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Recombinant Human Thyrotropin Reduces Endothelium-Dependent Vasodilation in Patients Monitored for Differentiated Thyroid Carcinoma

Department of Internal Medicine, University of Pisa, 56126 Pisa, Italy

Address all correspondence and requests for reprints to: Fabio Monzani, M.D., Department of Internal Medicine, University of Pisa, via Roma 67, 56126 Pisa, Italy. E-mail: fmonzani{at}med.unipi.it.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Aim: We evaluated endothelial-dependent vasodilation after administration of recombinant human TSH (rhTSH) in patients monitored for differentiated thyroid carcinoma. The role of inflammation and oxidative stress was also assessed.

Protocol: Twenty-four patients (21 women, mean age 40.5 ± 9.2 yr) received rhTSH (0.9 mg daily) on 2 consecutive days. At baseline and the day after the second rhTSH injection, endothelium-dependent vasodilation as flow-mediated dilation (FMD, induced by 5 min of forearm ischemia) and endothelium-independent vasodilation (glyceril trinitrate 25 µg, sublingual) were evaluated by high-resolution ultrasound in the brachial artery. At each experimental time, blood was drawn for the evaluation of thyroglobulin, TSH, free T₃, free T₄, as well as IL-6, C reactive protein, TNF, lipoperoxides, and ferric reducing antioxidant power levels as markers of inflammation and oxidative stress.

Results: At baseline, patients’ serum TSH values were below the normal range [0.12 mIU/liter (range 0.01–0.30)] in the face of normal free T₄ and free T₃ levels; FMD (8.9 ± 3.4 vs. 9.2 ± 3.1%, respectively) and response to glyceril trinitrate (11.0 ± 4.3 vs. 10.8 ± 4.7%, respectively) were similar in patients and controls. All the patients had serum thyroglobulin value less than 1 ng/ml, suggesting the absence of cancer recurrences. Besides the expected elevation of serum TSH, rhTSH induced a significant impairment of FMD (7.4 ± 3.0 vs. 8.9 ± 3.4%; P < 0.01) along with a significant elevation of blood IL-6 (P = 0.01), TNF (P < 0.001), and lipoperoxide levels (P = 0.01), as well as a reduction of ferric reducing antioxidant power (P = 0.01).

Conclusions: rhTSH administration acutely impaired endothelium-dependent vasodilation, possibly through the induction of low-grade inflammation and reduced nitric oxide availability by oxidative stress.

	Introduction

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HYPOTHYROIDISM PROMOTES SYSTEMIC arterial hypertension and hypercholesterolemia, and their combination enhances the atherosclerotic process (1, 2). Endothelial dysfunction is an early step in the development of atherosclerosis and is associated with an increased risk of cardiovascular events (3). Endothelium-dependent vasodilation impairment is characterized by decreased nitric oxide (NO) availability and, frequently, increased oxidative stress (4, 5). The measurement of flow-mediated dilation (FMD) in the brachial artery may be considered as an indirect index of NO bioavailability (6). A progressive impairment of FMD in hypothyroid patients as well as in subjects with high-normal serum TSH levels has been reported (7). To date, the pathophysiological role of TSH per se on endothelial dysfunction is still an unresolved question. Several studies have demonstrated the presence of functional TSH receptors in bone marrow cells (8), in cardiomyocytes (9), in human coronary artery smooth muscle cells (10), and in human endothelial cells (11). Furthermore, recent studies reported that TSH is able to induce IL-6 and TNF secretion in vitro (12, 13), and may be involved in the modulation of vascular function by increasing NO metabolites in vivo (14), thus suggesting a possible link between TSH increase and a low grade of inflammation and oxidative stress.

The aim of the present study was to evaluate in vivo the endothelial-dependent vasodilation as assessed by FMD after administration of recombinant human TSH (rhTSH) in patients monitored for differentiated thyroid carcinoma (DTC). The role of inflammation and oxidative stress in inducing a possible endothelial dysfunction was also assessed.

	Patients and Methods

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The study group included 24 patients (21 women) who had undergone total thyroidectomy for DTC followed by ablative radioiodine treatment and routine rhTSH (Thyrogen; Genzyme, Cambridge, MA) testing and whole-body ¹³¹I scanning. All the patients received TSH suppressive L-T₄ therapy (1.7–2.2 µg/kg); none had positive serum antithyroglobulin (Tg-Ab) antibody titers. Obese patients [body mass index (BMI) > 30 kg/m²], smokers, and patients affected by diabetes mellitus, systemic hypertension, prior cardiovascular events, renal and hepatic failure, or other systemic diseases were excluded. All women were premenopausal with regular menses; no patient received lipid-lowering agents or other drugs affecting vascular function during the study period.

Twenty euthyroid subjects, matched to the patient group for sex, age, BMI, blood pressure, and lipid profile, were recruited among staff and relatives of patients and served as the control group. All study subjects gave written informed consent to the study protocol, which was approved by the local ethical committee.

