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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 6 1968-1973
Copyright © 1997 by The Endocrine Society

Experimental Studies

Increased Concentration of Vascular Endothelial Growth Factor/Vascular Permeability Factor in Cyst Fluid of Enlarging and Recurrent Thyroid Nodules1

Kanji Sato, Megumi Miyakawa, Noritaka Onoda, Hiroshi Demura, Tetsuji Yamashita, Masakazu Miura, Takeshi Kasajima, Kazuko Yamazaki and Takao Obara

Department of Medicine(K.S., Me.M., N.O., H.D.) and Department of Surgery (K.Y., T.O.), Institute of Clinical Endocrinology, Tokyo Women’s Medical College; Second Department of Pathology (T.K.), Tokyo Women’s Medical College, Shinjuku-ku, Tokyo 162; and Mitsubishi-Kagaku Biochemical Laboratories (T.Y., Ma.M.) Itabashi-ku, Tokyo 111, Japan

Address all correspondence and requests for reprints to: Kanji Sato, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical College, Kawada-cho 8–1, Shinjuku-ku, Tokyo 162, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human thyrocytes produce vascular endothelial growth factor (VEGF), a potent angiogenic factor, also known as vascular permeability factor (VPF), which increases vascular permeability. Based on the assumption that VEGF/VPF is involved in fluid accumulation in thyroid cysts, we determined the VEGF/VPF concentration in cyst fluids of thyroid nodules from 79 patients. VEGF/VPF was found to be abundantly present in the cyst fluids (0.02–183 ng/mL). There was no significant difference of VEGF/VPF concentration in the cyst fluid obtained from thyroid adenoma or from adenomotous goiter with cystic degeneration. Immunoreactive VEGF/VPF in cyst fluid was eluted mainly at 45 kDa, and stimulated endothelial cell proliferation, which was partially blocked by anti-VEGF/VPF antibody. The VEGF/VPF concentration in the cyst fluid obtained from patients who required repeated aspiration or underwent surgical resection because of recurrent accumulation (84.8 ± 58.3 ng/mL, mean ± SD, n = 18) was significantly higher than that in the cysts that regressed or disappeared after a single aspiration (4.3 ± 4.4 ng/mL, n = 12, P < 0.001).

These in vitro and clinical findings suggest that VEGF/VPF is at least partly involved in the accumulation of cyst fluid in thyroid nodules, and that a high VEGF/VPF concentration predicts rapid accumulation of the cyst fluid, possibly necessitating interventional treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASCULAR endothelial growth factor (VEGF) is a potent angiogenesis factor that exclusively stimulates the proliferation of endothelial cells in vitro and in vivo (1). Recently, we demonstrated that human thyrocytes express VEGF messenger RNA (mRNA), the steady state level of which is increased by TSH and Graves’ disease IgG (2). VEGF, a secretable angiogenesis factor (3), stimulates endothelial cell proliferation in a paracrine manner, resulting in hypervascularity of the thyroid gland in Graves’ disease (2).

VEGF is also known as vascular permeability factor (VPF), which stimulates vascular permeability and promotes accumulation of fluid in the peritoneal and pleural cavities (4). Indeed, VEGF/VPF is present at a concentration of 10-11–10-10 M in ascites and pleural fluid of patients with malignant or nonmalignant disorders (5). Cyst fluid in thyroid nodules is frequently seen in adenomatous goiter and thyroid adenoma with cystic degeneration (6). When a thyroid nodule composed mainly of a cyst is large enough to cause discomfort in the cervical region and elicits cosmetic problems, or when a malignant disorder is suspected, the cyst fluid is frequently aspirated. Cystic nodules may disappear completely in some patients after a single aspiration, whereas they may recur rapidly and require repeated aspiration in others (7).

On the assumption that VEGF/VPF is also involved in cyst fluid accumulation in thyroid nodules, we measured the VEGF/VPF concentration in the cyst fluid of thyroid adenoma and multinodular goiter in 79 patients who visited our hospital complaining chiefly of thyroid nodules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cyst fluid from thyroid nodules

Cyst fluid was obtained from 79 patients (age 24–79 yr), who visited the Institute of Clinical Endocrinology, Tokyo Women’s Medical College Hospital, with chief complaints of thyroid nodules, between October 1994 and August 1996. When the thyroid nodules (adenomatous goiter and adenoma) were composed mainly of cyst fluid and the patients had cosmetic complaints, or when a malignant lesion was suspected in parenchymatous tissue, the cyst fluid was aspirated. Informed consent was obtained from all patients. After aspiration under echography, the cyst fluid (2–100 mL) was kept in an icebox and then centrifuged at 3000 rpm for 15 min. The supernatants were stored at -20 C until assayed for VEGF/VPF and thyroglobulin.

