Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2β to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.

PKD activity with a pan-PKD inhibitor increased AP2␤ nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2␤ nuclear localization through a Ser 258 -and Ser 277 -dependent mechanism. Furthermore, substitution of Ser 277 in AP2␤ increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.
Vascular endothelial growth factor (VEGF) 5 is the principal angiogenic growth factor that modulates physiological and pathological angiogenesis and plays a key role in cancer, ischemic and inflammatory diseases, and wound repair (1,2). VEGF receptor 2 (VEGFR-2) is recognized as the prominent receptor mediating VEGF-stimulated angiogenesis, vascular permeability, and remodeling (1). Upon binding of VEGF, VEGFR-2 undergoes dimerization and autophosphorylation to initiate activation of downstream signaling pathways (2,3). Numerous previous studies have contributed to a remarkable knowledge of VEGFR-2-mediated signaling cascades (4); however, the molecular mechanism controlling the expression levels of VEGFR-2 in endothelial cells remains unclear.
Protein kinase D (PKD), a family of serine/threonine kinases, regulates a variety of cellular functions, including proliferation, migration, and protein transport (5,6). In humans, three isoforms of PKD (PKD1, PKD2, and PKD3) have been described, of which PKD1 and PKD2 are predominantly expressed in endothelial cells (7). PKD is activated mostly by cell growthpromoting substances such as phorbol ester, platelet-derived growth factor, and G protein-coupled receptor ligands (8). PKD1 and PKD2 have been shown to function downstream of VEGFR-2 to directly phosphorylate heat shock protein 27 (HSP27), histone deacetylases (HDACs), and cAMP response element-binding protein (CREB) to modulate endothelial cell proliferation and migration (7,9,10), but the role of PKD1 and PKD2 in the regulation of VEGFR-2 expression remains poorly understood.
Transcription factor activator protein-2 (AP2, TFAP2) transcription factors constitute a family of closely related and evolutionarily conserved sequence-specific DNA-binding proteins. The five murine ap2 genes (AP2␣, AP2␤, AP2␥, AP2␦, and AP2⑀) are expressed in developing limbs, epithelia, and neuroectoderm, including neural crest-derived tissues such as facial mesenchyme (11,12). The importance of AP2 family members is highlighted by the embryonic or perinatal lethality of AP2 knockout mice (13)(14)(15)(16)(17). AP2 family members also play an integral role in developmentally controlled sleep behavior (18). AP2 proteins form homo-and heterodimers with other AP2 family members and can function as either transcriptional activators or repressors of genes involved in mammalian development, differentiation, and carcinogenesis (19). The transcriptional activity of AP2 is regulated by several interaction partners, subcellular localization, and posttranslational modifications (19,20). Although AP2 binding sites on the VEGFR-2 promoter have been predicted by computational modeling approaches (21), the specific AP2 family member (AP2␣, AP2␤, or AP2␥) that interacts with the VEGFR-2 promoter and its mode of transcriptional regulation has not been identified in endothelial cells.
In this study, we demonstrate that PKD1 and PKD2 directly regulate VEGFR-2 transcription through transcription factor AP2␤ in endothelial cells. We demonstrate that silencing PKD1 or PKD2 enhances association of AP2␤ with the VEGFR-2 promoter, decreasing VEGFR-2 transcription. The repressive effect of AP2␤ on VEGFR-2 expression was confirmed in AP2␤-overexpressing and silenced endothelial cells as well as AP2␤ knockout mouse embryos. Furthermore, we identified that PKD directly phosphorylates AP2␤ at serine 258 and 277 and that PKD-mediated phosphorylation controls subcellular localization and association with the VEGFR-2 promoter of AP2␤.

