Repair-Related Activation of Hedgehog Signaling in Stromal Cells Promotes Intrahepatic Hypothyroidism

Thyroid hormone (TH) is important for tissue repair because it regulates cellular differentiation. Intrahepatic TH activity is controlled by both serum TH levels and hepatic deiodinases. TH substrate (T4) is converted into active hormone (T3) by deiodinase 1 (D1) but into inactive hormone (rT3) by deiodinase 3 (D3). Although the relative expressions of D1 and D3 are known to change during liver injury, the cell types and signaling mechanisms involved are unclear. We evaluated the hypothesis that changes in hepatic deiodinases result from repair-related activation of the Hedgehog pathway in stromal cells. We localized deiodinase expression, assessed changes during injury, and determined how targeted manipulation of Hedgehog signaling in stromal cells impacted hepatic deiodinase expression, TH content, and TH action in rodents. Humans with chronic liver disease were also studied. In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, whereas stromal expression of D3 increased, particularly in myofibroblasts. Conditionally disrupting Hedgehog signaling in myofibroblasts normalized deiodinase expression. Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression. In patients, this was reflected by increased serum rT3. Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

affect downstream tissue-specific TH signaling and local action. During early embryogenesis, for example, D3 is highly expressed to limit cellular exposure to T 3 (3,4). After birth, D3 expression declines and expression of D1 and D2 increases, allowing for T 3 -induced tissue maturation and cellular differentiation (4). The reciprocal developmental functions of activating and inactivating deiodinases explain why D3 knockout mice exhibit growth restriction, neonatal mortality, and abnormal thyroid status (4). Interestingly, selective reexpression of D3 occurs during adult tissue injury, as evidenced by the up-regulation of hepatic D3 after partial hepatectomy, induction of D3 in cardiac muscle after myocardial infarction, and increased D3 expression in injured brain and skeletal muscle (5)(6)(7). Conversely, hepatic D1 activity generally falls in many chronic illnesses (8). Because liver D1 plays a significant role in regulating peripheral conversion of T 4 to T 3 , this contributes to the decreases in liver and systemic T 3 levels that occur in the euthyroid sick syndrome. Despite considerable evidence that tight control of TH homeostasis is necessary for normal tissue growth and differentiation and the acknowledged importance of deiodinases in regulating local TH activity, the mechanisms that control D1 and D3 during adult liver repair remain poorly understood.
Evidence that D3 expression predominates during adult liver regeneration suggested to us that this enzyme might localize in stromal cell types because these cells accumulate in response to factors produced during liver injury. We further theorized that injury-related Hedgehog pathway activation in the liver stromal compartment triggers reacquisition of a more fetal-like pattern of hepatic deiodinase expression because developing tissues in early embryos are particularly enriched with D3, and certain developmental morphogenic signaling pathways, such as Hedgehog, must become locally reactivated in hepatic stromal cells for injured adult livers to regenerate (9). To evaluate this hypothesis, we compared expression of D1 and D3 in various types of rodent liver cells and liver tissue before and after adult liver injury and assessed how the targeted manipulation of Hedgehog signaling in liver stromal cells influenced hepatic deiodinase expression, TH content, and tissue TH action. Humans with chronic liver disease were also studied to verify the translational applications of our findings. Our results confirm the hypothesis that the injured liver is in a state of intrahepatic hypothyroidism, identifying previously unsuspected roles for he- patic stromal cells and the canonical Hedgehog pathway as key regulators of TH homeostasis during adult liver injury. As such, our findings provide novel insight into both the mechanisms for, and the implications of, the euthyroid sick syndrome.

Animal models of liver injury
To assess the effect of liver injury on deiodinases, rats (n ϭ 18) underwent sham surgery or bile duct ligation (BDL), and were killed on days 3, 7, or 14. To address the role of Hedgehog signaling in regulating hepatic deiodinases, adult male double transgenic (DTG) ␣SMA-CreERT2 ϫ SMO/flox homozygote mice that have previously been fully characterized (15) were subjected to sham surgery (n ϭ 8 mice) or BDL (n ϭ 22 mice/group). Tamoxifen (TMX) was administered postoperatively to activate Cre recombinase and conditionally disrupt Hedgehog signaling (15). Results were compared with vehicle-treated controls. Livers were harvested 14 days after BDL. The Animal Care and Use Committee and the Duke University Institutional Animal Care and Use Committee approved animal care and surgical procedures.

