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Department of Internal Medicine, University of Pisa, 67-56100 Pisa, Italy
Address all correspondence and requests for reprints to: Stefano Taddei, M.D., Department of Internal Medicine, University of Pisa, Via Roma, 67-56100 Pisa, Italy. E-mail: s.taddei{at}med.unipi.it.
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Abstract |
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Background: sHT patients are characterized by peripheral endothelial dysfunction and low-grade inflammation.
Methods: In 53 sHT and 45 healthy subjects, we studied the forearm blood flow (strain-gauge plethysmography) response to intrabrachial acetylcholine (Ach) (0.15–15 µg/min·dl) with and without local vascular COX inhibition by intrabrachial indomethacin (50 µg/min·dl) or nitric oxide synthase blockade by N-mono methyl arginine (L-NMMA) (100 µg/min·dl) or the antioxidant vitamin C (8 mg/min·dl). The protocol was repeated 2 h after systemic nonselective COX inhibition (100 mg indomethacin) or selective COX-2 blockade (200 mg celecoxib) oral administrations.
Results: sHT patients showed higher C-reactive protein and IL-6 values. In controls, vasodilation to Ach was blunted by L-NMMA and unchanged by vitamin C. In contrast, in sHT, the response to Ach, reduced in comparison with controls, was resistant to L-NMMA and normalized by vitamin C. In these patients, systemic but not local indomethacin normalized vasodilation to Ach and the inhibition of L-NMMA on Ach. Similar results were obtained with celecoxib. When retested after indomethacin administration, vitamin C no longer succeeded in improving vasodilation to Ach in sHT patients. Response to sodium nitroprusside was unchanged by indomethacin or celecoxib.
Conclusions: In sHT patients, low-grade chronic inflammation causes endothelial dysfunction and impaired nitric oxide availability by a COX-2-dependent pathway leading to increased production of oxidative stress.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Thus, the present study was designed to assess whether a systemic inflammatory mechanism might be responsible for impaired endothelium-dependent vasodilation in patients with Hashimoto’s thyroiditis-derived sHT. In addition, the role of cyclooxygenase (COX) and oxidative stress was also explored.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study population included 53 sHT patients with Hashimoto’s thyroiditis, who were positive for both antithyroid peroxidase antibody (TPO-Ab) and antithyroglobulin antibody (Tg-Ab) (Table 1
). Only patients with sHT (TSH > 3.6 mIU/liter) documented for at least 6 months before the study were enrolled. As controls, 45 euthyroid subjects, matched to the patient group for sex, age, blood pressure (BP), body mass index (BMI), and lipid profile were recruited among staff and relatives of patients. Women were premenopausal with regular menses, and none was pregnant. Obese (BMI > 30 kg/m2) subjects, smokers, and patients with hypertension, diabetes mellitus, dyslipidemia [cut-off level for low-density lipoprotein (LDL) cholesterol and triglycerides, 160 and 180 mg/dl, respectively], or other systemic diseases were excluded from the study. No individual was following any drug regimen. All study subjects gave their signed informed consent to the study, which was approved by the Institutional Ethics Committee.
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Vascular reactivity was assessed by the perfused forearm technique, as already described (4). Briefly, the brachial artery was cannulated for drug infusion at systemically ineffective rates, and intraarterial BP and heart rate were monitored. Forearm blood flow (FBF) was measured in both forearms by strain-gauge venous occlusion plethysmography. Circulation to the hand was excluded 1 min before FBF measurement by inflating a pediatric cuff around the wrist at suprasystolic BP. Forearm volume was measured by the water displacement method.
