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Increased Serum Vascular Endothelial Growth Factor Levels and Intrathyroidal Vascular Area in Patients with Graves’ Disease and Hashimoto’s Thyroiditis

Fourth Department of Internal Medicine, Saitama Medical School, Saitama 350-0495, Japan

Address all correspondence and requests for reprints to: Makoto Iitaka, M.D., Ph.D., Fourth Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan. E-mail: miitaka{at}saitama-med.ac.jp

	Abstract

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Vascular endothelial growth factor (VEGF) is one of the angiogenic factors. We examined both thyroid volume and intrathyroidal vascular area by color flow Doppler ultrasonography in patients with Graves’ disease (GD), Hashimoto’s thyroiditis (HT), and subacute thyroiditis. The serum concentrations of thyroid hormones, TSH, TSH receptor antibodies, and VEGF were also examined. There was a significant increase in serum VEGF levels in patients with untreated GD and goitrous HT compared with those in healthy subjects. The serum VEGF levels in untreated patients with subacute thyroiditis were significantly higher than those in patients with untreated GD or HT. There was a significant correlation between serum VEGF levels and the ratio of intrathyroidal vascular area and thyroid area in untreated patients with GD who had a goiter larger than or equal to 40 cm³. There was also a significant correlation between serum VEGF and TSH levels in patients with HT who were hypothyroid and had a goiter. Serum VEGF levels decreased significantly in these patients after treatment; this was accompanied by a significant decrease in intrathyroidal vascular area and thyroid volume. Our study demonstrates that VEGF appears to play an important role in intrathyroidal angiogenesis in patients with GD and goitrous HT.

	Introduction

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IT IS WELL known that there is marked enlargement of the blood capillaries and an increase in blood flow in the thyroids of hypothyroid animals (1, 2, 3) and patients with untreated Graves’ disease (GD) (4, 5). Thyroid volume increases rapidly because of stimulation by TSH or thyroid-stimulating antibodies in hypothyroid animals or patients with GD, respectively. These stimuli primarily act on the thyroid epithelial cells, suggesting that these cells may produce an angiogenic factor(s).

Recently, it has been demonstrated in vitro that vascular endothelial growth factor (VEGF), an endothelial cell-specific angiogenic factor (6), is produced by thyroid follicular epithelial cells in response to stimulation of the TSH receptor (7, 8, 9, 10, 11, 12). Secreted VEGF then stimulates Flt receptors (receptors for VEGF) on endothelial cells in a paracrine manner, leading to proliferation of the endothelial cells and hypervascularity of the thyroid gland. VEGF, therefore, may be one of the important thyroid angiogenic factors.

In this study we have evaluated serum VEGF levels in patients with various thyroid diseases. Intrathyroidal vascularization has also been assessed using color flow Doppler ultrasonography. We found a significant increase in serum VEGF levels in patients with untreated GD, Hashimoto’s thyroiditis (HT), and subacute thyroiditis (SAT). Furthermore, there was a close relationship between serum VEGF levels and the intrathyroidal vascular area or serum TSH levels in patients with GD or HT, respectively.

	Subjects and Methods

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Patients

Forty-nine patients (10 men and 39 women; 34.7 ± 11.9 yr old, mean ± SD) with GD, 24 patients (3 men and 21 women; 38.6 ± 11.9 yr old) with HT, 23 patients (3 men and 20 women; 45.4 ± 6.8 yr old) with SAT, and 37 healthy subjects (11 men and 26 women; 35.7 ± 11.2 yr old) were examined. There was no significant difference in age and sex between the healthy subjects and patients with thyroid diseases, except that patients with SAT were significantly older. The diagnosis in each case was made by established clinical and laboratory criteria. Patients with GD were treated with antithyroid drugs, whereas patients with HT were treated with L-T₄, and those with SAT were treated with prednisolone. Serum samples were obtained from these patients before and after treatment.

Serum VEGF concentrations

Serum concentrations of VEGF were determined using commercially available enzyme immunoassay kits (human VEGF ELISA system, Amersham International, Aylesbury, UK). This kit contains Sf21-expressed recombinant human VEGF₁₆₅ and antibodies raised against the recombinant protein. The assay range for serum samples was about 31.2–2000 ng/L. The intra- and interassay variances were 4.8% and 9.7%, respectively.