Human studies
Liver and serum samples from 2 cohorts of patients in our Non-Alcoholic Fatty Liver Disease (NAFLD) Clinical Database and Biorepository were analyzed for TH content and deiodinase expression. Among the discovery cohort (n ϭ 70 patients), 42 individuals had histologically proven mild NAFLD fibrosis [stage 0 -1 hepatic fibrosis by the Nonalcoholic Steatohepatitis Clinical Research Network scoring system (16)], and 28 patients had advanced NAFLD fibrosis (stage 3-4 hepatic fibrosis). The 2 groups were matched for gender, age, and body mass index (kilograms per square meter) and analyses corrected for potential confounders. A second validation cohort (n ϭ 30 mild, n ϭ 30 advanced) was separately analyzed.

mRNA isolation and analysis
Total RNA was extracted from rodent liver tissue/cells and analyzed in triplicate by quantitative RT-PCR (qRT-PCR) as described (see Supplemental Methods). Liver RNA from the discovery patient cohort was analyzed by microarray analysis; differences in TH-responsive protein (THRSP; a TH inducible gene) were determined using limma (R/Bioconductor statistical package), followed by qRT-PCR validation (17).

Western blotting
Protein lysates were prepared with radioimmunoprecipitation assay and/or Laemmli buffer, separated by SDS-PAGE, and transferred to nylon membranes for detection by the Bio-Rad Laboratories ChemiDoc MP imaging system.

Flow cytometry
D1 or D3 proteins were evaluated in primary rat HSCs using methods of flow cytometry as previously described (18).

Liver histology
All human liver biopsy specimens were stained with hematoxylin-eosin and Masson's trichrome stains and were graded and staged by a hepatopathologist (C.D.G.) according to the Nonalcoholic Steatohepatitis Clinical Research Network Scoring System (16).

Liver immunohistochemistry (IHC) and morphometry
Rodent and human liver specimens were prepared for IHC as described (18).

Intrahepatic TH measurements
Concentrations of T 4 and T 3 were determined in homogenized whole livers from male rats (n ϭ 6) and male DTG ␣SMA-Cre ϫ SMO/flox homozygote mice (n ϭ 9) treated with sham surgery or BDL surgery using an Agilent 6410 triple quadruple tandem mass spectrometer (liquid chromatography and tandem mass spectrometry, LC/MS-MS) using established methods (19).

Serum TH measurement
Rodent serum TH levels were measured using established methods (17). Human serum TH measures including the following: TSH, free T4 (fT 4 ), free T3 (fT 3 ), and rT 3 ; fT 3 to rT 3 and fT 4 to rT 3 ratios were obtained on serum collected from patients on the same day as the liver biopsy.

Statistical analysis
Significance (P Ͻ .05) was assessed by a Student's t test, Pearson's 2 test, Wilcoxon rank-sum test, and/or univariate/multivariate analysis where appropriate. All analyses were done using JMP statistical software version 7.0 (SAS Institute Inc).
See Supplemental Tables 1-6, Supplemental Figure 1, and Supplemental Methods for complete details.

Differential expression of D1 and D3 in hepatocytes and liver stromal cells
Primary HCs and various types of liver stromal cells (eg, HSCs, KCs, and LSECs) from healthy, adult mice were analyzed for mRNA expression of DIO1 and DIO3, the main deiodinases that regulate T 4 activation/deactivation in adult liver ( Figure 1A). Although all cell types expressed DIO1 mRNA, levels of this transcript were particularly enriched in hepatocytes, which expressed high levels of DIO1, but were barely detectable DIO3 ( Figure 1B). In contrast, the expression of DIO1 and DIO3 transcripts was more balanced in liver stromal cells, with HSCs demonstrating the highest levels of DIO3 mRNA among the various liver cell types ( Figure 1B). In healthy liver, IHC confirmed ubiquitous strong expression of D1 protein but a much weaker expression of D3 protein that was mainly localized in stromal cells [ Figure 1C (ϫ10 and ϫ40) and Figure 1D (ϫ10 and ϫ40)].