Experimental design
The experimental design is summarized in Fig. 1. All the study experiments were carried out after an overnight fast, and subjects refrain from caffeine or other possible vasoactive substances for at least 48 h before the study. Endothelium-dependent vasodilatation was estimated by performing a dose-response curve to acetylcholine (Ach; infusion rates of 0.15, 0.45, 1.5, 4.5, and 15 µg/min·dl, 5 min each dose). In addition, to assess the NO contribution to endothelial responses, Ach was repeated in the presence of N-mono methyl arginine (L-NMMA) (100 µg/min·dl) to block NO synthase (20). To avoid L-NMMA-induced basal flow modification, the NO clamp (which allows assessment of endothelial agonists in the presence of NO synthase blockade without changes in basal flow) was applied (21). Thus, after 10 min of L-NMMA infusion, sodium nitroprusside (SNP) was coinfused (0.4 and 0.3 µg/min·dl for 5 min in control subjects and sHT, respectively) to neutralize the L-NMMA-induced vasoconstriction and restore baseline FBF. Details concerning the method as performed in our laboratory have already been published (21). Endothelium-independent vasodilatation was assessed with a dose-response curve to SNP (1, 2, and 4 µg/min·dl, 5 min each dose). These rates were selected to induce vasodilatation comparable with that obtained with Ach. A 30-min washout was allowed between each dose-response curve.
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To assess the effect of systemic inflammation on endothelial function, in 12 patients with sHT and 12 healthy controls, vasodilation to Ach with and without L-NMMA and to SNP was performed in basal conditions and repeated 2 h after indomethacin oral administration (50 mg) to block systemic COX. In addition, to distinguish between the effect of systemic inflammation and local production of COX-dependent endothelium-derived contracting factors (EDCFs), in another group of 12 sHT patients and 12 healthy controls, vasodilation to Ach was performed in the absence and presence of intrabrachial indomethacin administration (50 µg/min·dl) in basal conditions and repeated 2 h after indomethacin oral administration (50 mg). The infusion rate of intraarterial indomethacin was chosen on the basis of previous evidence demonstrating its efficacy in reversing EDCF-derived endothelial dysfunction in patients with essential hypertension or old age (22, 23).
COX-2 contribution to impaired endothelium-dependent vasodilation and NO availability in patients with sHT
In a further group of 20 sHT patients and 12 healthy controls, the role of COX-2 in endothelial function was studied by performing Ach infusion, with and without L-NMMA, in basal conditions and 2 h after oral administration of celecoxib (200 mg), a selective COX-2 inhibitor. Moreover, to compare the effect of nonselective and selective COX inhibition, in eight of 20 sHT patients, FBF response to Ach was repeated 2 h after oral administration of indomethacin (50 mg).
Role of oxidative stress as a mechanism responsible for inflammation-induced impairment in endothelium-dependent vasodilation
To assess whether oxygen free radical production might be responsible for impaired endothelium-dependent vasodilation caused by systemic inflammation in sHT, we studied a final group of nine sHT patients and nine control subjects in whom Ach was infused in the absence and presence of intrabrachial administration of the antioxidant vitamin C (8 mg/min·dl). This protocol was performed at baseline and repeated 2 h after indomethacin oral administration (50 mg).
At baseline after an overnight fast, blood samples were taken for the determination of serum fT3 and fT4, TSH, Tg-Ab, TPO-Ab, total and high-density lipoprotein (HDL)-cholesterol, triglycerides, high-sensitive C-reactive protein (CRP), and IL-6 levels. Samples were also repeated after indomethacin or celecoxib oral administration for CRP and IL-6 determination.
Analytical measurements
Serum fT3 and fT4 levels were measured by specific RIA (Techno-Genetics Recordati, Milan, Italy). TSH was determined with an ultrasensitive immunoradiometric assay method (Cis Diagnostici, Tronzano Vercellese, Italy). Tg-Ab was measured by specific immunoradiometric assay (Biocode, Sclessin, Belgium), and TPO-Ab was measured by specific RIA (Sorin Biomedica, Saluggia, Italy). Total serum cholesterol, triglycerides, and HDL-cholesterol were assayed by enzymatic methods (Roche Diagnostic, Mannheim, Germany). LDL-cholesterol level was calculated by Friedenwald’s equation. CRP was determined with a highly sensitive latex-based immunoassay (Dade Behring, Newark, DE; sensitivity, 0.05 mg/liter). IL-6 was measured by an ELISA kit (R&D Systems, Minneapolis, MN). Normal ranges in our laboratory are as follows: fT4, 5.3–15.5 pg/ml (6.8–20 pmol/liter); fT3, 2.8–5.6 pg/ml (4.3–8.6 pmol/liter); TSH, 0.30–3.6 mIU/liter; Tg-Ab, less than 50 U/ml; TPO-Ab, less than 10 U/ml; and CRP, less than 3 mg/liter.