Measurement of thyroid volume and intrathyroidal vascular area

An ALOKA SSD 520 echo camera and an ALOKA SU-46 ultra scanner were employed for the measurement of thyroid volume as reported previously (13). Color flow Doppler ultrasonography was also examined using an ALOKA SSD 2000 and a 7.5-MHz linear transducer. The threshold for color flow signal was set individually for each patient by raising the threshold control to a level slightly above the point at which random color noise disappeared. Images were obtained in the transverse and parasagittal planes by ultrasonographers who did not know the thyroid status of the patients. Color flow Doppler images were photographed with a set-up designed for thyroid vascularization. Intrathyroidal vascularization was quantified by a modification of the method of Shimosawa et al. (14). The area occupied by blood vessels (the vascular area) and the thyroid gland (the thyroid area) were obtained using Adobe Photoshop J 3.0 (Adobe Systems Inc., Tokyo, Japan) from the color images in the parasagittal plane. Each area was then calculated using NIH Image. The thyroid vascular index was obtained as the ratio of the vascular area and the thyroid area.

Assay for hormones and autoantibodies

Serum free T₃ (FT₃) and free T₄ (FT₄) concentrations were measured by RIA (Amerlex MAB FT₃ RIA and FT₄ RIA kit, Ortho-Clinical Diagnostics, Tokyo, Japan). The reference ranges of serum FT₃ and FT₄ were 4.0–7.4 and 9–24 pmol/L, respectively. Serum TSH levels were measured by a highly sensitive radioimmunometric assay (Riagnost hTSH, Behring Co., Marburg, Germany). The reference range was 0.3–3.5 mU/L. TSH receptor antibodies (TRAb) were assayed using a commercially available kit (Cosmic Co., Tokyo, Japan).

Statistical analysis

Scheffe’s F test (Fig. 1), paired t test (Fig. 2), Kruskal-Wallis test (Fig. 3), and Wilcoxon signed rank test (Fig. 4) were used. Correlation coefficients for multiple parameters were analyzed by stepwise regression analysis. P < 0.05 was considered statistically significant.

View larger version (21K): [in this window] [in a new window]   Figure 1. Serum VEGF levels in patients with untreated GD, HT, and SAT.

View larger version (28K): [in this window] [in a new window]   Figure 2. Serum VEGF levels before and after treatment in patients with GD, HT, and SAT.

View larger version (15K): [in this window] [in a new window]   Figure 3. The thyroid vascular index in patients with untreated GD and HT. Significantly higher indexes were obtained in these patients compared with those in healthy subjects (GD > HT > healthy subjects, by Kruskal-Wallis test).

View larger version (24K): [in this window] [in a new window]   Figure 4. The thyroid vascular index before and after treatment in patients with GD and HT.

	Results

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Laboratory data for patients before and after treatment are shown in Table 1. All patients with GD and SAT were hyperthyroid, and those with HT were hypothyroid before treatment. All patients except for 13 with GD and 6 with HT were euthyroid after treatment. Five patients with treated GD had subclinical hyperthyroidism, whereas the other 8 patients had subclinical hypothyroidism (i.e. normal serum FT₃ levels with suppressed or increased serum TSH levels). Two of the patients with treated HT had subclinical hyperthyroidism, whereas the other 4 had subclinical hypothyroidism. The thyroid volumes in patients with untreated GD (n = 49) and HT (n = 24) were 48 ± 29 and 52 ± 22 cm³, respectively. The serum TSH and TRAb levels decreased significantly after treatment in patients with HT and GD, respectively.