Liver injury provokes reciprocal changes in net hepatic expression of D1 and D3
The pattern of deiodinase expression changed dramatically during liver injury. After bile duct ligation in rats, for example, staining for D1 virtually disappeared, while D3 protein was strongly increased (Figure 2, A and B). Western blot analysis of whole liver protein confirmed the immunohistochemistry data ( Figure 2C). Changes in D1 and D3 protein expression generally paralleled changes in the respective whole liver mRNA levels ( Figure 2D). Because TH substrate (T 4 ) is converted to the more biologicallyactive TH (T 3 ) by D1, whereas D3 converts T 4 into a biologically-inert TH (reverse T 3 , rT 3 ) and further inactivates T 3 by conversion to T 2 ( Figure 1A), injury-related changes in D1 and D3 are predicted to limit hepatic accumulation of both bioactive forms of TH (ie, T 4 and T 3 ).
Indeed, liquid chromatography and tandem mass spectrometry measurement of TH concentrations before and after BDL confirmed that intrahepatic and serum concentrations of both T 4 and T 3 decreased significantly by day 14 post-BDL ( Figure 2E and Supplemental Table 5), a time when there is considerable cholestatic liver injury and fibrosis (Supplemental Figure 1). T 4 levels fell significantly more in the liver than in the serum after BDL (53.7% vs 20.7%, P Ͻ .01), suggesting that increased hepatic D3 was important in regulating liver TH content during liver injury. To determine whether these changes in TH concentrations were accompanied by changes in hepatic TH activity, we evaluated expression of representative THregulated genes. After BDL, mRNA levels of ME1 and THRSP (TH responsive genes) both decreased significantly ( Figure 2F). Thus, injured liver tissue reacquires a deiodinase expression profile that is reminiscent of developing tissue in which there is limited exposure to TH.

Stromal cells become the dominant deiodinaseexpressing cells in injured liver
To determine whether the observed changes in deiodinase expression reflected changes in residual resident liver cells, as opposed to nonspecific effects caused by liver cell loss and/or infiltrating inflammatory cells, additional im- munostaining was done ( Figure 3). Surprisingly, the types of liver cells producing deiodinases changed dramatically after liver injury. In injured livers, hepatocytes were no longer the dominant D1-expressing cell. Rather, D1 accumulated in ductular type cells ( Figure 3A). D3 protein remained in stromal cells ( Figure 3B) and mainly colocalized with markers for HSCs (desmin, Figure 3C), KCs (CD68, Figure 3D), and LSECs (CD31, Figure 3E). Desmin, CD68, and CD31 costains were performed by IHC and revealed that, although stellate cells were the major producers of D3 after injury (comprising Ͼ60% of D3 expressing cells), approximately 30% of D3-positive cells were CD68 positive and 10% were CD31 positive. In both instances, CD68-and CD31-producing cells increased in a time-dependent manner after BDL injury, with most cells seen by day 7. Hence, the accumulation of these stromal cells likely contributed to the injury-related shift from D1 to D3 that was demonstrated by analysis of whole liver mRNA and protein (Figure 2, A-D). Thus, liver injury recapitulates the fetal-like state of D3 predominance due not only to a loss of D1 in hepatocytes but also because D3 is dramatically increased in the hepatic stromal compartment.

Activated hepatic stellate cells are major sources of D3 during liver injury
Among liver stromal cells, injury-activated HSCs play particularly pivotal roles in hepatic wound healing/regen-eration. In addition to becoming a major source of fibrogenic myofibroblasts, activated HSCs produce various hepatocyte growth factors, chemokines, and mediators of angiogenesis, and there is strong experimental evidence that inhibiting HSC activation drastically impairs liver regeneration (20,21). Double immunostaining for desmin, a stellate cell-specific marker (22), and D3 confirmed that many of the D3-expressing cells that accumulated after BDL were HSCs ( Figure 4A), raising the possibility that HSC might up-regulate their expression of D3 as they activate during injury. To evaluate this more directly, we compared D3 expression in primary HSC at different time points during culture-induced activation. Both DIO3 mRNA ( Figure 4B) and D3 protein ( Figure 4C) levels increased as quiescent HSCs became myofibroblasts. This activation-related induction of D3 expression was accompanied by a reduced expression of DIO1 mRNA and D1 protein (Figure 4, B and C). Thus, HSC activation is characterized by reciprocal changes in the expression of DIO1 and DIO3, with the resultant predominance in DIO3 predicted to reduce accumulation of biologically-active TH.

