Hypothyroidism and Endothelial Function: A Marker of

Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2008, 2, 79-96

Hypothyroidism and Atherosclerosis?

Endothelial

Function:

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Angela Dardano and Fabio Monzani* Department of Internal Medicine, Endocrinology and Metabolism Section, University of Pisa, Italy Received: April 7, 2008; Accepted: April 21, 2008; Revised: May 14, 2008

Abstract: Endothelial dysfunction represents an important pathway thereby cardiovascular risk factors promote the development and progression of atherosclerosis. Hypothyroidism is associated with an increased cardiovascular risk, and the assessment of endothelium function is recognised an effective tool for the detection of evidence of preclinical cardiovascular alterations. Both vascular smooth muscle cells and endothelium play pivotal roles in modulating vascular tone and both are potential targets of thyroid hormone action. The pathogenesis of the association between endothelial dysfunction and hypothyroidism is complex and still not well established. The presence of traditional and emerging risk factors may contribute to the development of endothelium impairment, generating a chronic state of injury that triggers abnormal endothelial response. Levothyroxine replacement therapy is currently used for restoring euthyroidism and improving cardiovascular risk of hypothyroid patients. The decision to treat patients with subclinical hypothyroidism should depend on the presence of risk factors, rather than on a TSH threshold. However, the actual effectiveness of thyroid hormone substitution in reducing the risk of cardiovascular events, especially in subclinically hypothyroid patients, remains to be elucidated. Large multicenter, placebo-controlled prospective trials are necessary to address the issue. The article also discusses recent patents in this field.

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Keywords: Hypothyroidism, endothelium, cardiovascular risk, levothyroxine, DITPA, flow mediated dilation, atherosclerosis, dyslipidemia.

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INTRODUCTION

The endothelium is a monolayer of cells located between the blood lumen and vascular smooth muscle cells. Far from to be a static barrier, the endothelium is a dynamic endocrine organ, emerging as a major regulator of vascular function and remodelling through production of vasodilator and vasoconstrictor substances [1-3]. The key mediator of endothelial functions and the most important vasodilator substance produced by the endothelium is nitric oxide (NO) [1]. When the balance between vasodilation and vasoconstriction is upset, “endothelial dysfunction” or “endothelial activation” occurs and initiates a cascade of events that promote atherosclerosis, including NO breakdown, increased endothelial permeability, platelet aggregation, leucocyte adhesion and generation of pro-inflammatory cytokines [4]. It is well established that endothelial dysfunction represents an important pathway thereby cardiovascular risk factors promote the development and progression of atherosclerosis and is associated with an increased risk of cardiovascular events [5-8]. Hypothyroidism is associated with an increased cardiovascular risk [9-14], and the assessment of endothelium function is recognised an effective tool for the detection of evidence of preclinical cardiovascular alterations in early thyroid failure.

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In this article, we analyse the English literature on the association between endothelium dysfunction, characterized mainly by reduced NO availability, and hypothyroidism. We also speculate on the mechanisms involved in the pathogenesis of impaired endothelial function and the possible *Address correspondance to this author at the Department of Internal Medicine,University of Pisa,Via Roma 67, 56126 Pisa, Italy; Tel:++39 050993490; Fax: ++39 050553235; E-mail: [email protected] 1872-2148/08 $100.00+.00

effect of restoration of euthyroidism, while discussing recent patents in this field. NO AND ENDOTHELIAL FUNCTION

The healthy endothelium not only modulates the tone of the underlying vascular smooth muscle but also inhibits several proatherogenic processes, including monocyte and platelet adhesion and aggregation, oxidation of low-density lipoproteins, synthesis of inflammatory cytokines as well as smooth muscle proliferation and migration [15]. The key mediator of endothelial functions and the most important vasodilator substance produced by the endothelium is NO, originally identified as endothelium-derived relaxing factor [1]. NO derived from the conversion of its precursor Larginine into citrulline via the activity of the constitutive enzyme NO-Synthase (NOS), which is located in “caveolae” (invaginations in cell membranes). Three NOS isoenzymes are known: two are constitutively expressed in neurological tissue (nNOS) and endothelial cells (eNOS) and one is specially expressed in macrophage and endothelial cells through the effect of pro-inflammatory cytokines [inducible NOS (iNOS)]. Short term NO production by eNOS is induced by vasodilator substances likewise acetylcholine while, long-lasting NO synthesis by iNOS occurred when the stimulus comes from pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) [16,17]. The protein caveolin-1 binds to calmodulin to inhibit activity of eNOS, just as the binding of calcium to calmodulin displaces caveolin-1, activating eNOS and leading to NO production. This reaction requires a number of cofactors, including tetrahydrobiopterin and nicotinamide adenine dinucleotide phosphate [18]. Once formed NO, that is a very lipophilic compound, crosses the intima and © 2008 Bentham Science Publishers Ltd.

80 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2008, Vol. 2, No. 2

diffuses into the underlying smooth muscle cells, where it reacts with the heme group of soluble guanylate cyclase, resulting in enhanced synthesis of cyclic guanosine monophoshate (cGMP) [19]. cGMP activates protein kinase G, which in turn stimulates Ca2+ ATPase dependent refilling of intracellular calcium stores, thus mediating smooth muscle cell relaxation [20]. Besides acting as a vasodilator, NO also reduces vascular permeability, monocyte and platelet adhesion, tissue oxidation and inflammation, thrombogenic factors’ activation, cell growth, proliferation and migration as well as proatherogenic and pro-inflammatory cytokines’ expression [21-23]. The most important stimulation for NO release comes from shear stress, that is caused by the rise in blood velocity and leads to vasodilation proportional to the amount of NO released by the endothelium [24]. The endothelial cells contain membrane ion channels, such as Ca2+- activated K+ channels, opened in response to shear stress [25]. This vasodilation process is called “endothelium-dependent” (ED) dilation. Studies have identified hemodynamic shear stress as an important determinant of endothelial function and phenotype. Arterial-level shear stress (>15 dyne/cm2) induces endothelial quiescence and an atheroprotective gene expression profile, while low shear stress (6 mIU/L), had mean blood pressure significantly higher than controls; however, the results were not adjusted for age [100]. Other cross-sectional studies failed to demonstrate a significant difference in mean blood pressure or increased prevalence of hypertension in sHT as compared to euthyroid subjects [101-107]. In two case-control studies, Luboshitzky et al. [108,109] observed a significantly higher mean diastolic blood pressure in middle-aged sHT women than euthyroid controls. More recently, a significant increase of both diastolic blood pressure and brachial-ankle pulse wave velocity, a parameter of arterial stiffening, in sHT patients have been reported [110]. However, other casecontrol studies did not shown any association between sHT and increased blood pressure [11, 46, 87,111-113]. On the other hand, in line with the observation that subjects with high normal serum TSH value had either endothelial dysfunction or increased cholesterol levels, higher serum TSH levels in hypertensive compared with normotensive euthyroid subjects have been reported [51,114,115]. These results were confirmed in a recent large population-based study, including 5872 patients [116]. A familial aggregation of high-normal TSH concentrations in hypertensive families has also been reported [117]. Accordingly, Kanbay et al. [118] demonstrated that non-dipper hypertensive patients had lower free triiodo-thyronine (FT3) value compared to dipper patients; and, in the final regression model, the only independent predictor of non-dipper hypertension was FT3 . However, mean TSH level of non-dippers was 3.5+5.0 mIU/L, so including patients with mild hypothyroidism.

association between TH and lipoprotein profile [122,123], the largest being the Colorado Thyroid Disease Prevalence Study, which found a negative relationship between thyroid function and total (TC) and LDL cholesterol (LDLc) and triglyceride levels [124]. The relationship between overt hypothyroidism and dyslipidemia is well documented [10,124-131], and the prevalence of newly diagnosed cases of overt hypothyroidism in patients referred to a lipid clinic is approximately two times that in the general population [132]. On the other hand, the association of sHT with changes in serum lipid levels still remains controversial [10,122]. The relationship between sHT and serum TC and LDLc levels has been investigated in some population-based studies with negative results [100,101,106,133-136]. However, several crosssectional and case-control studies found a variable increase of both total and LDL cholesterol level with discordant changes in high-density lipoprotein cholesterol (HDLc) value [107,108,124,125,128,130, 137-152]. In the crosssectional analysis from the Third National Health and Nutritional Examination Survey [134], although partially limited by the somewhat unusual definition of sHT (serum TSH range of 6.70 to 14.99 mIU/L), mean cholesterol values and rates of elevated TC levels were higher in subjects with sHT than in euthyroid control group (TSH, 0.36-6.7 mIU/L). Nevertheless, when adjusted for age, race, sex and the use of lipid-lowering drugs, sHT was not related to elevations in TC levels [134]. In the Rotterdam Study [101], no difference was found between sHT and euthyroid women in terms of mean serum TC value, although other atherogenic lipid parameters [LDLc, triglycerides and Lp(a)] were not measured. In the New Mexico Elder Health survey, although no significant increase of TC, HDLc and triglycerides value was observed in sHT patients, women with serum TSH >10 mIU/L had LDLc and HDLc higher than euthyroid subjects [107]. At variance, in the community-based study from Busselton, including 2108 subjects, both serum TC and LDLc levels were significantly higher in sHT patients than in euthyroid subjects [153]. Moreover, in a cross-sectional study of a general population from a Danish primary health care centre, sHT patients had significantly higher serum triglyceride levels than euthyroid controls [102]. Another cross-sectional study evaluated the prevalence of sHT for different levels of TC in 1200 patients selected from a survey on risk factors for cardiovascular diseases [154]. After correction for age, an increase of 1.0 mIU/L in serum TSH was associated with a rise in TC value of 0.09 mmol/L in women. A similar trend was found in men (0.16 mmol/L) but the statistical significance was not reached due to the lower overall prevalence of sHT in males (1.9% Vs 7.6% in women). In the recent survey from Tromsø, including 5143 subjects, a significant and positive correlation between serum TSH levels and serum TC and LDLc values was found in both genders, even if in the females this did not reach statistical significance after adjusting for age and body mass index [155]. Accordingly, a very recent cross-sectional study from Brazil showed a positive relationship between serum TSH and lipoprotein values, confirming sHT as an intermediate state between euthyroidism and overt hypothyroidism in terms of lipid profile [152].

Overall, these data suggest that besides the well-recognized relationship between hypothyroidism and hypertension (mainly diastolic), the influence of thyroid function on blood pressure homeostasis extends also into the euthyroid range. The interaction between TH and blood pressure may, therefore, contribute to the development of endothelial dysfunction of hypothyroid patients. LIPOPROTEIN PROFILE Lipoproteins are fundamental 'players' in the initiation of platelet signaling pathways and endothelial dysfunction [119]. Thyroid hormones affect the synthesis, mobilization and breakdown of lipids: two major target points are the expression of low-density lipoprotein (LDL) receptors and subsequent cellular uptake of LDL and very low-density lipoprotein (VLDL) [120]. Indeed, a deficit in the expression of the hepatic LDL receptor gene decreases the rate of LDLcholesterol clearance, and is claimed to be responsible for elevated serum lipid levels in patients with hypothyroidism [121]. Many epidemiological studies have examined the

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Although most studies observed significant reductions in serum cholesterol levels, the effectiveness of LT4 replacement in lowering serum lipid levels of sHT patients is still controversial and, generally, based on after-before analysis, not on comparison between placebo versus treatment [132,141,148-151,155-171].Two meta-analyses addressed the issue [158,160]; in the first one, considering intervention studies cited in the Medline database from January 1976 until January 1995, thyroid substitution treatment decreased TC by 0.4 mmol/L independently of the baseline value, although plasma TC levels remain elevated in most sHT patients [158]. In the second one, LT4 replacement lowered serum TC by 0.20 mmol/L and LDLc by 0.26 mmol/L, while no significant effects on serum HDLc or triglyceride concentrations were observed [160]. Among the eight placebo-controlled, randomized clinical trials [141,151,161,162,172-175], four showed a significant reduction of serum TC and LDLc levels during LT4 therapy [141,151,161,175]. Meier et al. [161] showed that, after 48 weeks of LT4 treatment, serum TC decreased by 0.24 mmol/L (or 3.8%) and LDLc by 0.33 mmol/L (or 8.2%). The lipid lowering effect of LT4 replacement was more pronounced in sHT patients with high serum TSH values (>12 mIU/L) or elevated pretreatment total (>6.2 mmol/L) and LDLc (>4.0 mmol/L) levels. In a strictly selected group of patients (mainly premenopausal women) with slightly elevated serum TSH level and positive anti-thyroid antibody titers, we reported a mean serum reduction of 0.47 mmol/L (or 8.0%) for TC and 0.41 mmol/L (or 10.2%) for LDLc after six months of restored euthyroidism by LT4 replacement [151]. Interestingly, in a subsequent double-blind placebo-controlled study from our laboratory LT4 replacement was able to induce a significant improvement of both lipoprotein profile and mean carotid artery intima-media thickness, a surrogate index of systemic atherosclerotic disease and cardiovascular events [141]. In a recent randomized, double blind, crossover study of LT4 and placebo, including 100 patients with stable sHT, LT4 therapy significantly reduced serum TC and LDLc levels (5.5% and 7.3% decrease, respectively); intriguingly, LT4 replacement therapy induced a significant increase of ED dilation as measured by brachial artery FMD, without any change in either baseline vessel diameter or blood flow [175]. Although comparable data are not available for women, the Helsinki Heart Study has shown that, in men, a decrease of 7% in LDLc levels is associated with 15% reduction in the incidence of coronary artery disease (CAD) [176]. Moreover, giving that endothelium dependent vasodilation is emerging as an independent predictor of future cardiac events a significant improvement in FMD could translate into reduction in cardiovascular morbidity and mortality, although this has yet to be proved [177,178]. It is noteworthy that in a recent placebo-controlled study carried out in 33 patients with sHT and CAD, LT4 replacement therapy was effective in improving lipid alteration in those patients with shorter history of CAD, higher cholesterol levels and lower body mass index at baseline [179].

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hypothyroidism can be attributed to increased LDLc levels [180] or, as recently suggested, to reduced serum paraoxonase 1 activity [181]. oxLDL contains lyso-phosphatidylcholine, a potent chemo-attractant for macrophages, leading to generation of foam cells; moreover, oxLDL up-regulates the expression of endothelial vascular cell adhesion molecules, increasing monocyte adhesion [182]. So, oxidative modification of LDLc promotes a proinflammatory response and the development of atherosclerotic lesions [183]. Accordingly, in patients with overt hypothyroidism an increased susceptibility to oxidation of LDLc has been reported, that was normalized after restoration of euthyroidism [184-186]. Duntas et al. [122], in a single uncontrolled clinical study, confirmed increased plasma circulating oxLDL levels in overt hypothyroidism, while oxidized LDL were in the upper normal range in mild thyroid failure, suggesting that the degree of the TH deficiency may play a pivotal role in the extent of LDL oxidation. However, LT4 replacement significantly reduced oxLDL levels only in patients with overt hypothyroidism (-11%) while a slight reduction was obtained in sHT patients (-5.8%): a longer treatment course might be necessary to obtain a significant decrease in sHT patients too. Conversely, Brenta et al. [187] did not find significant difference in LDL susceptibility to oxidation between sHT patients and controls, however, sHT patients showed lower hepatic lipase activity than controls, inversely correlated with serum TSH levels. The reduced enzyme activity modified the conformation of LDL particles, enriching them of triglycerides; this qualitative alteration may contribute to the early atherogenic process of sHT patients.

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Besides the association with hypercholesterolemia, hypothyroidism can lead to intrinsic qualitative changes in circulating lipoproteins, thereby enhancing their atherogenicity [10]. The formation of oxidized LDL (oxLDL) in

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Small, dense LDL particles are more concentrated in arterial walls, more prone to oxidation, and have a reduced affinity for LDL receptors compared with larger LDL particles, resulting particularly atherogenic [188]. Few studies have evaluated the effects of thyroid function on LDL subparticle size. Roscini et al. [189] found no difference in LDL size in 50 overt hypothyroid postmenopausal women as compared to controls. In a recent cross-sectional cohort analysis and prospective clinical study, including 28 patients with short-term hypothyroidism and 2944 Framingham Offspring cohort subjects, a shift toward less atherogenic large LDL, small VLDL, and large HDL subparticle sizes was observed in hypothyroid women [190]. Other qualitative lipid alterations, such as abnormal catabolism of remnant lipoproteins, have been described in overt hypothyroidism [191]. Serum concentrations of remnant-like particle cholesterol were corrected by T4 replacement therapy [191]. The same authors, in a following clinical study, showed similar alterations also in sHT patients, reversed by LT4 replacement in most patients [168]. Several studies have suggested that alterations in postprandial lipid metabolism, particularly the number, size and density of particles contain-ing triglyceride-rich lipids, may lead to atherosclerosis [192, 193]. Recently, Tanaci et al. [194] found that post-prandial lipemia was increased as compared to controls in both overt and subclinical hypothyroid patients; moreover, fasting triglyceride levels directly correlated with serum TSH values. Accordingly, Weintraub et al. [195] showed that LT4 replacement therapy is able to enhance the clearance rate of chylomicron

Hypothyroidism and Endothelial Function

Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2008, Vol. 2, No. 2

remnants in hypothyroid patients. Non-HDLc, a measure of the difference between TC and HDLc levels, has been shown to be predictive of cardiovascular disease [196]. Recently, in a case-control study, Ito et al. [131] showed that LT4 replacement is able to induce reduction of non-HDL levels in both overt and subclinical hypothyroidism. Altered serum non-HDLc concentration may be related to the disturbed metabolism of LDLc, remnant lipoprotein and apolipoprotein B, suggesting that the determination of serum non-HDLc levels may provide relevant information on the cardiovascular risk of hypothyroid patients. Lipoprotein particles are carriers of several enzymes, including human plasma platelet-activating factor acetylhydrolase (PAF-AH), an enzyme associated with both LDLc and HDLc, which may play an important role in the pathogenesis of atherosclerosis [196,197]. Milionis et al. [170] showed that sHT patients exhibit increased plasma PAF-AH activity and low HDL-associated PAF-AH activity; LT4 treatment had no effect on plasma PAF-AH activity but resulted in a significant elevation of HDL-associated PAF-AH activity, which contributes substantially to the antioxidant and antiinflammatory effects of HDL. As a whole these data, although not homogeneous and not equally noticeable, especially in mild thyroid failure, suggest that hypothyroidism is associated with lipoprotein profile alterations, at least partially reversed by LT4 replacement. Dyslipidemia acts as an important mechanism involved in the development of hypothyroid associated endothelial dysfunction and contributes to the increased cardiovascular risk.

mIU/L [146,215]. However, Milionis et al. [163] showed raised Lp(a) concentrations in sHT patients with serum TSH levels >4.5 mIU/L, and Tzotzas et al. [149] found elevated Lp(a) levels in a sub-group of post-menopausal women with sHT, regardless of their serum TSH value. Most clinical trials failed to demonstrate an effect of levothyroxine replacement on Lp(a) levels in sHT patients [120,131, 141,149-151,164,166] although few studies [148,163,165] showed an overall significant reduction of Lp(a) values after treatment. These conflicting results could be interpreted according to the hypothesis that mechanisms other than thyroid function may independently affect Lp(a) metabolism as suggested by Lee et al. [212]. In this setting, we found a significant association between positive family history for cardiovascular heart disease and/or diabetes mellitus and elevated Lp(a) levels which, remained unchanged six months after restoring euthyroidism by LT4 replacement [151]. Thus, suggesting that the altered Lp(a) levels may reflect a genetic influence rather than a reduced TH action. The possible role of thyroid autoimmunity per se in the Lp(a) metabolism should be not overlooked. Indeed, Lotz et al. [216] found an association between thyroid autoimmunity and increased Lp(a) levels in males and post-menopausal women with serum TSH value within the normal range. Interestingly, Xiang et al. [217] recently showed significantly elevated Lp(a) levels in euthyroid patients with thyroid autoimmunity and, on multiple regression analysis, anti-thyroid peroxidase antibody (TPOAb) and Lp(a) value emerged as independent determinants of ED vasodilation. Therefore, whatever the pathogenesis, elevated Lp(a) levels may conspire with other risk factors of untreated hypothyroid patients to enhance endothelial impairment and cardiovascular risk.

Lp(a), a particularly atherogenic LDL variant, is synthesized mainly in the liver and consists of a carbohydrate-rich apolipoprotein, covalentely linked to a low density lipoprotein (LDL)-like particle [198]. Lp(a) has a striking homology to plasminogen [199] and is associated with increased risk of both atherosclerosis and thrombogenesis [200]. Moreover, several studies indicate that Lp(a) levels are independent of other cardiovascular risk factors as causes of endothelial dysfunction [201, 202]. The mechanisms implicated in the atherogenicity of Lp(a) are complex, including the ability to increase PAI-1 expression and inactivate tissue-type plasminogen activator as well as tissue factor pathway inhibitor [203]. Also, Lp(a) could contribute to a prothrombotic state by promoting platelet aggregation [204]. The relationship between overt hypothyroidism and Lp(a) levels are still controversial and data on the effect of LT4 replacement therapy on Lp(a) levels are not homogeneous [131, 205-213]. Previous studies found elevated Lp(a) levels in hypothyroid patients, inversely related to serum T4 levels, which was significantly reduced by LT4 replacement therapy [210, 211]. While, more recent reports failed to demonstrate an association between hypothyroidism and increase of Lp(a) values without any effect of LT4 replacement [131,213]. Studies on the association between sHT and Lp(a) levels have generally yielded consistently negative results [120,122,128,141,145,146,148-151,161, 163,164,214]. In two reports, increased Lp(a) values were found only in sHT patients who had TSH values above 12

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C-REACTIVE PROTEIN AND OXIDATIVE STRESS Growing evidence supports the hypothesis that atherosclerosis is an inflammatory process, and CRP level has been suggested as a strong predictor of cardiovascular risk [218, 219]. Moreover, recent studies suggest that CRP, besides being a marker of inflammation, may also directly contribute to endothelial dysfunction, reducing NO produc-tion and downregulating eNOS expression [220-222]. Several studies have investigated the possible association between hypothyroidism and CRP value, with discordant results [102,109,130,166,171,194,212,213,223-226].

Christ-Crain et al. [130] firstly reported increased plasma CRP concentrations in patients with overt and subclinical hypothyroidism however, CRP levels did not correlate either with the extent of hypothyroidism or thyroid autoimmunity and, were unaffected by LT4 replacement. Accordingly, in a recent cross-sectional study the presence of positive TPOAb titer was not associated with several cardiovascular risk factors, among them CRP [227]. On the other hand, Tanaci et al. [194] showed only a not significant increasing trend in CRP values of hypothyroid patients, and Luboshitzky et al. [109] found a significant correlation between CRP and body mass index without differences between sHT patients and euthyroid controls. Accordingly, no significant increase in serum CRP value was observed in either cross-sectional or case-control studies [171,212,223,226]. Moreover, Beyhan et


al. [171] did not find any improvement of emerging cardiovascular risk factors after restoration of euthyroidism, even in the subgroup of patients with higher serum TSH level (>10 mIU/L), and Perez et al. [166] found no influence of euthyroidism restoration on CRP levels in 42 sHT patients, also in those with higher CRP values (>3 mg/L). At variance, other population-based and case-control studies confirmed an increased CRP value in both overt and subclinical hypothyroidism, and a positive relationship with serum TSH level [102, 224, 225, 228, 229]. Furthermore, serum CRP value was associated with NO reduction and carotid artery stiffness index beta, a marker of early atherosclerosis, which significantly improved after LT4 replacement [225,228]. Interestingly, in a large carefully selected population of euthyroid hyperlipidemic patients (n=429, 28% smokers), CRP value was inversely correlated with serum FT4 level in non-smoker subjects only, suggesting that serum low T4 represents a biomarker of cardiovascular risk in hyperlipidemic subjects even if euthyroid [230]. Albeit the reported data are still controversial, an association between thyroid function and low-grade inflammation may be suggested. To this purpose, we have recently demonstrated that sHT patients show higher plasma CRP and interleukin-6 values compared with euthyroid controls along with an impaired ED vasodilatation and reduced NO availability. Endothelial function was restored by systemic administration of indometacin, a cyclooxygenase inhibitor, or the local infusion of vitamin C as antioxidant [113]. Therefore, supporting the hypothesis that hypothyroidism is associated to low-grade inflammation and enhanced oxidative stress, which may have detrimental effects on endothelial cells. Free radicals accumulation in hypothyroidism could be due to various reasons such as decreased clearance of oxidants, impaired effective activity of the antioxidant defense mechanism, upregulation of iNOS and modulation of the nuclear signaling pathway. The interplay of such various mechanisms could finally result in oxidative stress. However, the association between hypothyrodism and increased oxidative stress is controversial. Conflicting results from animal models can be ascribed to the differences in experimental designs [231-234]. On the other hand, besides our above reported study [113], scanty and not homogeneous informations on the association between hypothyroidism and oxidative stress are available in humans. Some reports showed an increased oxidative stress in hypothyroidism [235,236] but, more recently, a 3-fold ROS increase in hyperthyroid as compared to hypothyroid patients and euthyroid controls has been demonstrated [237]. Conversely, Nanda et al. [238] showed an association between oxidative stress and atherogenic dyslipidemia in hypothyroid patients. Therefore, increased ROS levels in hypothyroidism may result in a pro-oxidant environment, which in turn could alter NO production leading to lipid peroxidation and endothelial dysfunction [239].

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munity and thyroid status in modulating endothelial function should be considered, as supported also by the demonstration of anti-endothelial cell antibodies in hypothyroidism [240]. HOMOCYSTEINE Plasma values of homocysteine (tHcy) are affected by lifestyle, physiological factors likewise age and gender, folic acid and cobalamin status as well as renal function [241243]. The effect of elevated tHcy levels includes endothelial cell desquamation, smooth muscle cells’ proliferation and intimal thickening [244, 245]. Therefore, increased tHcy levels may represent an independent risk factor of atherosclerosis [246]. Indeed, several studies both in vitro and in vivo have demonstrated that hyperhomocysteinemia impairs endothelial function [247,248]. The molecular mechanisms responsible for decreased bioavailability of NO by tHcy involve the increase of vascular oxidant stress along with the inhibition of important antioxidant capacity. Glutathione peroxidase-1 (GPx-1), a selenocysteine-containing antioxidant enzyme, may be a key target of tHcy's deleterious actions, and several experimental and clinical studies have demonstrated a complex relationship between plasma tHcy, GPx-1, and endothelial dysfunction [249].

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Concerning the mechanism responsible for the inflammatory process, a possible basis might involve a chronic activation of immune system. Accordingly, Xiang et al. [217] demonstrated an impaired ED dilation in euthyroid patients with autoimmune thyroiditis; TPOAb value resulting an independent determinant of endothelial dysfunction. Therefore, a joint pathway between inflammation, autoim-

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Most studies showed an association between overt hypothyroidism and elevated tHcy levels, reversed by LT4 replacement therapy; serum FT4 level acting as an independent determinant of tHcy concentrations [130,250256]. Only few studies failed to demonstrate the presence of elevated tHcy levels in overt hypothyroidism [194, 213]. A transient increase in plasma tHcy levels has also been demonstrated during short-term iatrogenic hypothyroidism [257]. TSH secretion suppressive LT4 therapy is able to decrease tHcy levels by 2-5 mol/l: a magnitude of decline sufficient to lower cardiovascular risk [251,253,258]. Normalisation of serum tHcy value is obtained only after reaching serum TSH level less than 2 mIU/L [229]. Most cross-sectional and case-control studies failed to demonstrate a relationship between sHT and hyperhomocysteinemia [108,109,130,134,141,171,194,255,259,260]. However, the third US National Health and Nutrition Examination Survey reported moderately elevated tHcy levels only in patients with serum TSH levels>20 mIU/L compared with euthyroid controls [129]. Accordingly, Sengul et al. [261] observed that, although in the normal range, tHcy values were significantly higher in 33 well-selected sHT women when compared with 25 healthy controls. Moreover, LT4 therapy caused a significant reduction in tHcy levels. On the other hand, tHcy does not appear to contribute to the potential increased risk of atherosclerosis in sHT patients. Indeed, in the New Mexico Elder Health Survey carried out on a randomly selected sample of Medicare recipients (aged65 years), after adjustment for gender, ethnicity, age, folate and vitamin B12 status as well as plasma creatinine levels, no significant difference in serum tHcy levels was detected between 112 patients with sHT and 643 euthyroid subjects (serum TSH value10 mIU/L) had a significantly higher prevalence of coronary heart disease when compared against euthyroid participants. Besides the study by Sengul et al. [261], no effect of LT4 replacement therapy on serum tHcy levels has been reported in several

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case-control studies [166, 171, 255, 260]. Also, in three double blind, placebo-controlled trials, tHcy levels were unaffected by LT4 therapy, either during fasting or after methionine challenge [130,141,263]. The question of the mechanism(s) behind hyperhomocysteinemia in overt hypothyroidism remains to be elucidated. The renal metabolism may play an important role in the overall tHcy homeostasis, and a reduction in the glomerular filtration rate has been reported during discontinuation of LT4 therapy [264]. Plasma folic acid and vitamin B12 levels are well known determinants of tHcy. Therefore, an alternative explanation for the elevation of tHcy value in hypothyroidism is the presence of low plasma folic acid levels, induced by a direct effect of TH on folatemetabolizing enzymes [250, 254, 258, 265]. Indeed, vitamin deficiency is more frequently observed in overt than subclinical hypothyroidism so it is possible that hyperhomocystinemia results from vitamin deficiency rather than a renal mechanism or TH lack per se. Whatever the pathogenesis, these data suggest that hyperho-mocysteinemia is faintly involved in the pathogenesis of endothelial dysfunction and early atherosclerotic disease of hypothyroid patients. HEMOSTATIC DYSFUNCTION

replacement [288]. On the contrary, in the recent study by Gullu et al. [169] overt hypothyroidism was associated with significant reversible abnormalities in clotting parameters such as increased bleeding time, PT, aPTT and clotting time and decreased factor VIII activity and vWF activity while, in sHT only minor reversible changes in factor VIII activity and vWF were observed. Moreover, either a case control or a population-based study did not find significant difference in coagulation/fibrinolysis parameters between sHT patients and controls [278, 289]. On the other hand, in the TromsØ study, sHT patients had factor VIIa levels 10% lower than controls, TSH value being a significant negative predictor of VIIa levels [289]. In this setting, it is worth to note that patients suffering from moderate hypothyroidism (TSH from 10 to 50 mIU/L) showed an increased risk of thrombosis contrasting with the bleeding tendency of those presenting severe hypothyroidism (TSH>50 mIU/L) [276, 290].

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Increased levels of fibrinogen, factor VII activity, factor VII activity/factor VII antigen ratio, PAI antigen as well as decreased antithrombin III activity were described in sHT patients compared to euthyroid controls [275, 277]. Moreover, six months of restored euthyroidism by LT4 replacement induced a significant decrease in PAI antigen and factor VII levels [277]. Accordingly, data by Guldiken et al. [287] are consistent with a reduced global fibrinolytic capacity in patients with sHT than in euthyroid controls, suggesting a relative hypercoagulable state. Very recently, elevated levels of thrombin-activatable fibrinolysis inhibitor (TAFI) antigen were found in both overt and subclinical hypothyroid patients; increased TAFI levels were related to the degree of thyroid dysfunction and reduced by LT4

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Overall, these data, although not homogeneous and usually of limited direct clinical relevance, support the hypothesis that alterations in coagulation profile might contribute to endothelial dysfunction and to the potential development of atherosclerosis in hypothyroid patients.

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Hemostatic system is recognized to have critical importance in the pathogenesis of endothelial dysfunction and atherosclerosis [266]. Although the link between hemocoagulation and thyroid function has been known since the middle of the past century, data are still controversial. Both increased and decreased platelet adhesiveness as well as a hypo- or hypercoagulable state have been reported in hypothyroid patients [267-279]. Hypothyrodism is also associated with acquired vW syndrome, LT4 replacement being able to induce disappearance of the bleeding diathesis, with normalization of coagulation factors [279-281]. The mechanisms relating to the alterations of the coagulation system in hypothyroidism are not well established, but a direct role of TH in the regulation of inflammatory and clotting profile has been proposed [282,283], and seems to be dependent on the severity of thyroid failure [284]. Moreover, a possible involvement of thyroid autoimmunity in modifying coagulation process should be taken into consideration [285]. A recent systematic review of the effects of TH on the haemostatic system underlined the intrinsic difficulty to have high-quality studies on this complex issue [286].

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Adiponectin (AnP) is a major adipocyte-secreted adipokine abundantly present in the circulation. In addition to its role as an insulin sensitizer, growing evidence suggests that AnP is an important player in maintaining vascular homoeostasis. Numerous epidemiological studies have identified hypoadiponectinaemia as an independent risk factor for endothelial dysfunction, hypertension, coronary heart disease, myocardial infarction and other cardiovascular complications [291]. Conversely, elevation of circulating AnP levels improves vascular dysfunction in animal models [291]. AnP exerts its protective effects through its direct action in the vascular system, such as increasing endothelial NO production, inhibiting endothelial cell activation and endothelium-leucocyte interaction, enhancing phagocytosis, and suppressing macrophage activation, macrophage-tofoam cell transformation and platelet aggregation [291].

Data regarding the relationship between AnP levels and thyroid function are controversial [213, 292-295]. Most studies reported no significant difference in AnP levels between hypothyroid patients and euthyroid controls, either at baseline or after LT4 replacement therapy. Changes in plasma AnP levels are strongly linked with insulin resistance [296]; the metabolic syndrome (MS) being associated with high degree of inflammation and increased cardiovascular risk [297]. Considering that some cardiovascular risk factors associated with hypothyroidism cluster within MS, several studies have evaluated a possible relationship between thyroid function and MS with conflicting results. In short term, hypothyroidism no alteration of insulin sensitivity as evaluated by homeostasis model of assessment-insulin resistance (HOMA) has been recently reported [298]. In the study by Tuzcu et al. [224], sHT patients had increased fasting insulin as compared to euthyroid controls although no difference in the HOMA index was observed. The authors suggest that hypothyroidism and hyperinsulinemia could be associated before insulin resistance becomes evident. In a cross-sectional cohort study, including 2703 euthyroid


Dardano and Monzani

subjects and investigating a possible association between thyroid function and MS, both FT4 and TSH were significantly associated with HOMA index that was higher in the lowest FT4 tertile. Moreover, FT4 was significantly related to four of five components of the metabolic syndrome such as abdominal obesity, blood pressure and serum triglyceride and HDLc values [299]. Accordingly, a very recent study [300] showed a marked reduction in insulin sensitivity by euglycemic clamp in both overt and subclinical hypothyroidism in the face of normal fasting insulin and elevated free fatty acids (FFA) levels. The elevation of circulating FFA, confirming previous data both at rest and during effort [301], contributes to the observed changes in insulin sensitivity by attenuation of glucose uptake and glucose oxidation.

those in normal controls. It was further suggested that the increase in serum OPG levels in hypothyroid patients was independently associated with vascular injury, as reflected by increased plasma vWF levels, but not with the severity of hypothyroidism. Subsequently, elevated levels of OPG were found also in sHT patients as compared to controls; moreover, the absolute changes in OPG significantly correlated with the changes in TSH (p