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(Circulation Research. 2004;94:1500.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Proangiogenic Action of Thyroid Hormone Is Fibroblast Growth Factor–Dependent and Is Initiated at the Cell Surface

Faith B. Davis, Shaker A. Mousa, Laura O’Connor, Seema Mohamed, Hung-Yun Lin, H. James Cao, Paul J. Davis

From the Department of Veterans Affairs Medical Center (F.B.D, H.-Y.L., P.J.D.); Albany College of Pharmacy (S.A.M., L.O., S.M.); Ordway Research Institute, Inc. (F.B.D., H.-Y.L., H.J.C., P.J.D.); Wadsworth Center of the New York State Department of Health (P.J.D.); and Albany Medical College (P.J.D.), Albany, NY.

Correspondence to Faith B. Davis, MD, Ordway Research Institute, Inc., Center for Medical Science, 150 New Scotland Ave, Albany, NY 12208. Email fdavis{at}ordwayresearch.org

	Abstract

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The effects of thyroid hormone analogues on modulation of angiogenesis have been studied in the chick chorioallantoic membrane model. Generation of new blood vessels from existing vessels was increased 3-fold by either L-thyroxine (T₄; 10^–7 mol/L) or 3,5,3'-triiodo-L-thyronine (10^–9 mol/L). T₄–agarose reproduced the effects of T₄, and tetraiodothyroacetic acid (tetrac) inhibited the effects of both T₄ and T₄–agarose. Tetrac itself was inactive and is known to block actions of T₄ on signal transduction that are initiated at the plasma membrane. T₄ and basic fibroblast growth factor (FGF2) were comparably effective as inducers of angiogenesis. Low concentrations of FGF2 combined with submaximal concentrations of T₄ produced an additive angiogenic response. Anti-FGF2 inhibited the angiogenic effect of T₄. The proangiogenic effects of T₄ and FGF2 were blocked by PD 98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor. Endothelial cells (ECV304) treated with T₄ or FGF2 for 15 minutes demonstrated activation of MAPK, an effect inhibited by PD 98059 and the protein kinase C inhibitor CGP41251. Reverse transcription–polymerase chain reaction of RNA extracted from endothelial cells treated with T₄ revealed increased abundance of FGF2 transcript at 6 to 48 hours, and after 72 hours, the medium of treated cells showed increased FGF2 content, an effect inhibited by PD 98059. Thus, thyroid hormone is shown to be a proangiogenic factor. This action, initiated at the plasma membrane, is MAPK dependent and mediated by FGF2.

Key Words: angiogenesis • thyroxine • basic fibroblast growth factor • mitogen-activated protein kinase • endothelial cell

	Introduction

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Control of angiogenesis is a complex process involving local release of vascular growth factors,¹ the extracellular matrix, adhesion molecules, and metabolic factors.² Mechanical forces within blood vessels may also play a role.³ The principal classes of endogenous growth factors implicated in new blood vessel growth are the fibroblast growth factor (FGF) family and vascular endothelial growth factor (VEGF).⁴ The mitogen-activated protein kinase (MAPK) signal transduction cascade (extracellular signal-regulated kinases 1 and 2 [ERK1/2]) is involved in VEGF gene expression and in control of proliferation of vascular endothelial cells.⁴

In the setting of L-thyroxine (T₄)-induced myocardial hypertrophy in the rat, coordinated angiogenesis and cardiac growth have been described by Chilian et al⁵ and by Tomanek.⁶ In the latter report, the appearance of increased capillary numerical density preceded hypertrophy. The same group also showed that diiodothyropropionic acid, a thyroid hormone analog,⁷ promoted angiogenesis in the hypertrophied heart that was subjected to experimental myocardial infarction.⁸ The evidence for a proangiogenic effect of systemically administered T₄ has been based on histological evidence developed in animal models,⁵ and the possible mechanisms involved have not been described.