Hedgehog signaling in HSC-derived myofibroblasts controls hepatic deiodinase expression and TH homeostasis during liver injury
HSC activation is controlled by canonical Hedgehog signaling. Increasing pathway activity stimulates qui- escent HSCs to become myofibroblasts, whereas the reducing pathway activity in myofibroblasts restores the quiescent phenotype (23). To determine whether Hedgehog signaling influences HSC expression of DIO3, rat HSC-derived myofibroblasts were treated with recombinant SHH-L, and the expression of DIO3 was evaluated 24 hours later. Compared with vehicletreated controls, myofibroblastic HSCs treated with SHH-L demonstrated 50% higher levels of the SHH-inducible transcription factor, Glioblastoma 2 (Gli2), and more than 6-fold increased expression of DIO3 (P Ͻ .05, and P Ͻ .01, respectively) ( Figure 5A). These findings are consistent with a previous report that Gli2 directly interacts with the DIO3 promoter to induce DIO3 expression in keratinocytes after Sonic Hedgehog exposure (24) and suggest that deiodinase expression in liver myofibroblasts may also be controlled by Hedgehog signaling.
To further investigate this issue, we used ␣smaCresmo flx/flx mice to conditionally delete Smoothened (Smo), an obligate component of the canonical Hedgehog pathway, in myofibroblasts after BDL and compared deiodinase expression, hepatic TH levels, and hepatic expression of TH-regulated genes in mice with and without canonical signaling in HSCs. Previously we reported that treating such ␣smaCre-smo flx/flx mice with tamoxifen after BDL deleted Smo from myofibroblastic HSCs and demonstrated that this targeted inhibition of Hedgehog signaling decreased multiple fibrotic and regenerative responses that are typically provoked by cholestatic liver injury (15). Here we show that hepatic TH homeostasis was severely disrupted in the BDL mice that lacked Smo in HSC-derived myofibroblasts compared with BDL controls with intact HSC Hedgehog signaling. Both the induction of DIO3 and the fall in DIO1 mRNA that normally follow BDL were partially blocked by disrupting Hedgehog signaling in myofibroblasts ( Figures 5B). Surprisingly, changes in deiodinase expression were not limited to the stromal compartment. Immunostaining confirmed that disrupting Hedgehog signaling in myofibroblasts reduced accumulation of D3-positive stromal cells but also revealed relative restoration of D1 expression in hepatocytes ( Figure  5C). This finding suggests a previously unsuspected role for stromal cells in regulating net hepatic deiodinase expression, and thereby local TH activity, during liver injury. Because limiting myofibroblast accumulation during liver injury tended to restore the D1-predominant state that is typical of uninjured liver, this might prevent injuryrelated depletion of intrahepatic TH. Consistent with that concept, hepatic concentrations of T 4 were found to be about twice as high in the Smo-deficient (myofibroblast depleted) mice as in control mice 14 days after BDL (8.99 vs 4.99 ng T 4 per gram of liver, P Ͻ .05), despite continued euthyroid sick pattern of suppressed serum TH levels after BDL injury in both models (57% and 62% decrease in serum T 4 in mice after BDL Ϯ TMX compared with sham, respectively). The increase in intrahepatic TH levels was accompanied by an increased hepatic expression of the TH-responsive gene, ME1 (Figure 5, D and E). Intrahepatic T 3 concentrations and hepatic expression of the T 3 -responsive gene, THRSP, were similar in Smo-deficient mice and controls 14 days after BDL (data not shown). Serum rT 3 levels increased significantly (P ϭ .026) after BDL injury and fell to baseline levels with the addition of TMX and the resultant disruption of Hedgehog signaling in myofibroblasts, paralleling changes in hepatic D3 expression (Supplemental Table 6). Taken together, our findings demonstrate that Hedgehogresponsive myofibroblasts are important regulators of hepatic deiodinase expression during liver injury and are partially responsible for the intrahepatic hypothyroidism that develops after BDL in mice.

Injury-related changes in hepatic deiodinases parallel hepatic accumulation of Hedgehogresponsive myofibroblasts and contribute to tissue hypothyroidism in humans
Recently we reported that the hepatic gene expression profiles of NAFLD patients with advanced liver fibrosis reflected active tissue remodeling, including relative enrichment with various Hedgehog-regulated genes (17).
Here we compared serum TH profiles in these same age-, gender-, and body mass index-matched NAFLD subjects with either advanced or mild fibrosis and found that subjects with advanced NAFLD fibrosis had lower levels of fT 3 and higher levels of rT 3 (ie, a reduced fT 3 to rT 3 ratio) ( Table 1). Indeed, in both the discovery and validation cohorts, the fT 3 to rT 3 ratio and the fT 4 to rT 3 ratio correlated significantly with the stage of liver fibrosis as determined by multivariate logistic regression analysis. Given that D1 converts T 4 to T 3 and then clears rT 3 , whereas D3 converts T 4 to rT 3 and clears T 3 , this TH pattern suggests that a switch from D1 predominance to D3 predominance occurs during advanced NAFLD fibrosis in humans.
To assess the potential contribution of hepatic deiodinases to the differences in serum TH levels that we ob- served between mild and advanced NAFLD, we used IHC to compare expressions of D1 and D3 in the livers of a randomly selected subset of these NAFLD patients with either mild (F0 -1; n ϭ 6) or advanced (F3-4; n ϭ 6) liver fibrosis and healthy controls without known liver disease (n ϭ 4). Compared with healthy controls, NAFLD patients demonstrated reduced expression of D1 and increased expression of D3. In the mild NAFLD group, the intensity of D1 staining was generally less than observed in healthy controls, and rare patches of parenchyma that completely lacked hepatocyte D1 staining were noted in some patients. Moreover, in the group with advanced NAFLD, large areas of D1-negative hepatocytes were common ( Figure 6, A and C). Conversely, both the intensity and extent of D3 staining increased in parallel with fibrosis stage in NAFLD patients, with consistently strong sinusoidal accumulation of D3-positive cells in all patients with advanced NAFLD fibrosis ( Figure 6, B and C). Thus, like mice with BDL-induced liver fibrosis, humans with NAFLD-related liver fibrosis acquire a pattern of hepatic deiodinase expression that is reminiscent of developmental organogenesis. Because this D1/D3 switch is predicted to limit local exposure to T 3 , we evaluated hepatic expression of THRSP (a T 3 responsive gene) in our entire NAFLD cohort. THRSP mRNA levels in patients with advanced NAFLD fibrosis were significantly lower than in patients with mild NAFLD fibrosis ( Figure 6D), reflecting fibrosis-related changes in D1 and D3 and worsened intrahepatic hypothyroidism with advancing fibrosis (Table 1).

