ABSTRACT

Thyroid hormones (THs) regulate growth, development, metabolism. This chapter aims to provide a comprehensive overview of the molecular and cellular mechanism(s) for intracellular signaling by TH. At the cellular level, THs bind to thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor family.  TRs act as ligand-activated transcription factors that bind to their cognate thyroid hormone response elements (TREs) in the promoters of target genes. TRs regulate gene transcription by employing TR-interacting protein complexes containing coactivators (CoAs) or  corepressors (CoRs). Coactivator and corepressor complexes include histone modifying enzymes known as histone acetyltransferases (HATs) or histone deacetylases  (HDACs), respectively, that induce epigenetic changes in the chromatin structure of target gene promoters to enhance or repress the transcriptional efficiency of RNA polymerase on TH-responsive genes. TRs also can mediate transcriptional effects indirectly by binding to other transcription factors or activating cell signaling cascades.  Additionally, emerging evidence suggests that THs may bind to cell membrane proteins other than TRs to activate cell signaling pathways. The important role of TH on metabolism, has  spawned interest the pharmacological use of TH and its analogs/metabolites for the treatment of metabolic diseases such as hypercholestronemia, hypertriglycerimia obesity, and non-alcoholic fatty liver disease (NAFLD).

INTRODUCTION

The thyroid hormones (THs, thyroxine (T₄) and triiodothyronine (T₃)) have important effects on development, growth, and metabolism (1-3). Some of the most prominent effects of TH occur during fetal development and early childhood. In humans, the early developmental role of TH is illustrated by the distinctive clinical features of cretinism observed in iodine-deficient areas. In childhood, lack of TH can cause delayed growth. However, in this latter case, many of the effects of TH may be metabolic rather than developmental, as growth is restored rapidly after the institution of TH treatment. In adults, the primary effects of THs are manifested by alterations in metabolism. These effects include changes in oxygen consumption, protein, carbohydrate, lipid, and vitamin metabolism. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs.

At the clinical level, identification of quantitative markers of TH action has been difficult (4). At the extreme ends of the clinical spectrum, which extends from hypothyroidism to hyperthyroidism, the diagnosis of a thyroid abnormality is usually apparent. Clinical suspicion of a thyroid abnormality can be confirmed using laboratory tests for THs and thyroid stimulating hormone (TSH). However, more subtle forms of thyroid dysfunction, such as subclinical hypothyroidism or hyperthyroidism, pose a greater challenge. Although the level of circulating TSH provides a sensitive and quantitative indicator of TH action at the level of the hypothalamic-pituitary axis, there are few reliable peripheral or intracellular markers of TH action (5,6). The effect of TH on basal metabolism has been re-evaluated using measurements of resting energy expenditure (REE). In hypothyroid patients taking varying levels of TH replacement, there is a strong inverse correlation between REE and the TSH level (6). Nevertheless, TSH remains the most sensitive and useful clinical indicator of TH action. As discussed below, tissue-selective metabolism of THs, and variable tissue sensitivity to their effects, underscores the need to develop additional markers of TH activity in peripheral tissues.

Since the initial description of TH effects on metabolic rate more than 100 years ago (7), many theories have been proposed to explain its mechanism of hormone action. The proposed models include: uncoupling oxidative phosphorylation, stimulation of energy expenditure by the activation of Na+-K+ ATPase activity, and direct modulation of TH transporters and enzymes in the plasma membrane and mitochondria (8). Recently, there has been increasing evidence for non-genomic actions (see later under non-genomic actions of TH) (8); however, the major effects of TH occur via nuclear receptors that mediate changes in gene expression.

In 1966, Tata proposed that TH increased gene expression with attendant increases in protein synthesis and enzyme activity (9). In 1972, high affinity nuclear binding sites for TH were documented (Kd approximately 10-10 M for T₃) (10,11). The receptor-binding affinity of various THs and analogues correlated with their biologic potencies, consistent with the view that most biologic effects are mediated via the nuclear receptor (11– 14). Over the past 25 years, there has been a dramatic surge of new information on TH action resulting from the cloning of the TH receptors (15,16), the identification of regulatory DNA elements in TH responsive genes (1-3), the generation of TR isoform knockout mice (17,18), and the discovery and phenotype characterization of patients with mutations in TRα and TRβ (5,19,20). In this chapter, we will focus on our current understanding of nuclear TH receptor action.

BINDING OF THs TO NUCLEAR RECEPTORS

In many respects, T₄can be regarded as a prohormone for the more potent hormone, T₃. Most of the TH bound to receptors is in the form of T₃, either secreted into the circulation by the thyroid gland or derived from T₄to T₃ conversion by 5' monodeiodinases (see Chapter 3C). There are three distinct deiodinases- type I, type II, and type III (21,22). The distribution and regulation of these enzymes can have important effects on TH action. For example, Type II deiodinase has high affinity for T₄(Kd in the nanomolar range) and is found primarily in the pituitary gland, brain, and brown fat where conversion of T₄to T₃ modulates the intracellular concentration of T₃. Thus, tissues that contain type II deiodinase can respond differently to a given circulating concentration of T₄(by intracellular conversion to T₃) than organs that only can respond to T₃(23,24). Additionally, it appears that both type 1 and type II deiodinase regulate the circulating T₄and T₃levels (25). Recently, MCT8, OATP-1, and System L amino acid transporters have been identified as TH transporters which regulate T₄and T₃uptake into cells (26,27). Mutations in the former have been involved in a number of syndromes of x-linked mental retardation and neurologic deterioration (27,28).

T₃binds to its receptors with approximately 10 fold higher affinity than T₄. The dissociation constants for liver nuclear receptors measured in vitro are 2 x 10^-9M for T₄and 2 x 10^-10M for T₃(1,2). Nuclear receptors are approximately 75% saturated with TH in brain and pituitary and 50% saturated with TH in liver and kidney. It is notable that the extent of TH receptor occupancy varies in different tissues, providing a mechanism for alterations in circulating TH levels to alter receptor activity. In contrast to the related steroid hormone receptors, TRs are mostly nuclear both in the absence and presence of TH (1,2,30). In fact, TH receptors are tightly associated with chromatin (1-3,30), consistent with their proposed role as DNA-binding proteins that regulate gene expression.

CLONING, STRUCTURE, AND EXPRESSION OF TH RECEPTORS

Cloning of TRs

TH receptors (TRs) were first cloned in 1986 and belong to the nuclear hormone receptor superfamily that includes the glucocorticoid, estrogen, progesterone, androgen, aldosterone, vitamin D, retinoic acid (RARs), retinoid X (RXRs) and "orphan" (unknown ligand and/or DNA target) receptors (1,2,15,16,30-33). TRs are the cellular homologs of v-erbA, a viral oncogene product involved in chick erythroblastosis. TRs are encoded at two genomic loci (α and β) located on human chromosomes 17 and 3, respectively and their gene products result in two major isoforms, TRα and TRβ .

Structure of TRs

Like other members of the nuclear receptor superfamily, the TRs have a central DNA-binding domain (DBD) and a carboxy-terminal ligand-binding domain (LBD) (Figure 3d-1). The two major TR isoforms have high amino acid sequence homology in their respective DBDs and LBDs. Dimerization domains also are found in both DBDs and LBDs. The amino-terminal regions are more variable between TRα and TRβ (1-3), and contain ligand-independent activation domains. In contrast, multiple sub-regions are located in the LBD for ligand-dependent transcriptional activation and basal repression of target genes.

Figure 1. Functional domains of the TH receptor (TR). The TH receptor (TR) is depicted schematically. The zinc finger DNA-binding domain (DBD) is denoted along with the carboxy terminal ligand-binding domain (LBD). Other functional domains and interaction sites are indicated.

The DNA-binding domains of the nuclear receptors are comprised of two distinct zinc fingers that are separated by a 15-17 amino acid linker sequence. The crystal structure of the DNA-binding domains of the TH receptor and its heterodimeric partner, RXR has been determined. The two heterodimer partners interact with a direct repeat of the receptor binding site in a head-to-tail manner (34-37).

A small stretch of amino acids at the base of the first finger (referred to as the P-box) dictates the DNA sequence specificity of the receptor (35-39). The P-box sequence of the TH receptor is shared by other receptors that bind to similar or identical DNA recognition sites (AGGTCA). The underlined amino acids in the P-box (EGCKG) of the TH receptor are also found in the retinoic acid receptors, the retinoic acid X receptors, the rev-erbA protein, the vitamin D receptor, and NGFI-B. Of note, the steroid hormone receptors have a different P box sequence and bind as homodimers to a different consensus DNA half-site sequence (AGAACA). The region between the DBD and LBD is called the hinge region and contains the nuclear localization signal, typically a basic amino acid‑rich sequence, first described in viral nuclear proteins.

X‑ray crystallographic studies of the liganded rat TRα-1 show that TH is embedded in a hydrophobic "pocket" lined by discontinuous stretches of amino acid sequences within the LBD. Additionally, there are several hydrophobic interfaces within the LBD that contribute to the TR homo- and heterodimerization with RXR (39). There are twelve amphipathic helices in the LBD and specific helices among them provide the critical contact surfaces for protein-protein interactions with co-activators and co-repressors (helices 3,5,6,12 and 3,4,5,6, respectively) (40-43). Ligand-binding to TR causes a major conformational change in the LBD, particularly in helix 12. This, in turn, facilitates TR discrimination between co-activators and co-repressors (see below).

Splicing variants of TRs

The carboxy-terminal hormone-binding domain of the TRα gene is alternatively-spliced to generate several protein products (Figure 3d-2, below). One variant, referred to as α-2, is identical to TRα-1 through the first 370 amino acids, but then its sequence diverges completely, owing to splicing of alternate exons (44-47). Another splicing variant, referred to as TRvII or α-3, is similar to α-2 except that it lacks the first 39 amino acids found in the unique region of α-2 (45). α -2 cannot bind TH because of the replacement of critical amino acids at the extreme carboxy-terminal end of the protein due to alternative splicing (48), and thus cannot mediate ligand-dependent gene transcription (49– 51). The amino acid replacements in α-2 also alter its dimerization properties and reduce DNA-binding affinity (52-55). The α-2 splicing variant is highly expressed in many tissues such as brain, testis, kidney, and brown fat, but its function remains poorly understood (56). The α-2 isoform has been proposed to be an endogenous inhibitor of TH receptor function as it inhibits TRα and β activity in transient gene expression assays (44,54). The mechanism by which α-2 antagonizes TR action is controversial. Some studies indicate that α-2 competes for active receptor complexes at DNA target sites (57,58). Other studies indicate that α-2 inhibits TR activity independent of DNA-binding (59). It is likely that the inhibitory effects of α-2 involve more than one mechanism. Amino acid substitutions in the carboxy-terminal region of α-2 also prevent its interactions with transcriptional corepressors (see below) (55), and may provide an explanation as to why α-2 is not a more potent inhibitor of TR activity. Additionally, the phosphorylation state of α-2 may modulate its inhibitory activity (60). Given the foregoing features, the TRα-1 and α-2 system represents one of the few examples in mammals whereby multiple mRNAs generated by alternative splicing encode proteins that are antagonistic to each other.

Figure 2. TH receptor isoforms. The TH receptors (TR) β and α are expressed from separate genes. Each TR gene can be expressed as distinct isoforms, reflecting the use of alternate promoters and exons. The central zinc finger DNA-binding region is indicated and unique domains are shown by distinct patterns of shading. The TRβ-2 isoform, which is expressed predominantly in the pituitary and hypothalamus, contains a unique amino-terminus. The TRα-2 isoform contains unique carboxy-terminal sequences that eliminate hormone binding. The DNA- and T3-binding properties and transcriptional activity of the various isoforms are shown at the right.

A receptor-like molecule, Rev-erbA, is, surprisingly, encoded on the opposite strand of the TRα gene locus (61, 62). Rev-erbA mRNA contains a 269-nucleotide stretch which is complementary to the α-2 mRNA due to its transcription from the DNA strand opposite of that used to generate TRα-1 and α-2. This protein also is a member of the nuclear hormone receptor superfamily, and is highly expressed in adipocytes and muscle cells. Rev-erbA, contains a DBD that is homologous to the TR DBD. However, Rev-erbA does not bind TH and its putative LBD has minimal homology with other nuclear hormone receptors. Since no cognate ligand has been identified for Rev-erbA, it is categorized as an "orphan nuclear receptor. " It can act as a transcriptional repressor for nuclear hormone receptors and other transcription factors (63-65). Since Rev-erbA shares an exonic segment of the bidirectionally transcribed TRα gene, it is possible that it modulates the expression or splicing of TRα-1 and α-2 (66, 67) as parallel increases in rev-erbA mRNA and TRα-1 mRNA expression occur relative to α2 mRNA expression.

The major variant of the TRβ gene, TRβ-2, has a different amino-terminus than TRβ 1 (Figure 3d-2, above) (68). The distinct amino-terminal region of the TRβ-2 is due to transcription from a tissue-specific promoter. The function of the amino-terminus of the TH receptor is not known, but it likely plays a role in transcriptional control (69,70). The TRβ-1 and TRβ-2 isoforms function similarly in most transient gene expression assays (69, 71), although differences in the transcriptional activities of the TRβ 1 and TRβ 2 isoforms have been noted with respect to certain target genes (69-72). It is likely that tissue-restricted expression of the TRβ-2 isoform contributes to unique patterns of TR expression, which in turn, may modulate target gene regulation.

Recently, short isoforms of TRα and TRβ have been described (73, 74). The novel TRα isoforms arise from translational start sites in the 7th intron and yield shortened TRα 1 and α 2 isoforms that have dominant negative activity on WT TR. Novel short TRβ isoforms arise from alternative splicing of TRβ. It is possible that these isoforms may modulate T₃-responsiveness in a tissue- and/or developmental stage-specific manner.