The availability of a chick chorioallantoic membrane (CAM) model of angiogenesis⁹ provided us with a system to quantitate angiogenesis and to study mechanisms involved in induction of angiogenesis by thyroid hormone. In this article, we describe a proangiogenic effect of T₄ at physiological concentrations that approximates that of basic fibroblast growth factor (FGF2) in the CAM model. This effect can enhance the action of submaximal concentrations of FGF2. We also provide evidence that the hormone effect is initiated at the endothelial cell plasma membrane and is mediated by activation of the ERK1/2 signal transduction pathway. The mechanism also involves increased FGF2 expression by endothelial cells and release of FGF2 protein into the cell medium.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
T₄, 3,5,3'-triiodo-L-thyronine (T₃), tetraiodothyroacetic acid (tetrac), T₄–agarose, and 6-N-propyl-2-thiouracil (PTU) were obtained from Sigma; PD 98059 from Calbiochem; and CGP41251 was a gift from Novartis Pharma (Basel, Switzerland). Polyclonal anti-FGF2 and monoclonal anti-ß-actin were obtained from Santa Cruz Biotechnology and human recombinant FGF2 from Invitrogen. Polyclonal antibody to phosphorylated ERK1/2 was from New England Biolabs and goat anti-rabbit IgG from DAKO.

Chick CAM Model of Angiogenesis
Neovascularization was examined by methods described previously.^9–12 Ten-day-old chick embryos were purchased from SPAFAS (Preston, CT) and incubated at 37°C with 55% relative humidity. A hypodermic needle was used to make a small hole in the shell concealing the air sac, and a second hole was made on the broad side of the egg, directly over an avascular portion of the embryonic membrane that was identified by candling. A false air sac was created beneath the second hole by the application of negative pressure at the first hole, causing the CAM to separate from the shell. A window 1.0 cm² was cut in the shell over the dropped CAM with a small-crafts grinding wheel (Dremel, division of Emerson Electric Co.), allowing direct access to the underlying CAM. FGF2 (1 µg/mL) was used as a standard proangiogenic agent to induce new blood vessel branches on the CAM of 10-day-old embryos. Sterile disks of No. 1 filter paper (Whatman International) were pretreated with 3 mg/mL cortisone acetate and 1 mmol/L PTU and air dried under sterile conditions. Thyroid hormone, hormone analogues, FGF2 or control solvents, and inhibitors were then applied to the disks and the disks allowed to dry. The disks were then suspended in PBS and placed on growing CAMs. Filters treated with T₄ or FGF2 were placed on the first day of the 3-day incubation, with antibody to FGF2 added 30 minutes later to selected samples as indicated. At 24 hours, the MAPK cascade inhibitor PD 98059 was also added to CAMs topically by means of the filter disks.

Microscopic Analysis of CAM Sections
After incubation at 37°C with 55% relative humidity for 3 days, the CAM tissue directly beneath each filter disk was resected from control and treated CAM samples. Tissues were washed 3x with PBS, placed in 35-mm Petri dishes (Nalge Nunc), and examined under an SV6 stereomicroscope (Zeiss) at x50 magnification. Digital images of CAM sections exposed to filters were collected using a 3-charge–coupled device color video camera system (Toshiba) and analyzed with Image-Pro software (Media Cybernetics). The number of vessel branch points contained in a circular region equal to the area of each filter disk were counted. One image was counted in each CAM preparation, and findings from 8 to 10 CAM preparations were analyzed for each treatment condition (thyroid hormone or analogues, FGF2, FGF2 antibody, PD 98059). In addition, each experiment was performed 3 times. The resulting angiogenesis index is the mean±SEM of new branch points in each set of samples.

FGF2 Assays
ECV304 endothelial cells were cultured in M199 medium supplemented with 10% fetal bovine serum. ECV304 cells (10⁶ cells) were plated on 0.2% gel-coated 24-well plates in complete medium overnight, and the cells were then washed with serum-free medium and treated with T₄ or T₃ as indicated. After 72 hours, the supernatants were harvested and assays for FGF performed without dilution using a commercial ELISA system (R&D Systems).