Discussion
This study demonstrates, for the first time, that liver myofibroblasts and canonical Hedgehog signaling are key regulators of significantly contribute to intrahepatic changes in thyroid hormone homeostasis observed in a euthyroid sickness model after liver TH homeostasis during adult liver injury. These findings provide novel insight into both the mechanisms for, and the implications of, the euthyroid sick syndrome, showing that it is caused, in part, by stromal cell responses to tissue injury that limit exposure to T 3 , a factor that normally promotes cellular differentiation. Moreover, the results reveal that the liver fibrosis-associated euthyroid sick state is a mild form of peripheral tissue consumptive hypothyroidism mediated by the liver stromal cell up-regulation of D3 and reflected by detectable changes in serum levels of the D3 product rT 3 . D1 and D3 are known to be differentially expressed during tissue injury (5)(6)(7)(8). By studying mice and humans with fibrosing liver injury, we discovered that this reflects cell type-specific changes in deiodinase production. Specifically we showed that hepatocytes, the predominant D1-expressing cell type in healthy livers, down-regulate their expression of D1 during liver injury. In addition, we found that expression of this enzyme is also suppressed in various liver stromal cell types, whereas ductular cells accumulate D1. Moreover, we demonstrated that concomitant injury-related increases in D3 are caused by the upregulation of DIO3 expression in stromal cells and hepatic accumulation of such cells because hepatocytes remain predominantly D3 negative in chronically injured livers. This switch from TH-activating to TH-deactivating enzyme predominance during liver fibrosis may have important implications for liver repair because D3 predominance has been noted in relatively undifferentiated tissues, including developing embryos and various cancers (3,25,26). The concept that increased D3 activity regulates tissue construction is further supported by evidence that knocking down D3 in adult zebrafish inhibited fin regeneration after amputation (27). Although the roles of TH during adult liver injury remain to be determined, it is noteworthy that significant up-regulation of D3 has been reported to occur acutely in rats after partial hepatectomy (5). In that study, however, concomitant suppression of DIO1 was not observed. On the other hand, in rodents and humans with chronic liver injury and fibrosis, we demonstrated that D1 and D3 were reciprocally regulated and found that the net effect was increased accumulation of rT 3 relative to active T 3 . Reduced hepatic exposure to T 3 was accompanied by the suppression of TH-regulated gene expression, supporting the concept that local exposure to that important differentiating factor was limited. It is important to note that, although a majority of cells in advanced fibrosis remain hepatocytes, they represent a smaller proportion of liver cells per area secondary to an increase in nonparenchymal cell populations after injury. Therefore, it is pos-sible that a small component of the 80% decrease in THRSP with advanced fibrosis in both rats and humans could be secondary to smaller proportions of hepatocytes per liver area.
Indeed, our work identified Hedgehog, an injury-induced signaling pathway, to be a key regulator of net hepatic deiodinase expression during liver injury. Treating cultured liver myofibroblasts with Hedgehog ligand increased their expression of DIO3 mRNA. Conversely, targeted disruption of Hedgehog signaling in liver myofibroblasts of ␣sma-Cre/smo flx/flx mice suppressed their myofibroblastic phenotype and not only prevented injuryrelated inhibition of D1 and induction of D3 in these cells but also abrogated loss of D1 expression in neighboring hepatocytes. The latter finding suggests that Hedgehogresponsive myofibroblasts ordinarily inhibit hepatocyte D1 expression during liver injury, possibly by limiting availability of T 3 . Further research is needed to clarify this. Nevertheless, the available data in experimental animals indicate that Hedgehog-responsive myofibroblasts are key determinants of hepatic deiodinase expression and thereby, local TH activity during liver injury.
Consistent with that concept, we found that intrahepatic concentrations of TH were higher after liver injury in mice that were depleted of Hedgehog-responsive myofi- broblasts. Like mice, humans demonstrate Hedgehog pathway activation during cholestatic liver injury, NAFLD, and other forms of liver disease (28 -30). Indeed, in both species, the level of Hedgehog pathway activity generally parallels the severity of myofibroblast accumulation and liver fibrosis (28). More significantly, in humans with an NAFLD-related liver injury but normal serum levels of T 4 , we demonstrated strong correlations between advanced liver fibrosis (a state of increased hepatic Hedgehog pathway activity and liver myofibroblast accumulation) (29), hepatic D3 induction and serum rT 3 accumulation, suppression of intrahepatic T 3 levels, and the inhibition of T 3 -responsive hepatic gene expression. The aforementioned Hedgehog-directed changes in deiodinase activity might not be unique to injured liver. Dentice et al (24) demonstrated a Gli2-responsive promoter element in the DIO3 gene and reported that DIO3 expression is Hedgehog dependent in keratinocytes. This suggests that T 4 might be preferentially converted to rT 3 , a relatively inert TH whenever Hedgehog pathway activity increases, thereby engendering localized pockets of peripheral tissue consumptive hypothyroidism in individuals with normal thyroid glands. Moreover, because the liver generates as much as 30%-50% of serum T 3 (4), various types of liver disease that cause progressive liver fibrosis and sustained local hepatic activation of D3 might cause clinically significant systemic consumptive hypothyroidism. This possibility is supported by evidence that serum TSH elevations and frank hypothyroidism are common in many types of chronic liver disease (31,32). An analogous situation has been reported to occur in young children with infantile hepatic hemangioendothelioma. In those individuals, D3-expressing tumor-associated stromal cells generate an enormous requirement for T 4 that necessitates TH supplementation, and systemic hypothyroidism results when TH replacement is insufficient (33).
In summary, Hedgehog-regulated hepatic stromal cell responses that occur during adult liver repair shift the balance of local deiodinase expression to favor accumulation of biologically inert TH at the expense of biologically active TH. Although additional research is needed to clarify exactly how this process impacts liver repair, evidence suggests it might alter hepatic differentiation (3)(4)(5) as well as systemic TH homeostasis, thereby contributing to negative outcomes of fibrosing liver injury. As such, D3 may be a new therapeutic target in liver fibrosis, and its product, rT 3 , a novel biomarker of that process.