Statistical analysis
Data were analyzed in terms of changes in FBF. Because arterial BP did not change significantly during the FBF study, increments or decrements in FBF were taken as evidence of local vasodilation and vasoconstriction, respectively. Vascular data are expressed as mean ± SEM, whereas biochemical features are expressed as mean ± SD or median and range as appropriate. Student’s unpaired or paired t test, 
2, and ANOVA for repeated measures were used as appropriate. Linear regression analysis was carried out by standard techniques (SPSS version 11.0, SPSS, Chicago, IL). Significance was assumed for P < 0.05.
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Results |
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Serum TSH levels were significantly higher in sHT patients than controls, whereas fT3 and fT4 levels, within the normal range, were similar between the two groups. Patients with sHT were also characterized by significantly higher CRP and IL-6 values than control subjects. Lipid profile was within the normal range and similar between the two groups (Table 1
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Response to Ach, SNP, and NO availability in normal subjects and sHT patients
The FBF increase induced by Ach was significantly smaller in sHT patients (from 3.4 ± 0.2 to a maximum of 15.2 ± 0.9 ml/min·dl; +351 ± 24% increase above baseline) compared with healthy controls (from 3.4 ± 0.2 to a maximum of 25.2 ± 1.6 ml/min·dl; +648 ± 38% increase, P < 0.01 vs. sHT) (Fig. 2). In contrast, vasodilation to SNP was not significantly different between sHT patients (from 3.6 ± 0.5 to a maximum of 18.0 ± 3.2 ml/min·dl; +402 ± 42% increase) and controls [from 3.3 ± 0.3 to a maximum of 17.3 ± 1.7 ml/min·dl; +420 ± 49% increase, P = not significant (NS) vs. sHT]. In healthy subjects, as expected, vasodilation to Ach was significantly blunted by L-NMMA (baseline, 3.4 ± 0.2; L-NMMA, 2.0 ± 0.1; L-NMMA plus SNP, 3.4 ± 0.2; L-NMMA plus SNP and Ach, 15.8 ± 2.0 ml/min·dl; +370 ± 29% increase) (Fig. 2). In contrast, in sHT patients, the NO synthase inhibitor had no effect on Ach (baseline, 3.4 ± 0.2; L-NMMA, 2.7 ± 0.1; L-NMMA plus SNP, 3.4 ± 0.2; L-NMMA plus SNP and Ach, 15.0 ± 2.1 ml/min·dl; +341 ± 14% increase, P = NS vs. Ach alone) (Fig. 2). In the sHT group, a significant relationship was found between maximal response to Ach and serum TSH level (r = 0.57, P < 0.05); no other relationship was observed between FBF response to Ach and blood parameters.
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Systemic indomethacin administration did not affect plasma CRP and IL-6 values, either in sHT patients or healthy subjects (data not shown). In sHT patients, the vascular response to Ach, which was resistant to L-NMMA, was significantly increased by systemic oral indomethacin. In the presence of indomethacin, LNMMA also recovered its ability to inhibit the response to Ach (Table 2 and Fig. 3, A and B).