Vascular ultrasonographic scans were performed in the morning, with subjects supine, in a quiet air-conditioned room (22–24 C). A B-mode scan of the right brachial artery was obtained in longitudinal section between 5 and 10 cm above the elbow using a 7.0-MHz linear array transducer (AU5; ESAOTE, Florence, Italy) as already described (15). Briefly, the transducer was held at the same point throughout the scan by a stereotactic clamp. End-diastolic frames (electrocardiogram-triggered) were acquired every second on a personal computer using a commercial software program (miroVIDEO DC30/plus; Pinnacle Systems GmbH, Braunschweig, Germany). Arterial flow velocity was obtained by pulsed Doppler signal at 70 degrees to the vessel with the range gate (1.5 mm) in the center of the artery. A cuff was placed around the forearm just below the elbow.

All the patients received two consecutive im injections of rhTSH (0.9 mg/day), 24 h apart. At baseline and the day after the second administration of rhTSH, blood samples were collected for the determination of serum thyroglobulin (Tg), TSH, free T₄ (FT₄), free T₃ (FT₃), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Moreover, at each time point, IL-6, C reactive protein (CRP), and TNF levels were assayed as markers of systemic inflammation whereas, lipoperoxide (LOOH) levels as well as ferric reducing antioxidant power (FRAP) were assessed as markers of oxidative stress. Blood samples were stored at –80 C and assayed in the same run for each parameter.

Vascular responses were evaluated at baseline and the day after the second rhTSH injection. Endothelium-dependent vasodilation was assessed as dilation of the brachial artery induced by forearm reactive hyperemia (RH) (cuff inflated for 5 min at 250 mm Hg and then deflated). Endothelium-independent dilation was obtained by administration of a low sublingual dose (25 µg) of glyceril trinitrate (GTN), a dose previously tested to induce a vasodilation similar to FMD.

FMD was calculated as the maximal percent increase in diameter of the brachial artery above baseline after RH. The area under the curve of percent increase in brachial artery during 3 min after cuff release was also calculated. The response to GTN was calculated as the maximal percent increase in diameter of the brachial artery above baseline after GTN administration.

Measurements were performed on acquired frames by a computerized edge detection system

Blood flow volume was calculated by multiplying Doppler flow velocity (corrected for the angle) by heart rate and vessel cross-sectional area (*r²). Flow velocity was measured at baseline and within 15 sec after cuff release. RH was calculated as percent increase in flow after cuff release compared with baseline flow. In our laboratory, the coefficient of variation for FMD repeated measures is 8%

Serum FT₃ and FT₄ levels were measured by specific RIA, and TSH by ultrasensitive immunoradiometric assay (IRMA) (Techno-Genetics, Milan, Italy). Serum Tg-Ab values were evaluated by specific RIA and Tg levels by IRMA (SELco anti-Tg and SELco Tg, Berlin, Germany). Plasma IL-6 levels were evaluated by specific IRMA (BioSource, Nivelles, Belgium); TNF and CRP values were measured by ELISA (R&D Systems Gmbh, Wiesbaden-Nordenstadt, Germany). Plasma LOOH levels were measured by a colorimetric method, and FRAP was measured by spectrophotometric assay (16). Oxidative stress parameters’ variability was assessed in 40 healthy subjects: intraassay variability was found to be 3% for FRAP and 7% for LOOH, whereas interassay variability was 10 and 12%, respectively. Normal ranges are: FT₄ = 8.6–18.6 pg/ml (11.0–23.9 pmol/liter); FT₃ = 2.1–4.6 pg/ml (3.2–7.1 pmol/liter); TSH = 0.3–3.6 mU/liter.

Data are expressed as mean ± SD or median and range as appropriate. Statistical analysis was performed using the Student’s t test, ANOVA, Mann-Whitney U test, and Spearman correlation test, as appropriate (SPSS version 11.0; Chicago, IL). Significance was assumed for P < 0.05.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
At baseline, patients and controls were well matched with respect to age, sex, BMI, lipid profile, and blood pressure (Table 1).

FMD (8.9 ± 3.4 vs. 9.2 ± 3.1%, respectively) and response to GTN (11.0 ± 4.3 vs. 10.8 ± 4.7%, respectively) were similar in patients and controls. No difference was found in both RH (patients, 537 ± 328%; controls, 540 ± 257%) and brachial artery diameter (patients, 3.2 ± 0.6 mm; controls, 3.3 ± 0.5 mm).

As expected, rhTSH administration induced a remarkable elevation of serum TSH levels (P < 0.0001), whereas both FT₄ and FT₃ values did not change (Table 1). Serum Tg levels were unaffected by rhTSH (0.4 ± 0.6 vs. 0.3 ± 0.3 ng/ml), thus confirming the absence of functional thyroid tissue. Similarly, serum lipid profile and blood pressure values remained unchanged.

After rhTSH administration a significant elevation of serum IL-6 (P = 0.01) and TNF levels (P < 0.001) along with a slight not significant increase of CRP values was observed. Concomitantly, plasma LOOH levels were significantly increased (P = 0.01), whereas FRAP was significantly reduced (P = 0.01) (Table 1).

Brachial artery diameter (from 3.2 ± 0.6 to 3.2 ± 0.5 mm) and RH (from 537 ± 328 to 515 ± 258%) were unaffected by rhTSH testing. Similarly, endothelium-independent vasodilation in response to GTN remained unchanged (11.5 ± 4.3 vs. 11.0 ± 4.3%). On the contrary, a significant reduction of endothelium-dependent vasodilation expressed either as FMD (7.4 ± 3.0 vs. 8.9 ± 3.4%; P < 0.01) (Fig. 1) or as area under the curve (505.1 ± 316.2 vs. 684.7 ± 367.6 U; P < 0.001) was elicited by rhTSH.

View larger version (13K): [in this window] [in a new window]   FIG. 1. FMD expressed as maximal percent increase in diameter of the brachial artery after RH at baseline and after rhTSH testing in 24 patients with differentiated thyroid cancer. Results are reported either in each patient (left panel) or as the mean (±SD) value (right panel). *, P < 0.01 vs. baseline.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The current study assessed the acute effects of TSH on endothelial function and tested the possible involvement of inflammation and oxidative stress. Patients submitted to rhTSH testing for the follow-up of DTC represent an ideal model to study the acute effects of TSH per se because these patients receive long-lasting L-T₄ suppressive therapy and maintain thyroid hormone levels well within the normal range both at baseline and after rhTSH administration. Moreover, vascular parameters were studied shortly (48 h) after rhTSH testing, in the absence of significant variations of both clinical and lipid parameters. Because thyroid follicular cells can synthesize and secrete proinflammatory cytokines (17), we selected patients without evidence of functional thyroid tissue (i.e. Tg levels lower than 1 ng/ml, both at baseline and after rhTSH testing). Because it is well established that ¹³¹I induces in vivo oxidative injury by increasing isoprostanes levels (18) and in vitro by inducing micronuclei in peripheral lymphocytes (19), we evaluated vascular responses and oxidative stress parameters before ¹³¹I administration.

Our data demonstrate that acutely raising serum TSH into the supraphysiological range leads to an acute impairment of endothelium-dependent vasodilation, possibly through the induction of low-grade inflammation and reduced NO availability by oxidative stress. Although it cannot be excluded that the extrathyroidal effects of TSH and rhTSH may differ, these findings imply that TSH per se may play an independent role in the early atherosclerotic process of hypothyroid patients.

In particular, short-term rhTSH administration induced a significant reduction of FMD without affecting the endothelium-independent response. Moreover, rhTSH produced a significant increase in blood IL-6, TNF, and LOOH levels, as well as a significant decrease in total antioxidant power. It is noteworthy that these changes in parameters of oxidative stress and inflammation occurred in the absence of other clinical modifications. Thus, it is conceivable that the proinflammatory process relies essentially on raised serum TSH level. This interpretation is in keeping with the evidence that TSH directly induces TNF secretion by bone marrow cells (13) and IL-6 by adipocytes (12). TNF is a pivotal NO-controlling cytokine, and elevated TNF levels may promote the expression of inducible NO synthase, leading to increased oxidative stress (20). This mechanism may be implicated in the increased NO metabolites seen in patients undergoing rhTSH testing for the follow-up of DTC (15). In this setting, a direct effect of thyroid hormones on endothelial function can be excluded because patients did not stop LT₄ therapy, and serum thyroid hormone concentrations remained unchanged throughout the study.

In conclusion, the current results indicate that rhTSH administration acutely induces a significant impairment of endothelium-dependent vasodilation with a concomitant reduction of plasma antioxidant capacity and increased production of proinflammatory cytokines in the face of normal thyroid hormone levels. Whether these pharmacological responses reproduce the effects of smaller but protracted elevations in TSH—such as those obtained in chronic hypothyroidism—remains to be proven.

	Footnotes

 
These data were partly presented as oral communication at the 13th International Thyroid Congress, Buenos Aires, Argentina, October 30 to November 4, 2005.

Disclosure statement: The authors have nothing to disclose.

First Published Online July 25, 2006

Abbreviations: BMI, Body mass index; CRP, C reactive protein; DTC, differentiated thyroid carcinoma; FMD, flow-mediated dilation; FRAP, ferric reducing antioxidant power; FT₃, free T₃; FT₄, free T₄; GTN, glyceril trinitrate; IRMA, immunoradiometric assay; LOOH, lipoperoxide; NO, nitric oxide; rhTSH, recombinant human TSH; RH, reactive hyperemia; Tg, thyroglobulin.

Received February 27, 2006.

Accepted July 19, 2006.

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