Increased Tissue Endothelin-1 and Endothelin-B Receptor Expression in Temporal Arteries from Patients with Giant Cell Arteritis

Increased Tissue Endothelin-1 and Endothelin-B Receptor Expression in Temporal Arteries from Patients with Giant Cell Arteritis Ivan Dimitrijevic, MD,1 Christina Andersson, MT,2 Pehr Rissler, MD,2 Lars Edvinsson, MD, PhD1 Purpose: Endothelin (ET)-1 has been implicated in the atherosclerotic process and during inflammation. Similarity in the development process of giant cell arteritis (GCA) and atherosclerosis exists. Several ET receptor antagonists have been developed, principally to target cardiovascular disease states. High doses of corticosteroids currently are used in the treatment of GCA, whereas other treatments are not as reliably effective. The present study was performed to elucidate the role for ET-1, ETA, and ETB receptors in GCA. Design: Experimental, retrospective immunohistochemical study of temporal arteries using archival formalinfixed, paraffin-embedded tissue. Participants: The study included 10 patients with GCA and 10 control patients with clinically suspected GCA but diagnosed not to have GCA. Methods: Immunohistochemistry, with anti ET-1, anti-ETA, and anti-ETB antibodies, was performed on formalin-fixed and paraffin-embedded temporal arteries. Main Outcome Measures: Endothelin-1, ETA, and ETB receptor immunostaining intensities were quantified. Results: Temporal arteries from the patients with GCA showed the typical histologic features, including intimal thickening, disruption or loss of the elastic lamina, and inflammatory infiltrates of lymphocytes, macrophages, and multinucleated giant cells. These features were associated with increased ET-1 and ETB receptor immunoreactivity in the medial layer of the temporal arteries and endothelial cells in patients with GCA compared with the controls. The increased ET-1 and ETB receptor immunoreactivity occurred in vascular smooth muscle cells (SMCs) and multinucleated giant cells. The ET-1 and ETB receptor immunoreactivity correlated with the degree of systemic inflammation. No changes were observed in ETA receptor expression in SMCs or endothelial cells compared with controls. Conclusions: The results suggest a role for ET-1 and ETB receptors in GCA. Inhibiting the ET system may provide a corticosteroid-sparing alternative in the treatment of GCA. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2010;117:628 – 636 © 2010 by the American Academy of Ophthalmology.

Giant cell arteritis (GCA) is a vascular disease characterized by granulomatous pan arteritis of large and medium-sized arteries.1 The inflammation is often clinically limited to cranial branches of the aorta with involvement of the temporal arteries. Clinical presentation varies and involves fever of unknown origin to scalp necrosis and jaw claudication. Systemic manifestation is related to the inflammatory process, whereas organ involvement is mainly related to vascular occlusion.2 The erythrocyte sedimentation rate (ESR) is elevated, whereas a normal ESR value indicates less likelihood of disease. Corticosteroids are the drugs of choice in GCA, but even prolonged therapy does not always induce complete remission and side effects are common. Several corticosteroid-sparing medications have been investigated, but so far alternative immunosuppressive agents have been remarkably ineffective in treating GCA. Vascular wall cell injury in GCA induces proliferation of vascular smooth muscle cells (SMCs) and local inflamma-

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© 2010 by the American Academy of Ophthalmology Published by Elsevier Inc.

tion with production of promoting cytokines and chemokines. One important factor is that the vasoactive peptide endothelin (ET)-1, normally produced in the endothelial cells, has both proatherogenic and proinflammatory capacities.3 The effects of ET-1 are mediated by activation of ETA and ETB subtypes in vessels.4 Endothelin-1 may also increase the sensitivity of blood vessels to the action of other vasoconstrictive circulating hormones, such as noradrenaline,5 serotonin, and angiotensin II, thus augmenting vasoconstriction.4 The plasma concentrations of ET-1 are increased in atherosclerosis, inflammatory diseases,4 and GCA6; in addition, there is altered expression of ET receptors in systemic disease.7 On the basis of these observations, it has been suggested that the inflammatory process in vessels of GCA and atherosclerosis are similar. In inflammation, nuclear factor (NF)-␬B plays a critical role in the transcription of various genes implicated in immune responses, such as cytokines and adhesion molecules. EndothelinISSN 0161-6420/10/$–see front matter doi:10.1016/j.ophtha.2009.07.043

Dimitrijevic et al 䡠 Endothelin and Giant Cell Arteritis Table 1. Background Characteristics of Study Patients

Median age (range yrs) Gender (men/women) Headache Jaw claudication Polymyalgia rheumatica Temporal artery pain Abnormal temporal artery on palpation Any visual symptom Diplopia Scalp tenderness Weight loss Anorexia Fever Ophthalmologic disorder Malignancy Infection Myalgia Arthralgia ESR ⬍ 50 mm/h ESR 50–100 mm/h ESR ⬎ 100 mm/h CRP Anemia Abnormal arterial biopsy

Controls n ⴝ 10

GCA n ⴝ 10

75 (69–89) 5/5 3 1 1 2 1 4 0 2 1 0 1 4 3 2 2 0 10 0 0 19⫾11 1 0

78 (64–94) 5/5 6 3 3 10 5 4 1 4 2 3 3 0 0 0 3 2 0 6 4 152⫾31 2 10

CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; GCA ⫽ giant cell arteritis.