Patients with untreated GD, HT, and SAT had significantly higher serum VEGF levels than healthy subjects (130 ± 85 ng/L) as shown in Fig. 1 and Table 1. Serum VEGF levels in patients with untreated SAT were significantly higher than those in patients with untreated GD or HT. When patients with GD were divided into two groups according to their goiter size (using a volume of 40 cm³ as the cut-off), there was no significant difference (317 ± 211 ng/L for patients with a goiter volume 40 cm³vs. 224 ± 160 ng/L for patients with a goiter volume <40 cm³; P = 0.0768). Both values, however, were significantly higher than the control values (P < 0.0001 and P = 0.0328, respectively.) There was no significant difference in serum VEGF levels between patients with GD and HT. The serum VEGF levels in these patients decreased significantly after treatment (Fig. 2). Although there was no difference in serum VEGF levels between patients with treated HT or SAT and healthy subjects, patients with GD whose goiters were equal to or larger than 40 cm³ had significantly higher serum VEGF levels (205 ± 109 ng/L; n = 25; P = 0.001, by Scheffe’s F test) than healthy subjects even after they became euthyroid. It is of interest to note that there was a weak, but significant, correlation between serum VEGF and TSH levels (R = 0.642; P = 0.045) only in patients with untreated HT. Other parameters, including serum FT₃, FT₄, TRAb levels, and thyroid volume, did not correlate with serum VEGF levels in any of the patients.

Intrathyroidal vascularization in various thyroid diseases

Color flow Doppler ultrasonography demonstrated an increase in intrathyroidal blood flow in patients with GD and goitrous HT. There was little or no intrathyroidal blood flow in patients with nongoitrous HT and SAT (data not shown). We used the thyroid vascular index for the evaluation of intrathyroidal vascularization.

In 28 patients with untreated GD, the thyroid vascular index was significantly higher than that in 15 healthy subjects (12.98 ± 9.02 vs. 0.24 ± 0.33; P < 0.0001; Fig. 3). The thyroid vascular index decreased significantly after treatment (P = 0.0069; n = 10; Fig. 4). We then examined the correlation between the thyroid vascular index and other parameters, such as serum FT₃, FT₄, TRAb, serum VEGF levels, and thyroid volume. When examined by the stepwise regression analysis, there was no correlation between any of these parameters. However, when patients with a goiter volume greater than or equal to 40 cm³ were examined, serum VEGF levels correlated significantly with the thyroid vascular index (R = 0.877; P = 0.0042). There was no correlation between thyroid volume and other parameters, although it correlated weakly, but significantly, with serum TRAb levels when examined by simple regression analysis (r = 0.358; P = 0.048).

In 15 patients with untreated goitrous HT, the thyroid vascular index was significantly higher than that in healthy subjects (6.09 ± 7.22 vs. 0.24 ± 0.33; P < 0.0001; Fig. 3). After treatment with L-T₄, it showed a significant decrease (P = 0.028; n = 7; Fig. 4) along with a significant decrease in thyroid volume (53 ± 26 cm³ before treatment vs. 36 ± 19 cm³ after treatment; P = 0.0003; n = 15; by paired t test). The thyroid vascular index in patients with untreated HT showed a significant correlation only with serum TSH levels (R = 0.674; P = 0.0082). Other parameters, including serum FT₃, FT₄, VEGF levels, and thyroid volume, did not correlate with the thyroid vascular index.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Progress in the study of angiogenesis in the thyroid gland has been made using thiouracil-fed hypothyroid rats and mice. In these animals, the thyroid gland undergoes hypertrophy with marked enlargement of the capillaries and increased blood flow (1, 2, 3). Vascular enlargement is restricted to the thyroid vessels. In 1978, Wollman et al. (3) hypothesized that intrathyroidal angiogenesis was due to the action of one or more substances produced by neighboring epithelial cells on the endothelium.

It has now been demonstrated that angiogenic factors such as basic fibroblast growth factor (bFGF) (15), placenta growth factor (8, 11), and VEGF are produced by thyroid epithelial cells (7, 8, 9, 10, 11, 12). In this study, we found that there was a significant elevation of serum VEGF levels in patients with untreated GD, HT, and SAT. Serum VEGF levels in these patients decreased significantly after treatment. There was a significant increase in intrathyroidal vascular area in patients with untreated GD and HT, although no intrathyroidal blood flow was observed in patients with untreated SAT. This suggests that the mechanism of the increase in serum VEGF in these patients may be different.

