Enhancement of Vascular Endothelial Function by Recombinant ...

ORIGINAL E n d o c r i n e

ARTICLE R e s e a r c h

Enhancement of Vascular Endothelial Function by Recombinant Human Thyrotropin Raffaele Napoli, Bernadette Biondi, Vincenzo Guardasole, Carolina D’Anna, Antonietta De Sena, Concetta Pirozzi, Daniela Terracciano, Claudia Mazzarella, Margherita Matarazzo, and Luigi Sacca` Departments of Internal Medicine and Cardiovascular Sciences (R.N., V.G., C.D., A.D.S., C.P., M.M., L.S.), Clinical and Molecular Endocrinology and Oncology (B.B.), and Cellular and Molecular Pathology (D.T., C.M.), University Federico II, 80131 Naples, Italy

Context: The cardiovascular consequences of thyroid diseases are attributed to the altered secretion of thyroid hormones. The possibility that TSH also affects the cardiovascular system has been poorly explored. Endothelial cells and vascular smooth muscle cells possess TSH receptors. Objective: The study was designed to determine whether TSH exerts any effect on vascular homeostasis. Subjects and Methods: Two different double-blind, controlled studies were performed, one in eight healthy volunteers and the other in six thyroidectomized patients. Recombinant human (rh) TSH (or saline) was infused intrabrachially (1 mU/min) to raise TSH to severe hypothyroidism levels (⬃100 ␮U/ml). Endothelium-dependent and -independent vasodilation was tested by intraarterial infusion of acetylcholine and sodium nitroprusside, respectively, and forearm blood flow was measured by plethysmography. Results: Endothelium-dependent vasodilation was potentiated by rhTSH (P ⬍ 0.05 for the treatment effect; general linear model). The dynamics of the response was also profoundly affected by rhTSH because the dose-response curve was much steeper than in controls (P ⬍ 0.02 for the interaction between TSH and acetylcholine). rhTSH had no effect on endothelium-independent vasodilation (P ⫽ NS for both treatment and interaction). During rhTSH infusion, free T3 levels increased slowly from 2.3 ⫾ 0.2 to 3.6 ⫾ 0.2 pg/ml. In thyroidectomized patients, rhTSH potentiated endothelium-mediated vasodilation to an extent similar to that of healthy subjects (P ⫽ 0.05 for the treatment effect and P ⫽ 0.01 for the interaction), without affecting the response to nitroprusside. In these patients, thyroid hormones remained unchanged during rhTSH infusion. Conclusions: rhTSH exerts marked effects on the resistance vessels by enhancing endothelialmediated vasodilation, independent of changes in thyroid hormone concentration. (J Clin Endocrinol Metab 93: 1959 –1963, 2008)

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hereas there is solid evidence for a role of thyroid hormones in cardiovascular physiology (1), the question of whether TSH exerts any effect on the heart and vascular system has been poorly investigated. Several human cell lines, including endothelial and vascular smooth muscle cells, are endowed with TSH receptors (2– 4). This raises the important question whether TSH binding to these receptors has any physiologic relevance. Indeed, when in vitro cultured human endothelial cells are ex-

posed to TSH excess, cAMP concentration rapidly increases in conjunction with an increase in nitric oxide (NO) and a fall in the vasoconstrictor endothelin (4). In line with these findings, a significant increase in the circulating NO levels is observed in thyroidectomized patients treated with recombinant human (rh) TSH in the context of their clinical follow-up (5). Translated into circulatory physiology, these observations make TSH a likely candidate to affect endothelial function in vivo and to exert rel-

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Abbreviations: Ach, Acetylcholine; FBF, forearm blood flow; FMD, flow-mediated dilation; FVR, forearm vascular resistance; NO, nitric oxide; NP, sodium nitroprusside; rh, recombinant human.

Printed in U.S.A. Copyright © 2008 by The Endocrine Society doi: 10.1210/jc.2007-2797 Received December 19, 2007. Accepted February 27, 2008. First Published Online March 11, 2008

J Clin Endocrinol Metab, May 2008, 93(5):1959 –1963

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evant hemodynamic effects. Definite proof of this TSH action would be of interest not only from a physiologic point of view but also to disclose a potential contribution of altered TSH levels, independent of thyroid hormones, to the cardiovascular abnormalities associated with thyroid diseases. The aim of the present study was to determine whether changes in circulating TSH levels affect vascular homeostasis. The classic approach based on the forearm technique and plethysmographic measurement of blood flow was used to assess the effect of TSH on endothelial- and nonendothelial-dependent mechanisms and the vascular sensitivity to vasoactive agents.