PKD regulates VEGFR-2 expression, proliferation, and migration of endothelial cells and angiogenesis
To evaluate the role of PKD in VEGFR-2 expression in endothelial cells, human umbilical vein endothelial cells (HUVECs) were transfected with PKD1 siRNA or PKD2 siRNA. Knockdown of PKD1 or PKD2 down-regulated VEGFR-2 protein and transcript (Fig. 1, A and B). The inhibitive effect of PKD on VEGFR-2 was also observed in human blood outgrowth endothelial cells (BOECs) (Fig. 1). Administration of a pan-PKD inhibitor, CRT0066101 (CRT), reduced the phosphorylation of HSP27, a well-established downstream target of PKD1 and PKD2, and significantly inhibited the expression of VEGFR-2 ( Fig. 1C), suggesting that activation of PKD suppresses the expression control of VEGFR-2. Indeed, overexpression of WT PKD1, a constitutively active form of PKD1 (PKD1.CA), and PKD2 in HUVECs increased the expression of VEGFR-2, among which PKD1.CA exhibited the greatest effect on VEGFR-2 up-regulation (Fig. 1D). Furthermore, in accordance with a published report (7), down-regulation of PKD1 or PKD2 significantly inhibited VEGF-induced endothelial proliferation and migration (Fig. 1, E and F). The effect of PKD on angiogenesis in vivo was further examined in a Matrigel plug assay (Fig.  1G). Matrigel containing PBS vehicle, VEGF alone, or a combination of VEGF and CRT, was subcutaneously implanted in mice. Our results showed that inhibition of PKD activity with CRT suppressed VEGF-induced angiogenesis in vivo. Taken together, these results suggest that endogenous PKD1 and PKD2 control expression of VEGFR-2 and positively regulate the proliferation and migration of endothelial cells as well as angiogenesis in vivo.

Knockdown of PKD2 enhances binding of AP2␤ to the VEGFR-2 promoter
We sought to determine the molecular mechanism through which PKD modulates VEGFR-2 expression. Based on bioinformatics data, three AP2 binding sites exist within the VEGFR-2 promoter between bp Ϫ143 to Ϫ134, Ϫ102 to Ϫ89, and Ϫ69 to Ϫ60 (21). Thus, we hypothesized that PKD may down-regulate VEGFR-2 through AP2-mediated transcriptional repression of the VEGFR-2 promoter. The results of our ChIP assay showed that AP2␣ bound the VEGFR-2 promoter but did not show differences among control, PKD1 knockdown, and PKD2 knockdown groups ( Fig. 2A). However, increased AP2␤ bound the VEGFR-2 promoter upon PKD1 knockdown, and this effect was most pronounced in PKD2-ablated HUVECs, as evidenced by the CHIP assay ( Fig. 2A).

AP2␤ negatively regulates the transcriptional activity of VEGFR-2
To demonstrate AP2␤-specific inhibition of the VEGFR-2 promoter, serial deletion luciferase constructs of the VEGFR-2 promoter containing three, two, one, or no AP2␤ binding sites were transfected into HUVECs with or without an AP2␤-GFP expression plasmid. Maximum inhibition of luciferase activity was observed with the construct containing three AP2␤ binding sites, and the number of AP2␤ binding sites positively correlated with increasing inhibition, confirming negative regulation of VEGFR-2 transcription by AP2␤ (Fig. 2B).

AP-2␤ controls the expression of VEGFR-2 in vitro and in vivo
Next, to confirm the regulatory role of AP2 in expression VEGFR-2, we demonstrated that siRNA-mediated knockdown of AP2␤ enhanced VEGFR-2 expression (Fig. 3A). Conversely, overexpression of AP2␤ decreased VEGFR-2 protein expression (Fig. 3B). We also performed qPCR and confirmed that knockdown of AP2␤ increased but overexpression of AP2␤ decreased the mRNA levels of VEGFR-2 (Fig. 3C). Furthermore, immunohistochemistry was performed to examine the expression of VEGFR-2 in genomic AP-2␤ knockout mouse embryos. Increased VEGFR-2 protein expression was observed in the spinal cord of AP-2␤ knockout mice at embryonic day 13 compared with similarly aged WT control embryos (Fig. 3,

Protein kinase D controls endothelial VEGFR-2
D-G). These results suggest that endogenous AP-2␤ suppresses VEGFR-2 expression in vitro and in vivo.