Tissue- and development-specific expression of TRs

Most studies of TR isoform expression have employed mRNA analyses rather than protein measurement (30). In general, the α and β receptor isoforms are distributed widely and exhibit overlapping patterns of expression (30,75). TRα 1 mRNA is expressed in skeletal and cardiac muscle whereas TRβ-1 mRNA is predominant in liver, kidney, and brain. Α-2 mRNA is most prevalent in brain and testis. In contrast, TRβ-2 mRNA has the most tissue-restricted expression, and is present in the anterior pituitary gland, hypothalamus, and cochlea (75-79).

The TRs also are expressed in specific stages during development, and are subject to regulation by hormones and other factors (78, 79). For instance, TRα-1 mRNA is expressed early whereas TRβ 1 mRNA is expressed later during embryonic brain development. In the rat pituitary gland, TH decreases TRβ 2, TRα-1, and α-2 mRNAs while slightly increasing TRβ-1 mRNA. However, in most other tissues, TH decreases TRα-1 and α-2, but not TRβ-1 mRNA. Isoform-specific knockout mice of each of the TR isoforms display distinct phenotypes (17,18). However, lack of significant TR isoform-specific gene expression was observed in cDNA microarrays of hepatic genes in TR isoform knockout mice (80). Given the apparent redundancy in TR isoform function, it is possible the different KO phenotypes may be due to absolute TR expression levels in critical tissues and developmental stages.

TRANSCRIPTIONAL REGULATION BY TRS

TH receptors bind to TH response elements (TREs) in specific target genes (Figure 3d-3, below). After binding TH, the receptor induces changes in gene expression by either increasing or decreasing the transcriptional activity of target genes. Examples of the target genes that are positively- and negatively-regulated by TH are summarized in Table 1. cDNA microarrays have been employed to study TH regulation of hepatic genes in mice, and led to the identification of a large number of novel target genes (both positively- and negatively-regulated) (81,82). These studies demonstrated that TH affected gene expression in a wide range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. Although many of the TH-responsive genes were regulated directly by TRs, others were probably regulated indirectly through intermediate genes. Indirect regulation of TH-mediated transcription is suggested when the time course for induction is slow (hours) and when it is blocked by protein synthesis inhibitors. Although TH acts mainly at the level of transcription, it also can affect mRNA stability, translational efficiency, and miRNA regulation (83,84). Thus, TH acts at multiple levels to alter protein expression.

Figure 3. Mechanism of TH action via its nuclear receptor. TH is transported across plasma membrane and likely diffuses through nuclear membrane to bind to its receptor. The TH receptor (open circle) is localized almost exclusively in the nucleus where it associated with DNA as a homodimer or as a heterodimer with RXR (stippled box). The hormone-activated receptor binds to TH response elements (TREs) to alter rates of gene transcription and consequently levels of mRNA.

Table 1. Examples of Genes Positively-regulated by T3.

Table 2. Examples of Genes Negatively-regulated by T₃.

TR binding to TH response elements (TREs)

Detailed analyses of thyroid response elements (TREs) have led to the identification of a canonical TRE half-site sequence (1, 2,85) (Figure 3d-4). The TRE half-site is generally considered to be a hexamer (AGGTCA), but TR binding is optimal with a more extended binding site (37,88,89). Specifically, the sequence TAAGGTCA is optimal for TR binding and T₃-responsiveness. However, inspection of TREs from many different target genes reveals there is a relatively low degree of sequence conservation among these elements. This finding suggests the possibility that naturally-occurring TREs may have diverged from an ideal consensus element during evolution as a means to modulate the degree of TH responsiveness.

TR interactions with DNA are quite different from those observed with steroid receptors, which bind to palindromic DNA sequences as homodimers. Although TR also can bind to certain TREs in vitroas a homodimer, it binds preferentially to most TREs as a heterodimer with the retinoid X receptors (RXRs) (1-3,30). The TR-RXR heterodimer binds to half-sites that are arranged in several different configurations. These include palindromic arrangements (head-to-head), direct repeats (head-to-tail), and inverted repeats (tail-to-tail). Most naturally occurring TREs are direct repeats (Figure 3d-4), typically separated by four nucleotides. The ability of TR dimers to bind to TRE’s in different configurations suggests a flexible protein structure, or the possibility that distinct protein surfaces are involved in the formation of dimers (34,39,90). Taken together, the specificity and affinity for the TR-RXR heterodimer is primarily determined by sequences within the half-site, the length of the spacer region between the half sites, and the sequence context within the spacer region.

Figure 4. Consensus thyroid response element (TRE). Studies of TRE’s in many different promoters has allowed the derivation of a "consensus" TRE comprised of a direct repeat of the hexameric sequence, AGGTCA, spaced by four nucleotides (n). Of note, there is considerable diversity in the sequences of half-sites, orientation of half-sites, and bases that form the spacers between half-sites (see text).

Although TR can interact with a wide variety of other nuclear receptors and transcriptional adaptor proteins (see below), the RXR proteins (α, β, and g) represent its most important heterodimeric partners (1-3). The RXR proteins enhance TR binding to DNA and reduce the rate of receptor dissociation from DNA (91). RXR binds to the 5’ sequence and TR binds to the 3’ sequence of TREs in which half-sites are arranged as direct repeats (92, 93). The DNA-binding domains interact with the major grooves of the half-sites on the same face of the DNA (34,92,93). The carboxy-terminal end of the TR DNA-binding domain forms an α -helical structure that interacts with the spacer region in the DNA minor groove between the TRE half-sites. Although protein-protein contacts between the RXR and TR DNA-binding domains are important for dimerization, the major sub-regions involved in dimerization reside in the carboxy-terminii of the receptors (34). The dimerization surface of the TR appears to involve residues that lie along the surfaces of helices 10 and 11. T₃binding enhances the formation of TR-RXR heterodimers (94). On the other hand, T₃dissociates TR-TR homodimers (95). These findings raise the possibility that T₃binding might induce disruption of TR homodimers and induce the formation of TR-RXR heterodimers. The RXRs bind a stereoisomer of all trans retinoic acid, 9-cis retinoic acid (96,97). which variably alters transcriptional activity depending on the nature of the TH responsive gene (1,2). Additional studies are required to clarify the functional roles of RXRs and their ligands in TH action and interaction with other transcription factors.  Recently, Hollenberg and colleagues recentlyanalyzed the hepatic TRβcistrome of hyper- and hypothyroid mice using Chip-seq technology (98).  They found that the majority of TRβ-1 binding sites were not in the proximal promoter region but in other portions of target genes. Interestingly, by comparing the TR binding sites with previous Chip-seq data for RXRa, they found that some target genes may be regulated by TR homodimers rather TR/RXR heterodimers. Additionally, T₃increased TRβ-1 binding to DNA sites that, in turn, was correlated with T₃-induced gene expression. DR-4 and DR-0 motifs were significantly enriched at the DNA binding sites where T₃increased or decreased in TRβ1 binding, and were associated with positive and negative transcriptional regulation by T₃. Interestingly, in another study using Chip-chip methodology (99), some TH-regulated genes were identified that had little TR binding, despite the presence of putative TREs suggesting that other mechanisms such as receptor cross-talk, non-genomic effects, or indirect signaling mechanisms (see below) may be involved in regulating these genes.

TRs can have cross-talk with other nuclear hormone receptors owing to their common abilities to heterodimerize with RXRs.  TR crosstalk with peroxisome proliferator-activated receptor (PPAR) and LXR signaling via heterodimerization with RXR is a prominent example.   PPARg regulates the expression of its target genes by binding to the PPAR response element (direct repeat 1; DR1) as a heterodimer with RXR. Recently, it was shown that TRβ1 competes with PPARg for binding to DR1 as a heterodimer with RXR in vitroand in vivoto repress the transcriptional activity of PPARg (100). Since  PPARg plays a key role in lipid metabolism, carcinogenesis, and cardiovascular diseases (101.102), this mode of TR may exert some of its effects by crosstalk with PPARs. Recently, cell-based studies indicate that TRβ inhibits the activity of LXR-α transcription activity of the CYP7A1 promoter which shares a common DR-4 element with TR (103).  These studies show that TR cross-talk with other nuclear hormone receptor-mediated signaling expands TR effects beyond those target genes directly regulated by TRs (104).

Basal repression/Transcriptional corepressors

After binding to DNA, TR alters transcriptional activity by interacting directly or indirectly with a complex array of transcriptional cofactors. These proteins include corepressors (CoRs), coactivators (CoAs), integrators like CREB-binding protein (CBP), and general transcription factors (GTFs) (reviewed in 1-3, 31). Many of these factors have been identified by protein-protein interaction assays such as the yeast two-hybrid and glutathione-s-transferase pull down assays.

In the absence of TH, TR represses basal transcription in proportion to the amount of receptor and the affinity of receptor binding sites in positively-regulated target genes.This phenomenon also is referred to as transcriptional silencing (105-108). (Figure 3d-5, below). The addition of TH reverses basal repression and increases transcriptional activation above basal levels seen in the absence of receptor. Our understanding of the molecular mechanism for basal repression of transcription by unliganded receptor was advanced significantly by the discovery of a family of repressor proteins that bind selectively to unliganded TRs and RARs. This corepressor family includes silencing mediator for retinoid and TH receptors (SMRT) and nuclear receptor corepressor (NCoR) (105,109,110). These corepressors are 270 kD proteins that contain three transferable repression domains and two carboxy-terminal α -helical interaction domains. They are able to mediate basal repression by TR and RAR, as well as orphan members of the nuclear hormone receptor family such as rev-erbAα and chicken ovalbumin upstream transcription factor (COUP-TF). They have little or no interaction with steroid hormone receptors and therefore do not mediate basal repression by these receptors. Another protein, small ubiquitous nuclear co-repressor (SUN-CoR) enhances basal repression by TR and rev-erbA (31). This 16kD protein may form part of a co-repressor complex as it interacts with NCoR.

Within the interaction domains of NCoR and SMRT are consensus LXXI/HIXXXI/L sequences which resemble the LXXLL sequences that enable co-activators to interact with nuclear hormone receptors (40-42) (see below). Interestingly, these motifs allow both corepressors and co-activators to interact with similar amino acid residues on helices 3, 5, and 6 which are part of the ligand-binding pocket of TR. Differences in the length and specific sequences of the co-repressor and co-activator interaction sites coupled with the conformational changes in the LBD upon ligand binding, determine whether corepressor or coactivator binds to TR.

Recently, it has been shown that corepressors can form a complex with other repressors such as Sin 3 and histone deacetylases that are mammalian homologs of well-characterized yeast transcriptional repressors RPD1 and RPD3 (1-3,31). Thus, local histone deacetylation likely plays a critical role in the basal repression by unliganded TR/corepressor complex by maintaining local chromatin structure in a state that decreases basal transcription. Upon T₃binding, TR undergoes a conformational change that dissociates CoRs and recruits an array of coactivators (CoAs). Thus, hormone binding relieves repression and stimulates transcription by altering receptor binding to distinct classes of cofactors. Additionally, DNA-methylation may play a role in basal repression as methyl-CpG-binding proteins can associate with a co-repressor complex containing Sin3 and histone deacetylases (111,112). This repression was relieved by the deacetylase inhibitor, trichostatin A. These findings suggest that two repression processes, DNA methylation and histone deacetylation, may be linked via methyl-CpG-binding proteins.

The fact that TR alters the level of gene transcription in both the absence and presence of T₃has important implications for TH physiology. At low hormone concentrations, such as hypothyroidism, the unliganded receptor is predicted to repress transcription rather than function as an inactive, passive receptor. In some respects, this model is borne out by targeted inactivation of the TRα and TRβ genes. The phenotype of these double knockout mice are, for the most part, much less pronounced than the clinical features of congenital hypothyroidism (113,114). Thus, basal repression of transcription may explain why absence of receptor has less deleterious effects than absence of hormone (80,113,114).

Figure 5. TH receptor-mediated transcriptional silencing and activation. (A) Positively regulated genes. In the absence of hormone, the unliganded TH receptor represses or "silences" transcription in a process that involves TR interactions with a corepressor complex. Binding of T3 releases corepressors, relieving silencing and inducing the recruitment of coactivators that mediate transcriptional stimulation. (B) Negatively regulated genes. In the absence of hormone, the unliganded receptor activates transcription in a process that involves corepressors. Addition of TH dissociates corepressors and recruits coactivators. In the case of negatively regulated genes, this T3-mediated exchange of corepressors and coactivators inhibits transcription.

Figure 6. Role of corepressors and coactivators in the control of T3-regulated genes. In the absence of T3, the RXR-TR heterodimer recruits corepressors (CoR), which in turn, assemble additional components of a repressor complex that includes histone deacetylase (HDAC). Deacetylation of histones induce transcriptional repression. In the presence of T3, the corepressor complex dissociates and coactivators (CoA) bind to TR. The coactivator complex can include steroid receptor co-activators (SRCs)/p160, CREB-binding protein (CBP), p300/CBP associated factor (P/CAF), and proteins with histone acetyltransferase (HAT) activity. Vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP) complex can also interact with liganded TR, and may cycle with SRC/p160 complex. The general transcription factors (GTFs) are also indicated.

Transcriptional activation/Coactivators

A large and growing number of co-factors have been shown to interact with liganded nuclear hormone receptors and enhance their transcriptional activation. These include: steroid receptor coactivator 1 (SRC1); SRC2/transcriptional intermediary factor 2 (TIF2) / glucocorticoid receptor interacting protein 1 (GRIP1); SRC3/ amplified in breast cancer 1 (AIB1)/ receptor associated coactivator 3 (RAC3)/ p300/CBP cointegrator associated protein (p/CIP)/ nuclear receptor coactivator (ACTR)/ thyroid receptor activator molecule 1 (TRAM 1); peroxisome proliferator activated protein binding protein (PBP); TR accesory proteins (TRAPs) /vitamin D receptor interacting proteins (DRIPs); p300/CBP associated factor (p/CAF), and cAMP response element binding protein (CREB) binding protein (CBP)/ p300. among others (reviewed elsewhere (1-3,31).

