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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 6 2890-2896
Copyright © 2004 by The Endocrine Society

Angiogenesis and Lymphangiogenesis in Parathyroid Proliferative Lesions

N. Garcia de la Torre, I. Buley, J. A. H. Wass, D. G. Jackson and H. E. Turner

Endocrinology Department (N.G.d.l.T., J.A.H.W., H.E.T.), The Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, United Kingdom; and Histopathology Department (I.B.) and Wetherall Institute of Molecular Medicine (D.G.J.), John Radcliffe Hospital, Headington, Oxford OX3 9DZ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Helen Turner, Department of Endocrinology, The Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, United Kingdom. E-mail: helen.turner{at}orh.nhs.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis and lymphangiogenesis are involved in tumoral growth and metastatic spread. There is little information on angiogenesis and no available data on lymphangiogenesis in parathyroid glands (PTG). Using immunohistochemistry for CD34, LYVE-1 (specific markers for vascular and lymphatic endothelium, respectively), vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, this study analyzes microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic growth factors in 13 normal PTG, 77 parathyroid adenomas (PTA), and 17 primary parathyroid hyperplasia (PPH). MVD was higher in PPH and PTA, compared with PTG (P < 0.001). There was no difference in VEGF-A expression among groups. In contrast, FGF-2 expression was higher in PPH, compared with PTA and PTG (P < 0.0001). FGF-2 scores and MVD were significantly correlated (r = 0.43). LVD did not differ among groups, and VEGF-C expression was unrelated to LVD. There was no relationship between MVD and tumor behavior (adenoma size, PTH, or calcium).

In conclusion, this study shows increased angiogenesis in parathyroid proliferative lesions compared with normal glands and suggests that FGF-2 is proangiogenic in parathyroid tissue. In PTA, tumor behavior is not related to angiogenic phenotype. This is the first demonstration of lymphatic vessels in PTG, but the lack of correlation with VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic factor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRIMARY HYPERPARATHYROIDISM (pHPT) was described by Fuller Albright and others (1, 2) in the 1930s and is characterized by hypercalcemia and elevated circulating PTH. The most common lesion found in pHPT is the solitary parathyroid adenoma (PTA), occurring in 80% of patients. Multiple PTA have been reported in 2–4% of cases. In approximately 15% of patients with pHPT, all four parathyroid glands (PTG) are involved in a process that is seen histologically as hyperplasia. Parathyroid tumors may occur as an isolated and sporadic endocrinopathy or as a part of an inherited syndrome, such as MEN-1 and -2a. Parathyroid carcinoma accounts for less than 0.5% of cases of pHPT (3).

Tumor growth is a multifactorial process that, in addition to mutations of oncogenes or tumor suppressor genes and environmental stimuli, requires an optimal supportive environment. Folkman and co-workers (4) demonstrated that tumors implanted into isolated perfused organs failed to develop, but in contrast, the same tumors implanted within 6 mm of blood vessels induced angiogenesis, grew, and metastasized, therefore proposing that solid tumors are dependent on angiogenesis for growth further than a few millimeters in size. There are now several experimental (5) and clinical data (6) showing that growth of solid tumors is angiogenesis-dependent.

Aside from roles in immunity and fluid homeostasis, the lymphatics are an important route for early metastasis in cancer. Since the discovery of the LYVE-1 antibody binding the hyaluronan receptor on lymphatics, it has been possible to demonstrate the occurrence of intratumoral lymphangiogenesis (7). Two members from the vascular endothelial growth factor (VEGF) family, VEGF-C and VEGF-D, have been identified as the molecular link between tumor lymphangiogenesis and metastatic spread (8, 9).

VEGF-A plays a key role in both physiological and pathological angiogenesis through the proliferation and migration of endothelial cells (10) and increasing endothelial permeability by inducing fenestrations in the endothelium (11). VEGF-A also functions as an antiapoptotic factor promoting the survival of endothelial cells in newly formed vessels (12). Fibroblast growth factor (FGF)-2 is another potent angiogenic factor produced by endothelial, stromal, and tumoral cells as well as being released from the extracellular matrix. FGF-2 stimulates proliferation of endothelial cells (13, 14). Tissue expression of VEGF-A and FGF-2 is associated with poor prognosis in a wide variety of tumors (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27).