PKD activates AP2␤ at Ser 258 and Ser 277 and suppresses nuclear accumulation of AP2
We hypothesized that PKDs could directly regulate AP2␤ function by serine phosphorylation at positions 258 and 277. Previous studies have suggested Ser 258 as a phosphorylation site in AP2␤ using structural analysis (22). However, attempts to reliably detect serine phosphorylation of AP2␤ using phosphoserine-specific antibodies have proven challenging. Consequently, the effects of PKD1 and PKD2 on AP2␤ phosphorylation have not been examined. Therefore, to determine whether PKD1 is the upstream kinase for these sites, we performed Phos-tag TM assays with lysates from HEK293 cells that were cotransfected with vector, WT AP2␤, or mutated AP2␤-S258A or AP2␤-S277A together with PKD1.CA or vector control.
Expression of PKD1.CA led to a shift of WT AP2␤, indicating its phosphorylation by PKD1. This was decreased when Ser 258 or Ser 277 were blunted by serine-to-alanine substitutions, identifying both as PKD1 phosphorylation sites (Fig. 4A). To further examine the effect of endogenous PKD on AP2␤ phosphorylation, we collected lysates of HUVECs, performed immunoprecipitation with an AP2␤ antibody, and probed the immunoprecipitates with a pMOTIF antibody that specifically recognizes PKD-phosphorylated substrates. Our results confirmed that endogenous PKD phosphorylates AP2␤ and that this phosphorylation is reduced when the pan-PKD inhibitor CRT is present in the medium (Fig. 4B).
To define the functional consequence of PKD-mediated phosphorylation of AP2␤, the cellular localization of AP2␤ was examined using a fusion protein, AP2␤-GFP, in 293T cells. As shown in Fig. 4, C and D, administration of the pan-PKD inhibitor CRT significantly increased nuclear accumulation of AP2␤. . C, HUVECs were incubated with CRT (2.5 M) or DMSO as a control for 24 h, and then protein was collected and analyzed by Western blotting. D, HUVECs were infected with a lentivirus expressing LacZ as a control, WT PKD1, PKD1.CA, and WT PKD2 for 24 h, and then protein was collected for Western blotting. E and F, siRNA-transfected HUVECs as described in A were serum-starved and stimulated with VEGF (10 ng/ml). Migration was examined with a Boyden chamber assay 4 h after VEGF stimulation (E). Proliferation was determined using a [ 3 H]thymidine incorporation assay 20 h after VEGF stimulation (F). G, the Matrigel plug assay was performed by diluting Matrigel in EBM-2 medium containing heparin in the presence of PBS vehicle, VEGF (10 ng/ml), or a combination of VEGF and CRT0066101 (10 M). Matrigel was injected into 8-to 10-week-old mice subcutaneously. Fourteen days after implantation, mice were euthanized, and Matrigel plugs were removed, photographed, imaged, and quantitated for vessels as a measure of angiogenesis. Western blot images are representative of three independent experiments. *, p Ͻ 0.05; ***, p Ͻ 0.001. Scale bar for the Matrigel plug in G ϭ 5 mm; scale bar for H&E in G ϭ 50 m.

