likely that different Class IIa HDACs might also have distinct targets and functions, although they are regulated similarly in response to VEGF. Among these HDACs, HDAC7 might play a dominant role in regulating EC function.

Therapeutic Implications.

Our finding that VEGF acts through PKD to modulate HDAC7 phosphorylation and EC functions raises interesting prospects for pharmacological manipulation of the VEGF-PKD-HDAC7 axis in the settings of vascular disorders. Up-regulation of VEGF-A underlies angiogenesis associated with diabetic retinopathy, tumor growth, metastasis, arthritis, and atherosclerosis. Antiangiogenic therapy has shown promise in suppression of tumor growth and other diseases related to abnormal angiogenesis (2). Our findings suggest that pharmacological inhibition of PKD should prevent HDAC7 phosphorylation, thereby repressing EC proliferation and migration. In this regard, development of specific PKD inhibitors or inhibitors of HDAC phosphorylation may hold potential in antiangiogenic therapy.

Materials and Methods

EC Culture and Immunocytochemistry.

HAEC (Clonetics) and HUVEC (American Type Culture Collection) cells were grown in EC growth medium (Clonetics/Cambrex) supplemented with 2% FBS. Kinase inhibitor and/or growth factor treatments of adenovirus infected cells are described in SI Text. For immunocytochemistry, cells were plated on gelatin-coated glass coverslips and fixed and stained as described (13).

Adenovirus Generation and Infection.

Recombinant adenoviruses were generated as described in SI Text or in refs. 13, 14, and 17.

Western Blot Analysis and Immunoprecipitation.

Western blot analysis was performed according to standard procedures by using the ECL detection kit (Amersham Pharmacia Biotech). Immunoprecipitation was performed as described (36) and outlined in SI Text.

siRNA Transfection and Real-Time Quantitative RT-PCR.

Details of transfection with siRNA and real-time PCR are described in SI Text. Primers are listed in SI Text.

EC Proliferation Assay and Scratch-Wound Assay.

EC proliferation assays were carried out as described (33). HAEC cell migration was detected by using an “in vitro scratch assay” (37) and as described in SI Text.

Supplementary Material

Supporting Information
0802857105_index.html (715B, html)

Acknowledgments.

We thank S. Chang, M. Avkiran, and T. McKinsey for scientific input and advice. We thank Jose Cabrera for graphics and Jennifer Brown for editorial assistance. E.N.O. is supported by grants from the National Institutes of Health, the Donald W. Reynolds Cardiovascular Clinical Research Center, and the Robert A. Welch Foundation. S.W. was supported by a fellowship grant from the American Heart Association.

Note Added in Proof.

During the preparation of this manuscript, Ha et al. published a report (38), consistent with our data, showing that HDAC5 regulates VEGF signaling and angiogenesis.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802857105/DCSupplemental.

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