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). Effect of systemic celecoxib administration on response to Ach and NO availability in healthy subjects and sHT patients
Systemic celecoxib administration failed to modify plasma CRP and IL-6 values, both in sHT patients and healthy subjects (data not shown). Similarly to the findings obtained with indomethacin, systemic celecoxib administration also potentiated vasodilation to Ach and restored the inhibition by L-NMMA on response to the muscarinic agonist in patients with sHT (Table 3 and Fig. 3, C and D) In healthy subjects, systemic celecoxib administration failed to affect either vasodilation to Ach or inhibition by L-NMMA on the response to Ach (Table 3). The vascular response to SNP was not affected after celecoxib administration, either in sHT patients or healthy controls (data not shown). Finally, when in the same group of sHT patients, the effect of celecoxib was compared with indomethacin, it was found that the potentiating activity of the COX-2-selective inhibitor on the maximal vascular response to Ach (81 ± 4%) was similar to that observed with the nonselective COX blocker (79 ± 10%) (Fig. 4).
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In patients with sHT, intrabrachial vitamin C infusion significantly potentiated the vasodilation to Ach (saline, from 3.4 ± 0.2 to 14.9 ± 1.8 ml/min·dl, +337 ± 20%; vitamin C, from 3.4 ± 0.2 to 25.4 ± 1.3 ml/min·dl, +635 ± 31%). When Ach was repeated after systemic indomethacin administration, its vasodilating effect was significantly increased (from 3.4 ± 0.2 to 24.3 ± 1.7 ml/min·dl, +623 ± 33%; P < 0.001), whereas the facilitating effect of vitamin C was completely prevented (from 3.4 ± 0.3 to 24.3 ± 2.1 ml/min·dl, +624 ± 34%; P = NS vs. Ach after indomethacin).
In healthy subjects, intrabrachial vitamin C infusion failed to modify vasodilation to Ach (data not shown).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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To address this possibility, in patients with sHT and normal lipid profile, we tested the effect of systemic administration of an antiinflammatory dose of indomethacin, a nonselective COX inhibitor. We observed an increased response to Ach with no modification in the response to SNP. In addition, and no less important, the COX inhibitor restored the inhibiting effect of L-NMMA on vasodilation to Ach. Because indomethacin did not change vasodilation to SNP, these results indicate that the COX inhibitor is able to increase endothelium-dependent vasodilation and restore NO availability in patients with sHT. When investigating the mechanism whereby indomethacin can improve endothelial function, two different hypotheses must be taken into account. The first is related to the well-documented association between inflammation and endothelial dysfunction. Thus, it might be speculated that indomethacin could restore endothelial function through its classical antiinflammatory effect. Another tentative explanation concerns the fact that COX is a source of EDCFs, both in animals (24, 25) and humans (22, 26). However, this line of inquiry can probably be excluded by the demonstration that intrabrachial indomethacin has no effect on vasodilation to Ach in patients with sHT. Because in clinical conditions characterized by EDCF production, such as essential hypertension, intrabrachial indomethacin at the same dose was able to block EDCF effects (26), it is likely that in patients with sHT COX-dependent endothelial-derived vasoconstrictors play no major role in the pathogenesis of endothelial dysfunction. It is therefore conceivable that the beneficial effect of systemic indomethacin administration in sHT could be exerted against systemic inflammation.
There is growing evidence that both acute and chronic inflammation can lead to impaired endothelium-dependent vasodilation in humans (12, 13, 14). In agreement with previous evidence (18), our patients with sHT are characterized by low-grade inflammation, as confirmed by the slight, but significant, increase in high-sensitive CRP and IL-6 in the present study population. The mechanism responsible for the inflammatory process might be related to the increased TSH levels. This hypothesis is supported by the demonstration that TSH induces TNF-
production by bone marrow cells (27), but it is not in agreement with previous evidence indicating that levothyroxine replacement and the consequent normalization of TSH and lipid profile is not associated with complete restoration of endothelium-dependent vasodilation (17). Thus, an alternative account might involve chronic activation of the immune system due to Hashimoto’s thyroiditis. Such a hypothesis is in line with the demonstration that after levothyroxine replacement, autoantibody title and erythrocyte sedimentation rate remained the only parameters not corrected by the therapy and therefore at least potentially responsible for the residual endothelial dysfunction (17). However, a recent case control study excluded a significant relationship between low-grade chronic inflammation of sHT patients and autoimmune thyroiditis (28).