Protein kinase D controls endothelial VEGFR-2
The effect of PKD1 on AP2␤ nuclear localization was further examined in 293T cells transfected with mCherry-PKD1 or mCherry-PKD1.CA (Fig. 4, E and F). In cells transfected with PKD1, mutation of either Ser 258 or Ser 277 increased AP2␤ nuclear accumulation, as shown by enriched GFP signals in nuclei. Notably, overexpression of PKD1.CA decreased the nuclear localization of WT AP2␤ but not AP2␤ with mutations. Similar results were observed in PKD2-overexpressing 293T cells ( Fig. 4G and Fig. S1). In these experiments, substitution of both Ser 258 and Ser 277 did not significantly increase the nuclear localization of AP2␤ compared with single Ser 258 or Ser 277 substitution, suggesting that Ser 258 and Ser 277 play redundant roles in the AP2␤ nuclear localization. Nuclear protein was also collected from HUVECs stimulated with CRT and exhibited increased nuclear accumulation of AP2␤ (Fig. 4H). Overexpression of PKD1.CA in HUVECs was shown to decrease the nuclear AP2␤ protein level (Fig. 4I), suggesting that active PKD promotes cytoplasmic retention of AP2␤. Correspondingly, AP2␤ with a Ser 277 substitution showed significantly increased binding to the VEGFR-2 promoter (Fig. 4J). These results indicate that PKD-mediated phosphorylation of AP2␤, especially Ser 277 , controls the nuclear localization and DNA binding of AP2␤.

Discussion
In this study, we report that PKD regulates VEGFR-2 expression through AP2␤ in endothelial cells. It has been well-established that VEGF activity is controlled autonomously by its own expression levels through various circulating sequestering proteins and via VEGFR-2, its cognate tyrosine kinase receptor, predominantly responsible for transducing the angiogenic effects of VEGF (23). Several small-molecule VEGFR-2 inhibitors and VEGFR-2 monoclonal antibodies (24) have been devel-oped to therapeutically target tumor angiogenesis in cancer patients. Although VEGF is secreted by a variety of cell types, the expression of VEGFR-2 is mainly restricted to vascular endothelial cells. Here our results identify a novel mechanism through which PKD phosphorylates AP2␤ and regulates the cytoplasmic localization and DNA binding of AP2␤ to control transcription of VEGFR-2 in endothelial cells.
Because of the important role of VEGFR-2 in endothelial cells, multiple mechanisms have been reported to control the expression of VEGFR-2. Although endocytosis of VEGFR-2 was originally thought to induce its degradation and down-regulation, recent studies have indicated that VEGFR-2 endocytosis and trafficking in endosomal compartments are essential for VEGF-stimulated extracellular signal-regulated kinase activation but not down-regulation of VEGFR-2 (25). Previous studies identified that c-Cbl and activated PKC-mediated protein modification are important for down-regulation of VEGFR-2 levels (26,27). The promoter of VEGFR-2 was cloned, and positive regulatory elements were identified between bp Ϫ225 to ϩ127 (21). Important subsequent studies identified several molecules or protein complexes capable of increasing the transcriptional activity of VEGFR-2, including nuclear focal adhesion kinase, Sp1, and complexes consisting of mutant p53 bound to switching/sucrose non-fermenting (28 -31). AP2 was predicted to bind the promoter region of VEGFR-2 between bp Ϫ143 to Ϫ134, Ϫ102 to Ϫ89, and Ϫ69 to Ϫ60 (21); our promoter luciferase assay (Fig. 2) confirmed that all three binding regions are involved in AP2␤-suppressed VEGFR-2 expression. Although AP2 is generally considered a transcriptional activator, it has been shown to negatively regulate the transcription of several genes through interaction or competition with other positive transcription factors, such as Sp1 (32)(33)(34)(35)(36)(37). Indeed, the three identified AP2-binding elements in the VEGFR-2 promoter are either near or overlapping with Sp1-binding sites (21). Thus, it is possible that AP2␤ may compete or interact with Sp1 to repress transcription of VEGFR-2. Notably, although VEGFR-2 is often up-regulated during oncogenesis and recognized as a promising marker of several types of cancer, VEGFR-2 expression is negatively controlled by the inflammatory cytokines tumor necrosis factor ␣ and interleukin-1␤ (38), ␤-amyloid (39), and oxidized low-density lipoprotein (40), all of which induce endothelial cell dysfunction. Our results raise the possibility that AP2␤-mediated suppression of VEGFR-2 might be involved in endothelial cell dysfunction.
Positive feedback in the regulation of VEGFR-2 expression has been supported by several previous studies. VEGFR-2 translocates to nuclei and interacts with several nuclear proteins, including Sp1, to directly regulate its transcription (31). VEGF binding to membrane-bound VEGFR-2 induces an increase in gene transcription and protein expression of VEGFR-2 (41). Endogenous VEGF also activates and maintains the expression of VEGFR-2 in endothelial cells (42,43). Previous studies have indicated that PKD1 and PKD2 are required for VEGF-stimulated endothelial cell migration, proliferation, and tubulogenesis by mediating phosphorylation of HSP27, CREB-binding protein/p300, and HDACs (9, 10, 44). Our results demonstrate that genetic knockdown or pharmacological inhibition of PKD decreases VEGFR-2 expression in endo- A, HUVECs were transfected with control siRNA or PKD1 or PKD2 siRNA for 48 h, and then cross-linked chromatin-protein complexes were immunoprecipitated (IP) using AP2␣ or AP2␤ antibodies as well as control IgG. After reverse cross-linking, DNA fragments were isolated, and the VEGFR-2 promoter was amplified by PCR using promoter-specific primers and run on a 2% agarose gel. 5% of the input was run as a positive control. B, HUVECs were transfected with VEGFR-2 firefly luciferase (Luc) promoter constructs as indicated, with or without AP2␤-GFP and an internal Renilla luciferase control vector. After 48 h, luciferase activity was determined using the DualGlo luciferase kit, and data were normalized with respect to Renilla activity. *, p Ͻ 0.05; **, p Ͻ 0.01. N.S., not significant.