Convincing evidence for tumor lymphangiogenesis began to accumulate with the discovery of VEGF-C as a potent lymphangiogenic factor, which induces proliferation of lymphatic endothelial cells in vitro (28) and lymphatic hyperplasia in vivo (29). Thus far, VEGF-C levels in primary tumors have been shown to correlate significantly with lymph node metastasis in breast, thyroid, prostate, gastric, colorectal, lung, cervix, esophageal, and head and neck squamous cell carcinomas (30).

Parathyroid tissue has the ability to spontaneously induce angiogenesis in vitro (31) and in vivo models (32). Autotransplantation of parathyroid tissue after thyroidectomy maintains calcium homeostasis (33) because the transplanted parathyroid tissue spontaneously revascularizes (34). There is a single previous study analyzing vascularity in parathyroid proliferative lesions (35), which showed that the endothelial component of hyperplastic PTG was increased in glands from six patients with pHPT associated with MEN-1 syndrome compared with those from six patients with secondary hyperparathyroidism associated with chronic renal failure. There are no currently available data on the presence of lymphangiogenesis or lymphangiogenic growth factors in PTG.

The aims of this study were: 1) to analyze microvascular density (MVD) as a measure of angiogenesis using CD34 staining, which is a specific marker for detection of all vascular endothelial cells (mature and immature), and lymphatic vascular density (LVD) as a measure of lymphangiogenesis using LYVE-1 staining, which is a specific marker for detection of lymphatic vascular endothelial cells, in a large cohort of parathyroid proliferative lesions compared with normal tissue; 2) to assess whether this correlates with aspects of tumor behavior (biochemical activity of the disease and tumoral size) in PTA; and 3) to investigate the expression of the proangiogenic and lymphangiogenic factors VEGF-A, FGF-2, and VEGF-C in parathyroid tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor collection

A database of carefully characterized parathyroid proliferative lesions was created at the start of the project. Consecutive parathyroid tissue samples from patients of both sexes, ranging in age from 14 to 93 yr, were obtained from archival records from January 1995 to August 2001 in the Histopathology Department at the John Radcliffe Hospital, Oxford, UK. Each set of notes was studied and the information recorded on each patient, including the clinical and hormonal status at presentation, imaging studies, and pathology reports. Excluded were: referred cases in which biochemical determinations were not carried out in the John Radcliffe Hospital, and referred blocks in which the biochemical data and surgical reports were missing. The study was approved by the Oxfordshire Clinical Research Ethics Committee. PTA specimens removed from seventy-four patients with pHPT were assessed. There were three patients with double adenoma; thus, a total of 77 PTA were investigated. No recurrences were recorded during the follow-up. One patient was not cured after the removal of one PTA and needed a second operation in which a second PTA was removed; this was considered a double adenoma case. Seventeen glands with primary hyperplasia were obtained from six different patients. Three of these patients had MEN-1, and the other three had sporadic primary hyperplasia. Thirteen specimens of normal PTG were obtained during surgery for pHPT where an adenoma and a normal gland were removed (10); toxic multinodular goiter (1), squamous cell carcinoma of the larynx (1), and medullary carcinoma of the thyroid (1) were also studied. All the tissue had been fixed in 4% buffered formalin, dehydrated, and embedded in paraffin. Histological examination had been performed previously and, together with the clinical, biochemical, and radiological data, were used to characterize each tumor. The tumor size assessed by weight was determined from the initial surgical pathology report.

Biochemical data

The serum preoperative levels of corrected calcium and PTH were obtained from clinical records. The values corresponding to the first hospital visit before any medical treatment, and off drugs that could alter serum calcium, were chosen. The serum intact PTH values were measured by an immunometric assay (Immulite 2000, DPC, Los Angeles, CA). The normal range was 0.8–7.8 pmol/liter, with intra- and interassay coefficients of variation of 3.4% and 7.2%, respectively. The serum calcium and albumin levels were analyzed using a calcium-arsenazo III dye complex and an albumin-bromcresol green colored complex, respectively, which are measured spectrophotometrically (Aeroset System, Abbott Laboratories, Chicago, IL). The normal range for calcium was 2.10–2.55 mmol/liter, with intra- and interassay coefficients of variation of 0.6% and 0.8%, respectively. The normal range for albumin was 35–50 g/liter, with intra- and interassay coefficients of variation of 0.7% and 0.4%. The serum adjusted calcium levels were obtained using the formulas: corrected Ca = serum calcium + 0.02 per each gram of albumin less than 40; corrected Ca = serum calcium – 0.02 per each gram of albumin above 40.

Immunohistochemistry

Four-micron sections were mounted on X-Tra slides (Surgipath Europe Ltd, Peterborough, UK), dewaxed, and rehydrated. Sections for CD34 did not require pretreatment. Slides for staining with LYVE-1, VEGF-C, and FGF-2 were pretreated by microwave in Dako Target Retrieval Solution (Dako Ltd, Cambridgeshire, UK) for 20 min. Slides for staining with VEGF-A were pretreated by microwave in Dako High pH Target Retrieval Solution (Dako Ltd) for 20 min. The horseradish peroxidase Envision System (Dako Ltd) was used for CD34, LYVE-1, VEGF-A, VEGF-C, and FGF-2 staining. Endogenous peroxidase activity was blocked using Dako blocking reagent as per kit (Dako Ltd) for CD34, LYVE-1, VEGF-A, VEGF-C, and FGF-2 cases. The primary antibodies were applied for 30 min at room temperature. For CD34 and VEGF-A staining, mouse monoclonal antibodies provided by the Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK, were applied at a dilution of 1:100 and 1:2, respectively (36, 37). Mouse antibodies generated against human LYVE-1 (37A ) were used at a dilution of 1:2. The rabbit antihuman polyclonal antibodies for FGF-2 and VEGF-C (Biotechnology, Inc; Santa Cruz, CA) were used at a dilution of 1:250 and 1:50, respectively. After two washes in Tris-buffered saline for CD34 and Tris-buffered saline with 0.01% Tween for LYVE-1, VEGF-A, VEGF-C, and FGF-2 secondary antibody bound to horseradish peroxidase via a dextran backbone (Dako Ltd) was applied as per kit for 30 min at room temperature, followed by two washes. Color development was performed with metal-enhanced diaminobenzidine (Dako Ltd) applied for 5 min. The slides were lightly counterstained with hematoxylin (Sigma diagnostics, St. Louis, MO). Breast carcinoma sections were used as positive controls for CD34, VEGF-A, and FGF-2 staining, and tonsil sections were used as positive controls for LYVE-1 and VEGF-C staining. Negative controls were obtained by omitting the primary antibodies.

Assessment of vascular and lymphatic density

All scoring and interpretations of immunohistochemical results were made by one examiner (N.G.d.l.T.) without prior knowledge of histological diagnosis or size. The Chalkley point technique was used for assessment of vascular and lymphatic densities (38). The three most densely vascular areas (known as hot spots) were determined at low magnification. Because LYVE-1 staining was much more sparse, only two hot spots were determined. A 25-point Chalkley eyepiece graticule was orientated so that the maximum number of points was on, or within, areas of highlighted vessels at x400 magnification. The mean of the counts for the three most angiogenic areas and for the two most lymphangiogenic areas was recorded. A subjective semiquantitative grading system at low power (x100) was also used (1 and 2, low and low moderate vascular density; 3 and 4, high and very high vascular density) (39) to assess overall vascular density. Assessments of vascular density using mean Chalkley count and grade were highly correlated (r = 0.84 for CD34 and r = 0.75 for LYVE-1, P < 0.001 for both).

Assessment of VEGF-A, VEGF-C, and FGF-2 staining

Four high-power fields (x400 magnification) from a single representative tissue section, chosen to reflect the area of highest intensity of staining and highest percentage of tumor cells that were positive, were scored and the mean recorded. Each tumor field was assigned a value from 0 (negative) to 4 (maximum staining) for VEGF-A, VEGF-C, and FGF-2 staining.

Inter- and intraobserver reliability

Twenty percent of the slides were rescored by a second examiner (H.E.T.) and a second time by the main examiner (N.G.d.l.T.), both of them blinded to the first reading to assess inter- and intraobserver reliability. Inter- and intraobserver reliability was very good, with a 100% agreement for high and low grades for CD34, LYVE-1, VEGF-A, FGF-2, and VEGF-C staining.

Statistical analysis

The results were expressed as mean ± SD. Comparisons among the three different groups [PTG, PTA, and primary parathyroid hyperplasia (PPH)] were performed by the ANOVA test. When two groups were compared, the unpaired two-tailed Student’s t test was used. To measure the strength of the relationship between variables, the Pearson’s correlation coefficient was used. When the relationship between weight or PTH and other variables was analyzed, log-transformed data that were normally distributed were used. To investigate the relation between calcium and PTH levels with glandular size, MVD, and LVD in the cases with double adenoma, the mean weight, MVD, and LVD between the two adenomas were obtained. There was no difference in primary hyperplasia between patients without MEN and patients with MEN-1 regarding MVD and LVD; therefore, all the glands with primary hyperplasia were studied as a single group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MVD

Homogeneous staining through the vascular system of the entire gland was apparent for CD34, with blood vessels fairly evenly distributed through the parathyroid tissue, with no differences between central and peripheral areas (Fig. 1AGo). This even distribution was present in normal glands, adenomas, and hyperplasia. MVD was higher in primary hyperplasia compared with adenomas, and in adenomas compared with normal glands (Table 1Go). When two or more hyperplastic glands were available from the same patient, there was no difference between individual gland MVD, except in one of the six patients, in which the four hyperplastic glands had the following vascular densities: 20, 19.33, 18, and 13.33, respectively.



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  		FIG. 1. CD34 staining in blood vessels (A), LYVE-1 staining in lymphatic vessels (B), VEGF-A cytoplasmic staining in chief cells (C) and blood vessels (D), FGF-2 nuclear staining in chief cells (E) and blood vessels (F), and VEGF-C cytoplasmic staining in chief cells (G) and blood vessels (H). All the images are shown at x400 magnification. Arrows, Positive staining.

 

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  		TABLE 1. Blood and lymphatic vascular density and VEGF-A, FGF-2, and VEGF-C staining in normal PTGs, PTA, and PPH compared by ANOVA

 
LVD

LYVE-1 staining demonstrated lymphatics localized at the periphery of the lesions or in the vascular hilum and much less frequent or undetectable in the central portion (Fig. 1BGo). There was no difference in LVD among the three groups compared by ANOVA (Table 1Go).

Correlation MVD/LVD in parathyroid proliferative lesions

There was no significant correlation between MVD and LVD in normal glands (r = 0.17), adenomas (r = 0.08), or hyperplasia (r = 0.04).

Growth factors

VEGF-A positive staining was present in the parathyroid tissue (Fig. 1CGo) and endothelium of blood vessels (Fig. 1DGo). VEGF-A staining was heterogeneous within the parathyroid tissue, with areas of very strong cytoplasmic staining mainly in the periphery and weak or undetectable staining in the reminder gland. FGF-2 staining was more homogeneous and also localized in both chief cells (Fig. 1EGo) and endothelial cells (Fig. 1FGo). VEGF-C was expressed mainly in the cytoplasm of chief cells (Fig. 1GGo) but was occasionally expressed in blood vessels (Fig. 1HGo). The pattern of subcellular localization was different for FGF-2, which showed nuclear as well as cytoplasmic localization, compared with VEGF-A and VEGF-C, which accumulated only in the cytoplasm of chief cells. VEGF-A expression was not significantly different among the three groups, despite a weak correlation between MVD assessed by Chalkley count and VEGF-A expression in four fields (r = 0.29, P < 0.001). However, FGF-2 expression was higher in primary hyperplasia compared with adenomas and in adenomas compared with normal glands (3.91 ± 0.21 vs. 2.74 ± 1.10 vs. 2.11 ± 0.86, P < 0.0001). In addition FGF-2 scores and MVD assessed by Chalkley count were significantly correlated (r = 0.43, P < 0.001). There was no difference in VEGF-C expression between PTA and normal glands; nevertheless, VEGF-C expression was significantly decreased in primary hyperplasia compared with normal glands and adenomas (0.64 ± 0.47 vs. 1.26 ± 0.80 vs. 1.36 ± 0.69, P < 0.001). There was a very weak correlation between VEGF C expression and LVD (r = 0.21, P < 0.01). There was no relationship between VEGF-A and LVD or VEGF-C, or between VEGF-C and MVD or FGF-2.

Biochemical activity and tumor size in PTA

The mean weight of the adenomas was 1.51 g (range, 0.12–9.7), and the mean PTH and corrected calcium levels were 19.15 pmol/liter (range, 6.2–131) and 2.92 mmol/liter (range, 2.41–4.17), respectively. Preoperative serum intact PTH concentration was significantly related to serum adjusted calcium concentration (r = 0.57, P < 0.0001). Weight of PTA weakly correlated with preoperative serum intact PTH (r = 0.35, P < 0.01), but there was no relationship with preoperative serum adjusted calcium (r = 0.19). There was no relationship between MVD or LVD and size of PTA or biochemical severity of pHPT, assessed by preoperative serum levels of PTH and calcium.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first study assessing MVD and LVD in parathyroid proliferative lesions. Our data are in agreement with studies in other tissues where benign proliferative lesions have higher MVD than normal tissue. Premalignant lesions have been shown to be more vascular than normal tissue, and precarcinoma of the cervix and breast exhibit high levels of angiogenesis (40, 41, 42). There was no demonstrable relationship between tumor behavior and angiogenic phenotype. One possible reason for this lack of relationship could be that the vascularization of PTA is sufficient to not limit activity and growth. In other endocrine-secreting adenomas, such as ACTH-secreting and GH-secreting pituitary adenomas, MVD is not related to tumor size or preoperative levels of ACTH, cortisol, or GH. In contrast, macroprolactinomas are significantly more vascularized than microprolactinomas, and the preoperative level of prolactin is correlated with MVD (43). The finding of a higher vascularization in PPH compared with PTA is unlikely to be related to the cellular proliferation of the glands, because several studies have shown higher cellular proliferation index in PTA than in primary hyperplasia (44, 45). Although previous reports showed an increased proliferative activity in the central areas compared with the periphery of PTA (46), we found an even distribution of the vascular net through the entire gland. In addition, several studies performed in different endocrine tumors showed a lack of relationship between cell proliferation and angiogenesis (47, 48, 49). We did not find differences in the even distribution of blood vessels between different histological groups; thus, heterogeneity in vascularization between different regions of the gland is not likely to affect the results. It is possible that angiogenesis is impaired with aging (50), and the usually older age of patients with pHPT due to an adenoma may influence the potential for new vessel formation. The difference in MVD fits with the clinical observation that PPH is a distinct clinical entity from PTA, and there is no evidence of progression from PPH to adenoma.

Carter et al. (51) showed that human parathyroid tissue is capable of inducing angiogenesis through the paracrine activity of VEGF-A, because parathyroid tissue up-regulated production of VEGF-A mRNA, and soluble VEGF receptor-1 completely blocked parathyroid tissue-stimulated microvessel growth in vitro. However, angiogenesis induced by parathyroid tissue exceeded that induced by VEGF-A alone, suggesting that parathyroid-induced angiogenesis is mediated by other factors in addition to VEGF-A, possibly FGF-2. FGF-2 has a synergistic effect on VEGF-A in cultured endothelial cells (52) and has been shown to be expressed, with its receptor, in PTA and secondary PPH (53). In contrast to VEGF-A, FGF-2 expression was higher in adenomas than in normal glands and higher in primary hyperplasia than in adenomas. There was a significant correlation with MVD, suggesting that it is an important factor regulating angiogenesis in parathyroid tumors. This up-regulation in the FGF-2 expression in hyperplastic glands is in agreement with previous in vitro studies that showed that the plasma of subjects with MEN-1 syndrome had a mitogenic effect on cultures of bovine parathyroid tissue (54). Further investigations with cloned bovine parathyroid endothelial cells suggested that the parathyroid mitogenic component from plasma of patients with MEN-1 was selective for the endothelial cells of parathyroid tissue and was likely to be FGF-2 (55). However, in the Brandi et al. study (54), mitogenic activity in plasma from the patients with MEN-1 was greater than that in plasma from patients with MEN-2 or sporadic primary hyperplasia. In contrast to previous studies (54), we found no difference in MVD glands from patients with MEN and sporadic hyperplasia.

Our study shows that VEGF-C expression is significantly higher in adenomas compared with primary hyperplasia, but unassociated with differences in LVD. VEGF-C binds VEGF receptors 2 and 3, but not 1; therefore, these results are compatible with previous data regarding up-regulation of VEGF receptor 2 and 3 precursors in PTA vs. hyperplasia (56). In addition to differences in receptor expression, another possible explanation for the lower expression of VEGF-C in hyperplasia could be increased VEGF-C in response to proinflammatory cytokines (57). VEGF-C and VEGF receptor 3 are prominently expressed by activated macrophages (58), suggesting a role in the inflammatory response, and it is possible to speculate that the growing PTA compress normal tissue and provoke a stronger inflammatory response than hyperplasia. Normal tissue also had higher expression of VEGF-C than hyperplasia. Most of the normal glands in our study were adjacent to a PTA; and in two cases, they were next to malignant tumors of the larynx and thyroid. Therefore, it is possible that proinflammatory cytokines were released to local circulation.

The lack of correlation between MVD and LVD in normal glands, adenomas, and primary hyperplasia indicates that growth factors regulating angiogenesis and lymphangiogenesis are likely to be different. The lack of relationship between LVD and histological diagnosis suggests that the presence of lymphatic vessels in parathyroid tissue possibly plays a role only in immunity and fluid homeostasis. There are no previous studies comparing MVD and LVD in the same tissue. However, in vivo studies showed that a soluble form of VEGF receptor 3, an inhibitor of VEGF-C and VEGF-D, led to abnormalities in the dermal lymphatic vessels in the skin of transgenic mice but not in blood vessels (59), indicating that in vivo angiogenesis and lymphangiogenesis are separately controlled by different growth factors.

In contrast to secondary hyperparathyroidism due to chronic renal failure, where variations in PTG size are the major contributor to the excessive PTH secretion (60, 61), in pHPT adenoma, size is not a determinant of disease severity, because there was no correlation between preoperative serum-adjusted calcium levels and tumor weight. This lack of relationship between biochemical data and adenoma size may be due to the biphasic cell kinetics in PTA (62, 63): 1) a rapid initial growth, via an increase in the secretory set-point as the clone of mutants cells behaves as if they were in a hypocalcemic environment; and 2) slowing down as tumor size reaches an asymptotic value corresponding to the total rate of hormone secretion needed to raise the plasma calcium to the new set-point. Nevertheless, the fact that, in this study, vitamin D deficiency was not excluded might somehow mask the results, because suboptimal vitamin D nutrition stimulates PTA growth by a mechanism unrelated to hypocalcemia and reduces the calcemic response to PTH (64).

In this study, although a standard technique to assess MVD was used, the anti-CD34 antibody stains all blood endothelial cells, including mature and immature cells, and does not allow the distinction between newly formed vessels, responsible for angiogenesis, and preexisting vessels. In addition, angiogenesis and lymphangiogenesis are dynamic processes, and immunohistochemistry offers only a static view of the remodeling of the vasculature. Although MVD was high in PPH, compared with PTA, these data do not provide a useful tool for the histological distinction between hyperplasia and adenoma, because the levels of MVD were overlapping in both groups, and there was not a cutoff value. Due to the small number of double PTA and the lack of recurrences during the follow-up period, we were unable to study the relationship between MVD and these aspects of more aggressive tumor behavior. However, the finding of higher vascularization in parathyroid proliferative lesions, compared with normal glands, and the possible role of FGF-2 in parathyroid angiogenesis show the importance of future research in this field, because there are no currently available data on the presence of angiogenesis and angiogenic growth factors expression on parathyroid carcinomas. Another limitation of this study is that most of the so-called normal PTG are from patients with pHPT and are not strictly normal, because they are likely to be suppressed with respect to both secretory and proliferative activity compared with normal glands (46). However, we showed no relationship between vascularization and biochemical activity, and several studies in different endocrine tumors showed a lack of relationship between cell proliferation and angiogenesis (47, 48, 49); thus, we do not think the suppression of these normal glands influences the MVD. It is possible to hypothesize that angiogenesis, as in many other human cancers, plays a role in the progression of parathyroid carcinomas and that MVD and/or angiogenic growth factors could be used as a prognostic marker, or even further, as a potential therapeutic target. Studies are needed to confirm this hypothesis. Future research in this field is also needed to look for genotype-phenotype interactions, especially in patients with MEN, because a relationship between other endocrine genetic tumoral syndromes and an angiogenic phenotype has been shown, e.g. Von-Hippel-Lindau and increased MVD and VEGF-A expression (65).

In conclusion, this study shows, for the first time, increased angiogenesis in parathyroid proliferative lesions compared with normal glands, and in hyperplasia compared with adenomas. The correlation of MVD with FGF-2 expression suggests that FGF-2, rather than VEGF-A, is proangiogenic in parathyroid tissue. In addition, this is the first demonstration of lymphatic vessels in PTG. The lack of correlation between LVD and VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic growth factor in parathyroid tissue. Finally, in PTA, tumor behavior (secretory status and tumor size) is not related to angiogenic phenotype.


   Acknowledgments
 
We gratefully acknowledge Dr. Helen Turley for the donation of CD34 and VEGF-A antibodies.


   Footnotes
 
This work was supported by The Oxford Radcliffe Hospital Charitable Fund (Grant 1057295) and the Novo Nordisk Foundation.

Abbreviations: FGF, Fibroblast growth factor; LVD, lymphatic vascular density; MVD, microvascular density; pHPT, primary hyperparathyroidism; PPH, primary parathyroid hyperplasia; PTA, parathyroid adenoma(s); PTG, parathyroid gland(s); VEGF, vascular endothelial growth factor.

Received September 22, 2003.

Accepted March 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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