Another important finding of the present study is the major role played by the COX-2 subtype. Celecoxib, a selective COX-2 inhibitor, was able to increase vasodilation to Ach and restore the ability of L-NMMA to inhibit the relaxing effect of the endothelial agonist, while not affecting vascular responses in healthy controls. It is also important to observe that the degree of celecoxib-mediated potentiating effect on vasodilation to Ach was similar to that observed with indomethacin, a nonselective COX inhibitor. Taken together, these findings indicate that COX-2 is the isoform responsible for enhanced production of autocoids that mediate inflammation and, as a consequence, endothelial dysfunction. This interpretation is in agreement with different lines of evidence. Apart from a large body of experimental evidence demonstrating an association among COX-2, inflammation, and endothelial dysfunction (29, 30), recently Chenevard et al. (31) demonstrated that 2-wk treatment with celecoxib improves conduit artery endothelial dysfunction in patients with coronary artery disease.
To further investigate the mechanism linking systemic inflammation and COX activity to endothelial dysfunction, we investigated the potential role of oxidative stress. To address this issue we used vitamin C, an antioxidant compound capable of scavenging superoxide anions at high concentrations (32). In baseline conditions, vitamin C significantly increased vasodilation to Ach. However, indomethacin administration potentiated the response to Ach by a similar extent to that exerted by the antioxidant and, moreover, prevented the facilitating effect of vitamin C. It can therefore be suggested that COX activity, exerted mainly through the COX-2 isoform, could lead to endothelial dysfunction by oxidative stress production. Support for this view comes from experimental data indicating that COX-2 may be a source of oxygen free radicals (33, 34) and clinical data indicating that COX-2 inhibition by celecoxib treatment reduces plasma concentrations of ox-LDL, a marker of lipid peroxidation (31). We are aware that other COX-independent enzymes might act as a source of oxidative stress (i.e. the NADPH oxidase or xanthine oxidase). Such possibilities were not explored in our experiments. However, if we consider that systemic COX blockade was able to normalize NO availability, it is conceivable that in sHT COX activity might be likely considered a predominant source of oxidative stress.
In conclusion, these results indicate that in patients with sHT, low-grade chronic inflammation causes endothelial dysfunction and impaired NO availability by a COX-2-dependent pathway, bringing about increased production of oxidative stress. This vascular alteration may have clinical relevance in view of the cumulating evidence that sHT is associated with increased risk for atherosclerosis and ischemic heart disease. Because endothelial dysfunction is an independent promoter of cardiovascular events, it is likely that impaired endothelium-dependent vasodilation could be one of the early mechanisms promoting atherosclerosis and cardiovascular disease. The demonstration that chronic inflammation not only leads to an alteration in lipid profile but can also represent a trigger for endothelial dysfunction may suggest a possible alternative therapeutic strategy to prevent cardiovascular disease in patients with sHT. Unfortunately, chronic administration of selective COX-2 inhibitors does not seem to ensure adequate cardiovascular protection (35). Other specific tools that may be developed in the near future to block the inflammation process might represent an adjunctive possibility in the therapeutic approach to cardiovascular disease.
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Footnotes |
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First Published Online September 12, 2006
Abbreviations: Ach, Acetylcholine; BMI, body mass index; BP, blood pressure; COX, cyclooxygenase; CRP, C-reactive protein; EDCF, endothelium-derived contracting factor; FBF, forearm blood flow; fT3, free T3; fT4, free T4; HDL, high-density lipoprotein; LDL, low-density lipoprotein; L-NMMA, N-mono methyl arginine; NO, nitric oxide; NS, not significant; sHT, subclinical hypothyroidism; SNP, sodium nitroprusside; Tg-Ab, antithyroglobulin antibody; TPO-Ab, antithyroid peroxidase Ab.
Received May 18, 2006.
Accepted August 31, 2006.
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References |
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