Protein kinase D controls endothelial VEGFR-2
thelial cells, suggesting that PKD1 and PKD2 act as a positive feedback mechanism to maintain the expression of VEGFR-2 upon stimulation of VEGF.
Previous studies have shown that knockdown of PKD1 phosphorylates AP2␣ at Ser 258 to negatively regulate expression of the ABC transporter in THP-1 cells (22). A follow-up study reported that knockdown of PKD1 leads to decreased phosphorylation levels of both AP2␣ and AP2␤ in 3T3-L1 preadipocytes (45). The Ser 258 in the basic domain of AP2, which is conserved among species of all five subtypes of AP2, exists within an LXRXXS/T sequence, a phosphorylation motif of PKD1 (45). The LXRXXS (Ser 277 ) motif of the AP2␤ basic domain is completely identical to that of AP2␣ (45). Given that Ser 258 and Ser 277 are functionally important and well-conserved phosphorylation sites among AP2 family members, it stands to reason that PKD activates AP2␤ at Ser 258 and Ser 277 to regulate nuclear localization and DNA binding of AP2␤.
Indeed, our results showed that PKD controlled the cytoplasmic retention of AP2␤, as shown by the increased nuclear localization of AP2␤ upon inhibition of PKD (Fig. 4). Typically, PKD1 and PDK2 reside in the cytosol as well as in intracellular compartments such as the Golgi and mitochondria. A recent report demonstrated that stimulation of cholecystokinin-2 promotes nuclear PKD2 localization in epithelial cells (46). PKD has also been shown to directly interact with and control the nuclear export of HDAC5 (47). Our results showed that two phosphorylated sites, Ser 258 and Ser 277 , are required for PKDcontrolled cytoplasmic retention of AP2␤. Furthermore, our results showed that mutation of AP2␤ Ser 277 increased the association of AP2␤ with the VEGFR-2 promoter. However, the molecular mechanism by which PKD-mediated phosphorylation controls cellular localization of AP2␤ and how the two phosphorylated sites of AP2␤ synergistically control the biolog-ical function of AP2␤ still requires further investigation. Our study does not exclude the possibility that PKD phosphorylates AP2␤ in both the cytosol and nucleus. Our results (data not shown) do not support the hypothesis that PKD forms complexes with AP2␤ in endothelial cells. Although PKD phosphorylates multiple downstream targets to control their cellular localization, not all phosphorylated substrates form complexes with PKD (5). Thus, we anticipate that PKD phosphorylates AP2␤ and rapidly dissociates from it. In future studies, we will examine the association of AP2␤ and other candidate molecules, such as 14-3-3 protein, which have been shown to associate with PKD-phosphorylated substrates and sequester them to the cytosol (49,50).
In conclusion, our results identified a novel signaling cascade of PKD-AP2␤ that controls the transcription of VEGFR-2 in endothelial cells. Our results provide insights for therapeutic strategies targeting VEGFR-2 in several diseases, including cancer, diabetic retinopathy, and ischemic diseases.