At present, the precise roles of all these putative coactivators are not known; however, it appears that there are at least two major complexes involved in ligand-dependent transcriptional activation: the steroid receptor co-activator (SRC) complex and the vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP complex) (Fig. 3d-6). SRCs (SRC-1,SRC-2, and SRC-3) are 160 kD proteins that associate with nuclear hormone receptors, including TRs, and enhance their ligand-dependent transcription (115-117). SRCs also interact with the CREB-binding protein (CBP), the co-activator for cAMP-stimulated transcription as well as the related protein, p300, which interacts with the viral co-activator E1A (118-121). Recent studies also have shown that CBP/p300 can interact with PCAF (p300/CBP-associated factor), the mammalian homolog of a yeast transcriptional activator, general control nonrepressed protein 5, GCN5. Like GCN5, PCAF has intrinisic histone acetyltransferase activity (HAT) activity. Both PCAF and CBP interact with TBP associated factors (TAFs) and RNA pol II. Thus, PCAF and CBP possess dual functional roles both as adaptors of nuclear receptors to the basal transcriptional machinery as well as enzymes that can alter chromatin structure by histone acetyl transferase (HAT) activity. SRC‑1 and CBP may coordinate with TRs to synergize further the actions of TH, and also allow for the convergence of plasma membrane and nuclear hormone receptor signaling pathways in the cell.

The DRIP/TRAP complex also interacts with liganded VDRs and TRs (122-125). However, none of the subunits are members of the SRC family or their associated proteins. Instead, several DRIP/TRAP components are mammalian homologs of the yeast Mediator complex, which associates with RNA Pol II. Thus, TR recruits DRIP/TRAP complex which, in turn, may recruit or stabilize RNA Pol II holoenzyme via their shared subunits. It is noteworthy that DRIP/TRAP complex does not appear to have intrinisic HAT activity. Recent chromatin immunoprecipitation assays of proteins bound to hormone response elements (HREs), suggest that there may be a sequential, possibly cyclical recruitment, of co-activator complexes to hormone response elements by liganded nuclear hormone receptors (126-129). Studies of co-activator recruitment to TH-regulated genes showed distinct temporal patterns of recruitment. Last, other co-factors such as SW1/Snf and BRG-1 may be involved in early chromatin remodeling before the co-activator complexes are recruited to the TREs (130,131).

Negative regulation by TRs

In contrast to positively-regulated target genes, negatively-regulated genes can be stimulated in the absence of TH and repressed by TH (Figure 3d-5, above). Regulation of TRH and the TSH α  and β -subunit genes have been studied most extensively as models of negatively-regulated genes. From a physiological perspective, negative-regulation of these genes represents a critical aspect of feedback control of the TH axis. The T₃-responsive regions of these negatively- regulated genes have been localized to the proximal promoter regions (132-134). However, TR binding to putative TREs in these promoters is relatively weak in comparison to the binding sites in positively-regulated genes.

There are several different potential mechanisms for negative regulation by TH. Negative regulation may involve receptor interference with the actions of other transcription factors or with the basal transcription apparatus (135,136). For instance, TR can inhibit the activity of AP-1, a heterodimeric transcription factor composed of Jun and Fos. T₃-mediated repression of the prolactin promoter has been proposed to occur by preventing AP-1 binding (137). The TR also interacts with other classes of transcription factors, including NF-1, Oct-1, Sp-1, p53, Pit-1, CTCF, and GATA (138-144). By binding to these, or other positive transcription factors, the TH receptor may be able to inhibit gene expression by protein-protein interactions. Negative regulation may also occur by TR directly binding to DNA. A negative TRE from the TSHβ gene resides in an exon downstream of the start site of transcription (134) raising the possibility that it occludes the formation of a transcription complex. (Figure 3d-6, above) Additionally, liganded TRs may potentially recruit positive cofactors off DNA (squelching), which in turn, could lead to decreased transcription of target genes.

Transcriptional CoRs and CoAs, or even novel co-factors, may be involved in the control of negatively regulated genes. In contrast to the basal repression by unliganded TR in the case of positively regulated genes, CoRs cause basal activation of the TSH and TRH genes (132-134,145,146). CoAs also play an apparently paradoxical role in T₃-dependent repression of negatively regulated genes (146,147). Moreover, both SRC-1 knockout mice and knockin mice which express a TRβ mutant with a mutation in the helix 12 region (that interacts with CoAs) have defective negative regulation of TSH (148,149).  Interestingly, histone acetylation can be increased in the T₃- mediated negative regulation of TSHawhereas it is decreased in regulation of TSHβ and TRH (150,151).

Epigenetic modifications by TRs

Transcriptional regulation by TRs is a multistep process involving: (1) association of TRs with regulatory sites in the genome (usually within the targe gene promoters) in the context of chromatin, (2) ligand-dependent recruitment and function of coregulators to modify chromatin and thereby regulating RNA Pol II recruitment to the target genes, and (3)co-valent modifications of histones to alter chromatin structure, recruit RNA pol II complex, and to mediate transcription. In particular, the site-specific acetylation of histone tails induces local relaxation of chromatin, which enhances the binding of some transcriptional regulators and facilitates the recruitment and functioning of the general transcriptional machinery. Recent studies have demonstarted that thyroid hormone-positively regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation (129). However the epigenetic changes associated with negetively regulated gene seems to be much more complex. For instance, histone acetylation of H3K9 and H3K18 sites, two modifications usually associated with transcriptional activation, occur in negative regulation of TSHapromoter. T₃also caused the release of a corepressor complex composed of histone deacetylase 3 (HDAC3), transducin b-like protein 1, and nuclear receptor coprepressor (NCoR)/ silencing mediator for retinoic and thyroid hormone receptor from TSHapromoter in chromatin immunoprecipitation assays. These findings demonstrate the critical role of NCoR/HDAC3 complex in negative regulation of TSHagene expression and show that similar complexes and overlapping epigenetic modifications can participate in both negative and positive transcriptional regulation (150).  Of note, histone deacetylation has been observed in T₃-mediated negative regulation of several target genes (150-152).  Moreover, abberant histone modification at the TRH and TSHagenes has been implicated in the inappropriate TSH secretion observed in resistance to thyroid hormone (RTH) syndrome (150,151)). Other coregulators may be involved in T₃-mediated regulation as RIP140, a coregulator that can decrease transcription by some nuclear hormone receptors, mediated T₃repression of Crabp1 gene via chromatin remodeling during adipocyte differentiation (153). Interestingly, the use of HDAC inhibitors to counteract the effects of basal repression of target genes have restored some transcriptional activity in hypothyroidism associated with RTH syndrome and hypothyroidism (154,155). Although nuclear CoRs play a prominent role in T3 nuclear action (156,157), NCoR-independent signaling may account for basal repression by unliganded TRs for a significant number of target genes (158).

Another mechanism for T₃-mediated epigenetic signaling is regulation of small non-coding microRNAs. MicroRNAs act as negative regulators of gene expression by inhibiting the translation or promoting the degradation of target mRNAs. Since individual microRNAs often regulate the expression of multiple target genes with related functions, modulating the expression of a single microRNA can, in principle, influence an entire gene network and thereby modify complex disease phenotypes (159). Thyroid hormone have been shown to regulate the levels of microRNA pair miR-206/miR-133b in human skeletal muscles (160), miR 208a in heart (161), miR21 and miR181d in liver (162,163).  These miRNAs regulate important cellular events by TH such as differentiation, contractility, and metabolism.  MicroRNAs thus are a novel mechanism for thyroid hormone signaling which may regulate mRNA levels of target genes in which TRs are not recruited to their promoters or directly affect their transcription (164).   Last, it recently has been reported that miRNA 27a can modulate the expression of target gene, b-MHC in cardiac myocytes by decreasing TRb mRNA expression (165) and multiple miRNAs may also regulate TRb expression in papillary thyroid cancer (166) suggesting direction regulation of TR expression may be another mechanism for modulating target gene expression by TH.

Novel indirect pathways for TH action

It has been assumed that early transcriptional activation of target genes are mediated by direct transcriptional effects by TRs owing to their abilities to bind to TREs and recruit co-activators (167,168). Previous studies have suggested that TH may have non-genomic signaling activation that may result in rapid transcriptional changes (3, see below). However, several groups have shown that TH can activate SIRT-1 activity by a TR-dependent process, that in turn, can lead to deacetylation and activation of transcription factors such as PGC1a and FoxO1a.  These findings raise the possibility that TRs can activate some target genes without TREs through activating other transcription factors (169).  On the other hand, TRs can interact with SIRT-1 directly so it is possible that it can recruite deacetylase activity that can act on transcription factors as well as modulate transcription through histone modification (170-72).

Thyroid Hormone Receptors and Carcinogenesis

There are many reports providing evidence that reduced TR expression and/or alterations in TH levels are common events in human cancer (173, 174). These alterations include loss of heterozygosity, gene rearrangements, promoter methylation, aberrant splicing and point mutations (173,175). Tumors, including lung, breast, head and neck, melanoma, renal, uterine, ovarian and testicular tumors, present high frequencies of somatic deletions and mutations in  both TR alpha/beta loci (176-178). Aberrant TRs have also been found in more than 70% of human hepatocellular carcinomas The tendency for TR expression to disappear as malignancies progress suggests that TR can act as a tumor suppressor in human cancers; therefore, loss of expression and/or function of this receptor could result in cell transformation and tumor development (179). In fact, TR overexpression in hepatoma cell lines shows repression of various tumor promoting genes such as PTTG1 (180),  and activation of  anti-tumorogenic TGF-beta. However certain mutant TRs like TRb^PV/PVmey even enhance tumor growth by non-genomicaly activating beta-catenin and PI3K pathways (181,182). Last, it recently has been reported that miRNAs can downregulate the expression of dio 1 in renal cell carcinoma, and TRbin papillary thyroid, carcinoma.  Clarifying the molecular mechanisms by which TRs influence tumor progression and elucidating the epigenetic modifications ofT₃target genes in cancers would perhaps lead to a better understanding of the treatment regime in humans.

PHENOTYPIC EFFECTS OF TRa AND TRβ KNOCKOUTS AND KNOCKINS (CLINICALLY RECOGNIZED DISORDERS RELATED TO TR DYSFUNCTION ARE DESCRIBED IN CHAPTER 16D)

Recently, targeted gene inactivation or knockout (KO) of TR isoforms, and “knockin” of mutant TRs to their native TR genomic locii have provided new information on the mechanisms of TH action (16,17). The ability to disrupt TR genes by targeted mutagenesis has been particularly challenging given there is more than one gene encoding TRs, multiple splicing variants (TRα-1, α-2, TRβ-1, TRβ-2), and an additional transcript (Rev-erbA) derived from the opposite strand of the TRα gene (12,16,17). Two TRα knockout mouse lines have been generated that display different phenotypes (175, 176). It is likely this difference is due to the different sites in the TRα gene locus used for homologous recombination to generate the knockout mice. The TRα gene is complex as it encodes TRα-1, α-2 (which cannot bind T₃), and rev-erbA (generated from the opposite strand encoding TRα) (12, 16, 17). KO mice in which both TRα-1 and α-2 were deleted (TRα ^-/-) had a more severe phenotype with hypothyroidism, intestinal malformation, growth retardation, and early death shortly after weaning (175). T₃injection prevented the early death of pups. KO mice that lacked only TRα-1 (TRα-1^-/-) had a milder phenotype with decreased body temperature and prolonged QT intervals on electrocardiograms (183). The phenotypic effects of the loss of TRα 1 are relatively mild (184-185). Unexpectedly, there is no evidence of resistance to TH, as occurs with the TRβknockout. Disruption of the TRα-1 causes lower heart rates (19% reduced) and prolonged QRS and QT durations. These cardiac effects persist after hormone replacement. No changes were found in the levels of known TH-responsive genes in the heart (e.g., sarcoplasmic Ca²⁺ ATPase, Na⁺-K⁺ATPase, β-adrenergic receptors). The bradycardic effect of the TRα-1 knockout may result from alterations in the sympathetic or parasympathetic nervous systems or it could result from an intrinsic defect in cardiac myocytes. The TRα-1-deficient mice also have a 0.5 ^oC reduction in body temperature that is independent of TH levels. The mice have normal amounts of brown adipose tissue.

Samarut and co-workers have reported generation of short TRα isoforms from intronic transcriptional start sites which have dominant negative activity on TR function (73), and it is likely these short TRα isoforms are responsible for the more severe phenotype of the TRα ^-/-mice. In this connection, TRα KO mice which did not express either TRα-1 and α-2 (TRα^o/o), had a milder phenotype than TRα ^-/-mice which expressed only the short TRα isoforms (186). Interestingly, TH stimulation of some target genes was increased, perhaps due to the absence of α-2 which inhibits normal TR-mediated transcription (57,58).

Targeted disruption of the TRβ locus created a mouse deficient in both TRβ-1 and TRβ-2 (16,17). These mice had elevated circulating TSH and T₄levels, thyroid hyperplasia, as well as hearing defects (187,188). These findings are similar to the index patients with resistance to TH who were later shown to have homozygous deletion of TRβ (4,152). Thus, the mouse model appears to faithfully reproduce some of the features seen in humans with resistance to TH who are lack TRβ or express a dominant negative mutant TRβ (189). TRβ-2-selective knockout mice also have been generated and exhibited elevated levels of TH and TSH suggesting TRβ-2 plays the major role in regulating TSH (190). TRβ-2-selective knockout mice also have abnormal color discrimination and suggest TRβ-2 may play a role in cone development of the retina (191).

The relatively mild phenotypes of the TRα-1 and TRβ KO mice suggest the two isoforms have redundant roles in the transcriptional regulation of many target genes. In this connection, microarray studies of TRα and TRβ KO mice showed similar gene regulation profiles in the absence and presence of T₃in liver (82).  Recently, a study employing TRα or TRbreceptor over-expressing cell lines also showed that both these receptor isoforms mostly share a common gene repertoire but with varying degrees of induction or repression of target genes (192).

