Key words
VEGF - VEGF receptors 1 and 2 - goiter - recurrent - multinodular - Graves’ disease
Introduction
In 1989 vascular endothelial growth factor (VEGF) was identified as an angiogenic
factor [1]. VEGF is a potent stimulator of endothelial cell growth and a critical regulator
of both physiological and pathological angiogenesis [2].
VEGF binds to 2 different tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR),
mainly expressed on endothelial cells [3].
VEGFR-1 may play a more regulatory role in angiogenesis, whereas VEGFR-2 is responsible
for signaling [4]. In addition, VEGF mediates inflammatory reactions [5].
Via a paracrine mechanism VEGF signaling plays a critical role in tumor angiogenesis,
hence, high expression of VEGF and its receptors was shown in a number of human cancers
such as gastric cancer [6], breast cancer [7], colon cancer [8], ovarian cancer [9] and prostate cancer [10].
Increased expression of VEGF was even linked to poor outcome and increased risk for
metastasis and tumor recurrence [11]
[12]
[13]
[14]. On the other hand, inhibition of VEGF, its receptors or signaling pathways leads
to suppression of tumor angiogenesis and growth [15]
[16].
Several studies have demonstrated expression of VEGF and its receptors in benign and
malignant thyroid tissue [17]
[18]
[19].
High expression of VEGF in thyroid cancer was found to be correlated with advanced
tumor stage, lymph node metastasis and increased risk of recurrence [20]
[21]
[22]. Therefore, VEGF inhibitors are approved for the treatment of advanced thyroid cancers
[23].
Furthermore, VEGF seems to be involved in goiter development by inducing endothelial
cell proliferation and angiogenesis. Recently, in clinical studies it was demonstrated,
that treatment with tyrosine kinase inhibitors leads to hypothyroidism in over 40%
of patients, indicating a functional role of the VEGF signaling pathway even in normal
thyroid tissue [24]
[25].
To date, the role and expression of VEGF and its receptors in normal and benign diseases
of the thyroid have rarely been investigated.
This study was performed to investigate the expression pattern of VEGF and its receptors
in thyrocytes from normal thyroid tissue and benign thyroid diseases.
Materials and Methods
Patients
50 patients (♀=38, ♂=12) were enrolled in the clinical study. The protocol was approved
by the ethics committee of the University Hospital of Frankfurt and preoperative written
consent was obtained from all patients. The patients were divided into 4 groups according
to the diagnosis, 11 patients with uninodular goiter (UN), 15 with multinodular goiter
(MN), 10 with Graves’ disease (G) and 14 patients with recurrent nodular goiter (R)
were operated on and included.
Recurrent goiter was defined as development or regrowth of nodules after the first
operation of the thyroid gland as revealed by the patient’s history. The indication
for reoperation was one or more nodular growths as detected by ultrasound. All patients
fulfilled these criteria.
Tissue samples and cell culture
Thyroid tissue was obtained after surgical resection. All tissue samples were examined
and characterized by a pathologist and when possible, nodular and adjacent non-tumor
or paranodular tissues were acquired. In the case of Graves’ disease only paranodular
tissue was investigated.
The thyroid tissue was minced into small fragments, washed 3 times with HBSS (Hank’s
balanced salt solution) containing 50 U/ml penicillin and 50 μg/ml streptomycin followed
by enzymatic dissociation with 246 U/ml collagenase I for 60–90 min at 37°C. After
digestion, thyrocytes were filtered through a 100 μm nylon mesh (Becton Dickinson;
Heidelberg, Germany), pelleted and washed with HBSS.
Thyrocytes were resuspended and counted in Neubauer counting chamber before plating.
Cell viability was assessed using trypan blue exclusion (>98%). Thyrocytes were plated
at a density of 50 000 cells/well in 6-well plates and were cultured in DMEM supplemented
with 10% fetal calf serum, 200 mM Hepes, 50 U/ml penicillin and 50 μg/ml streptomycin.