Blood was obtained on the same day, before cyst fluid was aspirated, and serum levels of T4, T3, TSH, and thyroglobulin were determined using RIA or radiometric assay kits at Mitsubishi-Kagaku Biochemical Laboratories (Tokyo, Japan).

Assays for VEGF/VPF and thyroglobulin

VEGF/VPF concentration was determined using a solid-phase enzyme linked immunosorbent assay (ELISA) (R & D Systems, Minneapolis, MN). This assay contains insect cell Sf 21-expressed recombinant human VEGF165 and antibodies raised against the recombinant protein, and its sensitivity is <15 pg/mL. The intra- and interassay variances are 5.6% and 9.5%, respectively. Preliminary experiments revealed that VEGF/VPF was very stable in the cyst fluid. Freezing and thawing of the cyst fluid up to five times hardly affected the immunoreactive VEGF/VPF concentration (data not shown). Thyroglobulin concentration was determined using an ELISA kit (Fuji-Rebio, Tokyo, Japan).

Biochemical and biological properties of VEGF/VPF in the cyst fluid

To determine the molecular weight of VEGF/VPF, 2 mL cyst fluid containing a high VEGF/VPF concentration was applied to a Sephadex G-75 column (2.2 cm x 85 cm), which had been equilibrated with phosphate-buffered 0.9% NaCl solution containing 0.02% sodium azide. Each fraction (7 mL/tube) was collected and assayed for VEGF/VPF.

To investigate the proliferative effect of cyst fluid on human endothelial cells, cyst fluid containing a high VEGF/VPF concentration (50 ng/mL) was centrifuged at 10,000 x g for 15 min and then filtered through a Millipore filter (0.45 µm; Millipore Corp., Bedford, MA). The filtrate was then dialyzed against 0.9% NaCl solution, and sterilized by filtering through a Millipore filter. Human umbilical vein endothelial cells (HUVEC) purchased from Kurabou (Endocell, Neyagawa-shi, Japan) were cultured in 24-multiwell dishes in the supplemented medium containing 2% FBS, human epidermal growth factor (10 ng/mL), hydrocortisone (1 µg/mL), gentamycin (50 µg/mL), and bovine brain extract (0.4%, vol/vol). Brain extract contains VEGF/VPF, which is indispensable for endothelial cell proliferation. When the cells reached 50% confluence, the medium was discarded and replaced with 1 mL {alpha}-MEM supplemented with 10% FCS. Preliminary experiments revealed that the cells proliferated for at least 2 days under these culture conditions without the addition of VEGF/VPF. The sample or 0.9% NaCl solution containing 0.2% BSA (0.2 mL) was then added. In some experiments, polyclonal anti-VEGF-antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added (8). The cells were precultured for 24 h, and then [3H]thymidine was added. After an additional 18 h of culture, the cell monolayer was washed and [3H]thymidine incorporated into HUVEC was counted in a liquid scintillation counter.

Northern blot analysis

In a few experiments, the cyst fluid was immediately centrifuged for 15 min. After the supernatant had been transferred to another tube, 4 M guanidinium isocyanate solution was added to the pellets. Total RNA was extracted according to the method of Chomczynski and Sacchi (9), and RNA samples (10 µg) were size-fractionated on 1.0% agarose gel containing 1.2 M formaldehyde and transferred to nylon membranes. The integrity of the RNA was assessed by ultraviolet shadowing. Using human VEGF complementary DNA (cDNA) as a probe, Northern blot hybridization was performed as described previously (2). The filter was then rehybridized with 32P-labeled human glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Immunohistochemistry