Protein kinase D controls endothelial VEGFR-2
Thermo Fisher and used for the ChIP assay. The pMOTIF antibody was from Cell Signaling Technology (51). The PKD1/2 antibody (2052, Cell Signaling Technology) that was initially used for immunoblotting (Fig. 1A) to examine PKD1 protein levels has been discontinued. Subsequently, the mouse monoclonal PKD1 antibody developed by Storz and co-workers (52) Protein kinase D controls endothelial VEGFR-2 was used in the Western blot depicted in Fig. 1D. Light-chainspecific secondary antibodies (211-032-171, Jackson Immu-noResearch Laboratories) were used in the Western blots of immunoprecipitation. Nuclear proteins were collected as described previously (53).

Phos-tag TM SDS-PAGE
Phos-tag TM SDS-PAGE was performed according to the manufacturer's instructions (Wako Chemicals USA Inc., Richmond, VA). In brief, 2 g of protein per sample was separated by electrophoresis at 50 V per gel on an 8% SDS-PAGE gel containing Phos-tag TM reagent (50 mol/liter) and MnCl 2 (50 mol/liter). Prior to protein transfer, gels were incubated in transfer buffer containing 1 mM EDTA for 10 min (twice) and then incubated in transfer buffer for 10 min. Proteins were transferred to a PVDF membrane and detected by standard immunoblotting techniques using Odyssey (LI-COR, Lincoln, NE).

Migration assay
5 ϫ 10 4 HUVECs were transfected with the indicated siRNA, serum-starved, and treated with or without 10 ng/ml VEGF. Following 4-h incubation at 37°C, cells that remained in the upper chamber were gently removed. Cells that had migrated through the filter were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet dissolved in 2% ethanol. Migration was quantitated by counting the number of cells in four separate fields on the filter using bright-field optics with a Nikon Diaphot microscope.

Real-time PCR
Total RNA was isolated from cells with the RNeasy Mini Kit (Qiagen). RNA was reverse-transcribed by using oligo(dT) priming using the iScript cDNA Synthesis Kit (Bio-Rad). Realtime PCR was performed with a TaqMan SYBR Green Master Mix (Applied Biosystems). The comparative cycle threshold method was used to calculate the relative abundance of mRNA compared with that of ␤-actin expression.

Protein kinase D controls endothelial VEGFR-2 ChIP assay
Cells were cross-linked with 1% formaldehyde followed by sonication. The sheared chromatin was immunoprecipitated with Dynabeads conjugated with control IgG or GFP primary antibodies. The eluted immunoprecipitates were incubated at 65°C for 6 h for reverse formaldehyde cross-linking. DNA was extracted with a Qiagen PCR purification kit and subjected to PCR with specific primers (Table S1).

Matrigel plug assay
The Matrigel plug assay was performed by diluting Matrigel in EBM-2 medium containing heparin in the presence of PBS vehicle, VEGF (10 ng/ml), or a combination of VEGF and CRT0066101 (10 m). Matrigel was injected into 8-to 10-week-old mice subcutaneously. Fourteen days after implantation, mice were euthanized, and the Matrigel plugs were removed, photographed, imaged, and quantitated for vessels as a measure of angiogenesis. All animals were maintained in accordance with Institutional Animal Care and Use Committee guidelines.

Statistics
Statistics was performed using Kruskal-Wallis ANOVA with Dunn's multiple comparisons test and Student's t test. Data are represented as mean Ϯ S.D. and considered statistically significantly different at p Ͻ 0.05.