The monocultures were maintained in a standard humidified incubator at 37°C in a 5%
CO2 atmosphere. On day 1 medium was changed to remove cell debris, on day 4 harvesting
was performed. Thyrocyte culture by this procedure has no detectable stromal or endothelial
contamination, as attested to by immunohistochemistry and PCR.
Immunofluorescence
Primary thyrocyte cultures were washed with PBS (phosphate buffered saline) and fixed
with ice cold acetone for 10 min. After fixation, cells were washed again followed
by incubation with primary antibody for 60 min at 4°C. Primary antibodies were as
follows: mouse monoclonal anti-cytokeratine 18 (clone Ks18.04) and 19 (clone Ks19.10)
(Progen Biotechnik; Heidelberg, Germany, diluted 1:2), mouse monoclonal anti-fibroblast
antibody (Acris antibodies; Hiddenhausen, Germany, diluted 1:100), mouse anti-von
Willebrand factor (Dako; Hamburg, Germany, diluted 1:50). After incubation with primary
antibodies, cultures were washed twice with wash buffer (0.5% BSA in PBS without Ca++
and Mg++) followed by incubation with secondary antibody for 60 min at 4°C (NL493
[NorthernLights] – conjugated donkey anti-mouse IgG, diluted to 1:100, (R&D Systems;
Wiesbaden-Nordenstadt, Germany). All antibodies were diluted with 0.5% BSA (bovine
serum albumin) in PBS. Cell cultures were rinsed 3 times in wash buffer and finally
mounted with Prolong antifade Kit (Molecular Probes; Eugene, Oregon). Staining was
visualized microscopically by Zeiss Z-1 Observer (Zeiss; Göttingen, Germany). A negative
control was also carried by incubating cell cultures with mouse IgG1 (Dako Cytomation,
Denmark). All controls were consistently negative.
RNA isolation, cDNA synthesis, semiquantitative RT- PCR, quantitative PCR
Total RNA was extracted using RNeasy kit (Qiagen GmbH; Hilden, Germany) according
to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA with
Oligo (dT) Primer using AffinityScript QPCR cDNA synthesis kit (Stratagene; CA, USA)
according to the manufacturer’s instructions. Thyroid peroxidase (TPO), TSH receptor
(TSHR), Thyreoglobulin (Tg), VEGF-A and VEGFR-1 and -2 transcripts were analyzed by
performing reverse-transcriptase PCR (RT-PCR) ([Table 1]). RNA amount and RNA quality were analyzed using Nanovue (GE Healthcare, USA) and
Bioanalyzer (Agilent Technologies, USA) respectively. Semi-quantitative RT-PCR was
carried out in a 50 μl reaction mixture for each assay containing 1 μl of DNA template,
5 μl of 10× Titanium Taq PCR Buffer, 1 μl of 50× Titanium Taq DNA polymerase (Takara
Bio Europe/Clontech; Saint Germain-en-Laye, France), 1 μl of dNTPs (10 mM of each
dATP, dCTP, dGTP, dTTP from Takara Bio Europe/Clontech) and 1 μl of primer mix – 50 pmol,
of each forward and reverse primer ([Table 1]) using a Tpersonal Thermocycler (Biometra GmbH; Goettingen, Germany). PCR parameters
were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles 30 s
denaturation at 95°C; 30 s of annealing at 64°C for VEGF-A, VEGFR-1, and VEGFR-2,
58°C for GAPDH and 55°C for TPO, TSHR and Tg; 30 s elongation at 72°C and, finally,
a further elongation step at 72°C for 10 min. Ethidium bromide stained amplified fragments
were separated by agarose gel electrophoresis (2%), visualized and analyzed by Gel
Doc 100 with molecular analyst software (BioRad Laboratories; Munich, Germany).