Sections (3.5 mm) of formalin-fixed paraffin-embedded tissues were deparaffinized in lemosol (Wako-Junyaku, Tokyo, Japan), dehydrated in graded ethanol, and then immersed in distilled water. For enzymatic treatment, sections were immersed 0.01 N hydrochloric acid (HCl) solution containing 0.4% pepsin at 37 C for 40 min and then washed with PBS. Sections were incubated in normal swine serum for 10 min at room temperature and then incubated with a rabbit polyclonal antibody against human VEGF at a concentration of 0.5 µg/mL in PBS with 1% BSA at 4C in a humidified chamber. On the following day, the sections were washed in PBS and exposed to biotinylated goat antirabbit IgG for 30 min at room temperature. After sufficient washes with PBS, the sections were incubated with streptavidin-horseradish peroxidase complex (Vector Labs., Burlingame, CA) for 60 min. After incubation, the sections were washed thoroughly, and immersed in 0.5 M Tris-HCl buffer containing 3,3'-diaminobenzidine and 0.01% hydrogen peroxide. After detection of the brown reaction products, the light microscopy sections were washed with distilled water and counterstained with hematoxylin, and finally mounted under cover glasses in the routine way. Simultaneously, control studies was carried out using normal rabbit serum instead of the primary antibody.

Statistical analysis

Data are presented as the mean ± SD. Differences between groups were analyzed by unpaired Student’s t test where appropriate, or by ANOVA with pairwise comparison by Sheffe’s method, using Statview (Abacus Concepts, Berkley, CA). Statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VEGF/VPF concentration in cyst fluid

In all patients, the serum levels of T4, T3, and TSH were within the normal ranges, whereas the serum level of thyroglobulin was elevated (>35 ng/mL) in 38 of the 50 patients (76%).

VEGF/VPF was present in the cyst fluid of the thyroid nodules at various concentrations, ranging from 0.02–183 ng/mL. The VEGF/VPF concentration in the cyst fluid of patient with adenomatous goiter and thyroid adenoma with cystic degeneration was 26.6 ± 46.3 ng/mL (mean ± SD, n = 36) and 25.4 ± 39.4 ng/mL (mean ± SD, n = 42), respectively (Fig. 1Go). There was no significant difference between them (P > 0.1). Aspiration biopsy revealed papillary carcinoma cells in one patient. The VEGF/VPF concentration in the cyst fluid of this patient was 1.8 ng/mL.



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  		Figure 1. VEGF/VPF concentrations in the cyst fluid of thyroid nodules. VEGF/VPF concentrations were determined by ELISA as described in Materials and Methods. There was no significant difference in VEGF/VPF concentration between adenoma and adenomatous goiter with cystic degeneration.

 
As expected, thyroglobulin was present abundantly in the cyst fluid (497 ± 895 µg/mL, mean ± SD, n = 60). However, no significant correlation was found between cyst fluid levels of VEGF/VPF and thyroglobulin (data not shown), or between the serum level of thyroglobulin and VEGF/VPF concentration in the thyroid cysts (data not shown). There was also no significant correlation between VEGF/VPF concentration and fluid volume (data not shown).

The VEGF/VPF concentration in the bloody cystic fluid obtained from rapidly enlarging, tender thyroid nodules because of bleeding was 64.0 ± 19.6 ng/mL (mean ± SD, n = 4). Furthermore, the VEGF/VPF concentration in cyst fluid of thyroid nodules that were gradually enlarging and required repeated aspiration within 1 month or subjected to surgical resection because of rapid recurrent accumulation of cyst fluid was elevated (84.8 ± 58.3 ng/mL, mean ± SD, n = 18), whereas it was significantly decreased (4.3 ± 4.4 ng/mL, n = 12, P < 0.01) in cystic fluid in thyroid nodules that regressed or disappeared after a single aspiration (Fig. 2Go).



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  		Figure 2. VEGF/VPF concentrations in cyst fluid of enlarging and regressing thyroid nodules. VEGF/VPF concentrations in thyroid cysts, which were steadily increasing in size and necessitated repeated aspiration within 1 month because of recurrence, were determined as described in Materials and Methods. These concentrations were significantly higher than those in fluid of cysts that were decreasing in size or that disappeared after a single aspiration (P < 0.001).

 
Biochemical and biological properties of VEGF/VPF in cyst fluid

When the cyst fluid with high immunoreactive VEGF/VPF was applied to a Sephadex G-75 column, the immunoreactive VEGF/VPF was eluted mainly at 40–50 kDa, with a small peak in the void volume (Fig. 3Go).