When both TR isoforms were abolished, the resultant double knockout mice (TRα 1^-/-TRβ^-/-) were surprisingly viable (113, 114). Thus, the absence of TRs is compatible with life. These mice had markedly elevated T₄, T₃, and TSH as well as large goiters. They also showed decreased growth, fertility, heart rate as well as bone density and development. Interestingly comparison of cDNA microarrays of double KO and hypothyroid mice showed only partial overlap of their gene regulation profiles, confirming the observation that the absence of receptor can give a different phenotype than lack of hormone. It is likely that basal transcription occurs even in the absence of receptor whereas basal repression of target genes occurs in the absence of hormone.  When the phenotypes of TRa, TRb, and double KO are compared, it is apparent that each isoform may have isoform-specific function, perhaps in part due to different expression patterns of the isoforms as well as gene-specific actions by each isoform (114).

Cheng and colleagues have generated a “knock-in” mouse model in which a mutant TRβ from a patient with RTH (PV) was introduced into the endogenous TRβ gene locus (193). These mice have a phenotype similar to patients with RTH, as the heterozygous mice showed elevated serum T₄and TSH, mild goiter, hypercholesterolemia, impaired weight gain, and abnormal bone development. Homozygous mice had markedly elevated serum T₄and TSH, and a much more severe phenotype than heterozygous mice. Wondisford and colleagues also have generated a “knock-in” mouse that expresses mutant TRβ (194). These mice had abnormal cerebellar development and function, and learning deficits. These latter studies suggest that expression of mutant TRβ under the control of endogenous TRβ promoter produces many of the clinical features of RTH in mice. This same group also recently developed a knock-in of a mutant TRβ that cannot bind DNA. This model should be useful in distinguishing signaling and developmental patterns due to protein-protein interactions of TRs (as well as non-genomic pathways) from those that require TR binding to TREs of target genes (195). Knockin mice harboring a TRamutant at the same site as the TRβ^PVmutant gene decreased white adipose tissue (WAT) and liver mass (196). In contrast, TRβPV markedly induced hepatosteatosis and mass of liver but had little effect on white adipose tissue. The expression of lipogenic genes was decreased in white adipose tissue and liver of TRa^PVmice whereas it was increased in liver and normal in TRβ^PVmice. A recent study showed that the phenotype of impaired adipogenesis can be restored by crossing with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit the TR (197). These findings support the notion that the phenotypes in the TRa^PVmutant mice, and perhaps some patients with RTH with mutant TRa, may be due to aberrant repression of target gene (18, 198, 199).

NONGENOMIC PATHWAYS REGULATED BY TH

There is increasing evidence for non-genomic effects by TH (3) in addition to the transcriptional effects mediated by nuclear TRs. There is continuous shuttling of a small amount of TRs between the cytoplasm and nucleus (200), so non-genomic effects may be mediated by cytoplasmic TRs (see below). Recently, a TRavariant from alternative translation was shown to be palmitoylated and associated with the plasma membrane (201). TH binding to this receptor led to increased intracellular calcium, nitrous oxide, and cyclic guanine monophosphate (cGMP), which in turn activated PKGII, Src, ERK, and Akt signaling pathways.  Another recent study suggests that non-DNA-binding TRs that cannot stimulate transcription may have “non-canonical” thyroid hormone signaling to regulate important physiological effects such as serum glucose and triglyceride  levels, body temperature, and heart rate. (202).  However, it appears that many non-genomic effects by TH are  likely mediated by cellular binding proteins other than TRs. Evidence supporting this notion comes from the rapid time course of some TH effects (thus precluding transcription and protein synthesis), utilization of membrane-signaling pathways such as kinases or calmodulin, lack of dependence on the presence of nuclear TRs, and structure-activity correlations by TH analogs that are different than those observed for nuclear TRs (3). Several non-nuclear sites for TH binding have been identified in various cell systems although their functional significances are not well characterized. Some of these include: plasma membrane associated T₃transporters, actin, calcium ATPase, adenylate cyclase, and glucose transporters; an endoplasmic reticulum associated protein, prolyl hydroxylase; and monomeric pyruvate kinase  (3, 203-207). A useful guideline that describes transcriptional and non-transcriptional signaling via TR and non-TR mechanisms recently has been published (208)

TH also has profound effects on mitochondrial activity and cellular energy state. A 43 kD protein has been described in mitochondria which also could bind to TREs and could be recognized by antibodies against the TRα ligand-binding domain (209). Recently, it has been shown that TRβ can interact with the p85 subunit of PI3K and activate the PI3K-Akt/PKB signaling cascade; thus, the small subpopulation of cytosolic TRβmay be involved in cell signaling (210). This PI3K activation by T₃leads to both direct and indirect effects on the transcription of several genes involved in glucose metabolism (210, 211,) and provides a mechanism for cross-talk between TH and cell signaling pathways.

Recently, integrin α-Vβ-3, has been identified as a plasma membrane TH-binding site (212). Previously, T₄, but not T₃, was shown to promote actin polymerization and integrin interaction with laminin in neural cells (213). Additionally, both T4 and T₃activated mitogen-activated protein kinase (MAPK) activity, and led, among other events, to phosphorylation of TRβ (210). Using a chick chorioallantoic membrane (CAM) system, Davis et al. showed that both T4 and T₃stimulated angiogenesis (214). Since integrin α-Vβ-3 is involved in angiogenesis, T₄and T₃binding to it was examined, and T₄was found to bind to integrin α-Vβ-3 with high affinity. Tetraiodothyroacetic acid (tetrac) and antibodies against laminin blocked T₄binding (213). Moreover, siRNAs against the integrin α-V or β-3 subunits blocked MAPK activation by TH. These findings suggest that TH activates the APK cascade and stimulates angiogenesis via TH binding to integrin α Vβ 3. Additionally, thyroid hormone non-genomically suppresses Src thereby stimulating osteocalcin expression in primary mouse calvarial osteoblasts (215). A direct physical interaction of TRbPV with cellular proteins, namely the regulatory subunit of the phosphatidylinositol 3-kinase (p85alpha), the pituitary tumor transforming gene (PTTG) and beta-catenin, that are critically involved in cell proliferation, motility, migration, angiogenesis and metastasis suggest a novel mode of non-genomic action, whereby mutant TR isoform acts as an oncogene in thyroid carcinogenesis (216).

TH ANALOGS, METABOLITES, AND ANTAGONISTS

Several tissue-specific and TR isoform-specific compounds have been developed as potential treatments for hypercholesterolemia, obesity, and heart failure. An early prototypical compound was 3,5-dibromo-3-pyridazinone-L-thyronine (L-940901) that bound preferentially to the TRs in the liver over those in the heart (217). Although the relative affinity of this compound for the respective TR isoforms has not been reported, the selective action of L-940901 is likely due to tissue-specific uptake of the compound. Interestingly, mice treated with L-940901 had decreased serum cholesterol levels without cardiotoxicity. Recently, several other TH analogs have been described that have isoform-selective affinity for TRβ  compared to TRα (218-220). Since TRs in the liver are approximately 90% TRβ whereas those in the heart are mostly TRα, these isoform-selective compounds may serve as novel agents to lower serum cholesterol with minimal cardiotoxicity. N-[3,5-dimethyl-4-(4’-hydroxy-3’isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4(4’-hydroxy-3’-isopropylbenzyl)-phenoxy) acetic acid (GC-1), and 3,5-dichloro-4[(4-hydroxy-3-isoopropylphenoxy)phenyl] acetic acid (KB-141) all have been reported to lower total serum cholesterol and LDL-cholesterol (205-209). CGS 23425 also increases LDL receptor expression in HepG2 cells. Additionally, these compounds can increase serum apoA1 levels; however, the total serum high density lipoprotein (HDL) cholesterol level does not changes or may even decrease. In this connection, GC-1 decreased serum HDL; increased expression of HDL receptor, SR-B1; stimulated the activity of cholesterol 7α hydroxylase; and increased fecal excretion of bile acids in treated mice (221). Thus, GC-1 regulates important steps in the reverse cholesterol transport pathway (221). Recently, KB141 was shown to be a potential treatment for obesity by decreasing body weight via stimulation of metabolic rate and oxygen consumption (222). The TR agonist MB07811, which is converted to an active metabolite in the liver, has proven to be effective in reducing hepatic steatosis in rodents (223). The TRb-specific compound, KB215 recently was shown to be effective in decreasing LDL cholesterol, apoliprotein B, triglycerides, and Lp(a) in humans (224)when used in combination with statins.  These findings suggest that TH analogs may be useful in the treatment of a wide range of metabolic disorders (225).

TH analogs and derivatives also bind specifically to proteins other than TRs, and are involved in non-genomic cell signaling pathways, Thyronamines (3-T1AM, T0AM) are endogenous compounds derived from L-thyroxine or its intermediate metabolites. Activities of intestinal deiodinases and ornithine decarboxylase generate 3-T1AM (226). Significantly, this compound bound poorly to nuclear TRs. T1AM has interesting physiological actions as it produced a rapid drop in body temperature and heart rate when injected intraperitoneally in mice. T1AM also decreased cardiac output in an ex vivo working heart model. Although 3-T1AM have a weak affinity towards classical nuclear TH receptors a number of putative receptors, binding sites, and cellular target molecules mediating actions of 3-T1AM have been proposed. Among those are members of the trace amine associated-receptor family (TAR1), the adrenergic receptor ADRα2a, and the thermosensitive transient receptor potential melastatin 8 channel (226). Preclinical studies  using animal models are in progress, and more stable receptor-selective agonistic and antagonistic analogues of 3-T1AM are now being synthesiszed exerting marked cryogenic, metabolic, cardiac and central actions and represents a key lead compound linking endocrine, metabolic, and neuroscience research to advance development of new drugs (226).

TH can increase cardiac performance by increasing cardiac contractility and decreasing systemic vascular resistance (2); however, TH excess also can cause cardiotoxicity. 3,5-diiodothyropropionic acid (DITPA) is a TH-related compound with low metabolic activity and low affinity for nuclear TRs (Kd 10^-7M). DITPA was able to increase cardiac contractility and peripheral circulation without significant effects on heart rate in animal studies (227). Moreover, DITPA improved hemodynamic performance in animal models of congestive heart failure after myocardial infarction. Patients with heart failure treated with DITPA showed significant improvement in systolic cardiac index and systemic vascular resistance in preliminary studies (227). Thus DITPA or similar compounds may represent a novel class of drugs for the treatment of heart failure.

Recently, the naturally occurring analogs, 3,5,3'-triiodothyroacetic (TRIAC) and 3,5,3',5'-tetraiodothyroacetic TETRAC) acids decreased heat-induced albumin fibrillation suggesting they may have a protective effect against amyloid formation (228). Additionally, tetraiodothyroacetic acid (tetrac) caused radiosensitization of GL261 glioma cells (229).  The mechanisms for these effects are not understood at this time but likely involve non-genomic effects as TRIAC and TETRAC have weak binding affinity for TRs.  Similarly, 3,5-diiodothyronine (T₂) has also shown favorable effects in rodent models of fatty liver diseases (230,231).

SUMMARY

We have learned much about molecular mechanisms of nuclear TH action during the past 25 years. In particular, the identification and characterization of TRs, their heterodimeric partners, corepressors, coactivators, and TREs, generation of TR knockout mice, and discovering non-genomic pathways have provided new insight into TH action. It is expected that new information will be obtained from microarray and proteomic studies, structural biology approaches, and in vitro transcriptional systems. Such information should provide an even better understanding of the mechanisms of disease caused by abnormal circulating TH and/or altered intracellular TH levels, and provide targets for the development useful therapeutic agents for not only TH-related conditions but also for metabolic derangements such as hypercholesterolemia, non-alcoholic fatty liver disease, and obesity.

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ABSTRACT

Thyroid storm is an acute and life-threatening worsening of hyperthyroidism, characterized by an exacerbation of symptoms and signs of hyperthyroidism, with high fever, dehydration, marked tachycardia or tachyarrhytmias, heart failure, hepatomegaly, respiratory distress, abdominal pain, delirium, possibly seizures. It may occur in patients submitted to thyroidectomy or radioactive iodine treatment while hyperthyroid, or as a consequence of infections in unteated hyperthyroid patients. Treatment consists of antithyroid drug treatmnt, rest, sedation, fluid and electrolyte replacement, cardio-supportive therapy, oxygen therapy, antibiotics, cooling. Mortality is about 10%.

Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease, found in about 25% of patients at diagnosis, often mild and self-remitting. Removal of risk factors (refrain from smoking, correction of thyroid dysfunction, oral steroid prophylaxis after radioactive iodine therapy, antioxidant therapy with seleniomethionine) are fundamental to prevent progression of mild GO to more severe forms. In moderate-to-severe and active GO, intravenous glucocorticoids are the first-line treatment, second line treatments include cyclosporine, orbital radiotherapy, rituximab (controversial). Novel biologicals, such as teprotumumab and tocilizumab are under investigation. Rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often required. Thyroid dermopathy (pretibial myxedema) is a rare complication of Graves’ disease, usually observed in patients who also have severe GO. Topical glucocorticoids are usually effective. Thyroid acropachy (clubbing of fingers and toes, with swelling of hands and feet) is an extremely rare conditions, for which no treatment is available.

THYROID STORM

Thyroid (or thyrotoxic) storm is an acute, life-threatening syndrome due to an exacerbation of thyrotoxicosis. It is now an infrequent condition, because of earlier diagnosis and treatment of thyrotoxicosis, better pre- and postoperative medical management. However, acute exacerbation of thyrotoxicosis caused by intercurrent illness, especially infections, may still occur. Thyroid storm in the past most frequently occurred after surgery, but now it is usually a complication of untreated or partially treated thyrotoxicosis, rather than a postoperative complication.