Table 1 Primer sequences for RT-PCR and expected sizes.
|
Target genes
|
Sequence of forward and reverse primer (5′-3′)
|
Size (bp)
|
|
VEGF-A
|
5′-TGC CTT GCT GCT CTA CCT CC-3′
|
VEGF121-410
|
|
5′-TCA CCG CCT CGG CTT GTC AC-3′
|
VEGF145-480
|
|
|
VEGF165-540
|
|
|
VEGF189-610
|
|
|
VEGF209-660
|
|
VEGFR-1
|
5′-GAT GTT GAG GAA GAG GAG GAT T-3′
|
1 100
|
|
5′-AAG CTA GTT TCC TGG GGG TAT A-3′
|
|
|
VEGFR-2
|
5′-GAT GTG GTT CTG AGT CGG TCT-3′
|
560
|
|
5′- CAT GGC TCT GCT TCT CCT TTG-3′
|
|
|
GAPDH
|
5′-ATC TTC CAG GAG CGA GAT CC-3′
|
509
|
|
5′-ACC ACT CAC ACG TTG GCA GT-3′
|
|
|
TPO
|
5′-AGA TCT GCT GAG CAT CAT TG-3′
|
330
|
|
5′-CAT CAG GAG CTC AGA ATA GCG-3′
|
|
|
TSHR
|
5′-TAC TTC AGT CCA AGG ATA TG-3′
|
361
|
|
5′-GCA AGC TCT GCA TAC TGC TCT-3′
|
|
|
Tg
|
5′-GAT CTT ACT GAG TGG CTA CA-3′
|
682
|
|
5′-ACT GCA CCG CCT GAT AGT CG-3′
|
|
Densitometric analysis was performed and signal was evaluated as area (mm2). GAPDH was used as housekeeping gene and a 100 bp DNA ladder was used as molecular
weight marker.
The results were confirmed by real-time quantitative PCR (QPCR) using RT² SYBR-Green/Rox
qPCR master mix and gene specific primers ([Table 2]) (both from SABioscience, USA) according to the manufacturer’s protocol. Amplification
of genes was performed using MX3005P (Stratagene, USA). 100 ng of cDNA was used in
qPCR reaction in duplicates. The dissociation curve was checked at the end of each
PCR reaction. GAPDH and β-Actin were used as housekeeping genes and for normalization.
Gene expression (fold change) was calculated using ΔΔC
t method after normalizing nodular tissue with each corresponding paranodular or normal
adjacent tissue.
Table 2 Primers for QPCR from SABiosciences.
|
Target genes
|
Accession number
|
Reference Position
|
Size (bp)
|
|
VEGF-A
|
NM 003376
|
2 649
|
183
|
|
VEGFR-1
|
NM 002019
|
5 144
|
58
|
|
VEGFR-2
|
NM 002253
|
629
|
151
|
|
beta-Actin
|
NM 0011013
|
1 222
|
191
|
Protein isolation and western blot
Thyrocytes cultures were rinsed twice with ice-cold PBS and lysed for 10 min on ice
in lysis buffer (50 mM HEPES, 200 mM NaCl, 0.2 mM MgSO4, 0.4 mM phenylmethylsulfonyl
fluoride, 2% Triton-X-100, 10 μg/mL leupeptine, 10 μg/mL aprotinine, 0.02% soybean
trypsin inhibitor, 0.2 mM ortho-vanadate (Sigma-Aldrich; Taufkirchen, Munich, Germany).
Cell lysates were centrifuged for 10 min at 12 000×g at 4°C. Protein concentration
of the supernatants was determined by Coomassie Plus protein assay kit (Pierce; Rockford,
IL, USA) and were measured spectrophotometrically at 595 nm by Tecan Infinite® M 200 microplate reader (Tecan-Deutschland; Crailsheim, Germany). Protein was denatured
in Laemmli sample buffer (Bio-Rad Laboratories; Munich, Germany) with β-mercaptoethanol
(Sigma; Taufkirchen, Germany), boiled for 5 min, transferred on ice and subjected
to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (50 μg per
lane). PeqGold prestained protein marker IV (Peqlab Biotechnologie GmbH; Erlangen,
Germany) was used as molecular weight standards. After separation, protein was transferred
to a polyvinylidene difluoride membrane (Hybond P; GE Healthcare; Munich, Germany).