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  		Figure 3. Elution profile of VEGF/VPF on a gel filtration chromatography. Two milliliters cyst fluid containing approximately 100 ng VEGF/VPF was applied to a Sephadex G-75 column that had been equilibrated with phosphate-buffered 0.9% NaCl solution (pH 7.4). Protein concentration in each fraction (7 mL) was evaluated at OD280. VEGF/VPF concentration was determined as described in Materials and Methods. OD280 nm, {circ}; VEGF/VPF (pg/mL), •. V0, Void volume.

 
To investigate whether the immunoreactive VEGF/VPF was bioactive, the effects of recombinant human VEGF (1 ng/mL) and cyst fluid on endothelial cell proliferation were studied. Similar to recombinant human VEGF/VPF, the cyst fluid stimulated [3H]thymidine incorporation into HUVEC. Addition of anti-VEGF antibody partially inhibited the [3H]thymidine incorporation induced by the cyst fluid, suggesting that the immunoreactive VEGF/VPF in the cyst fluid was bioactive (Table 1Go).


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  		Table 1. Effects of cyst fluid and anti-VEGF/VPF antibody on endothelial cell proliferation

 
VEGF mRNA expression in cells obtained from cyst fluid

Total RNA obtained from the pellets of thyroid cysts was degraded to some extent, as demonstrated by use of ultraviolet light. However, consistent with our previous findings (2), Northern blot analysis revealed VEGF/VPF transcript of 3.9 kilobases (Fig. 4Go). In this cyst fluid (in patients 1–3), which was reddish-brown and turbid, the VEGF/VPF concentration was elevated (>30 ng/mL), whereas it was decreased (3.3 ng/mL) in the cyst fluid in which VEGF mRNA was not detected (patient 4).



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  		Figure 4. Expression of VEGF/VPF mRNA in cells floating in cyst fluid. Total RNA (10 µg) was extracted from pellets of cyst fluid. VEGF mRNA expression was demonstrated by Northern blot hybridization as described previously (2). Lower panel shows same blot after rehybridization with a 32P-labeled GAPDH cDNA.

 
Immunohistochemistry

Microscopically, various-sized cystic lesions were observed in the thyroid tissue. The cyst walls were covered with a thin monolayer of epithelial cells and partially protruded into the cyst lumen, accompanied by slightly adenomatous hypertrophic thyroid gland (Fig. 5AGo). Immunohistochemically, a moderately to intensively positive reaction for VEGF/VPF was detected in the lining of the epithelial cells and epithelial cells of thyroid glands distributed in the vicinity of the cyst walls (Fig. 5Go, B–E). Furthermore, a positive reaction was recognized in small blood vessels in the cyst walls. These positive reactions were observed as a fine, globular pattern in the cytoplasm and perinuclear region. No positive reaction was detected in sections with control staining described in Materials and Methods.



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  		Figure 5. Immunohistochemical staining for VEGF/VPF in cystic adenoma. A, Hematoxylin-eosin staining of a cystic lesion in thyroid tissue. B-E, Thyroid tissue stained by anti-VEGF/VPF antibody. There is a positive reaction (brown precipitate) in lining epithelium and in adenomatous and cystic growing follicular cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have for the first time shown that bioactive VEGF/VPF is present abundantly in the cyst fluid of thyroid nodules. Because the cyst fluid is surrounded by VEGF/VPF-positive thyrocytes, it is highly likely that VEGF/VPF is secreted by these cells, stimulating fenestration of endothelial cells in blood vessels (10) and increasing microvascular permeability in a paracrine manner, which leads to accumulation of cyst fluid. As expected, no significant difference in cyst fluid VEGF/VPF concentration was observed between adenoma and adenomatous goiter with cystic degeneration. Although there was no significant correlation between the level of VEGF/VPF and the volume of cyst fluid, this was not unexpected, because cyst volume is determined by a number of factors, such as degeneration of thyrocytes, bleeding into the cyst, and negative pressure in the cyst after aspiration of cyst fluid.