Sato et al. reported that TSH and thyroid-stimulating antibodies stimulated the expression of VEGF messenger ribonucleic acid in vitro in thyroid epithelial cells (7). The VEGF then stimulated vascular endothelial cells in the thyroid, resulting in increases in blood vessels and thyroid volume. The present study showed a significant correlation between serum VEGF levels and intrathyroidal vascular area in patients with untreated GD who had relatively large goiters. After treatment with antithyroid drugs, the intrathyroidal vascular area decreased significantly, accompanied by a decrease in serum VEGF levels. Although serum VEGF may not be derived solely from thyroid epithelial cells, VEGF appears to be one of the important angiogenic factors responsible for increasing intrathyroidal vascularization in patients with GD.

There was also a significant correlation between serum TSH levels and the thyroid vascular index in patients with untreated goitrous HT. Other studies (11, 16, 17, 18) have shown an increase in thyroidal blood flow after endogenous or exogenous TSH stimulation. These observations suggest the important role that TSH plays in thyroid angiogenesis. Similar to that in patients with GD, intrathyroidal vascularity decreased after treatment with L-T₄ along with a decrease in serum VEGF and TSH levels. However, there was no significant correlation between serum VEGF levels and intrathyroidal vascular area or thyroid volume in these untreated patients. In rats, the expression of VEGF messenger ribonucleic acid increased 3–7 days after thiouracil administration, but decreased to the control level on day 14 (7). Wollman et al. (3) demonstrated fusion of intrathyroidal vessels, rather than an increase in their numbers, in the thyroid after persistent TSH stimulation. In patients with treated GD, serum VEGF levels were still significantly higher than in healthy subjects. This suggests that TRAb may continue to stimulate thyroid epithelial cells to produce VEGF even after patients become euthyroid. Serum TSH levels decrease to normal soon after L-T₄ supplementation, in contrast to the gradual decrease in serum TRAb levels in patients with GD. Thus, TRAb in patients with GD may be able to stimulate thyroid epithelial cells to produce VEGF for longer than observed with TSH in patients with HT. TRAb may also induce another angiogenic factor, bFGF and/or placenta growth factor, although it was recently reported that serum bFGF levels in patients with GD were not elevated (19). Furthermore, VEGF may be produced in other organs apart from the thyroid in hyperthyroid patients. VEGF is known to be expressed by a number of normal adult tissues, including kidney, lung, uterus, ovary, brain, heart, skin, pituitary gland, and macrophages (6). However, vascular enlargement was restricted to the thyroid blood vessels in thiouracil-fed hypothyroid rats (3). In hypothyroid patients with HT, therefore, VEGF may be produced solely in the thyroid. The impaired MCR in hypothyroid patients may also contribute to the elevation of serum VEGF levels. These factors may explain the differences in serum VEGF levels and their correlation with intrathyroidal vascular area in patients with GD and HT. It is of interest that the intrathyroidal vascular area did not increase in hypothyroid patients with an atrophic thyroid. This suggests that even a high concentration of serum TSH observed when the thyroid gland is atrophic does not induce the production of large amounts of angiogenic factors from thyroid epithelial cells.

Serum VEGF levels were significantly higher in patients with SAT, although there was no blood flow in the thyroid. Serum VEGF levels decreased to normal after treatment. In contrast, serum VEGF levels in patients with silent thyroiditis were not elevated (Miura, S., M. Iitaka, K. Yamanaka, and S. Katayama; unpublished data). The increased level of serum VEGF in patients with SAT thus appears to be due to the systemic inflammatory reaction seen in other inflammatory diseases (20, 21).

Our study has demonstrated that VEGF produced by thyroid epithelial cells appears to play an important role in intrathyroidal angiogenesis in patients GD and hypothyroid goitrous HT. The decreases in serum TRAb and TSH levels appear to contribute to the reductions in serum VEGF levels, intrathyroidal vascular area, and thyroid volume.

	Acknowledgments

 
The authors thank Ms. Suzuki for her skillful technical assistance, and the ultrasonographers in the central ultrasound room. We also thank Dr. Volpé for his valuable comments.

Received April 16, 1998.

Revised July 1, 1998.

Accepted August 4, 1998.

	References

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