Blots were blocked with 10% low-fat milk for 1 h at room temperature followed by incubation
with primary antibodies from Santa Cruz Biotechnology (rabbit polyclonal VEGF [total
protein], 1:200; rabbit polyclonal VEGFR-1, 1:200; mouse polyclonal VEGFR-2, clone
A-3, 1:200 and mouse monoclonal β Actin, clone AC-15, 1:1 000; Sigma) overnight at
4°C. Blots were then washed 3× with Towbin buffer with 0.5% Tween 20 followed by incubation
for 30 min with secondary antibody from Millipore GmbH; Schwalbach/Ts, Germany (polyclonal
goat antirabbit IgG, 1:5 000; goat anti-mouse IgG, 1:5 000, both HRP conjugated) at
room temperature. All antibodies were diluted in Towbin Buffer with 0.5% Tween 20
and 0.5% bovine serum albumin. Blots were then washed and incubated with ECL (Enhanced
Chemiluminescence-ECL detection kit from GE Healthcare; Munich, Germany) and exposed
to ECL Hyperfilm (GE healthcare), developed in Curix 60 (Agfa; Dusseldorf, Germany)
and documented by Gel Doc 100. β Actin was used as loading control.
ELISA
Cell culture supernatant was quantified using VEGF ELISA-kit (R&D systems; Wiesbaden-Nordenstadt,
Germany). The assay was done according to the manufacturer’s instructions. Briefly,
200 μl samples (1:30), standard, and controls were dispensed into microplate wells
precoated with anti-VEGF monoclonal antibody, and then incubated for 120 min at room
temperature. After incubation, the wells were washed 5 times and 50 μl of horse radish
peroxidase (HRP) conjugated VEGF was added and incubated for 60 min at room temperature.
After 5 washes, 100 μl of chromogenic enzyme substrate solution was added and incubated
for 30 min at RT; 50 μl stop solution was added and spectrophotometrically measured
using Infinite® M 200 microplate reader (Tecan-Deutschland; Crailsheim, Germany) at 405 nm. The sample
concentration was calculated based on standard concentrations.
Statistical analysis
Data were analyzed using commercially available software Bias for Windows, Version
8.3.7 (H. Ackermann, Johann-Wolfgang Goethe University, Department of Mathematics).
Data are presented as means±SEM. Non-parametric tests were used due to irregularly
distributed data. The groups were compared using Kruskal-Wallis’test. Paired data
(nodular and paranodular tissue) were analyzed using Wilcoxon’s test. Statistical
significance was achieved at p<0.05.
Results
Characterization of thyrocyte cultures
The primary cell cultures showed the typical morphology of thyrocytes on microscopic
examination, i. e., small, elongated cells arranged as cellular isles ([Fig. 1a, b]).
Fig. 1 Characterization of primary culture of thyrocytes. a Primary thyrocyte culture appearing in clusters on day 1, b that reach 95% confluency on day 4 (both 10X). c Immunofluorescence staining in primary culture of thyrocytes showing positive cytokeratin
18 d and negative isotype control. e RT-PCR confirmed the presence of thyrocyte specific markers TSHR, TPO and Tg.
Immunohistochemical staining demonstrated the cells to be positive for cytokeratin
18 ([Fig. 1c, d]) and so to be of epithelial origin. The cells were negative for cytokeratin 19,
including fibroblasts specific antigen and Factor VIII (von Willebrand factor), showing
no contamination of stromal and endothelial cells, yielding primary thyrocyte culture
purity of greater than 98%.
In addition, using RT-PCR all cell cultures were positive for thyroid peroxidase (TPO),
TSH receptor (TSHR) and thyreoglobuline (Tg), namely indicating the presence of thyrocytes
([Fig. 1e]).
mRNA expression of VEGF-A, VEGFR-1 and -2 in human thyrocytes
VEGF-A isoforms were expressed by 99% of all thyrocyte cultures. Here expression of
VEGF145 was significantly higher in nodular tissue from uninodular goiter compared with corresponding
normal tissue (VEGF145=70±7 counts/mm2 [UN] vs. 46±7 counts/mm2 [P]; p<0.05) ([Fig. 2a]). In recurrent goiter we found higher amounts of VEGF145 mRNA in nodular tissue compared to paranodular tissue, but without statistical significance
([Fig. 2c]). VEGF-R1 and -R2 were significantly increased in nodular vs. paranodular tissue,
as shown in uninodular goiter (VEGF-R1=479±68 counts/mm2 [UN] vs. 133±47 counts/mm2 [P]; p<0.05; VEGF-R2=710±72 counts/mm2 [UN] vs. 361±52 counts/mm2 [P]; p<0.05) ([Fig. 2a]) and recurrent goiter (VEGF-R1=428±46 counts/mm2 [UN] vs. 72±20 counts/mm2 [P]; p<0.05; VEGF-R2=617±56 counts/mm2 [UN] vs. 174±45 counts/mm2 [P]; p<0.05) ([Fig. 2c]).
Fig. 2 Analysis of VEGF145, VEGFR-1 and -2 in primary culture of thyrocytes by RT-PCR. a–c Expression in nodular tissue of uninodular, multinodular and recurrent goiter (UN,
MN, R) vs. their corresponding paranodular tissue (P). Depicted were 5 representative
bands from each group. d Expression in paranodular tissue of Graves’ disease (G) vs. paranodular tissue of
uninodular, multinodular and recurrent goiter (UNP, MNP, RP). Depicted was 1 representative
band from each group.
In Graves’ disease we found the highest levels of VEGF145, VEGFR-1 and -2 mRNA compared to the paranodular tissue of the other 3 groups, but
without reaching statistical significance ([Fig. 2d]).
Interestingly, in tissue from patients with multinodular goiter we did not find any
significant difference in the expression levels of VEGF and its receptors between
nodular and paranodular tissue ([Fig. 2b]).
The results of RT-PCR were confirmed by performing QPCR. The results are presented
as fold change after normalizing nodular tissue with each corresponding paranodular
tissue ([Table 3a]), as well as after normalizing paranodular tissue of Graves’ disease with paranodular
tissue of the other 3 groups ([Table 3b]).
Table 3 Results of QPCR (fold change).
|
a) Nodular tissue vs. paranodular tissue
|
|
Group
|
VEGF
|
VEGFR-1
|
VEGFR-2
|
|
UN
|
+15,78 (upregulation)
|
+10,28 (upregulation)
|
+8,28 (upregulation)
|
|
MN
|
+1,57 (no regulation)
|
− 1,64 (no regulation)
|
+1,26 (no regulation)
|
|
R
|
+41,53 (upregulation)
|
+28,44 (upregulation)
|
+8,06 (upregulation)
|
|
b) Graves’ disease vs. paranodular tissue of UN, MN, R
|
|
Group
|
VEGF
|
VEGFR-1
|
VEGFR-2
|
|
UNP
|
+3,34 (upregulation)
|
+8,11 (upregulation)
|
+2,26 (upregulation)
|
|
MNP
|
+1,43 (no regulation)
|
3,18 (upregulation)
|
+1,68 (no regulation)
|
|
RP
|
+19,29 (upregulation)
|
+14,32 (upregulation)
|
+30,91 (upregulation)
|
Protein expression of VEGF (total), VEGFR-1 and -2 in human thyrocytes
The VEGF protein was highly expressed in all cell cultures obtained. Furthermore,
expression of the receptor proteins was demonstrated in over 90% of the isolated thyrocytes.
In uninodular and recurrent goiter, protein expression of VEGF was higher in nodular
compared to paranodular tissue as shown in [Fig. 3a]. The same results were detected for protein expression of VEGFR-1 and VEGFR-2 ([Fig. 3a]).
Fig. 3 Protein expression of VEGF, VEGFR-1 and -2 of cellular lysates from primary culture
of thyrocytes analyzed by western blotting. Depicted was 1 representative band from
each group. a Expression in nodular (UN, MN, R) vs. their corresponding paranodular tissue (P).
b Paranodular tissue of Graves’disease (G) vs. paranodular tissue of uninodular, multinodular
and recurrent goiter (UNP, MNP, RP).
Interestingly, again in multinodular goiter we could not find any difference in protein
expression between paranodular and nodular tissue as seen at mRNA level ([Fig. 3a]).
In tissue from Graves’ disease we found the highest protein levels of VEGF, VEGFR-1
and -R2. Protein expression of VEGF and VEGFR-1 was significantly increased in comparison
to the paranodular tissue of the other 3 groups (VEGF=0.7±[G] vs. 0.5±[UN]; 0.31 [MN];
0.32 [R]; p<0.05; VEGF-R1=0.7±[G] vs. 0.36 [UN]; 0.19 [MN]; 0.2 [R]; p<0.05) ([Fig. 3b]).
Secretion of VEGF by human thyrocytes
All primary cell cultures secreted VEGF protein in the supernatant. The secretion
level of VEGF was higher in nodular tissue of patients with recurrent goiter compared
to paranodular tissue, without reaching statistical significance ([Fig. 4]). Again, the highest levels of VEGF were measured in tissue obtained from Graves’
disease patients.
Fig. 4 VEGF expression in supernatant of primary culture of thyrocytes from nodular and
paranodular tissue determined by ELISA. n=(nodular=38: paranodular=36), results are
expressed as mean±SEM.
Discussion
VEGF, as an angiogenic factor, has been implicated in tumor angiogenesis and in progression
of different malignant tumors. Therefore, it is important to investigate its role
in thyroid cancer. So far, elevated VEGF expression seems to be associated with higher
risk of metastasis, of recurrence and with a poor outcome in thyroid cancer [26].
To date, the expression of VEGF in normal thyroid tissue and its role in the development
of goiter and Graves’ disease is not well determined.
In the present study, we were able to demonstrate co-expression of VEGF and its receptors
VEGFR-1 and -2 in normal human thyrocytes and in benign diseases of the thyroid. In
addition, we detected active secretion of VEGF protein by thyrocytes in the supernatant
and measured higher concentrations of VEGF protein in nodular tissue of uninodular
and recurrent goiter compared to the corresponding normal tissue. Furthermore, the
present data demonstrate large amount of VEGF protein in nodular and paranodular tissue
of multinodular goiter, that might contribute to the proliferating effect on thyrocytes
and its involvement in goiter development.
These findings contradict previous studies, which reported weak expression of VEGF
in benign nodules of the thyroid with no differences between normal thyroid tissue
and benign adenomas [27]
[28]. Itoh et al. did not even find any expression of VEGF in normal thyroid tissue and
only negative or weak expression in adenomatous goiter [29]. These discrepancies may be explained by the methodological approach used. In previous
studies, fresh frozen tissue or formalin-fixed paraffin-embedded tissue was primarily
used for the investigation while our study examined pure cultures of freshly isolated
thyrocytes, thus, excluding possible endothelial contamination and antigen masking
of targets of interest due to formalin-fixation.
With regards to the expression of the VEGF receptors, only immunohistochemistry staining
of VEGF receptors expression on human thyrocytes of benign thyroid diseases has been
shown so far. Jebreel et al. reported no differences in VEGFR-1 and -2 expression
between the different thyroid pathologies, so the author suggested that up-regulation
of VEGF and not of its receptors was important in the development of thyroid diseases
[30]. In contrast to our findings, we demonstrated significantly higher expression of
the 2 receptors in nodular tissue of uninodular and recurrent goiter compared to
corresponding normal tissue both in mRNA and protein level. These results might indicate,
that not only up-regulation of VEGF, but also up-regulation of its receptors plays
a critical role in the development of goiter.
In addition to receptor expression, we were able to detect significant amount of VEGF
in the supernatant of thyrocytes culture.
Consistent with our data, Sato et al. reported high levels of VEGF in cyst fluid of
thyroid nodules. They postulated that VEGF was secreted by thyrocytes and that it
was involved in the pathogenesis of cyst fluid accumulation [31].
In the present study, we were able to detect an active secretion of VEGF by human
thyrocytes and simultaneous expression of both VEGF receptors. This coexpression of
VEGF and its receptors raises the possibility that VEGF may act in an autocrine loop
in thyrocytes, as observed previously in thyroid cancer [32].
Perhaps, an autocrine stimulation plays a crucial role in proliferation of thyrocytes
and such up-regulation of the VEGF signaling might be involved in the development
of nodular goiter.
Besides, high levels of VEGF and its receptors in recurrent nodules may pose potential
risk for the development of goiter recurrence.
It is also known, that autocrine stimulation of VEGF receptors leads to enhanced tumor
growth [33]. This mechanism, initiated by increased VEGF expression, may also be involved in
the growth of thyroid nodules and goiter recurrence.
In our study, we did not find any differences in gene or protein expression of VEGF
and its receptors between nodular and paranodular tissue of patients with multinodular
goiter, but instead detected an increased concentrations of the growth factor in both
tissues. Therefore, we postulated, that in multinodular goiter the entire tissue of
the thyroid gland might be pathologically altered and are not only limited in the
circumscribed nodules. Although the cause of recurrence might be of multifactorial
origin, elevated expression of VEGF and its receptors might also contribute to the
high rate of recurrence of over 20% of multinodular goiter after subtotal resection
[34]
[35]
[36], Our findings are also in accord with clinical studies which assumed an involvement
of the whole thyroid parenchyma in benign disease and therefore, recommended total
thyroidectomy as a suitable treatment [37].
Furthermore, we examined tissue from 10 patients with Graves’ disease. In the literature,
contradictory data are reported on VEGF expression in this autoimmune disease of the
thyroid gland. However, several authors could not detect any expression of VEGF in
Graves’ disease in immunohistochemical staining [29] or only at lower levels compared to neoplastic tissue [30] and so argued that VEGF signaling plays only a minor role in the development of
autoimmune disease of the thyroid gland. On the other hand, Iitaka et al. reported
increased serum VEGF levels in patients with Graves’ disease compared to healthy persons
[38]. Furthermore, studies demonstrated a positive correlation between VEGF levels and
increased vascular density in Graves’ disease [39] as well as strong expression of VEGF in hyperplastic follicular cells and surrounding
capillaries [40], indicating a link between epithelial function and microcirculation.
In concordance with these data, we have detected in our study high levels of VEGF
and its receptors both in mRNA and protein in monocultures of thyrocytes obtained
from patients with Graves’ disease. Here, we have shown that the expression was significantly
increased compared with the other groups.
These results may indicate that VEGF is involved in the pathogenesis of Graves’ disease
and may have an influence on both epithelial proliferation and thyroid microcirculation,
causing a typical hypervascularization and hyperplasia seen in this autoimmune disease.
Moreover, this might be a first hint, that VEGF might also be involved in the control
of thyroid function.
Besides the up-regulation of VEGF in goiter, the present study was able to confirm
the expression of VEGF and its receptors in normal thyroid tissue. Clinical studies
have also reported, that over 40% of patients treated with tyrosine kinase inhibitors
(TKI) developed hypothyroidism [24]
[25]. Recently Sato et al. reported a sonographically detectable atrophy of the thyroid
gland caused by sunitinib therapy [41]. One hypothesis for TKI-related hypothyroidism is inhibition of the VEGF-pathway
resulting in reduction of capillary blood flow. These observations and data may signify
that VEGF signaling might participate not only in thyroid hyperplasia but is also
essential in normal thyroid function as well as physiological proliferation of thyrocytes.
Conclusion
In summary, our study showed an increased expression of VEGF and its receptors in
nodular tissue of uninodular and recurrent goiter as well as in the entire tissue
of multinodular goiter and Graves’ disease. Therefore, we hypothesized that VEGF may
be involved in the proliferation of thyrocytes and its up-regulation via autocrine
mechanism might contribute to the development of thyroid nodules and hyperplasia of
the thyroid gland.
Although, it is very likely that goiter recurrence is of multifactorial origin, VEGF
may serve as a proliferation marker in thyroid disease and may be used as a possible
target for future strategies for the treatment of neoplastic and autoimmune diseases
of the thyroid. Until now, the exact role of VEGF in thyroid function remains to be
determined. Hence, more future studies are eminent to elucidate the role of VEGF in
thyroid diseases.
