Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppressed TSH levels. The serum of these mice contained thyroid-stimulating activity, as measured in a classic assay using CHO cells expressing recombinant TSHr. In contrast, only 1 male out of 30 had moderately elevated serum total T4 with undetectable TSH values. The hyperthyroid animals had goiters with extensive lymphocytic infiltration, characteristic of a Th2 immune response. In addition, these animals displayed ocular signs reminiscent of Graves' ophthalmopathy, including edema, deposit of amorphous material, and cellular infiltration of their extraocular muscles. Our results demonstrate that genetic immunization of outbred NMRI mice with the human TSHr provides the most convincing murine model of Graves' disease available to date.

Early occurrence of metastatic differentiated thyroid carcinomas in transgenic mice expressing the A2a adenosine receptor gene and the human papillomavirus type 16 E7 oncogene.

We report here the characterization of a transgenic mouse model (Tg-A2aR/Tg-E7) resulting from the coexpression of two oncogenic transgenes in the thyroid. The two transgenes (Tg-A2aR and Tg-E7) were placed under control of the thyroid specific thyroglobulin gene promoter, and directed the expression of either the A2a adenosine receptor that constitutively activates the cAMP pathway, or the E7 protein of the human papillomavirus type 16, that binds and inactivates the retinoblastoma susceptibility gene product (Rb1). Transgenic mice expressing both transgenes were generated by interbreeding the Tg-A2aR and Tg-E7 transgenic lines, generated and characterized previously (Ledent et al., 1992, 1995). These mice develop a larger goiter than that of the two parental lines, and a severe hyperthyroidism comparable to that observed in the Tg-A2aR parental line. The main feature of the Tg-A2aR/Tg-E7 mice is the rapid occurrence of malignant lesions, and the dissemination of malignant thyroid tissue through the blood stream, generating multiple differentiated and functional metastases in the lung. These metastases appeared as early as 2 months after birth and their frequency increased to 75% over 3 months. They were associated with the presence of large vascular lakes in the thyroid. Electron microscopy of the malignant cells revealed nuclear features similar to those of human thyroid papillary carcinoma. These mice, in which two oncogenes are co-expressed in the thyroid, represent the first genetic animal model developing metastatic differentiated carcinomas of the thyroid with a high frequency.

Destruction of contrast microbubbles by ultrasound: effects on myocardial function, coronary perfusion pressure, and microvascular integrity.

BACKGROUND: Recent experimental data indicate that ultrasound-induced destruction of ultrasound contrast microbubbles can cause immediate rupture of the microvessels in which these microbubbles are located. METHODS AND RESULTS: To examine the functional and morphological significance of these findings in the heart, isolated rabbit hearts were perfused retrogradely with buffer containing ultrasound contrast agents and were insolated at increasing levels of acoustic energy with a broadband transducer emitting at 1.8 MHz and receiving at 3.6 MHz and operated in the triggered mode (1 Hz). At the end of each experiment, the hearts were fixed in glutaraldehyde and examined with light microscopy. Neither exposure to ultrasound alone or to contrast alone affected left ventricular developed pressure. By contrast, simultaneous exposure to contrast and ultrasound resulted in a reversible, transient mechanical index (MI)-dependent decrease in left ventricular developed pressure (to 83+/-5% of baseline at an MI of 1.6) and a transient MI-dependent increase in coronary perfusion pressure (to 120+/-6% of baseline at an MI of 1.6). Myocardial lactate release also showed significant increases with increasing MIs. Macroscopically, areas of intramural hemorrhage were identified over the beam elevation in hearts exposed to both contrast and high-MI ultrasound. Light microscopy revealed the presence of capillary ruptures, erythrocyte extravasation, and endothelial cell damage. The mean percentage of capillaries ruptured at an MI of 1.6 was 3.6+/-1.4%. CONCLUSIONS: Simultaneous exposure of isolated rabbit hearts to ultrasound and contrast agents results in an MI-dependent, transient depression of left ventricular contractile function, a rise in coronary perfusion pressure, an increase in lactate production, and limited capillary ruptures.

Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats.

The effect of selenium deficiency associated with various iodide intake was investigated in rats in order to better understand its possible role in the etiopathogeny of myxedematous cretinism. Groups of rat pups were fed from birth a low selenium diet (Se-) and submitted to goitrogenic treatment (1% perchlorate in water) for one month. Some animals were refed iodide after perchlorate withdrawal. The gland morphology was analyzed in correlation with the glutathione peroxidase (GPX) activity and the thyroid hormone plasma levels. In all Se- rats, the GPX activity was strongly reduced as compared to selenium sufficient (Se+) animals (P < 0.01). Goitrous rats were hypothyroid whatever the selenium intake. After iodide refeeding, plasma T4 and T3 levels were increased by 160% in Se- rats and by respectively 330% and 580% in Se+ rats. The thyroid morphology was different according to the selenium intake: necrotic cells were about three times more numerous in Se- than in Se+ rats (P < 0.01) and the inflammatory reaction was increased. These experimental data demonstrate the detrimental role of selenium deficiency in one experimental case of thyroid disease. Such reduction of cell defences could contribute to the thyroid failure of African myxedematous cretins.

The influence of thyrotropin and growth hormone on the thyroid gland in the hereditary dwarf mouse: a morphometric study.

Snell-type dwarf mice were injected with TSH, GH, or both hormones together for 6 days. GH induced an increase in body weight but not in the weight of the thyroid gland itself; on the contrary, TSH caused an increase in the weight of the thyroid but no increase in body weight. After TSH injection, the relative volume of the thyroid parenchyme was enhanced by 45% compared to that in untreated dwarf mice, and the radius of the follicles and follicular lumina increased by 50% and 48%, respectively. The major effect of TSH was an increase in cellular volume (+93%), and the mean number of cells in the average follicle was doubled, without a reduction in the number of follicles. GH had almost the same effect as TSH on the relative volume of the parenchyme and caused the radius of follicles and of the follicular lumina to increase by 61% and 69%, respectively. However, GH did not influence cellular volume. Its primary effect was to stimulate cellular division (cells were increased about 5 times in the average follicle) and to reduce the number of follicles. The nucleo-cytoplasmic ratio increased with GH but decreased with TSH. T4 serum levels increased to a much lesser extent with GH than with TSH, while normal values were obtained with both hormones together. At a morphological level, the combined administration of TSH and GH produced the same qualitative effects as separate administration, inducing an increase in cell volume and number which was less than the sum of the effects of each hormone administered separately.

Morphological and functional changes during thyroid hyperplasia and involution in C3H Mice: evidence for folliculoneogenesis during involution.

Involution of thyroid hyperplasia was induced in C3H mice by discontinuing a goitrogenic treatment (low iodine diet supplemented with 0.25% propylthiouracil) and refeeding a normal iodine diet. Thyroid involution was studied by morphological, histochemical, autoradiographic, and stereological methods. The onset of the involution was characterized by an early accumulation of colloid, the presence of necrotic cells in the follicular lumina, and the appearance of homogeneous microcavities in the epithelial layers. The intraepithelial microcavities had the same morphological and functional properties as the follicular lumina. They were limited by a membrane covered with microvilli; polysaccharides and peroxidase activity were detected on their membranes, and 125I-labeling was marked in their lumina. Thin serial sections demonstrated that the microlumens originated from the intercellular space; plasma membranes differentiated into junctional complexes, and a narrow lumen limited by a membrane covered with short microvilli was formed in the intercellular space between the junctions. Later on, the newly formed microlumens fused to form new follicles with a cloverleaf pattern. As a consequence of the folliculogenesis, the total number of follicles doubled after 8 days of involution. This increase in number was mainly due to the presence of a population of small follicles. The folliculogenesis was associated in the first 4 days of involution with an active cellular multiplication which compensated for the early cell necrosis and led to a doubled number of epithelial cells. The increase in the total number of follicles and cells could partially explain the persistence of a relatively high thyroid weight after involution of hyperplasia.

Increased follicular heterogeneity in experimental colloid goiter produced by refeeding iodine excess after thyroid hyperplasia.

Delayed morphological changes induced in mouse hyperplastic thyroid by refeeding iodine were analyzed by light and electron microscopy, stereology, and autoradiography. Thyroid hyperplasia was induced by a low iodine diet supplemented with 0.25% propylthiouracil for 10 days. Involution was obtained by discontinuing the propylthiouracil and returning either to a moderate iodine diet [(MID) 1 microgram I/day] or to an iodine-rich diet [(HID) 10 micrograms I/day] for 40 days. In other experiments, three cycles of hyperplasia (8 days) and subsequent involution (8 days) with MID or HID were brought about. Control animals were fed MID or HID. All animals were killed when 12-14 weeks old after injection of 10-50 microCi 125I. Double labeling, with repeated injections of [3H]thymidine from day 0 to day 7 of involution followed by 125I injection 4 h before killing, was also performed. When involutions were performed with MID, most morphological variables returned to control values. However, when involution was brought about with HID, the glandular weight, the number of follicles, and the relative volume of follicular lumina remained larger than in controls. Moreover, the 125I-labeling pattern of the follicles was altered. The proportions of unlabeled, and unevenly or partly labeled, follicles, which were fewer than 5% in control groups, represented 25-35% of all follicles after involution with HID, whereas they were unchanged with MID. In unlabeled follicles the epithelium was flattened, with a reduced number of microvilli. Partly labeled follicles were of two types. In some follicles a persistent ring reaction was observed, suggesting an abnormally slow mixing of thyroglobulin. In others, the 125I labeling was restricted to areas adjacent to the apex of a reduced number of cells, suggesting that some cells were iodinating thyroglobulin, whereas others were not. There was no relationship between the follicular 125I labeling and the frequency of [3H]thymidine-labeled cells. These results indicate that refeeding iodine excess after hyperplasia leads to the formation of a colloid goiter with new follicles, and to an increased heterogeneity of iodine metabolism among follicles and among cells.

Detection and identification of endothelin-1 immunoreactivity in rat and porcine thyroid follicular cells.

Endothelin-1 immunoreactivity (irET-1) was observed in rat and porcine thyroid glands. Using a radioimmunoassay for endothelin-1, the mean concentration in extracts of rat and porcine thyroid glands were 0.75 pg/mg +/- 0.03 (n = 4) and 1.5 pg/mg +/- 0.2 (n = 8) (mean +/- SE) respectively. Gel-filtration and reverse-phase HPLC showed that ir ET-1 eluted in a position identical to synthetic endothelin-1. In addition, immunohistochemical study showed that irET-1 is located within epithelial follicular cells. No immunostaining was seen in parafollicular C-cells nor in parathyroid.

Costimulation of adenylyl cyclase and phospholipase C by a mutant alpha 1B-adrenergic receptor transgene promotes malignant transformation of thyroid follicular cells.

Proliferation of thyroid follicular cells is controlled by three intra-cellular cascades [cAMP, inositol 1,4,5-triphosphate (IP3)/Ca2+/diacylglycerol (DAG), and tyrosine kinases] that are activated by distinct extracellular signals and receptors. We had previously generated a transgenic mouse model in which the cAMP cascade was permanently stimulated in thyroid cells by an adenosine A2a receptor (Tg-A2aR model). In the present work, we have generated a transgenic model characterized by the chronic stimulation of both adenylyl cyclase and phospholipase C in thyroid follicular cells. The bovine thyroglobulin gene promoter was used to direct the expression of a constitutively active mutant of the alpha 1B adrenergic receptor, which is known to couple to both cascades in transfected cell lines. The expression of the transgene resulted, as expected, in the activation of phospholipase C and adenylyl cyclase, as demonstrated by the direct measurement of IP3 and cAMP in thyroid tissue. The phenotype resulting from this dual stimulation included growth stimulation, hyperfunction, cell degeneracy attributed to the overproduction of free radicals, and the development of malignant nodules invading the capsule, muscles, and blood vessels. Differentiated metastases were found occasionally in old animals. The development of malignant lesions was more frequent and of earlier onset than in our previous Tg-A2aR model, in which only the cAMP cascade was stimulated. These observations demonstrate that the cAMP and IP3/Ca2+/DAG cascades can cooperate in vivo toward the development of thyroid follicular cell malignancies.

In vitro study of acute toxic effects of high iodide doses in human thyroid follicles.

The acute effects of increasing doses of sodium iodide were studied on human thyroid follicles isolated from normal paranodular tissue. After 24 h incubation in culture medium, follicles isolated from most thyroids maintained their capacity for 125I accumulation and organification and a normal cellular ultrastructure. 125I accumulation was significantly increased after addition of TSH, whereas 125I organification was not affected. In presence of TSH, numerous follicles had large empty-looking follicular lumina unlabeled on autoradiographies. Follicles incubated for 24 h in the presence of a low concentration (10(-7) M) of iodide retained their function and morphology. However, incubation with a high dose of iodide (10(-3) M) caused marked inhibition of 125I accumulation and organification reaching values similar to those obtained in presence of inhibitors of iodide trapping and organification. At high doses, iodide induced necrosis of thyroid epithelial cells: the percentage of necrotic cells was significantly increased with 10(-5) M and doubled with 10(-3) M as compared to values measured at 10(-7) M. Ultrastructural lesions such as apical blebbing, cytoplasmic fragments desquamation, endoplasmic reticulum vesiculation, and accumulation of lipofuscin in secondary lysosomes were also present. The necrotic effect and the ultrastructural alterations also occurred in the presence of TSH but were prevented by the addition of inhibitors of iodide trapping or organification. These results demonstrate a direct acute toxic effect of iodide in human thyroid cells. The nature of the ultrastructural alterations is in agreement with a mechanism of toxicity involving a free radical attack and lipid peroxidation as observed in other tissues.

Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation.

The thyroid gland is a highly vascular tissue, and its blood flow changes dramatically in various pathological conditions. Although the mechanisms regulating these changes in vascularity and blood flow are not well understood, candidate mediators include endothelin-1 (ET-1) and nitric oxide (NO). In the present study, we used a reverse transcriptase-polymerase chain reaction assay to determine which components of these vasoregulatory pathways are present in the thyroid and to analyze changes in gene expression in an experimental model of goiter formation and involution. Expression of messenger RNAs (mRNAs) encoding ET-1, ET receptors (ETA and ETB), ET-converting enzyme, and the three nitric oxide synthase (NOS) isoforms (NOS I, NOS II, and NOS III) was readily detected in the rat thyroid. After goiter formation was induced by thiouracil and a low iodine diet, there was increased expression of the genes encoding ET-related proteins (ET-1, 3.2-fold; ETA, 2.9-fold; ETB, 3.5-fold) as well as two of the three NOS isoforms (NOS I, 2.7-fold; NOS III, 4.9-fold). During iodide-induced involution, the ET-related mRNA levels remained elevated, whereas those of the two NOS isoforms returned to basal values. ET-converting enzyme, NOS II, and thyroglobulin mRNAs were minimally affected in this model, providing evidence for selective regulation of these genes. To assess whether NO plays a role in vascular changes during goiter formation, animals were treated with a NOS inhibitor, N-nitro-L-arginine methyl ester (NAME). NOS activity in the thyroid was inhibited by more than 75% after treatment with NAME. Thyroid hormone and TSH levels were unchanged. Although NAME had little effect on overall thyroid size, vascular expansion during goiter formation was decreased by 36%. We conclude that the thyroid gland expresses a complex network of vasoactive genes whose expression is regulated dynamically during thyroid goiter formation and involution. NO production and probably other locally produced vasoactive substances are involved in changes in thyroid vascularization.

Morphological and functional changes during thyroid hyperplasia and involution in C3H mice: effects of iodine and 3,5,3'-triiodothyronine during involution.

Involution of thyroid hyperplasia was induced in mice by discontinuing a goitrogenic treatment (low iodine diet plus 0.25% propylthiouracil for 10 days) and returning either to a moderate iodine diet (MID; 1 microgram I/day) alone or associated with T3 administration (1 microgram/day) or to a high iodine diet (HID; 10 micrograms I/day) alone or associated with T3 treatment. Thyroid involution was studied by morphological, stereological, and biochemical methods after 2, 4, 6, and 8 days of involution. Age-paired, HID-fed animals were used as controls. When the involution was induced by MID, the glands resumed a normal morphological aspect. The synthesis and secretion of T3 were highly stimulated on day 2, but decreased thereafter. Plasma T4 levels reached a plateau at 50% of the control value from days 2-8. The administration of T3 together with MID accelerated the involution of hyperplasia and colloid accumulation in the follicular lumina. The synthesis and secretion of T3 and T4 remained lower than those in controls. When the involution was induced by HID, the thyroid weight remained higher than that in controls or in any involuting groups. The number of follicles and epithelial cells as well as the glandular thyroglobulin content were twice the control values. A Wolff-Chaikoff effect was evident on day 4, and hypothyroidism persisted. When HID was supplemented with T3 treatment, glandular weight and morphology were normal, but the Wolff-Chaikoff effect occurred earlier. In conclusion, the iodine dose given after a goitrogenic treatment must be carefully controlled; a high but physiological dose can have deleterious effects, whereas a small dose is beneficial. T3 prevents the deleterious effects of HID, but the thyroid enters a resting state.

Structural changes in the angiofollicular units between active and hypofunctioning follicles align with differences in the epithelial expression of newly discovered proteins involved in iodine transport and organification.

In animals, as well as in humans, the thyroid gland is made of active follicles, with cuboidal cells and hypofunctioning follicles, with flattened cells. In this study, the functional status of human follicles was dissected out, based on immunohistochemical detection of TSH receptor, Na(+)/I(-) symporter, pendrin, thyroperoxidase (TPO), thyroid oxidases (ThOXs), and T(4)-containing iodinated Tg (Tg-I). To ascertain that angiofollicular units exist in the human, we studied the microvascular bed of each follicle, in correlation with detection of vascular endothelial growth factor (VEGF), of nitric oxide synthase III, and of endothelin in normal and goitrous thyroids. In hypofunctioning follicles, pendrin, TPO, and ThOXs were not detected, and there was no Tg-I in the colloid. At the opposite, in active follicles, pendrin, TPO, and ThOXs were detected in thyrocytes, and Tg-I was present in the colloid. In normal and goitrous thyroids, the capillary networks surrounding active follicles were larger than those surrounding hypofunctioning follicles. Immunoreactivity for nitric oxide synthase III and endothelin was solely detected in active follicles. Only a few follicles in normal thyroids were immunostained for VEGF, regardless of their functional status. In multinodular goiters, VEGF was detected in contact with the extracellular matrix at the basal pole of the cells. In conclusion, the present study endorses the likelihood of angiofollicular units in the human thyroids. Vascular changes are related to the functional status of thyrocytes.

Correlation between the loss of thyroglobulin iodination and the expression of thyroid-specific proteins involved in iodine metabolism in thyroid carcinomas.

Progress in biotechnology has provided useful tools for tracing proteins involved in thyroid hormone synthesis in vivo. Mono- or polyclonal antibodies are now available to detect on histological sections the Na(+)/I(-) symporter (NIS) at the basolateral pole of the cell, the putative iodide channel (pendrin) at the apical plasma membrane, thyroperoxidase (TPO), and members of the NADPH-oxidase family, thyroid oxidase 1 and 2 (ThOXs), part of the H(2)O(2)-generating system. The aim of this study was to correlate thyroglobulin (Tg) iodination with the presence of these proteins. Tg, T(4)-containing Tg, NIS, pendrin, TPO, ThOXs, and TSH receptor (TSHr) were detected by immunohistochemistry on tissue sections of normal thyroids and various benign and malignant thyroid disorders. Tg was present in all cases. T(4)-containing Tg was found in the adenomas, except in Hurthle cell adenomas. It was never detected in carcinomas. NIS was reduced in all types of carcinomas, whereas it was detected in noncancerous tissues. Pendrin was not expressed in carcinomas, except in follicular carcinomas, where weak staining persisted. TPO expression was present in insular, follicular carcinomas and in follicular variants of papillary carcinomas, but in a reduced percentage of cells. It was below the level of detection in papillary carcinomas. The H(2)O(2)-generating system, ThOXs, was found in all carcinomas and was even increased in papillary carcinomas. Its staining was apical in normal thyroids, whereas it was cytoplasmic in carcinomas. The TSHr was expressed in all cases, but the intensity of the staining was decreased in insular carcinomas. In conclusion, our work shows that all types of carcinomas lose the capacity to synthesize T(4)-rich, iodinated Tg. In follicular carcinomas, this might be due to a defect in iodide transport at the basolateral pole of the cell. In papillary carcinomas, this defect seems to be coupled to an altered apical transport of iodide and probably TPO activity. The TSHr persists in virtually all cases.

Effects of iodide and thyroxine on iodine-deficient mouse thyroid: a morphological and functional study.

The effects of iodide and thyroxine (T4) on female mice fed a low iodine diet (LID) for 8 weeks were analysed by morphological, stereological and biochemical methods. Iodide was given at a dose of 10 micrograms/day (HID) or 1 microgram/day (MID), either alone or together with daily injections of 1 microgram T4 for 8 or 40 days. With HID, the thyroid weight and the numbers of follicles and cells remained higher than in controls, although cell necrosis occurred. Colloid volume increased and iodine was stored within the gland: a colloid goitre with non-functioning follicles was produced. With MID, the glands resumed an almost normal appearance. With T4 and LID, progressive normalization occurred, but after 40 days thyroid weight and numbers of follicles and cells remained higher than in controls. Glandular iodine content slowly increased and reached control value. The proportions of 125I-labelled tri-iodothyronine (T3) and T4 in thyroglobulin were reduced. With T4 and HID, the glands resumed a normal appearance. Neither necrosis nor folliculoneogenesis was noted. The proportions of 125I-labelled T3 and T4 in thyroglobulin were reduced, but T3 and T4 serum levels were higher than with HID. With T4 and MID, a normal state was obtained as early as day 8. After 40 days the gland was morphologically and functionally inactive. In conclusion, the association of T4 and iodide seems to be the best way to obtain a rapid and complete involution of thyroid hyperplasia. The administration of T4 prevents the deleterious effects of an excess of iodine on follicular cells, and causes the gland to enter a slow-functioning state.

Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone non-obese diabetic mice: effects of iodine-induced cell necrosis.

The administration of a high iodide dose (HID; 10 micrograms/day) to goitrous mice is known to induce thyroid cell necrosis and inflammation, which, in most strains, is transient. In this study, we analyzed the effects of iodide in autoimmune prone non-obese diabetic (NOD) mice. Control NOD mice fed a standard diet (MID; 1 microgram I/day) or HID did not spontaneously develop thyroiditis. In NOD mice previously made goitrous, HID provoked thyroid cell necrosis and diffuse inflammation within 4 days. Inflammatory cells consisted of MHC-class II+ antigen-presenting cells, CD4+ T helper cells and CD8+ T suppressor/cytotoxic cells. After 96 days of treatment with HID, thyroiditis similar to Hashimoto's disease was obtained in 100% of the animals, with destruction of thyroid follicles, large clusters of T and B cells, and antithyroid antibodies in the plasma. When treating goitrous mice with MID, no cell necrosis was observed and no autoimmune thyroiditis was obtained. The early iodide-induced cell necrosis and inflammation may thus be considered as an important factor in the induction and persistence of autoimmune thyroiditis in individuals carrying a genetic susceptibility to autoimmune disease.

Antigoitrogenic effect of combined supplementation with dl-alpha-tocopherol, ascorbic acid and beta-carotene and of dl-alpha-tocopherol alone in the rat.

The effects of the vitamins dl-alpha-tocopherol, ascorbic acid and beta-carotene, free radical scavengers and lipid peroxidation inhibitors, were analyzed in male Wistar rats made goitrous by feeding a low iodine diet (< 20 micrograms iodine/kg) and perchlorate (1% in drinking water) for 4, 8, 16, and 32 days. Groups of control or goitrous rats received for at least 16 days before killing a diet containing 0.6% vitamin E (as dl-alpha-tocopherol acetate), 1.2% vitamin C (ascorbic acid) and 0.48% beta-carotene, either simultaneously (vitamin cocktail) or separately. This treatment led to a 5-fold increase of vitamin E in the thyroid gland, a 24-fold increase in the liver and a 3-fold increase in the plasma. In control rats, vitamin cocktail administration increased slightly the thyroid weight with little changes in thyroid function parameters. During iodine deficiency, administration of the vitamin cocktail or vitamin E alone reduced significantly the rate of increase in thyroid weight, and DNA and protein contents, as well as the proportion of [3H]thymidine labeled thyroid follicular cells, but not that of labeled endothelial cells. Plasma tri-iodothyronine, thyroxine, TSH levels, thyroid iodine content and concentration as well as relative volumes of glandular compartments were not modified. The proportion of necrotic cells rose from 0.5% in normal animals to about 2% after 16 days of goiter development. No significant protective effect of the vitamins was observed. These results suggest that these vitamins, particularly vitamin E, modulate one of the regulatory cascades involved in the control of thyroid follicular cell growth, without interfering with the proliferation of endothelial cells.

Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution.

Necrosis and apoptosis coexist in the thyroid during goitre development and involution, but little is known about their respective causes. To test the possible role of free radicals, we analysed separately necrosis and apoptosis in male Wistar rats with depressed or normal antioxidant protection. Vitamin E-deficient and -sufficient rats were made goitrous with perchlorate in drinking water; involution was induced by repeated injection of NaI, without or with methimazole. Increase of thyroid malondialdehyde concentration and decrease of glutathione peroxidase activity confirmed the depressed antioxidant protection in vitamin E-deficient rats. Plasma thyroxine and TSH levels were not modified. Necrosis (swollen cells) and apoptosis (pyknotic cells) were quantified on histological sections. In vitamin E-sufficient rats, dead cells were very rare in control thyroids, increased 3-fold in goitre and still further during involution. Necrotic epithelial cells predominated in the goitre and their number declined after iodide supplementation, without or with methimazole. In contrast, the number of apoptotic cells and the caspase-3 activity were increased in goitre and further increased after involution, with two-thirds of pyknotic cells being observed in the interstitium. Apoptosis was prevented by methimazole. Vitamin E deficiency significantly increased total cell death and epithelial cell necrosis and induced the occurrence of much cell debris in the follicular lumen during involution, with no modification of the apoptotic reaction. These results show that the type of cell death is differentially regulated during goitre development and involution: necrosis is related to the oxidative status of the cells, while apoptosis comes with iodine-induced involution.

Expression of the endothelin-1 gene in the rat thyroid gland and changes in its peptide and mRNA levels in goiter formation and iodide-induced involution.

Endothelin-1 (ET-1) is a major vasoconstrictor peptide, first found in endothelial cells, and later in many other tissues, including the thyroid gland. We analysed the expression of the ET-1 gene in the rat thyroid gland and changes in ET-1 mRNA and peptide levels in goiter development and involution, two circumstances characterised by vascular changes. Thyroid hyperplasia was induced in adult Wistar rats by feeding a low iodine diet (LID) supplemented with 0.25% thiouracil for 10 days, and LID alone for 2 further days (H.12 group). Involution was induced by injecting 100 micrograms iodide and refeeding a normal diet during 6 h, 12 h, and 24 h (I.6h, I.12h, I.24h groups). Rats fed a normal iodine diet were used as controls. A specific 488 bp cDNA corresponding to the known sequence of pre-pro ET-1 was found by RT-PCR from RNA extracts in all thyroid experimental groups, as well as in lung and kidney which were used as positive controls. RP-HPLC analysis showed that ET-1 immunoreactivity eluted similarly as mature ET-1. During hyperplasia, ET-1 mRNA and peptide levels were increased 3.5- and 5-fold respectively. The relative volume of the vascular bed was more than doubled. During iodide-induced involution, the glandular ET-1 mRNA level remained elevated. The concentration of ET-1 peptide increased and was significantly greater at 12 h involution than in the H.12 group. At this time, the capillary reticulum reverted to individual capillaries and the vascular bed was significantly reduced. These data demonstrate that the ET-1 gene is expressed in the rat thyroid gland and that the ET-1 mRNA and peptide levels are increased during thyroid hyperplasia and remain elevated during a phase of rapid iodide-induced involution. These data suggest that changes in ET-1 production may play a role in control of thyroid gland trophic regulation and vascularity.

Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease.

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.