Subjects and Methods

Assays and calculations

Subjects Two different studies were done to assess the vascular effects of TSH. The first was performed in eight healthy volunteers, recruited from the population of the medical school. The second study included six patients who had undergone total thyroidectomy 2–5 yr earlier. The patients were under replacement therapy with levothyroxine that was omitted the day of the study. The clinical characteristics of healthy subjects and patients are summarized in Table 1. The reason for thyroidectomy was papillary carcinoma in five patients and multinodular goiter in one patient. Thyroid cancer was classified as low risk in all five patients (undetectable thyroglobulin after TSH stimulation and negative ultrasound exploration of the neck). Total thyroidectomy provides a unique model that allows one to exclude with certainty that any potential effect of TSH on vascular function may be, even to a small extent, mediated by thyroid hormones. Informed written consent was obtained from all subjects and the study protocol was approved by the Ethics Committee of the Federico II University School of Medicine.

Experimental procedures The experimental procedures were identical in the two studies. The study design was double blinded and controlled, each subject or patient serving as his/her own control. The test (TSH) and control study (saline) were performed in random order and separated by a 3- to 4-wk interval. In the test study, rhTSH (Thyrogen; Genzyme, Cambridge, MA) was infused into the brachial artery, and forearm hemodynamics were monitored for 240 min. Then the responses of forearm blood flow (FBF) to vasoactive agents were measured while keeping the infusion of rhTSH ongoing. In the control study, the protocol was the same except that saline was infused instead of rhTSH. Both the patients and the plethysmographic operators were blinded to the infusate content (rhTSH or saline). The dose of rhTSH was 1 mU/min. Based on a FBF of 2.5 ml/100 ml/min, this dose is expected to raise TSH concentration in the forearm circulation to levels observed in severe primary hypothyroidism. All the experiments were performed in the morning in a quiet room kept at 22–24 C. A plastic cannula (20-gauge) was inserted into the brachial artery of the nondominant arm under local anesthesia and used for the infusion of the test substances and the monitoring of arterial blood pressure. Systolic and diastolic blood pressure and heart rate were recorded by a transducer connected to the arterial cannula. FBF was measured by a strain-gauge plethysmograph (Hokanson 045 EC4; PMS In-

TABLE 1.

struments, Berks, UK). The data were monitored continuously with McLab software. Further details of the procedure have been previously published (6, 7). Each subject underwent the following stepwise infusions into the brachial artery in this order: 1) acetylcholine (Ach) infused at a rate of 15, 30, 45, and 60 ␮g 䡠 liter of forearm⫺1 per minute⫺1 to assess endothelium-mediated vasodilation; and 2) sodium nitroprusside (NP), a direct NO donor, infused at the rate of 1, 3, and 9 ␮g 䡠 liter⫺1 䡠 min⫺1 to assess nonendothelium-mediated vasodilation. At least 30 min of washout time were allowed between each substance. Test substances were infused in the same order in all the subjects, and the infusion of each substance started only when the effect on FBF of the previous infusion was dissolved and near-baseline FBF restored. Each dose of the test substances was infused for 5.5 min, and FBF was measured during the last 1.5 min of infusion. Each FBF value represents the mean of six consecutive measurements performed at 10-sec intervals.

Serum levels of free T3 and T4 were measured by RIA and serum TSH by ultrasensitive immunoradiometric assay. Forearm vascular resistance (FVR) was measured as the ratio of mean arterial blood pressure to FBF. Comparison between TSH and the control study was performed by a two-way ANOVA for repeated measures (general linear model, version 13.0; SPSS Inc., Chicago, IL). Results are expressed as the mean ⫾ SEM.

Results The study was well tolerated by all subjects without any systemic reaction. In the study performed in healthy subjects, the local, intrabrachial infusion of rhTSH raised the plasma venous concentration of TSH in the ipsilateral forearm from 1.8 ⫾ 0.2 to 105 ⫾ 17 ␮U/ml after 4 h of infusion. No change in TSH concentration was observed in the saline control study. The systemic free T3 concentration showed a slight and progressive increase from the basal value of 2.3 ⫾ 0.2 to 3.6 ⫾ 0.2 pg/ml at 240 min during TSH infusion, whereas it remained unchanged in the control study. Arterial systolic and diastolic blood pressure, heart rate, and FBF did not show any change from baseline during the 4-h TSH infusion (Table 2). At baseline, the FVR values in the healthy subjects were higher in the placebo study due to lower values of FBF. However, the difference was not statistically significant. Likewise, no change in FVR was observed during the subsequent infusion period (Table 2). Figure 1 depicts the dose-response curves to vasoactive agents in healthy subjects. In the TSH study, the increment of FBF in response to the endothelium-dependent vasodilator Ach was more pronounced as compared with the control study (P ⬍ 0.05 for the effect of TSH treatment). The rate of the vasodilatory response was also profoundly affected by TSH, as evidenced by the significant interaction between TSH and Ach (P ⬍ 0.02). This indicates that TSH did not simply displace upward the doseresponse curve but was able to change the intrinsic dynamics of

Clinical characteristics of the healthy subjects (study I) and thyroidectomized patients (study II)

Study I (n ⫽ 8) Study II (n ⫽ 6)

Age (yr)

Sex (M/F)

BMI (kg/m2)

SBP (mm Hg)

DBP (mm Hg)

HR (beats/min)

fT3 (pg/ml)

fT4 (ng/ml)

TSH (␮U/ml)

Levothyroxine (␮g/d)

24 ⫾ 2 43 ⫾ 4

5/3 4/2

22.5 ⫾ 0.9 29.3 ⫾ 0.9

120 ⫾ 5 134 ⫾ 4

75 ⫾ 6 69 ⫾ 4

81 ⫾ 8 73 ⫾ 7

2.3 ⫾ 0.2 2.5 ⫾ 0.3

1.2 ⫾ 0.1 1.6 ⫾ 0.2

1.84 ⫾ 0.18 0.25 ⫾ 0.15

166 ⫾ 16

BMI, Body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; fT3, free T3; fT4, free T4.


TABLE 2.


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Effects of intrabrachial rhTSH infusion on FBF, mean blood pressure, heart rate, and vascular resistance

Study I (healthy subjects) FBF (ml per 100 ml/min) Mean blood pressure (mm Hg) Heart rate (beats/min) FVR (resistance units) Study II (thyroidectomized patients) FBF (ml per 100 ml/min) Mean blood pressure (mm Hg) Heart rate (beats/min) FVR (resistance units)

0 min

60 min

120 min

180 min

240 min

Placebo rhTSH Placebo rhTSH Placebo rhTSH Placebo rhTSH

1.6 ⫾ 0.2 2.5 ⫾ 0.5 89 ⫾ 5 92 ⫾ 5 81 ⫾ 8 81 ⫾ 9 61.8 ⫾ 11.7 46.7 ⫾ 10.3

1.7 ⫾ 0.2 2.5 ⫾ 0.5 88 ⫾ 4 92 ⫾ 5 76 ⫾ 10 67 ⫾ 3 56.7 ⫾ 7.4 49.2 ⫾ 9.5

2.2 ⫾ 0.4 2.8 ⫾ 0.6 88 ⫾ 5 94 ⫾ 6 81 ⫾ 11 71 ⫾ 6 48.1 ⫾ 6.0 56.2 ⫾ 17.3

2.1 ⫾ 0.4 2.3 ⫾ 0.4 89 ⫾ 5 95 ⫾ 5 76 ⫾ 9 71 ⫾ 5 57.1 ⫾ 12.3 54.3 ⫾ 12.6

2.0 ⫾ 0.3 2.7 ⫾ 0.5 89 ⫾ 5 97 ⫾ 6 76 ⫾ 9 71 ⫾ 4 53.3 ⫾ 8.4 43.8 ⫾ 7.5

Placebo rhTSH Placebo rhTSH Placebo rhTSH Placebo rhTSH

2.6 ⫾ 0.7 3.0 ⫾ 0.9 90 ⫾ 4 88 ⫾ 3 73 ⫾ 7 65 ⫾ 7 43.6 ⫾ 8.1 39.4 ⫾ 7.4

2.7 ⫾ 0.8 3.0 ⫾ 0.7 92 ⫾ 5 87 ⫾ 2 70 ⫾ 7 63 ⫾ 6 44.7 ⫾ 9.3 36.2 ⫾ 7.3

2.8 ⫾ 0.9 3.4 ⫾ 1.2 90 ⫾ 5 90 ⫾ 3 71 ⫾ 8 64 ⫾ 6 45.6 ⫾ 7.8 39.9 ⫾ 8.3

2.6 ⫾ 0.6 3.2 ⫾ 1.1 88 ⫾ 5 88 ⫾ 3 74 ⫾ 5 63 ⫾ 6 42.8 ⫾ 8.6 44.1 ⫾ 12.3

2.9 ⫾ 0.9 3.6 ⫾ 1.7 95 ⫾ 6 89 ⫾ 3 73 ⫾ 6 65 ⫾ 6 46.6 ⫾ 10.1 45.2 ⫾ 11.0

the endothelial response to the vasodilator stimulus. With regard to the response of FBF to NP, the dose-response curves were virtually identical in the TSH and control studies (P ⫽ NS for both the treatment effect and the interaction). As observed in healthy subjects, the 4-h, basal infusion of rhTSH in thyroidectomized patients did not cause any change in heart rate, arterial blood pressure, FBF, and FVR (Table 2). Figure 2 depicts the response to Ach and NP in thyroidectomized patients receiving rhTSH. rhTSH potentiated endothelium-mediated vasodilation to a similar extent to that observed in healthy subjects (P ⫽ 0.05 for the treatment effect and P ⫽ 0.01 for the interaction), without affecting the response to NP. TSH levels rose from 0.25 ⫾ 0.15 to 76 ⫾ 19 mU/liter, whereas free T3 and free T4 remained unmodified (T3: 2.38 ⫾ 0.28 and 2.20 ⫾ 0.29 pg/ml in the basal state and after TSH, respectively; T4: 1.62 ⫾ 0.18 and 1.76 ⫾ 0.39 ng/ml in the basal state and after TSH, respectively).

Discussion Patients with thyroid disease present with a variety of cardiovascular complications. In particular, hypothyroidism is characterized by increased peripheral vascular resistance, endothelial dysfunction, and propensity to develop atherosclerosis and clinical vascular events (8 –10). These effects have been attributed to the changes in thyroid hormone secretion or the inflammatory process associated with thyroid disease. The question of whether the abnormal TSH levels secondary to impaired thyroid hormone secretion also play a role has been poorly investigated. In general, it is not known whether TSH is able to affect vascular function. Such a possibility is suggested by several observations: 1) when rhTSH is administered to thyroidectomized patients for reasons inherent in their clinical follow-up, the circulating levels of NO rise significantly (5), and this finding is particularly pertinent, given the pivotal role of NO in vasomotion and vascular risk; 2) TSH receptors are expressed by several cell lines in the

cardiovascular system, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells (2– 4, 11); and 3) both in vitro and in vivo studies have shown that TSH stimulates the production of the inflammatory cytokines TNF␣ and IL-6 (12, 13), which may exert adverse effects on endothelial function. In view of this quite controversial background, we designed this study in an attempt to provide a clear answer to the question of whether an acute increase in TSH levels exerts any effect on the resistance vessels in humans. The results of the study performed in healthy subjects provide strong evidence for an effect of TSH to activate the endothelial component of vascular reactivity. This effect of TSH was remarkable in terms of both the difference in the absolute FBF values and the rate of the vasodilatory response. In contrast, the response to NP, an endothelial-independent vasodilator, remained unchanged during rhTSH infusion, indicating that the hormone has no direct effect on the smooth muscle cell compartment. Previous studies showed that TSH increased cAMP levels in isolated human aortic smooth muscle cells (2). This finding, supporting a direct vascular effect of TSH, is not incompatible with the lack of response of the smooth muscle cell compartment observed in the present study. The activation of cAMP in the smooth muscle cells may lead to many responses other than vasorelaxation, for which cGMP is the mediator. During rhTSH infusion, we observed a slight and progressive increase in free T3 concentration. Although this response was quite small, we could not exclude that it might have contributed, at least in part, to rhTSH effect to enhance endothelial reactivity. In a previous study from our laboratory, we showed that acute T3 increments activate endothelial function (7). This effect was observed in the presence of much higher T3 levels (10 –11 pg/ml) than those achieved in the present study during rhTSH infusion (3.6 pg/ml). Nevertheless, we wanted confirmation of our finding in normal subjects by means of a different model, in which a T3 response to rhTSH was impossible. For this purpose, we repeated the same protocol in a group of patients who had undergone total thyroidectomy and were under replacement therapy

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Acute TSH Effects on Vascular Function


30

Saline TSH

Forearm Blood Flow (ml/100ml/min)


25

20

15

10

25 20 15 10

5

5

0

0

0

15

30

45

Saline TSH

60

0

25



Saline TSH

20 15 10 5 0

0

1

3

30

45

60

Acetylcholine µg/l/min

Acetylcholine µg/l/min 25

15

9

Sodium Nitroprusside (µg/l/min) FIG. 1. FBF response to infusion of Ach or NP into brachial artery in rhTSH- or saline-treated subjects (n ⫽ 8). rhTSH or saline was infused for 4 h, and then vascular reactivity was tested while keeping the infusion ongoing. Data (means ⫾ SE) were analyzed by ANOVA for repeated measures (P ⬍ 0.05 for the effect of rhTSH in the Ach test and P ⬍ 0.02 for the interaction between rhTSH and Ach, and P ⫽ NS for both the treatment effect and the interaction in the NP test).

with levothyroxine. As expected, in this study rhTSH infusion was unable to cause any change in T3 concentration. Yet the vasodilatory response of the forearm resistance vessels was potentiated by rhTSH, and this response was entirely mediated by the endothelial-dependent component. The responsiveness of the smooth muscle cells was unaffected by rhTSH. These results are very similar to those observed in the protocol performed in healthy subjects, which supports the concept that rhTSH exerts endothelial effects through a mechanism independent of changes in the vascular exposure to thyroid hormones. In a previous study performed in thyroidectomized patients treated with rhTSH in the context of their clinical follow-up, flow-mediated dilation (FMD) of the brachial artery was found

Saline TSH

20 15 10 5 0

0

1

3

9

Sodium Nitroprusside (µg/l/min) FIG. 2. Response of FBF to infusion of Ach or NP into brachial artery in rhTSH- or saline-treated thyroidectomized patients (n ⫽ 6). rhTSH or saline was infused for 4 h and then vascular reactivity was tested while keeping the infusion ongoing. Data (means ⫾ SE) were analyzed by ANOVA for repeated measures (P ⫽ 0.05 for the effect of rhTSH in the Ach test and P ⫽ 0.01 for the interaction between rhTSH and Ach, and P ⫽ NS for both the treatment effect and the interaction in the NP test).

to be slightly reduced by rhTSH (13). The authors concluded that rhTSH acutely impairs endothelial function. There are several explanations for these divergent results. First, the FMD approach is entirely different from the approach used in the present study because FMD explores the conduit arterial system and not the resistance vessels. Second, only one measurement of FMD at a single point in time (48 h after rhTSH) was taken in the previous study and the possibility cannot be excluded that rhTSH might have exerted completely different effects at an earlier and really acute point after rhTSH administration. Thus, the conclusion previously reached that rhTSH acutely impairs endothelial function cannot be unequivocally made on the basis of the design


adopted. Finally, the view that rhTSH impairs endothelial function in large arteries is difficult to be reconciled with previous in vitro experiments showing that human aortic endothelial cells respond to TSH with a rapid increase in NO production (4). In summary, the results of the present study provide strong evidence for an acute effect of rhTSH to enhance vascular reactivity through a mechanism entirely involving endothelium-mediated vasodilation and independent of changes in thyroid hormones. Within the limitations of the acute setting of this study, our findings suggest that the augmented vascular risk associated with subclinical or clinical hypothyroidism is unlikely to involve the increased circulating levels of TSH. Finally, the present data point to rhTSH as a potential novel tool to induce quickly and safely a marked activation of endothelial function in vascular disease states.

Acknowledgments The authors thank Dr. V. Angelini, Dr. E. Zarra, and Dr. G. Albano for helpful discussions and recruiting some of the thyroidectomized patients. Address all correspondence and requests for reprints to: Raffaele Napoli, M.D., Department of Internal Medicine and Cardiovascular Sciences, Via Pansini 5, 80131 Napoli, Italy. E-mail: [email protected]. This work was presented as oral communication at the 78th Annual Meeting of American Thyroid Association, October 3–7, 2007, New York, New York. Disclosure Statement: The authors have nothing to disclose.

References 1. Klein I, Ojamaa K 2001 Thyroid hormone and the cardiovascular system. N Engl J Med 344:501–509


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