Clinical pattern

Classic features of thyroid storm are indicative of a sudden and severe exacerbation of thyrotoxicosis, with fever, marked tachycardia, tremor, nausea and vomiting, diarrhea, dehydration, restlessness, extreme agitation, delirium or coma. Fever is typical and may be higher than 105.8 F (41 C). Patients may present with a true psychosis or a marked deterioration of previously abnormal behavior. Sometimes thyroid storm takes a strikingly different form, called apathetic storm, with extreme weakness, emotional apathy, confusion, absent or low fever

Signs and symptoms of multiple organ failure may be present. Delirium is one example. Congestive heart failure may also occur, with peripheral edema, congestive hepatomegaly, and respiratory distress. Marked sinus tachycardia or tachyarrhythmias, such as atrial fibrillation, are common. Liver damage and jaundice may derive from congestive heart failure or a direct action of thyroid hormone on the liver coupled with malnutrition (Chapter 10). Fever and vomiting may produce dehydration and prerenal azotemia. Abdominal pain may be a prominent feature. The clinical picture may be masked by a secondary infection such as pneumonia, a viral infection, or infection of the upper respiratory tract. Death may be caused by cardiac arrhythmia, congestive heart failure, hyperthermia, or other unidentified factors.

Storm is typically associated with Graves' disease, but it may occur in patients with toxic nodular goiter (1, 2). At present, although still life-threatening, death from thyroid storm is rarer if it is promptly recognized and aggressively treated in an intensive care unit. In recent nationwide studies from Japan mortality rate was >10% (3, 4).

Incidence

In Nelson and Becker's series reported in 1969 (5), there were 21 cases of thyroid storm among 2,329 admissions due to thyrotoxicosis (about 1%). Other series, which included all cases with fever of 38.3 C or more in the postoperative period, reported an incidence of thyroid storm as high as 10% of patients operated on (6). Few patients are now seen with the classic pattern of thyroid storm, but patients are occasionally encountered with marked accentuation of symptoms of thyrotoxicosis in conjunction with infection. The incidence of thyroid storm currently may currently be as low as 0.2 cases/100,000 population (3).

Cause

Thyroid storm classically began a few hours after thyroidectomy performed on a patient prepared for surgery by potassium iodide alone. Many such patients were not euthyroid and would not be considered appropriately prepared for surgery by current standards. Exacerbation of thyrotoxicosis is still seen in patients sent too soon to surgery, but it is unusual in the antithyroid drug-controlled patient. Thyroid storm occasionally occurs in patients operated on for some other illness while severely thyrotoxic. Severe exacerbation of thyrotoxicosis is rarely seen following 131-I therapy for hyperthyroidism; some of these may be defined as thyroid storm (7). Thyroid storm appears most commonly following infection (1), which seems to induce an escape from control of thyrotoxicosis. Pneumonia, upper respiratory tract infection, enteric infections, or any other infection can cause this condition. The decreased incidence of thyroid storm can be largely attributed to improved diagnosis and therapy. In most cases, thyrotoxicosis is recognized early and treated by measures of predictable therapeutic value. Patients are routinely made euthyroid before thyroidectomy or 131-I therapy (8). Using thionamides preoperatively, thyroid glands have only minimal amounts of stored hormones, thus minimizing thyroid hormone release due to manipulation.

Diagnosis

Diagnosis of thyroid storm is made on clinical grounds and involves the usual diagnostic measures for thyrotoxicosis. Semi-quantitative scales and related scores evaluating the presence and severity of clinical manifestations may be of some help in confirming the diagnosis (1, 3, 9). There are no peculiar laboratory abnormalities. Free T4 and, if possible, free T3 should be measured. Serum total T3 may be not particularly elevated or even normal, due to reduced T4 to T3 conversion as observed in nonthyroidal illness (1). Electrolytes, blood urea nitrogen (BUN), blood glucose, liver function tests, and plasma cortisol should be monitored.

Therapy

Thyroid storm is an endocrine emergency that has to be treated in an intensive care unit (Table 12-1).

Table 1. Treatment of Thyroid Storm

If drugs cannot be given orally (e.g., in the unconscious patient), they can be administered by naso-gastric tube or enemas (1). In some European countries intravenous preparations have been used (10).  If the thyrotoxic patient is untreated, an antithyroid drug should be given. PTU, 150-250 mg every 6 hours should be given, if possible, rather than methimazole, since PTU also prevents peripheral conversion of T4 to T3, thus more rapidly reduces circulating T3 levels. Methimazole (15-25 mg every 6 hours) can be given orally, or if necessary, the pure compound can be made up in a 10 mg/ml solution for parenteral administration. Methimazole is also absorbed when given rectally in a suppository. An hour after a thionamide has been given, iodide should be administered. A dosage of 100 mg twice daily is more than sufficient. Unless congestive heart failure contraindicates it, propranolol or other beta-blocking agents should be given at once, orally or parenterally in large doses, depending on the patient's clinical status. Permanent correction of thyrotoxicosis by either 131-I or immediate thyroidectomy should be postponed until euthyroidism is restored. Other supporting measures should fully be exploited, including sedation, oxygen, treatment for tachycardia or congestive heart failure, rehydration, multivitamins, occasionally supportive transfusions, and cooling the patient to lower body temperature down. Antibiotics may be given on the presumption of infection while results of culture are awaited.

The adrenal gland may be limited in its ability to increase steroid production during thyrotoxicosis. If there is any suspicion of hypoadrenalism, hydrocortisone (100-200 mg/day) or its equivalent should be given. The dose can rapidly be reduced when the acute process subsides. Pharmacological doses of glucocorticoids (2 mg dexamethasone every 6 h) acutely depress serum T3 levels by reducing T4 to T3 conversion. This effect of glucocorticoids is beneficial in thyroid storm and supports their routine use in this clinical setting. Propranolol controls tachycardia, restlessness, and other symptoms.

Usually rehydration, repletion of electrolytes, treatment of concomitant disease, such as infection, and specific agents (antithyroid drugs, iodine, propranolol, and corticosteroids) produce a marked improvement within 24 hours. A variety of additional approaches have been reported, but indications for their use are not well defined. For example, oral gallbladder contrast agents such as ipodate and iopanoic acid in doses of 1-2 g, which inhibit peripheral T4 to T3 conversion, might have value. Unfortunately, these agents are no longer available. Peritoneal dialysis can remove circulating thyroid hormone, and plasmapheresis can do likewise, but at the expense of serum protein loss. Orally administered ion-exchange resin (20-30g/day as Colestipol-HCl) can trap hormone in the intestine and prevent recirculation. These treatments are rarely needed.

Antithyroid treatment should be continued until euthyroidism is achieved, when a final decision regarding antithyroid drugs, surgery, or 131-I therapy can be made.

GRAVES’ ORBITOPATHY

Graves’ orbitopathy (GO) is the main and most frequent extrathyroidal manifestation of Graves’ disease, although it may less frequently occur in patients with Hashimoto’s thyroiditis or apparently without thyroid abnormalities (so-called Euthyroid Graves’ disease) (11-15).

Epidemiology

Fig. 1: Prevalence of GO in a series of 346 patients with newly diagnosed Graves’ hyperthyroidism. Moderate-to-severe GO includes one case of sight-threatening dysthyroid optic neuropathy (DON). Derived from Tanda ML et al. (17).

Data on the incidence of GO are limited (11, 14). In a population-based setting in USA, an adjusted rate of 16 cases per 100.000 per year in women and 2.9 cases per 100.000 in men was reported (16). In a recent study of a large cohort of newly diagnosed Graves’ patients, about 75% had no ocular involvement at diagnosis, only 6% had moderate-to-severe GO, and 0.3% showed sight-threatening GO due to dysthyroid optic neuropathy (DON) (17) (Figure 1). In a Danish population, moderate-to-severe GO showed an incidence of 16.1/million per year (women: 26.7; men: 5.4) (18). Ocular involvement is in most cases bilateral, although often asymmetrical, but it may be unilateral in up to 15% of cases (12, 14). As recently reviewed by the European Group on Graves’ Orbitopathy (EUGOGO), the overall prevalence of GO in Europe is about 10/10,000 patients, but the prevalence of its variants (hypothyroid GO, GO associated with dermopathy, GO associated with acropachy, asymmetrical or unilateral GO) is much lower, and recently euthyroid GO has been listed as a rare disease in Europe (19). The onset of GO apparently has a bimodal peak in the fifth and seventh decades of life, but eye disease may occur at any age (20). It is more frequent in women, but men tend to have a more severe disease (21-23), as suggested by a decrease in the female/male ratio from 9.3 in mild GO, to 3.2 in moderately severe GO, and 1.4 in severe GO (20). There is a close temporal relationship between the onset of GO and the onset of hyperthyroidism. In approximately 85% of cases GO and hyperthyroidism occur within 18 months of each other (20), although GO may both precede (about 20% of cases) or follow (about 40% of cases) the onset of hyperthyroidism (20).

The natural history of GO is poorly understood. However, in a longitudinal cohort study, spontaneous amelioration was observed in two thirds of cases, while ocular involvement did not change with time in 20% and progressed in 14% (22). The observation that mild GO rarely progresses and often spontaneously remits was recently confirmed by a large prospective study of patients with recent onset Graves’ hyperthyroidism (17) and summarized in a review of published studies (24). It is worth noting that GO seems to be less frequent than in the past. A review of the first 100 consecutive patients seen at the same joint thyroid-eye unit in 1960 and 1990 revealed a decrease in the proportion of Graves’ patients with clinical relevant GO from 57% to 32% (23); likewise, a reduction in the proportion of severe forms of GO compared to milder forms was observed (18), likely reflecting an earlier diagnosis and treatment of both hyperthyroidism and orbitopathy. It should be noted that a multicenter study carried out by the European Group on Graves’ Orbitopathy (EUGOGO) reported that 40% of GO patients had mild disease, 33% had moderate GO, and 28% had severe eye disease (25). It should be noted that these figures were clearly influenced by the fact that EUGOGO centers are all referral centers where it is likely to see more complicated cases of GO. Accordingly, a recent single-center study confirmed that most patients newly diagnosed with Graves’ disease have mild GO (26), although whether these forms are chronic remitting or a transient disease (27) or whether GO ever disappears completely (28) is unsettled. In summary, based on recent studies and reviews of the available literature, it can be concluded that GO is a rare disease, particularly in its severe expressions (19).

An important epidemiologic feature of GO is its relation with cigarette smoking (29,30). The prevalence of smokers among Graves’ women with orbitopathy is much higher than that in Graves’ women apparently without GO or in normal controls (Figure 2) (31). Smoking is a predictor of Graves’ hyperthyroidism, with a hazard ratio of 1.93 in current smokers, 1.27 in ex-smokers, and 2.65 in heavy smokers (32). In a case-control study, the odds ratio of cigarette smoking for Graves’ hyperthyroidism without GO was 1.7, but raised to 7.7 for Graves’ disease with GO (33). Whether passive smoking may have the same impact as active smoking is unsettled; however, in a recent European survey of GO in childhood, the highest prevalence of Graves’ children with GO was found in countries where the prevalence of smokers among teenagers was also highest: since >50% of children were <10 years of age, it is likely that passive smoking rather than active smoking influenced GO occurrence (34). Mechanisms whereby smoking may affect the development and course of GO are unclear. In addition to direct irritative effects and modulation of immune reactions in the orbit (35), smoking was associated with an increase in the orbital connective tissue volume as assessed by MRI (36), and with an increased adipogenesis and hyaluronic acid production in in vitro cultured orbital fibroblasts (37). Whatever the mechanism(s) involved, cigarette smoking is strong (probably the strongest) predictor of GO occurrence in patients with Graves’ hyperthyroidism (38).

Figure 2. Prevalence of smokers among women with Graves’ disease with (GO) or without (GD) associated orbitopathy. NTG: Non-toxic goiter; C: controls. Derived from Bartalena et al (31).

Pathogenesis

Clinical manifestations of GO reflect remodeling of the orbital space related to the enhanced orbital volume, due to an increase in retroocular fibroadipose tissue and swelling of extraocular muscles (39-41). Orbital tissues, including muscles, are infiltrated by inflammatory cells, including lymphocytes, mast cells, and macrophages. Proliferation of orbital fibroblasts and adipocytes, both in the retroocular space and in the perimysial space, is also associated with an increased production of glycosaminoglycans, which are the ultimate responsible for edematous changes both in the connective tissue and the muscles, owing to their hydrophilic nature. The relative contribution of the increase in fibroadipose tissue volume and extraocular muscle swelling is not always the same, and a predominance of either component may be observed in different patients with similar clinical features (42). Furthermore, the increase in orbital fat might be a rather late phenomenon (43). Because the orbit is a rigid, bony structure anteriorly limited by the orbital septum, the increased orbital volume deriving from cell proliferation, inflammatory infiltration and edema, results into enhanced intraorbital pressure, forward displacement of the globe (proptosis or exophthalmos), extraocular muscle dysfunction causing diplopia and/or strabismus, soft tissue changes with periorbital edema, conjunctival hyperemia and chemosis. If proptosis, which can be considered a form of spontaneous decompression, is severe, subluxation of the eye may occur. Proptosis is responsible for corneal exposure which may be particularly dangerous at night for the incomplete eyelid closure (lagophthalmos), and may result into sight-threatening corneal ulceration. The enlarged muscle volume may cause optic nerve compression (dysthyroid optic neuropathy), especially if the orbital septum is tight and proptosis is minimal. Optic nerve compression is particularly evident at the orbital apex and may be responsible for sight loss. Orbital inflammation and related anatomical changes may cause venous and lymphatic congestion that contribute to periorbital edema and chemosis. With time inflammation subsides and muscle fatty degeneration and fibrosis may contribute to further extraocular muscle restriction and strabismus, which, at this stage, can only be corrected by surgery.

GO is an autoimmune inflammatory disorder related to the thyroid and triggered by the migration of autoreactive T-helper cells into the orbit, which is infiltrated by CD4+ T cells and, to a lesser extent, CD8+ T cells, B cells, fibrocytes, mast cells, and macrophages (40, 41). Orbital fibroblasts are the main target and key effector cells in the disease (44). After recognition of one or more antigens (shared with the thyroid) on fibroblast surface, facilitated by HLA class II antigen expression on antigen-presenting cells (B cells, macrophages), CD4+ T cells secrete cytokines which activate CD8+ T cells, autoantibody-synthesizing B cells (45) and stimulate orbital fibroblasts (46).  Fibroblasts proliferate, may differentiate into myofibroblasts and adipocytes, accumulate and secrete hyaluronic acid (HA), synthesize and secrete chemoattractants (interleukin-16, RANTES, CXCL10) and a number of cytokines (interleukin-1, interleukin-6, interferon-g, tumor necrosis factor-a, interleukin-8, interleukin-10, platelet-derived growth factor, transforming growth factor-b), which concur to auto-maintain the inflammatory process (47). HA is hydrophilic and thereby attracts water and causes edema of the extraocular muscles and orbital tissue (40). Orbital fibroblasts can be differentiated based on the expression or lack of expression of a cell surface glycoprotein (thymocyte antigen-1, thy-1). Thy-1+ fibroblasts are mostly represented in extraocular muscles and produce HA, thereby contributing to extraocular muscle edema and enlargement, whereas  thy-1- fibroblasts are mainly present in connective tissue and may differentiate into adipocytes, thereby contributing to fibroadipose tissue expansion (40).

The TSH receptor (TSH-R), the ultimate cause of hyperthyroidism due to Graves’ disease, is likely the shared antigen responsible for GO. TSH-R expression has been shown in the orbital tissue of GO patients, both at the mRNA and protein levels (48, 49); however, TSH-R is also expressed in several other tissues not involved in Graves’ disease and orbitopathy (50), and, although at lower levels, in normal orbital fibroadipose tissue samples and cultures (51). On the other hand, BALB/c mice injected with spleen cells primed either with a TSH-R fusion protein or with TSH-R cDNA developed thyroiditis with blocking-type TRAb, but also showed orbital pathological changes (lymphocytic and mast cell infiltration, edema, presence of glycosaminoglycans) similar to those seen in human GO (52).  This model could not be reproduced in other laboratories. More recently a novel preclinical female mouse model of GO was established (53). In this model, some of the mice immunized by human TSH A-subunit by TSH receptor plasmid in vivo electroporation showed large infiltrates surrounding the optic nerve, increased adipogenesis, orbital muscle hypertrophy, exophtahlmos, and chemosis (53, 54). The role of the TSH-R seems to be supported also by other animal models (55-57). Another putative antigen involved in the pathogenesis of GO is the IGF-1 receptor. As recently reviewed (58), increased IGF-1 receptor levels have been reported in orbital fibroblasts as well as in B and T lymphocytes from Graves’ patients, and immunoglobulins displacing IGF-1 from its binding to the IGF-1 receptor have also been found in these patients (59). Colocalization of TSH receptor and IGF-1 receptor has been shown both in orbital fibroblasts and thyrocytes, suggesting that the two receptors might form a functional complex (60). Stimulation of the TSHR by the monoclonal TSHR-stimulating antibody, M22, could be inhibited by an IGF-1R-blocking monoclonal antibody in orbital fibroblasts (61).  Involvement of the IGF-1R is not specific for Graves’ disease, since it is implicated in other autoimmune diseases, such as rheumatoid arthritis (62). Thus, although involvement of the IGF-1R in the pathogenesis of GO seems likely (63), it is tempting to speculate, for the time being, that autoimmunity to the TSH receptor be the initiating mechanism, while subsequent increased expression of the IGF-1R might be fundamental to maintain ongoing reactions in the orbit (64). Interestingly, two recent reports found that, at variance with TRAb, only a minority of patients with GO have circulating antibodies to the IGF-1R (65, 66). This may be related to the low sensitivity and specificity of tests used to detect such antibodies (62). Alternatively, it may be conceived that IGF-1 (and/or IGF-1R antibodies) locally produced in the orbit be relevant for the pathogenesis of GO (67).

Other autoantigens, including several eye muscle antigens, acetylcholine receptor, thyroperoxidase, thyroglobulin, alpha-fodrin, have been proposed as putative shared antigens, but their true role is, to say the least, uncertain (68).

The role of genetic factors in the pathogenesis of GO is not very well defined, and no striking differences have been observed between Graves’ patients with or without associated GO (69-73). An association between GO and Major Histocompatibility Complex (MHC), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) or intercellular adhesion molecule 1 gene polymorphisms has been looked for, but results are not unequivocal (74-76). GO likely stems from a complex interplay between endogenous factors and exogenous (environmental) risk factors (13, 77). The latter are probably more important and include cigarette smoking, thyroid dysfunction, and, in a subset of patients, radioiodine therapy for Graves’ hyperthyroidism (13, 77). The relationship between cigarette smoking and GO has been discussed above (see paragraph on Epidemiology). Both hyperthyroidism (78, 79) and hypothyroidism (80) seem to influence negatively the course of the orbitopathy. TRAb are independent risk factors for GO and can help to predict severity and outcome of eye disease (81). Radioiodine therapy for Graves’ hyperthyroidism is associated with GO progression in about 15% of cases, although this effect may be transient (82-86). This effect is more frequently observed in patients who already have GO prior to radioiodine therapy, smoke, have high TRAb levels, or whose post-radioiodine hypothyroidism is not promptly corrected by L-thyroxine replacement therapy (13, 77). Radioiodine-associated progression of GO can be prevented by a short course of prednisone (87). Lower doses of oral prednisone (0.2 mg/Kg bw) for 6 weeks are as effective in preventing RAI-associated progression of GO as higher doses used in the past (88). Neither thyroidectomy (typically partial) nor antithyroid drugs influence the course of the orbitopathy (89-91). The above observations have important practical implications in terms of GO prevention (Table 2), because GO patients should be urged to refrain from smoking, their thyroid dysfunction (both hyper- and hypothyroidism) should be promptly corrected, and, in the case of radioiodine therapy, a short course of oral prednisone should be administered (92).

Table 2. Risk factors for the occurrence/progression of Graves’ orbitopathy and preventive   measures

Clinical Features

Signs & Symptoms. Clinical features of GO include soft tissue changes, exophthalmos, extraocular muscle dysfunction, corneal abnormalities, and optic nerve involvement (Figures 3-6). The NOSPECS classification (Table 3) is a useful memory aid of GO abnormalities (93). Recommendations for GO assessment in clinical practice have recently been reviewed by EUGOGO (94) and other groups (VISA classification) (95). Soft tissue changes include eyelid edema and periorbital swelling, eyelid erythema, conjunctival hyperemia and chemosis, inflammation of the caruncle or plica: their assessment and grading can be done with the aid of a color atlas (96), which can be downloaded from EUGOGO website (www.eugogo.eu). Proptosis, i.e., protrusion of the eye (exophthalmos), is usually measured by Hertel exophthalmometer; normal values are usually less than 20 mm, but vary with race, age, gender, degree of myopia, and should be established in each center or institution. Extraocular muscle dysfunction is responsible for diplopia (double vision), which can be subjectively defined as intermittent (i.e., present only when fatigued or when first waking), inconstant (i.e., present only at extremes of gaze), or constant (i.e., present also in reading positions and primary gaze); objective assessment of extraocular muscle functioning can be done by several methods, including measurement of duction  in degrees (94). Palpebral aperture may be increased due to several factors, including upper and/or lower lid retraction, and proptosis. Lid retraction and proptosis are responsible for corneal exposure, which may lead to corneal epithelium damage (punctate keratopathy), corneal ulceration and perforation. The incomplete eye closure at night (lagophthalmos) and the absence of Bell’s phenomenon (no upward eye rotation on attempted eye closure) are risk factors for corneal damage (92, 94). Intraocular pressure is often increased, particularly in upward gaze, but this abnormality rarely progresses to true glaucoma. Dysthyroid optic neuropathy, due to optic nerve compression at the orbit apex by swollen extraocular muscles, or, less frequently, to optic nerve stretching in cases of marked proptosis or eye subluxation, is a sight-threatening expression of GO. It can be diagnosed by fundoscopy showing disc swelling, reduced visual acuity, abnormal color vision test, contrast sensitivity, perimetry, visual-evoked potentials, and pupillary responses (95).

Figure 3. Female patient with moderately severe GO. Note periorbital swelling, injection of conjunctival vessels, proptosis, marked lid retraction, and proptosis.

Figure 4. Male patient with moderately severe GO. Note marked periorbital swelling, conjunctival hyperemia, esotropia (strabismus) in the left eye.

Figure 5. Male patient with moderately severe GO. Note the superior eyelid edema, mild conjunctival vessel injection, marked proptosis, and marked upper lid retraction.

Figure 6. Female patient with severe GO. Note marked periorbital swelling, palpebral hyperemia, conjunctival hyperemia, proptosis (particularly in the left eye), caruncle edema. Eye motility was markedly reduced, lagophthalmos was present, there were two corneal ulcers in the left eye, and corneal punctate staining in the right eye, reduced visual acuity in the left eye (5/10). CT scan showed enlargement of extraocular muscles (particularly medial rectus and inferior rectus) in both eyes, but no relevant compression of the optic nerve at the orbit apex.

Table 3. NOSPECS classification of eye changes of Graves’ disease

Symptoms of GO (Table 4) include, in addition to changes in ocular appearance related to periorbital swelling and proptosis, excess lacrimation, photophobia, grittiness, pain in or behind the eyes, either spontaneous or with eye movements, diplopia of different severity with or without strabismus, blurred vision, which may clear with blinking (due to excessive lacrimation) or covering one eye (reflecting extraocular muscle impairment), or may persist (probably reflecting optic neuropathy, particularly if associated with gray areas in the field of vision). In addition to reduced visual acuity, optic nerve involvement can be heralded by decreased color perception. Diplopia may be absent if extraocular muscle involvement is symmetrical in both eyes.

Table 4. Symptoms associated with Graves’ orbitopathy

Clinical manifestations of GO have a profound negative impact on quality of life and daily activities of affected individuals (98). By the use of general health-related quality of life (HRQL) questionnaires, such as the SF-36 or its shorter forms, it was shown that GO is associated with significant changes in several functions, including physical functioning, role functioning, social functioning, mental health, general health perception, and bodily pain (99). Interestingly, these changes in HRQL parameters were similar to those found in patients with inflammatory bowel disorders, and even more marked than those observed in patients with diabetes mellitus, heart failure or emphysema (99). Since HRQL questionnaires are broad and may not address items specific for a given disease, a GO-specific quality of life (GO-QoL) questionnaire was developed and validated in clinical studies (99, 100-102). This questionnaire (dowloadable from EUGOGO website at www.eugogo.eu) is composed of 16 questions, 8 concerning the consequences of diplopia and deceased visual acuity on visual functioning, and 8 regarding the consequences of changes in physical appearance on social functioning. The Go-QoL is a useful tool for self-assessment of treatment outcomes for GO (103).

Activity & Severity. Definition of GO severity is somehow arbitrary and may reflect different views (11, 23). According to the most recent EUGOGO definition (92),  mild GO is characterized by one or more of the following features: minor lid retraction (<2 mm), mild soft tissue involvement, exophthalmos <3 mm above normal for race and gender, transient or no diplopia, and corneal exposure; the above features usually have a minor impact on daily life to justify immmuno-suppression or surgical treatment; moderate-to-severe GO have any one or more of the following: lid retraction >2 mm, moderate or severe soft tissue involvement, exophthalmos >3 mm above normal for race and gender, inconstant or constant diplopia; Patients in this category have an impact on daily life as to justify immunosuppression (if GO is active) or surgical intervention (if GO is inactive); sight-threatening GO is due to dysthyroid optic neuropathy (DON) or corneal breakdown, and warrants immediate intervention (Table 5) (92). Assessment of severity is particularly relevant to decide on whether a given patient should be treated by aggressive treatments (either medical or surgical) or simply by local or general supportive measures (see below).The other important feature of GO is its activity. Although, as stated above, GO natural history is not completely understood, it is commonly accepted that GO undergoes an initial phase of activity, characterized by progressive exacerbation of ocular manifestations until a plateau phase is reached; GO then tends to remit spontaneously, but remission is invariably partial. In the inactive phase (burnt-out GO), only residual ocular manifestations are present (e.g., proptosis, strabismus due to muscle fibrotic changes), but inflammation has subsided and it is unlikely that it may flare up. It is unknown how long this process takes to be completed, but it is widely believed that it takes between 6 months and two years. Recognition of the different phases of the disease is important, because active disease, basically characterized by the presence of inflammation, can respond to immunosuppressive treatments, which are largely ineffective when GO is burnt-out. Different indicators have been proposed to assess GO activity, including short duration of treatment (<18 months), positivity of octreoscan, decreased extraocular muscle reflectivity at orbital ultrasound, prolonged T2 relaxation time at MRI, increased urinary glycosaminoglycan levels, but they lack sufficient specificity and accuracy. A useful tool to assess GO activity is represented by the Clinical Activity Score (CAS), which can be calculated very easily and is recommended by EUGOGO in the assessment of GO in routine clinical practice, in specialist multidisciplinary clinics, and for clinical trials (92). In its original formulation (104) it included 10 items, which were subsequently reduced to 7 when revised by an ad hoc Committee of the four sister thyroid societies (105). (Table 6). If one point is given to each item when present, CAS, which basically reflect eye inflammation, may range from 0 (absent activity) to 7 (maximal activity); GO is generally defined active if CAS is >3.

Table 5. Assessment of severity of Graves’ orbitopathy

Derived from Bartalena & EUGOGO (92)

Table 6. Clinical Activity Score (CAS).

Diagnosis

Diagnosis of GO is usually easy on clinical grounds and by careful ophthalmological examination. Although not necessary in most Graves’ patients, CT scan or MRI of the orbit confirm diagnosis by showing enlarged extraocular muscles (without involvement of the tendon) and/or increased orbital fibroadipose tissue (106). Modest extraocular muscle enlargement and increased fibroadipose tissue volume are often found in Graves’ patients without clinical manifestations of ocular involvement. Orbital imaging is very useful to detect signs of optic nerve compression, which support the diagnosis of optic neuropathy. Imaging is required in asymmetrical or, particularly, unilateral forms of GO, to rule out that proptosis, periorbital swelling, inflammation, or diplopia be due to disorders other than GO (12, 106). The latter include primary or metastatic orbital tumors, vascular abnormalities (e.g., carotid-cavernous sinus fistula, carotid aneurysm, cavernous sinus thrombosis, subarachnoid hemorrhage, subdural hematoma), granulomatous disorders, IgG4-related ophthalmic disease (107). Occasionally, angiograms or venograms may be required for diagnosis. Octreoscan may be useful to identify patients with active GO (106), but its role in clinical practice is limited, also in view of its high cost.

Management

Management of GO is based on a multidisciplinary approach which involves endocrinologists, ophthalmologists, orbit surgeons, radiologists and radiotherapists. In a survey of GO management based on a questionnaire distributed among members of the European Thyroid Association, European Society of Ophthalmic Plastic and Reconstructive Surgery, and European Association of Nuclear Medicine, 96% of responders stated that a multidisciplinary setting for GO management is valuable, although 21% of patients were in the end not treated in a multidisciplinary setting (108). The therapeutic approach to a GO patient should be based on both severity and activity of the disease, the former being the feature to assess first.

Mild GO. Most patients have mild GO, which does not require particularly aggressive treatments and often is self-limiting (92, 110, 111). If GO activity is modest, simple local measured can be suggested to obtain symptomatic relief until GO is burnt-out (Table 7). Photophobia can be mitigated by sunglasses; grittiness due to corneal exposure can be controlled by artificial tears and topical lubricants, particularly indicated in the presence of lagophthalmos; the latter may require taping the eyelids shut at night; eyelid retraction can be controlled (with a variable degree of success) by b-blocking drops (useful for the increased intraocular pressure) or by botulinum toxin injections (112); elevation of the bed may be helpful to reduce periorbital swelling due to congestion; mild diplopia often is controlled by prisms (if they are tolerated). Reassurance is an important issue, and the patient must be informed that his/her eye disease is unlikely to progress to more severe forms, usually stabilizes, and often ameliorates spontaneously. Control of thyroid dysfunction is fundamental, because progression often is associated with hyper- or hypothyroidism (12, 92); refrain from smoking is also essential, because it is associated with a decreased chance of developing proptosis and diplopia (113), and decreases the likelihood to develop severe GO (29). Patients who do not succeed to quit smoking by themselves, should be helped by professional stop-smoking clinics, organizations, groups, where they can receive counseling, behavioral therapies, pharmacological treatments. In a subset of patients with mild GO, the impact of GO on the quality of life is so pronounced as to justify the risk of immunosuppression (or surgery) as for moderate-to-severe GO (92). A recent randomized controlled trial performed by EUGOGO in a large cohort of patients with mild GO showed that selenium supplementation for 6 months has beneficial effects on mild GO compared with placebo and can often prevent its progression to more severe forms (114). Thus, selenium, for its anti-inflammatory and immunomodulatory actions, should be considered both as a therapeutic tool for mild GO and a preventive measure (115). Whether selenium is also useful as an adjuvant therapy in patients with moderate-to-severe GO is presently unsettled.

Table 7. Management of mild Graves’ orbitopathy

Moderate-to-severe GO. Management of moderate-to-severe GO depends not only on severity, but also on activity of the orbitopathy (Table 8). Medical treatment is likely to be beneficial in patients with active GO, with florid signs and symptoms of inflammation, recent-onset extraocular muscle dysfunction, recent progression of the ocular abnormalities as a whole. On the contrary, in long-standing GO, with chronic proptosis and residual, stable diplopia and/or strabismus, but no no evidence of inflammation, medical treatment has little chances to produce favorable effects, and the surgical, rehabilitative approach is preferable (92). Dysthyroid optic neuropathy, the most severe expression of the orbitopathy, is a clinical, sight-threatening emergency, which requires immediate treatment. If there is no response to medical treatment (high-dose intravenous glucocorticoids), orbital decompression is warranted (92).

Table 8. Management of moderate-to-severe Graves’ orbitopathy

Glucocorticoids are the mainstay in the medical treatment of GO (12, 92, 116, 117). They have been used for decades because of their anti-inflammatory effects, but also because they exert immunosuppressive actions useful to control the course of the orbitopathy (12, 92, 116, 117). The latter include interference with the function of T and B lymphocytes, decreased recruitment of neutrophils and macrophages, down-regulation of adhesion molecules, inhibition of cytokine secretion, inhibition of glycosaminoglycan secretion. Locally (subconjunctivally or retrobulbarly) given glucocorticoids are less effective than systemically given glucocorticoids ) (11, 92), although favorable responses in terms of improvement of diplopia and reduction in extraocular muscle dysfunction have been reported with in a recent randomized clinical trial of periocular injections of triamcinolone acetate (118) . Glucocorticoids have for a long time been given mostly orally. This route of administration has several drawbacks: high doses are required every day (e.g., prednisone 60-100 mg daily as a starting dose, or equivalent doses of other steroids) (11), treatment lasts for several months (at least 5-6 months), recurrences are frequent upon drug tapering or withdrawal, side effects (particularly Cushing’s syndrome) are frequent (12, 92, 116, 117). In the last 20 years the intravenous route has become the most commonly used (101) and currently represents the first-line treatment for moderate-to-severe and active GO (92). Intravenous glucocorticoids are more effective, with a rate of favorable responses reported until few years ago of about 80-90% versus 60-65% with oral glucocorticoids (117, 119), and better tolerated than oral glucocorticoids (120). As a proof of principle, in a placebo-controlled, randomized trial, intravenous methylprednisolone (four cycles at a dose of 500 mg for 3 consecutive days at 4-week intervals) effected inflammatory changes and extraocular muscle dysfunction in 5 of 6 patients (83%) compared to only one of 11 placebo-treated patients (121) Glucocorticoids are most effective on soft tissue, inflammatory changes, recent-onset extraocular muscle dysfunction, and dysthyroid optic neuropathy, whereas proptosis and long-lasting eye muscle impairment are less responsive (11, 117). However, it should be noted that severe liver damage, heralded by a marked rise in serum concentrations of hepatic enzymes, was noted in 7 of about 800 treated patients (approximately 0.8%), three of whom died (122). The causes of this hepatotoxicity are unclear, but might include direct liver toxicity of glucocorticoids, precipitation of virus-induced hepatitis, sudden reactivation of the immune system upon drug withdrawal leading to autoimmune hepatitis. Statins are not a risk factor for liver damage associated with intravenous glucocorticoid pulse therapy for GO (123). The cumulative dose of glucocorticoids might also be important, since no cases of liver damage were reported in a recent randomized clinical trial in which lower, but equally highly effective, doses of glucocorticoids were employed (124). In this trial, the cumulative dose of methylprednisolone was 4.5 grams, subdivided in 12 weekly 2-hour infusions (500 mg for the first 6 infusions, 250 mg for the remaining 6 infusions) (124). A questionnaire-based surgery carried out among members of the European Thyroid Association showed a wide heterogeneity in the regimens of intravenous glucocorticoid therapy for moderate-to severe and active GO (125). A recent mullticenter, randomized clinical trial of a large cohort of patients with moderate-to-severe and active GO showed that a cumulative dose of about 7.5 g of methylprednisolone was associated with more favorable treatment outcomes than lower doses (about 5 g or 2.25 g), but also caused adverse events more frequently (126). Thus, the cumulative dose should somehow be tailored to the severity and activity of GO, reserving the highest dose to patients with most severe expressions of the disease.  In any case, the current recommendation is that the cumulative dose of glucocorticoids per course should not exceed 8 grams (92). Response to intravenous glucocorticoids may occur early in the course of the intravenous course, but also later; accordingly, the lack of response after the first 5-6 infusions is not an indication to stop the treatment (127). Adverse events of high-dose glucocorticoid treatment remain a relevant issue (128). Accordingly, patients should be treated in specialized centers under strict medical surveillance (92). Intravenous glucocorticoid treatment of Graves’ ophthalmopathy is not associated with secondary adrenocortical insufficiency. (129, 130),presumably because it is given for a limited period and intermittently.

Orbital radiotherapy is the other non-surgical mainstay in the management of GO (131). The rationale for its use and the indications are quite similar to those of glucocorticoids; in addition, irradiation exploits the radiosensitivity of T lymphocytes which infiltrate the orbit (131). Irradiation is currently carried out by linear accelerators, using a cumulative dose of 20 Gray fractionated in 10 daily 2-Gray doses over a 2-week period (131), although other regimens (and lower doses) might be equally effective (132). Favorable responses have been reported in about 60% of treated patients (131). Recent years have witnessed a lively debate on the true effectiveness of orbital radiotherapy (133, 134). However, the results of several randomized studies confirmed, with one exception, its efficacy (132, 135-138). In addition, orbital radiotherapy is a safe procedure devoid of relevant short-term and long-term side effects or complications (139, 140). Preexisting retinopathy associated with diabetes mellitus or hypertension represents a contraindication to its use (92). As for glucocorticoids, orbital radiotherapy is mostly effective on soft tissue inflammatory changes and recent-onset extraocular muscle dysfunction (131). Orbital radiotherapy can be used either alone or in combination with glucocorticoids. The association exploits the prompter effect of glucocorticoids and the more sustained action of irradiation; in two randomized prospective studies, combined therapy (using oral glucocorticoids) proved to be more effective than either treatment alone (141, 142). Whether the combination of intravenous glucocorticoids and orbital radiotherapy is more effective than intravenous glucocorticoids alone is presently unsettled. For this reason the recent guidelines for the management of moderate-to-severe and active suggest the combination of orbital radiotherapy and oral glucocorticoids as a second-line treatment in the case of a partial or absent response to intravenous glucocorticoids (92).

Cyclosporine, used in GO for its immunosuppressive properties, has been reported in only two randomized and controlled studies (143, 144) . Cyclosporine has a lower efficacy than glucocorticoids as a single-agent therapy, although a combination of both drugs might be more effective than either treatment alone (143, 144). Thus, the use of cyclosporine might be maintained in patients who are relatively resistant to glucocorticoids in whom persistent GO activity warrants continuing medical intervention (145). Side effects of cyclosporine are not negligible and should be carefully considered. As for orbital radiotherapy, no studies have compared the combination of cyclosporine and intravenous glucocorticoids. Therefore, the association of oral glucocorticoids and cyclosporine is considered an alternative option when the response to intravenous glucocorticoids is poor (92).

Rituximab is a CD20+ B-cell depleting monoclonal antibody originally used for B-cell non-Hodgkin lymphoma, but then utilized for autoimmune B-cell (and T-cell) driven autoimmune disorders. This drug has been used for GO in small and uncontrolled preliminary studies (reviewed in refs. 146 and 147). The rationale for using rituximab is sound, based on our understanding of pathogenesis of GO. The available data suggest that rituximab may have favorable effects on moderate-to-severe and active GO, with relatively low adverse event rate (146, 147). Two small, single-center, randomized clinical trials have recently been published. They produced conflicting results, because the first one (148) showed no difference in the effect of rituximab compared to placebo (148), whereas the second one (149) showed that rituximab was as effective as intravenous glucocorticoids in inactivating GO and was not associated with a flare-up of the disease, not infrequently seen after withdrawal of glucocorticoids. The reason for this conflicting results remains elusive and larger multicenter studies are warranted to clarify this issue (150), nevertheless rituximab may be considered as a possible second-line treatment in the case of a partial or absent response to intravenous glucocorticoids (92). Rituximab is contraindicated in patients with overt or impending dysthyroid optic neuropathy (92).

Two recent randomized clinical trials have evaluated the effect of mycophenolate, an immunosuppressant agent inhibiting both T cells and B cells, widely used for the prevention of organ transplant rejection, but also for autoimmune disorders. The first, single-center study showed that mycophenolate mofetil was more effective than intravenous glucocorticoids on moderate-to-severe and active GO, but the experimental design had several limitations (151). The second, large, multicenter study carried out by EUGOGO demonstrated that the combination of mycophenolate sodium and intravenous glucocorticoids was more effective than intravenous glucocorticoids alone (152). The safety profile at the dose used (720 mg/day for 6 months) appears to be good (153). Therefore mycophenolate sodium may represent a useful tool for the management of GO.

Teprotumumab is a monoclonal antibody inhibiting the IGF-1 receptor. Because the latter seems to be involved in the pathogenesis of GO (154), teprotumumab might play a role in the management of the disease. A recent placebo-controlled randomized clinical trial showed that teprotumumab is more effective than placebo in reducing the CAS (155). The most striking effect was marked reduction in the exophthalmos (155), a feature of GO which is very poorly modified by whatever medical treatment. This study is not devoid of limitations and further studies are warranted before it can be included among the tools for the management of active and moderate-to-severe GO (156).

Orbital decompression is a milestone in the management of GO. It is aimed at increasing the space available for the increased orbital content by removing part of the bony walls of the orbit and/or the orbital fibroadipose tissue (157). It is indicated in patients who have impending sight loss due to optic neuropathy and do not respond promptly to intravenous glucocorticoids (157). Other important indications for decompressive surgery are represented by corneal damage due to eyeball exposure in patients with marked proptosis, or by recurrent subluxation of the globe, which may stretch the optic nerve and cause sight loss (157). In recent years, thanks to the improved surgical techniques and the diminished surgical risk, the indications for orbital decompression have expanded, including also correction of residual cosmetic problems (157, 158). Several techniques of orbital decompression are available, aimed at removing part of one, two, three or four orbital walls (floor, roof, lateral wall, medial wall) as well as part of the retroorbital fibroadipose tissue. The different surgical options should be discussed with the patient, as well possible complications of the procedure, particularly the de novo occurrence or worsening of diplopia, particularly frequent after extensive removal of the orbital floor (157, 159). Removal of fibroadipose tissue can be done together with or without bone removal, but removal of fat alone is associated with a lower reduction of proptosis (159).

Rehabilitative surgery includes surgery for strabismus or eyelid retraction. Extraocular muscle surgery is aimed at correcting residual diplopia after medical and/or surgical treatment of GO. Timing of surgery is crucial, because it should not be performed when GO is active, but when it has been inactive for 6 months (160, 161). The goal of eye muscle surgery is to align the eyes, avoiding abnormal head posture and restoring single binocular vision in primary and reading positions; multiple operations may be required to achieve this goal. Eyelid surgery may rarely be an emergency procedures in patients with exposure keratitis and corneal ulcerations, but it usually is carried out to correct eyelid malposition after medical treatment or orbital decompression. Eyelid surgery usually constitutes the last step of rehabilitation (161).

Thyroid ablation. The question of whether in a patient with GO, Graves’ hyperthyroidism should be treated by non-ablative (i.e., thionamides) or ablative (i.e., radioiodine therapy, thyroidectomy, both) therapy is unanswered (162, 163). Supporters of thyroid ablation justify this approach by mentioning the pathogenic link between thyroid and orbit: removal of thyroid-orbit shared antigen(s) and autoreactive T lymphocytes might be beneficial to the eye (162); supporters of non-ablative thyroid treatment suggest that control of thyrotoxicosis by antithyroid drugs may be associated with a reduction of autoimmune phenomena which might be reflected by an amelioration of ocular conditions; furthermore, once triggered, GO might proceed independently of thyroid treatment (163). Two retrospective studies showed that total thyroid ablation (thyroidectomy followed by radioiodine therapy, as in thyroid cancer) was associated with an improvement of clinical GO (164, 165). A recent randomized, controlled clinical trial demonstrated that, as compared to total thyroidectomy alone, total thyroid ablation is followed by a better outcome of GO in patients given intravenous glucocorticoids (166). A follow-up study of this cohort recently showed that in the long run total thyroid ablation was no better than total thyroidectomy alone, although it was associated with a prompter achievement of the best possible result obtainable by medical treatment and with an earlier possibility to submit the patient to rehabilitative surgery (147). Other cohort studies emphasized the opportunity to postpone definitive treatment of hyperthyroidism until GO is cured (168, 169). Thus, the optimal thyroid treatment in patients with GO still is a dilemma (170-172).

THYROID DERMOPATHY AND ACROPACHY

Thyroid dermopathy (also called pretibial myxedema or localized myxedema) is an uncommon extrathyroidal manifestations of Graves’ disease (less frequently of chronic autoimmune thyroiditis) (13, 173). It almost always occurs in Graves’ patients who also have GO. In a review of 178 consecutive patients with thyroid dermopathy, only 4 patients had no evidence of eye disease (174). However, in a community-based epidemiologic study, only 4% of GO patients also had thyroid dermopathy, although the latter was more frequent in patients with severe GO (175). It is more common in older than in younger patients, with a large preponderance in women (176). Skin lesions are edematous and thickened plaques, typically localized in the pretibial area; however they can be less frequently found in other skin areas, such as feet, toes, upper extremities, shoulders, upper back, nose. Prevalent localization in the pretibial area is related to mechanical and dependent position. The occurrence of lesions in less common sites is often preceded (triggered?) by local trauma (177, 178). There can be three clinical types: nodular, diffuse, and elephantias-like.(179, 180) (Figures 7-9).

Fig. 7: Thyroid dermopathy. Courtesy of Dr. Vahab Fatourechi, Mayo Clinic

Fig. 8: thyroid dermopathy. Courtesy of Dr. Vahab Fatourechi, Mayo Clinic

Fig. 9: Thyroid dermopathy. Courtesy of Dr. Vahab Fatourechi, Mayo Clinic

Histopathologically, skin lesions are characterized by the accumulation of activated fibroblasts (and, to a lesser extent, mast cells), with a markedly increased production of glycosaminoglycans in the dermis and subcutaneous tissues (181). Whereas in normal skin approximately 5% of the acid mucopolysaccharides are hyaluronic acid, in pretibial myxedema this amount increases to 90%. Glycosaminoglycans are responsible for fluid retention, subsequent compression and occlusion of lymphatic vessels, and lymphedema (182). Thus, as in GO, fibroblasts seem to play a central role in the pathogenesis of localized myxedema. This notion is further supported by the finding of limited variability of T cell receptor V gene usage in pretibial myxedema, pointing to a primary immune response of antigen-specific T lymphocytes (183). Furthermore, as is the case with acropachy, lymphocytes do recognize local fibroblasts. IgG from patients with pretibial myxedema was shown to stimulate proteoglycan synthesis by human skin fibroblasts (182). As for GO, TSH-R has been implicated in the pathogenesis of localized myxedema. TSH-R is expressed in peripheral skin fibroblasts from patients with localized myxedema, both at the mRNA and protein level (13). However, TSHR is expressed also in skin from normal subjects (183, 184). Likewise, TSH-R immunoreactivity was detected in cultured fibroblasts from pretibial myxedema, although the specificity of this finding remains to be established. As mentioned above, IgG from Graves’ patients with localized myxedema was reported to stimulate glycosaminoglycan production in cultured skin fibroblasts (183), but this data is not unequivocal, because IgG from normal subjects were equally effective in other studies (185). To summarize, although pathogenic mechanisms remain to be fully elucidated, localized myxedema appear to result from autoimmune reactions leading to fibroblast proliferation and increased glycocosaminoglycan secretion.

From a clinical standpoint, localized myxedema presents as light-colored (sometimes yellowish brown) skin lesions, frequently with an orange peel texture (Figures 7-9). Skin lesions may be characterized by hyperpigmentation and hyperkeratosis. They usually represent only a cosmetic problem and are asymptomatic, but sometimes they may be associated with itching and pain, or may be functionally important, e.g., they may cause problems to wear shoes, especially the elephantiasic form of localized myxedema. Localized myxedema may remain stable, but frequently improves with time, partially or completely (13). Many cases of mild localized myxedema do not require any treatment, but in moderately severe lesions or when there is cosmetic concern, topical glucocorticoids applied with occlusive plastic dressing produce beneficial effects in a relevant proportion of patients (13). If necessary, treatment is repeated until clinical remission occurs (13). When localized mxedema is severe and extensive, steroid pulse therapy, or decongestive physiotherapy, a combination of manual lymphatic drainage, bandaging, exercise, and scrupulous skin care, may be tried (13). No substantial effect was reported by long-term octreotide treatment in three patients with localized myxedema (186). Two studies on the use of intravenous IgG in a small number of patients have reported discrepant effects (reviewed in 13). Thus, measures such as compression bandaging and topical glucocorticoids still are the most cost effective treatments for localized myxedema.

Acropachy is a very uncommon extrathyroidal expression of Graves’ disease, usually associated with severe GO (13) and localized myxedema (13), thus reflecting severity of the autoimmune process. It seems more common in women than in men (13). It is characterized by clubbing of fingers (Fig. 10) and toes, with concomitant soft-tissue swelling of hands and feet. These abnormalities are usually painless and may be asymmetric (13). As for GO, there seems to be a strong relation with cigarette smoking. X-ray of affected sites shows soft-tissue swelling and subperiosteal bone formation. There is currently no treatment that can solve the esthetic and (less frequent) functional abnormality of thyroid acropachy, which occasionally may remit spontaneously in the long-term.

Fig. 10: Thyroid acropachy. Courtesy of Dr. Vahab Fatourechi, Mayo Clinic

CLINICAL ABNORMALITIES OF THE HEART

The biochemical actions of thyroid hormone on the heart are described in Chapter 10.

Hyperthyroidism is usually associated with relevant cardiovascular symptoms and changes in cardiovascular hemodynamics (186, 187).Thyrotoxicosis increases the demands on the heart both by chronotropic and inotropic alterations. Cardiac output is markedly increased owing to increased stroke volume and rapid heart rate (186, 187). It is possible that the metabolic efficiency of heart muscle is decreased (186, 187). Irritability of the heart is increased. Investigation with stress echocardiography shows in hyperthyroidism impaired chronotropic, contractile, and vasodilatatory cardiovascular reserves, that are reversible upon conversion to euthyroidism (188). In a recent, large, matched case-control study, cardiovascular symptoms and signs, including palpitations, chest pain, dyspnea, cough, orthopnea, displaced apex, cardiac murmur, chest wheeze/crepitus were much more frequent in hyperthyroid patients than in controls, and some of them persisted despite effective restoration of euthyroidism by antithyroid drug treatment (182). This common finding of cardiovascular alterations in hyperthyroid patients may result from thyroid hormone excess itself, by hyperthyroidism-related worsening of preexisting cardiovascular disorders, or by the occurrence of novel cardiovascular abnormalities (186, 187). The importance of cardiovascular abnormalities is underscored by the observation that mortality of hyperthyroid patients is increased, mainly due to cardiovascular events (189, 190). Similar conclusions were reached also in a community-based study of elderly people (191), in which, however, definition of hyperthyroidism was based on the finding of low/suppressed serum TSH, which may not necessarily reflect thyroid hormone excess, but rather be the result of non-thyroidal illness syndrome.

Mitral valve prolapse was found more commonly in hyperthyroid patients (43%) than in controls (18%) (190). This increased incidence might be due to increased adrenergic tone, autoimmunity, or the augmented cardiac output associated with thyrotoxicosis. Most patients with thyrotoxicosis are adults. Many, especially those with toxic nodular goiter, are in the 50- to 70-years age group, which has a relatively high incidence of organic heart disease anyway (192). Thus, it is not surprising that cardiac abnormalities are prominent among the symptoms of thyrotoxicosis. Frequent premature beats and paroxysmal tachycardia sometimes appear in thyrotoxic patients and may be disturbing to the patient. Atrial fibrillation occurs in thyrotoxicosis with or without preexisting heart disease, but it is more frequent in older patients (193), probably reflecting an increase in the prevalence of underlying cardiac abnormalities of ischemic or different origin (194). It may be paroxysmal or persistent during the thyrotoxic period. Attempts to correct this arrhythmia to normal in patients with persistent atrial fibrillation are usually unsuccessful while they are hyperthyroid. Once euthyroidism has been restored, atrial fibrillation may revert spontaneously or may be converted pharmacologically or by electroconversion. About two-thirds of patients undergo spontaneous reversion to sinus rhythm after receiving therapy for thyrotoxicosis, usually within 4 months; later on, spontaneous conversion is unlikely (195). It is wise to always evaluate thyroid function in clinically euthyroid patients with atrial arrhythmias with or without heart disease, because in about 20% of patients TSH tests and/or FT4 point to an overactive thyroid and in 50% of these patients normal sinus rhythm resumes after treatment with antithyroid drugs (195). It is widely accepted that subclinical hyperthyroidism is associated, in individuals aged 60 years or more with a 3-to-5-fold increased risk of developing atrial fibrillation (195).

Congestive heart failure is a frequent complication in thyrotoxic patients with pre-existing organic heart disease, particularly if old (196-198). In the elderly hyperthyroid patient, cardiac symptoms may so dominate the clinical picture that diagnosis of thyrotoxicosis may be overlooked. Careful attention should be given to this possibility in all patients with congestive heart failure, especially if goiter is detected (196). Congestive heart failure may occur in patients who have no detectable preexisting organic heart disease (199). Overt hyperthyroidism may cause ventricular dilatation and persistent tachycardia, which may lead to heart failure and fatal events (200). It is often difficult to establish whether an underlying heart disease is present in a hyperthyroid patient who also has a disorder of rhythm, a cardiac murmur, or congestive heart failure, because all these conditions may be ascribed to thyrotoxicosis per se. It is frequently gratifying to observe normalization of cardiac findings once euthyroidism has been restored.

In hyperthyroidism, owing to the increased metabolic demand, angina can be worsened if pre-existing, or induced de novo (186, 201, 202). Evidence of myocardial lactate production when the heart is paced at an accelerated rate (203), and normal coronary arteries are found at angiography after episodes of angina or infarction (203), have suggested that changes in thyrotoxicosis are due to an imbalance between O2 demand and supply rather than to arterial obstruction. This possibility is corroborated by the finding that coronary artery spasm of an otherwise normal vessel may occur during thyrotoxicosis (202).

Cardiac abnormalities found in Graves' disease often are entirely reversible, except that longstanding atrial fibrillation due to hyperthyroidism is not always convertible after euthyroidism is restored. It has become evident that even in the mildest forms of thyrotoxicosis subtle cardiac abnormalities may be present. Thus, in patients with so-called "subclinical" thyrotoxicosis, i.e. suppressed TSH and normal serum free T4 and T3 concentrations, due to multinodular, autonomous goiter or TSH-suppressive T4 treatment, mean basal 24-h heart rate is increased, there is an augmented risk of atrial premature beats and atrial fibrillation, and left ventricular function and wall thickness are increased (204).There is controversy whether TSH suppressive T4 treatment leads to functional cardiac abnormalities (204, 205).

Treatment of heart failure in the presence of thyrotoxicosis does not differ from its treatment in euthyroid patients, but it may be more difficult. Rest, salt restriction, diuretic therapy, digitalization and administration of afterload-reducers, like angiotensin converting enzyme (ACE) inhibitors, betablockers, aldosterone antagonists and other specific measures, are in order (186, 196). Larger than normal doses of digoxin are required, but there is probably no change in the toxic-to-therapeutic dose ratio. Atrial fibrillation may be controlled by digoxin, propranolol, or both. Electroconversion is usually successful only after thyrotoxicosis has been resolved for a few months (206).

Hyperthyroidism should be controlled as expeditiously as possible. Congestive heart failure is a contraindication to operation. Most patients with thyrotoxicosis and clinically relevant heart disease are now treated with RAI. This treatment may be preceded by a 3-to-6-month course of antithyroid drug therapy to deplete their glands of stored thyroid hormone, a program that lessens any chance of an exacerbation of the heart disease caused by a radioiodine-induced release of thyroid hormone from the gland. Administration of 131I followed by antithyroid drugs, and potassium iodide or ipodate, that also inhibit T4 to T3 conversion, may be used in severely ill patients in whom a prompt response is needed. This method is described in Chapter 11.

Propranolol has been used successfully in the control of tachycardia, and also in patients with congestive heart failure if tachycardia appeared to be adding substantially to the problem. In these instances, possible depression of myocardial contractility by the drug was outweighed by the benefit derived from controlling the rate. In such circumstances, one must proceed with caution and often digoxin should be added.

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