The lack of correlation between the levels of VEGF and thyroglobulin in the cyst fluid is also reasonable, because VEGF/VPF mRNA expression is up-regulated by hypoxia, glucopenia, interleukin-1, interleukin-6, tumor necrosis factor-{alpha}, interferon-ß, transforming growth factor-ß, and epidermal growth factor (11, 12, 13, 14, 15, 16, 17), whereas most of these proinflammatory cytokines have inhibitory effects on thyroid function (18, 19, 20), accompanied by decreased synthesis of thyroglobulin (21). To our knowledge, only TSH and insulin-like growth factor-I simultaneously stimulate expression of VEGF/VPF and thyroglobulin mRNA (22, 23).

As expected, the concentration of VEGF/VPF was increased in the cyst fluid of thyroid nodules that were steadily increasing in size, or in cyst fluid that rapidly reaccumulated, necessitating repeated aspiration or surgical resection. This suggests that VEGF/VPF is at least partly involved in the pathogenesis of cyst fluid accumulation in thyroid nodules. Involvement of VEGF/VPF in fluid accumulation has also been reported in ovarian cysts, as well as in ovarian hyperstimulation syndrome (24, 25) .

When cyst fluid was subjected to gel filtration column chromatography, a small peak of immunoreactive VEGF was eluted in the void volume; this was probably inactive VEGF bound to macromolecular proteins such as {alpha}2-macroglobulin (26). However, the immunoreactive VEGF was eluted mainly at 45 kDa, suggesting that VEGF/VPF was eluted in the dimeric form (1). In line with these observations, the cyst fluid containing a high VEGF/VPF concentration stimulated proliferation of vascular endothelial cells, which was partially blocked by anti-VEGF antibody, as reported previously (8). Therefore, it is reasonable to speculate that VEGF/VPF in the cyst fluid also has bioactivity to increase vascular permeability, and that it is at least partly involved in the accumulation of cyst fluid. Incomplete inhibition by anti-VEGF antibody of HUVEC proliferation induced by cyst fluid would indicate that other growth factors for endothelial cells (27), such as basic fibroblast growth factor and insulin-like growth factor-I (28, 29), known to be produced by thyrocytes, are also present in the cyst fluid.

Previously, high levels of VEGF/VPF (1 x 10-11–1.4 x 10-10 M) have been reported in the pleural and peritoneal fluids of patients with malignant effusion, whereas lower levels of VPF (5.5–10 x 10-12 M) have been also found in patients with congestive heart failure, liver cirrhosis, or pneumonia (4). Also, the VEGF/VPF concentration in ocular fluid from patients with active diabetic retinopathy is higher (3.3 ± 6.3 ng/mL) than that of patients with nonproliferative diabetic retinopathy (0.1 ± 0.1 ng/mL)(30). An elevated VEGF/VPF concentration has also been reported in the synovial fluids of patients with rheumatoid arthritis (6.9–180.5 x 10-12 M) (31). In comparison with these reports, the VEGF/VPF concentration in the cyst fluid of thyroid nodules was considerably elevated, ranging from 1 x 10-12–8 x 10-9 M (0.02–183 ng/mL), which is comparable with the VEGF/VPF concentration in the cyst fluid of brain tumors (7.9–9.0 ng/mL, 20–163 ng/mL) (32, 33). It is likely that the small volume of the cyst fluid surrounded by parenchymatous thyroid tissue and the presence of VEGF mRNA-producing cells floating in the cyst fluid accounts for this extremely high concentration of VEGF/VPF.

In summary, we have demonstrated that bioactive VEGF/VPF is present in the cyst fluid of thyroid nodules, and that it is significantly increased in the enlarging or recurrent cysts compared with that in regressing cysts, suggesting that VEGF/VPF is at least partly involved in the accumulation of cyst fluid in the thyroid nodules. When cyst fluid reaccumulates rapidly after aspiration and VEGF/VPF is abundant in the cyst fluid, interventional treatment for thyroid cysts, such as ethanol injection or resection, may be recommended.


   Acknowledgments
 
We thank Prof. M. Shibuya (Institute of Medical Science, University of Tokyo) and Dr. Y. Murata (Nagoya University, Nagoya, Japan) for providing VEGF and GAPDH cDNA, respectively.


   Footnotes
 
1 This work was partly supported by a Scientific Research Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan (08671185). Back

Received November 12, 1996.

Revised February 24, 1997.

Accepted March 3, 1997.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals