Anne-Catherine Gérard

Iodine Deficiency Induces a Thyroid Stimulating Hormone-Independent Early Phase of Microvascular Reshaping in the Thyroid

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency. Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early TSH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angiogenic phase that supports thyrocyte proliferation.

Recent insights into the cell biology of thyroid angiofollicular units.

In thyrocytes, cell polarity is of crucial importance for proper thyroid function. Many intrinsic mechanisms of self-regulation control how the key players involved in thyroid hormone (TH) biosynthesis interact in apical microvilli, so that hazardous biochemical processes may occur without detriment to the cell. In some pathological conditions, this enzymatic complex is disrupted, with some components abnormally activated into the cytoplasm, which can lead to further morphological and functional breakdown. When iodine intake is altered, autoregulatory mechanisms outside the thyrocytes are activated. They involve adjacent capillaries that, together with thyrocytes, form the angiofollicular units (AFUs) that can be considered as the functional and morphological units of the thyroid. In response to iodine shortage, a rapid expansion of the microvasculature occurs, which, in addition to nutrients and oxygen, optimizes iodide supply. These changes are triggered by angiogenic signals released from thyrocytes via a reactive oxygen species/hypoxia-inducible factor/vascular endothelial growth factor pathway. When intra- and extrathyrocyte autoregulation fails, other forms of adaptation arise, such as euthyroid goiters. From onset, goiters are morphologically and functionally heterogeneous due to the polyclonal nature of the cells, with nodules distributed around areas of quiescent AFUs containing globules of compact thyroglobulin (Tg) and surrounded by a hypotrophic microvasculature. Upon TSH stimulation, quiescent AFUs are activated with Tg globules undergoing fragmentation into soluble Tg, proteins involved in TH biosynthesis being expressed and the local microvascular network extending. Over time and depending on physiological needs, AFUs may undergo repetitive phases of high, moderate, or low cell and tissue activity, which may ultimately culminate in multinodular goiters.

Oxidative Stress: A Required Condition for Thyroid Cell Proliferation

Goiter is associated with increased oxidative stress (OS). We studied the effects of an anti-inflammatory agent, 15 deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) and an antioxidant, N-acetylcysteine (NAC), on OS, thyroid function, and goiter expansion in a model of goiter induced by propylthiouracil (PTU) or perchlorate. OS was assessed by the immunodetection of 4-hydroxynonenal, thyroid function by measuring thyroxin (T4) and thyrotropin (TSH) plasma levels and detecting T4-rich thyroglobulin (Tg-I), and goiter expansion by weighing the thyroids and measuring cell proliferation (PCNA and cyclin D1 immunodetection). In both PTU and perchlorate-induced goiters, OS, TSH plasma levels, thyroid weight, and cell proliferation were strongly enhanced, whereas Tg-I expression was negative. All these parameters were reversed by NAC and 15dPGJ2 in PTU-goiters. In perchlorate-goiters, TSH plasma levels remained elevated and Tg-I-negative after NAC or 15dPGJ2 treatment. OS was reduced by NAC, but not by 15dPGJ2. In addition, NAC reduced PCNA and cyclin D1 immunostainings, as well as thyroid weight, whereas 15dPGJ2 influenced neither thyroid weight nor cell proliferation. In conclusion, NAC and 15dPGJ2 overcome PTU- but not perchlorate-induced effects. The retrieval of hormonal synthesis may result from direct chemical interactions between PTU and NAC/15dPGJ2. Although 15dPGJ2 has no effect in perchlorate-goiters, the reduction of OS by NAC is associated with altered goiter development, making OS a required condition for the growth of the thyroid gland.

Minimal oxidative load: a prerequisite for thyroid cell function

In addition to reactive oxygen species (ROS) produced by mitochondria during aerobic respiration, thyrocytes are continuously producing H(2)O(2), a key element for hormonogenesis. Because nothing is known about ROS implication in normal non-stimulated cells, we studied their possible involvement in thyrocytes incubated with a potent antioxidant, N-acetylcysteine (NAC). NAC, which blocked the production of intracellular ROS, also decreased dual oxidases, thyroperoxidase, pendrin, and thyroglobulin protein and/or gene expression. By contrast, Na(+)/I(-) symporter mRNA expression was unaffected. Among antioxidant systems, peroxiredoxin (PRDX) five expression was reduced by NAC, whereas peroxiredoxin three increased and catalase remained unchanged. In vivo, the expression of both dual oxidases and peroxiredoxin five proteins was also decreased by NAC. In conclusion, when intracellular ROS levels drop below a basal threshold, the expression of proteins involved in thyroid cell function is hampered. This suggests that keeping ROS at a minimal level is required for safeguarding thyrocyte function.

A Coherent Organization of Differentiation Proteins Is Required to Maintain an Appropriate Thyroid Function in the Pendred Thyroid

CONTEXT Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. OBJECTIVE To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed. RESULTS Three morphological zones were identified: nearly normal follicles with iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone 2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were localized at the apical pole, OS and cell apoptosis were absent, but ClC-5 expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly present and increased in the cytosol and associated with high OS, apoptosis, cell proliferation, and increased expression of peroxiredoxin-5, catalase, and dehalogenase-1 but moderate ClC-5 expression. CONCLUSION In conclusion, the absence of pendrin is accompanied by increased ClC-5 expression that may transiently compensate for apical iodide efflux. In more affected follicles, Duox and TPO are relocated in the cytosol, leading to abnormal intracellular thyroid hormone synthesis, which results in cell destruction presumably because intracellular OS cannot be buffered by antioxidant defenses.

Role of caveolin-1 in thyroid phenotype, cell homeostasis, and hormone synthesis: in vivo study of caveolin-1 knockout mice.

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, (127)I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of H(2)O(2), we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobulin were intracellular rather than at the apical membrane/extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart.

Iodide deficiency-induced angiogenic stimulus in the thyroid occurs via HIF- and ROS-dependent VEGF-A secretion from thyrocytes

Vascular supply is an obvious requirement for all organs. In addition to oxygen and nutrients, blood flow also transports essential trace elements. Iodine, which is a key element in thyroid hormone synthesis, is one of them. An inverse relationship exists between the expansion of the thyroid microvasculature and the local availability of iodine. This microvascular trace element-dependent regulation is unique and contributes to keep steady the iodide delivery to the thyroid. Signals involved in this regulation, such as VEGF-A, originate from thyrocytes as early TSH-independent responses to iodide scarcity. The question raised in this paper is how thyrocytes, facing an acute drop in intracellular stores of iodine, generate angiogenic signals acting on adjacent capillaries. Using in vitro models of rat and human thyroid cells, we show for the first time that the deficit in iodine is related to the release of VEGF-A via a reactive oxygen species/hypoxia-inducible factor-1-dependent pathway.

Differential Interactions between Th1/Th2, Th1/Th3, and Th2/Th3 Cytokines in the Regulation of Thyroperoxidase and Dual Oxidase Expression, and of Thyroglobulin Secretion in Thyrocytes in Vitro

Hypothyroidism, together with glandular atrophy, is the usual outcome of destructive autoimmune thyroiditis. The impairment in the thyroid function results either from cell destruction or from Th1 cytokine-induced alteration in hormonogenesis. Here, we investigated the impact of the local immune context on the thyroid function. We used two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes incubated with IL-1alpha/interferon (IFN) gamma together with IL-4, a Th2 cytokine, or with TGF-beta, or IL-10, two Th3 cytokines. We first observed that IL-4 totally blocked IL-1alpha/interferon gamma-induced alteration in dual oxidase and thyroperoxidase expression, and in thyroglobulin secretion. By contrast, TGF-beta and IL-10 had no such effect. They rather repressed thyrocyte function as do Th1 cytokines. In addition, IL-4 blocked IL-10-induced repression of thyrocyte function, but not that induced by TGF-beta. In conclusion, Th1 cytokine- and IL-10-induced local inhibitory actions on thyroid function can be totally overturned by Th2 cytokines. These data provide new clues about the influence of the immune context on thyrocyte function.

N-Acetylcysteine and 15 Deoxy-Δ12,14-Prostaglandin J2 Exert a Protective Effect Against Autoimmune Thyroid Destruction in Vivo but Not Against Interleukin-1α/Interferon γ-Induced Inhibitory Effects in Thyrocytes in Vitro

Reactive oxygen species (ROS) are crucial for thyroid hormonogenesis, and their production is kept under tight control. Oxidative stress (OS) is toxic for thyrocytes in an inflammatory context. In vitro, Th1 pro-inflammatory cytokines have already been shown to decrease thyroid-specific protein expression. In the present study, OS level and its impact on thyroid function were analyzed in vitro in Th1 cytokine (interleukin [IL]-1alpha/interferon [IFN] gamma)-incubated thyrocytes (rat and human), as well as in vivo in thyroids from nonobese diabetic mice, a model of spontaneous autoimmune thyroiditis. N-acetylcysteine (NAC) and prostaglandin, 15 deoxy-(Delta12,14)-prostaglandinJ2 (15dPGJ2), were used for their antioxidant and anti-inflammatory properties, respectively. ROS production and OS were increased in IL-1alpha/IFNgamma-incubated thyrocytes and in destructive thyroiditis. In vitro, NAC not only reduced ROS production below control levels, but further decreased the expression of thyroid-specific proteins in addition to IL-1alpha/IFNgamma-inhibitory effects. Thus, besides ROS, other intracellular intermediaries likely mediate Th1 cytokine effects. In vivo, NAC and 15dPGJ2 reduced OS and the immune infiltration, thereby leading to a restoration of thyroid morphology. It is therefore likely that NAC and 15dPGJ2 mainly exert their protective effects by acting on infiltrating inflammatory cells rather than directly on thyrocytes.

Involvement of Nitric Oxide in Iodine Deficiency-Induced Microvascular Remodeling in the Thyroid Gland: Role of Nitric Oxide Synthase 3 and Ryanodine Receptors

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10μM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.