MAPK Activation
ECV304 endothelial cells were cultured in M199 medium with 0.25% hormone-depleted serum¹³ for 2 days. Cells were then treated with T₄ (10^–7 mol/L) for 15 minutes to 6 hours. In additional experiments, cells were treated with T₄ or FGF2 or with T₄ in the presence of PD 98059 or CGP41251. Nuclear fractions were prepared from all samples by our method reported previously,¹³ the proteins separated by polyacrylamide gel electrophoresis, and transferred to membranes for immunoblotting with antibody to phosphorylated ERK 1/2. The appearance of nuclear phosphorylated ERK1/2 signifies activation of these MAPK isoforms by T₄.^13–15

Reverse Transcription–Polymerase Chain Reaction
Confluent ECV304 cells in 10-cm plates were treated with T₄ (10^–7 mol/L) for 6 to 48 hours and total RNA extracted using guanidinium isothiocyanate (Biotecx Laboratories). RNA (1 µg) was subjected to reverse transcription–polymerase chain reaction (RT-PCR) using the Access RT-PCR system (Promega). Total RNA was reverse transcribed into cDNA at 48°C for 45 minutes, then denatured at 94°C for 2 minutes. Second-strand synthesis and PCR amplification were performed for 40 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 60 s, and extension at 68°C for 120 s, with final ex-tension for 7 minutes at 68°C after completion of all cycles. PCR primers for FGF2 were as follows: FGF2 sense strand 5'-TGGTATGTGGCACTGAAACG-3', antisense strand 5'-CTCAATGACCTGGCGAAGAC-3'; the length of the PCR product was 734 bp. Primers for GAPDH included the sense strand 5'-AAGGTCATCCCTGAGCTGAACG-3', and antisense strand 5'-GGGTGTCGCTGTTGAAGTCAGA-3'; the length of the PCR product was 218 bp. The products of RT-PCR were separated by electrophoresis on 1.5% agarose gels and visualized with ethidium bromide. The target bands of the gel were quantified using LabImage software (Kapelan), and the value for [FGF2/GAPDH]x10 calculated for each time point.

Statistical Analysis
Statistical analysis was performed by 1-way ANOVA comparing experimental with control samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Thyroid Hormone on Angiogenesis
As seen in Figure 1A and summarized in Figure 1B, both L-T₄ and L-T₃ enhanced angiogenesis in the CAM assay. T₄, at a physiologic total concentration in the medium of 0.1 µmol/L, increased blood vessel branch formation by 2.5-fold (P<0.001). T₃ (1 nmol/L) also stimulated angiogenesis 2-fold. The possibility that T₄ was only effective because of conversion of T₄ to T₃ by cellular 5'-monodeiodinase was ruled out by the finding that the deiodinase inhibitor PTU had no inhibitory effect on angiogenesis produced by T₄ (data not shown). In the studies included in this article, PTU was applied to all filter disks used in the CAM model. Thus, T₄ and T₃ promote new blood vessel branch formation in a CAM model that has been standardized previously for the assay of growth factors.

View larger version (55K): [in this window] [in a new window]   Figure 1. Effects of L-T4 and L-T3 on angiogenesis quantitated in the chick CAM assay. A, Control samples were exposed to PBS and additional samples to 1 nM T3 or 0.1 µmol/L T4 for 3 days. Both hormones caused increased blood vessel branching in these representative images from 3 experiments. B, Tabulation of mean±SEM of new branches formed from existing blood vessels during the experimental period drawn from 3 experiments, each of which included 9 CAM assays. At the concentrations shown, T3 and T4 caused similar effects (1.9-fold and 2.5-fold increases, respectively, in branch formation). **P<0.001 by 1-way ANOVA, comparing hormone-treated with PBS-treated CAM samples.

Effects of T₄–Agarose and Tetrac
We have shown previously that T₄–agarose stimulates cellular signal transduction pathways initiated at the plasma membrane in the same manner as T₄ and that the actions of T₄ and T₄–agarose are blocked by a deaminated iodothyronine analogue, tetrac, which is known to inhibit binding of T₄ to plasma membranes.^13–15 In the CAM model, the addition of tetrac (0.1 µmol/L) inhibited the action of T₄ (Figure 2A), but tetrac alone had no effect on angiogenesis (Figure 2C). The action of T₄–agarose, added at a hormone concentration of 0.1 µmol/L, was comparable to that of T₄ in the CAM model (Figure 2B), and the effect of T₄–agarose was also inhibited by the action of tetrac (Figure 2B; summarized in 2C).

View larger version (51K): [in this window] [in a new window]   Figure 2. Tetrac inhibits stimulation of angiogenesis by T4 and agarose-linked T4 (T4-ag). A, A 2.5-fold increase in blood vessel branch formation is seen in a representative CAM preparation exposed to 0.1 µmol/L T4 for 3 days. In 3 similar experiments, there was a 2.3-fold increase. This effect of the hormone is inhibited by tetrac (0.1 µmol/L), a T4 analogue shown previously to inhibit plasma membrane actions of T4.13 Tetrac alone does not stimulate angiogenesis (C). B, T4-ag (0.1 µmol/L) stimulates angiogenesis 2.3-fold (2.9-fold in 3 experiments), an effect also blocked by tetrac. C, Summary of the results of 3 experiments that examine the actions of tetrac, T4-ag, and T4 in the CAM assay. Data (means±SEM) were obtained from 10 images for each experimental condition in each of 3 experiments. **P<0.001 by ANOVA, comparing T4-treated and T4–agarose-treated samples with PBS-treated control samples.

Enhancement of Proangiogenic Activity of FGF2 by a Submaximal Concentration of T₄
Angiogenesis is a complex process that usually requires the participation of polypeptide growth factors. The CAM assay requires at least 48 hours for vessel growth to be manifest; thus, the apparent plasma membrane effects of thyroid hormone in this model are likely to result in a complex transcriptional response to the hormone. Therefore, we determined whether FGF2 was involved in the hormone response and whether the hormone might potentiate the effect of subphysiologic levels of this growth factor. T₄ (0.05 µmol/L) and FGF2 (0.5 µg/mL) individually stimulated angiogenesis to a modest degree (Figure 3). The angiogenic effect of this submaximal concentration of FGF2 was enhanced by a subphysiologic concentration of T₄ to the level caused by 1.0 µg FGF2 alone. Thus, the effects of submaximal hormone and growth factor concentrations appear to be additive.

View larger version (55K): [in this window] [in a new window]   Figure 3. Comparison of the proangiogenic effects of FGF2 and T4. A, Tandem effects of T4 (0.05 µmol/L) and FGF2 (0.5 µg/mL) in submaximal concentrations are additive in the CAM assay and equal the level of angiogenesis seen with FGF2 (1 µg/mL in the absence of T4). B, Summary of results from 3 experiments that examined actions of FGF2 and T4 in the CAM assay (means±SEM) as in A. *P<0.05; **P<0.001, comparing results of treated samples with those of PBS-treated control samples in 3 experiments.

To define more precisely the role of FGF2 in thyroid hormone stimulation of angiogenesis, a polyclonal antibody to FGF2 was added to the filters treated with either FGF2 or T₄, and angiogenesis was measured after 72 hours. Figure 4 demonstrates that the FGF2 antibody inhibited angiogenesis stimulated either by FGF2 or by T₄ in the absence of exogenous FGF2, suggesting that the T₄ effect in the CAM assay was mediated by increased FGF2 expression. Control IgG antibody has no stimulatory or inhibitory effect in the CAM assay.¹²

View larger version (54K): [in this window] [in a new window]   Figure 4. Effect of anti-FGF2 on angiogenesis caused by T4 or exogenous FGF2. A, FGF2 caused a 2-fold increase in angiogenesis in the CAM model in 3 experiments, an effect inhibited by antibody (ab) to FGF2 (8 µg). T4 also stimulated angiogenesis 1.5-fold, and this effect was also blocked by FGF2 antibody, indicating that the action of thyroid hormone in the CAM model is mediated by an autocrine/paracrine effect of FGF2 because T4 and T3 cause FGF2 release from cells in the CAM model (Table 1). We have shown previously that a nonspecific IgG antibody has no effect on angiogenesis in the CAM assay.12 B, Summary of results from 3 CAM experiments that studied the action of FGF2-ab in the presence of FGF2 or T4. *P<0.01; **P<0.001, indicating significant effects in 3 experiments studying the effects of thyroid hormone and FGF2 on angiogenesis and loss of these effects in the presence of antibody to FGF2.

View this table: [in this window] [in a new window]   Table 1. Effect of T4 and T3 on Release of FGF2 From ECV304 Endothelial Cells

Stimulation of FGF2 Release From Endothelial Cells by Thyroid Hormone
Levels of FGF2 were measured in the media of ECV304 endothelial cells treated with either T₄ (0.1 µmol/L) or T₃ (0.01 µmol/L) for 3 days. As seen in the Table, T₃ stimulated FGF2 concentration in the medium 3.6-fold, whereas T₄ caused a 1.4-fold increase. This finding indicates that thyroid hormone may enhance the angiogenic effect of FGF2, at least in part, by increasing the concentration of growth factor available to endothelial cells.

Role of the ERK1/2 Signal Transduction Pathway in Stimulation of Angiogenesis by Thyroid Hormone and FGF2
A pathway by which T₄ exerts a nongenomic effect on cells is the MAPK signal transduction cascade, specifically that of ERK1/2 activation.^13–15 We know that T₄ enhances ERK1/2 activation by epidermal growth factor.¹⁶ The role of the MAPK pathway in stimulation by thyroid hormone of FGF2 expression was examined by the use of PD 98059 (2 to 20 µmol/L), an inhibitor of ERK1/2 activation by the tyrosine–threonine kinases MAPK kinase-1 (MEK1) and MEK2.^13–15 The data in the Table demonstrate that PD 98059 effectively blocked the increase in FGF2 release from ECV304 endothelial cells treated with either T₄ or T₃.

Parallel studies of ERK1/2 inhibition were performed in CAM assays, and representative results are shown in Figure 5. A combination of T₃ and T₄, each in physiologic concentrations, caused a 2.4-fold increase in blood vessel branching, an effect that was completely blocked by 3 µmol/L PD 98059 (Figure 5A). FGF2 stimulation of branch formation (2.2-fold) was also effectively blocked by this inhibitor of ERK1/2 activation (Figure 5B). This result is consistent with reports from other laboratories.^17,18 Thus, the proangiogenic effect of thyroid hormone begins at the plasma membrane and involves activation of the ERK1/2 pathway to promote FGF2 release from endothelial cells. ERK1/2 activation is again required to transduce the FGF2 signal and cause new blood vessel formation.

View larger version (63K): [in this window] [in a new window]   Figure 5. Effect of PD 98059, a MAPK (ERK1/2) signal transduction cascade inhibitor, on angiogenesis induced by T4, T3, and FGF2. A, Angiogenesis stimulated by T4 (0.1 µmol/L) and T3 (1 nmol/L) together is fully inhibited by PD 98059 (3 µmol/L). B, Angiogenesis induced by FGF2 (1 µg/mL) is also inhibited by PD 98059, indicating that the action of the growth factor is also dependent on activation of the ERK1/2 pathway. In the context of the experiments involving T4–agarose (T4-ag) and tetrac (Figure 2) indicating that T4 initiates its proangiogenic effect at the cell membrane, results shown in A and B are consistent with 2 roles played by MAPK in the proangiogenic action of thyroid hormone: ERK1/2 transduces the early signal of the hormone that leads to FGF2 elaboration and transduces the subsequent action of FGF2 on angiogenesis. C, Summary of results of 3 experiments, represented by A and B, showing the effect of PD 98059 on the actions of T4 and FGF2 in the CAM model. *P<0.01; **P<0.001, indicating results of ANOVA on data from 3 experiments.

Action of Thyroid Hormone and FGF2 on MAPK Activation
Stimulation of phosphorylation and nuclear translocation of ERK1/2 MAPKs was studied in ECV304 cells treated with T₄ (10^–7 mol/L) for 15 minutes to 6 hours. The appearance of phosphorylated ERK1/2 in cell nuclei occurred within 15 minutes of T₄ treatment, reached a maximal level at 30 minutes, and was still apparent at 6 hours (Figure 6A). This effect of the hormone was inhibited by PD 98059 (Figure 6B), a result to be expected because this compound blocks the phosphorylation of ERK1/2 by MAPK kinase.^13,14 The traditional protein kinase C (PKC)-, PKC-ß, and PKC- inhibitor CGP41251 also blocked the effect of the hormone on MAPK activation in these cells, as we have seen with T₄ in other cell lines.¹³

View larger version (28K): [in this window] [in a new window]   Figure 6. T4 and FGF2 activate MAPK in ECV304 endothelial cells. Cells were prepared in M199 medium with 0.25% hormone-depleted serum and treated with T4 (0.1 µmol/L) for 15 minutes to 6 hours. Cells were harvested and nuclear fractions prepared as described previously.13 Nucleoproteins, separated by gel electrophoresis, were immunoblotted with antibody to phosphorylated MAPK (pERK1 and pERK2, 44 and 42 kDa, respectively), followed by a second antibody linked to a luminescence-detection system.13 A ß-actin immunoblot of nuclear fractions serves as a control for gel loading in each part of this figure. Each immunoblot is representative of 3 experiments. A, T4 causes increased phosphorylation and nuclear translocation of ERK1/2 in ECV304 cells. The effect is maximal in 30 minutes, although the effect remains for 6 hours. B, ECV304 cells were treated with the ERK1/2 activation inhibitor PD 98059 (PD; 30 µmol/L) or the PKC inhibitor CGP41251 (CGP; 100 nmol/L) for 30 minutes, after which 10–7 M T4 was added for 15 minutes to cell samples as shown. Nuclei were harvested, and this representative experiment shows increased phosphorylation (activation) of ERK1/2 by T4 (lane 4), which is blocked by both inhibitors (lanes 5 and 6), suggesting that PKC activity is a requisite for MAPK activation by T4 in endothelial cells. C, ECV304 cells were treated with either T4 (10–7 mol/L), FGF2 (10 ng/mL), or both agents for 15 minutes. The figure shows pERK1/2 accumulation in nuclei with either hormone or growth factor treatment and enhanced nuclear pERK1/2 accumulation with both agents together.

Thyroid hormone enhances the action of several cytokines and growth factors, such as interferon-¹³ and epidermal growth factor.¹⁶ In ECV304 cells, T₄ enhanced the MAPK activation caused by FGF2 in a 15-minute coincubation (Figure 6C). Applying observations made in ECV304 cells to the CAM model, we propose that the complex mechanism by which the hormone induces angiogenesis includes endothelial cell release of FGF2 and enhancement of the autocrine effect of released FGF2 on angiogenesis.

RT-PCR in ECV304 Cells Treated With Thyroid Hormone
The final question addressed in studies of the mechanism of the proangiogenic action of T₄ was whether the hormone may induce FGF2 gene expression. Endothelial cells were treated with T₄ (10^–7 mol/L) for 6 to 48 hours, and RT-PCR–based estimates of FGF2 and GAPDH RNA (inferred from cDNA measurements; Figure 7) were performed. Increase in abundance of FGF2 cDNA, corrected for GAPDH content, was apparent by 6 hours of hormone treatment and was further enhanced by 48 hours.

View larger version (55K): [in this window] [in a new window]   Figure 7. T4 increases accumulation of FGF2 cDNA in ECV304 endothelial cells. Cells were treated for 6 to 48 hours with T4 (10–7 mol/L) and FGF2 and GAPDH cDNAs isolated from each cell aliquot. The levels of FGF2 cDNA, shown in the top blot, were corrected for variations in GAPDH cDNA content, shown in the bottom blot, and the corrected levels of FGF2 are illustrated below in the graph (mean±SE of mean; n=2 experiments). There was increased abundance of FGF2 transcript in RNA extracted from cells treated with T4 at all time points. *P<0.05; **P<0.01, indicating comparison by ANOVA of values at each time point to control value.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The CAM assay has been used to validate angiogenic activity of several growth factors and other promoters or inhibitors of angiogenesis.^9–12 In the present studies, this model was used to establish that thyroid hormone in a physiologic concentration is proangiogenic. The mechanism by which thyroid hormone causes angiogenesis in the CAM model is conceived by us to be a complex one that is initiated at the plasma membrane by the hormone and involves transduction of the hormone signal into an FGF2-dependent angiogenic response. The basis for the latter conclusion includes (1) increased abundance of FGF2 RNA in endothelial cells with T₄ treatment; (2) increased medium content of FGF2 with T₄ treatment of endothelial cells; (3) blocking of the angiogenic effect of T₄ by addition of anti-FGF2 to the CAM assay; and (4) enhancement by T₄ of the angiogenic activity of low concentrations of FGF2 in the CAM model. In studies of T₄-induced myocardial hypertrophy in the rat, the hormone has been shown by other investigators to increase abundance of myocardial FGF2 mRNA in 1 to 2 days.¹⁹

T₄ at 10^–7 M total hormone concentration was comparable in angiogenic activity to the maximal FGF2 effect in this model. T₃ at 10^–9 M was also an effective proangiogenic factor in the CAM model. Although new blood vessel growth in the rat heart has been reported to occur concomitantly with induction of myocardial hypertrophy by a supraphysiologic dose of T₄,⁶ thyroid hormone has not been regarded as an angiogenic factor.²⁰ This article establishes that the hormone in physiologic concentrations is angiogenic in a setting other than the heart.

Because the appearance of new blood vessel branch points in the CAM model requires several days, we assumed from preliminary studies with T₄ that the effect of thyroid hormone was wholly dependent on a genomic mechanism. That is, T₄ was very likely converted to T₃ by cellular 5'-deiodinase activity, and T₃ entered the cell and cell nucleus, formed a complex with nuclear thyroid hormone receptor (TRß1), and initiated transcription of 1 or more genes relevant to angiogenesis. The presence of PTU in the assay system did not diminish the action of T₄, however, indicating that production of T₃ via deiodination of T₄ did not contribute to the action of T₄. This did not exclude the possibility that T₄ entered the cell and activated TRß1, but T₄–agarose reproduced the effects of T₄, and this derivative of thyroid hormone is thought not to gain entry to the cell interior, and has been used in our laboratory to examine models of hormone action for possible cell surface-initiated actions of iodothyronines.^13,14 Further, experiments performed with T₄ and tetrac also supported the conclusion that the action of T₄ in the CAM model was initiated at the plasma membrane. We have shown previously that tetrac blocks membrane-initiated effects of T₄ but does not itself have agonist activity at the membrane.^13,14

Because we have shown previously that thyroid hormone nongenomically activates the MAPK (ERK1/2) signal transduction pathway, we examined the possibility that the action of the hormone on angiogenesis was MAPK mediated. When added to the CAM model, an inhibitor of the MAPK cascade, PD 98059,^13,21 inhibited the proangiogenic action of T₄. Although this result was consistent with an action on transduction of the thyroid hormone signal upstream of an effect of T₄ on FGF2 elaboration, it is known that FGF2 also acts via a MAPK-dependent mechanism.^17,18 In studies presented above, we demonstrate that T₄ and FGF2 individually cause phosphorylation and nuclear translocation of ERK1/2 in endothelial cells, and when used in submaximal doses, combine to enhance ERK1/2 activation further. To examine the possibility that the only MAPK-dependent component of hormonal stimulation of angiogenesis related exclusively to the action of FGF2 on vessel growth, we measured cellular release of FGF2 in response to T₄ in the presence of PD 98059. The latter agent blocked the hormone-induced increase in growth factor concentration and indicated that MAPK activation was involved in the action of T₄ on FGF2 release from endothelial cells, as well as the consequent effect of FGF2 on angiogenesis.

That thyroid hormone is proangiogenic in the CAM model permits speculation about possible clinical significance of the finding. Because ambient concentrations of thyroid hormone in the intact organism are relatively stable, the hormone may be a tonic permissive factor in settings of new blood vessel growth. In the case of angiogenesis in the heart in the presence of arterial narrowing, the proangiogenic effect of thyroid hormone may be desirable. In contrast, in proliferative neovascularization in the eye (eg, diabetic retinopathy), a proangiogenic contribution of the hormone would be undesirable. If thyroid hormone were a permissive factor in this setting, its action might be subject to inhibition by administration of tetrac. Angiogenesis around primary or metastatic tumors might also be tonically stimulated by iodothyronines. This also is undesirable and might be pharmacologically inhibitable. Cellular models are available in which to test these possible consequences of thyroid hormone action.

	Acknowledgments

 
This work was supported in part by the Department of Veterans Affairs (H.-Y.L., P.J.D.), the Candace King Weir Foundation, and the Charitable Leadership Foundation (Albany, NY).

	Footnotes

 
Original received November 13, 2003; revision received April 21, 2004; accepted April 22, 2004.

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