1 modulates the immune response via the production of NF␬B in human vascular smooth muscle increasing both interleukin-1 and 6, which have been implicated in the pathophysiology of GCA.2 Attempts to regulate the effects of ET-1 by the use of pharmacologic antagonists are currently under investigation.8 Taken together, ET-1 modulates vessel wall inflammation in both atherosclerosis and systemic diseases. To our knowledge, this is the first study to investigate the role of ET-1 and its receptors in temporal arteries of GCA in humans. We demonstrate the expression of ET-1, ETA, and ETB receptors in temporal arteries from patients with GCA and control subjects by using immunohistochemistry. Modulating the inflammatory response by inhibitors of the ET system may be an alternative approach for the treatment of GCA.

Materials and Methods Ethics The samples were handled in accordance with permission obtained from the Lund University Human Ethics Committee (LU 818-01), and the study conforms to the principles outlined in the Declaration of Helsinki.

Tissue Collection Temporal artery specimens were obtained from 20 patients who underwent biopsy because of suspicion of arteritis; the patients had no known ischemic cardiovascular disorder. Biopsies ranged from 2.5 to

4 cm in length and were taken within 5 days after initiation of corticosteroid treatment. The vessels were examined for histopathology according to clinical practice at the Department of Pathology, Lund University, Sweden. The patients’ laboratory and clinical data, obtained by retrospective chart review, are summarized in Table 1. The original pathology reports were reviewed to verify the GCA diagnosis. The diagnosis was based on the American College of Rheumatology 1990 criteria for the classification of GCA.1 The absence of inflammatory lesions in the sample led to a negative categorization of the sample. The histopathologic findings in the temporal arteries from patients with GCA are listed in Table 2.

Immunohistochemistry Sections of 4 ␮m were cut from formalin-fixed, paraffin-embedded blocks and dried at 60°C for 1 hour. After dewaxing and rehydration, the sections were treated with 10 mmol/L citrate buffer pH 6.0 in a microwave oven for 15 minutes for antigen retrieval. The immunohistochemistry was performed with mouse monoclonal ET-1 antibody (GTX22786, GeneTex, Inc., San Antonio, TX) diluted 1:250 and rabbit polyclonal ETA receptor (GTX71455, GeneTex, Inc.) diluted 1:200 and goat anti-human ETA (sc-21194, Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:100, ETB receptor (GTX78093 GeneTex, Inc.) diluted 1:200, goat antihuman ETB (sc-21196, Santa Cruz Biotechnologies), anti-CD68 (M876 Dako, Glostrup, Denmark) diluted 1:200, and anti-CD45 (M701 Dako) diluted 1:600, anti-human smooth muscle actin antibody diluted 1:500 (M0851 Dako), and anti-CD31 antibody diluted 1:50 (M823 Dako) and performed in an automated immunostainer, TechMate 500 (Ventana Biotek, Tucson, AZ) using the biotin-streptavidin-peroxidase method with diaminobenzidine as the chromogen (Dako REAL Detection System, peroxidase/diaminobenzidine⫹, rabbit/mouse). The specificity of the antibodies has been characterized.9,10 Mayer’s hematoxylin was used for counterstaining, and primary antibodies were omitted for negative control. Nonspecific binding of ET-1 antibodies was controlled by Table 2. Giant Cell Arteritis Was Defined Using The American College of Rheumatology (1990) Criteria for the Classification of Giant Cell Arteritis1

Presence of multinucleated giant cells Yes No Internal elastic lamina degeneration Intact Focal rupture Rupture of ⬍50% of the vessel circumference Rupture of ⬎50% of the vessel circumference Intimal hyperplasia Absent 0%–25% lumen occlusion 25%–50% lumen occlusion ⬎50% lumen occlusion Calcifications Histocytes Multinucleated giant cells

Controls n ⴝ 10

GCA n ⴝ 10

0 10

10 0

5 3 1 1

0 2 7 1

1 8 1 0 0 0 0

0 0 8 2 0 10 10

DAB ⫽ diaminobenzidine; GCA ⫽ giant cell arteritis. The absence of both lymphocytes and multinucleated giant cell infiltrates in the sample led to the categorization of a sample as negative. The histopathologic findings in the arteries from control patients and with GCA are listed.

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Table 3. Relative Diaminobenzidine Staining Intensity in Temporal Artery Walls in Controls and in Patients with Giant Cell Arteritis DAB Staining Intensity ET-1 Control (n) GCA (n) ETA Control (n) GCA (n) ETB Control (n) GCA (n)

0

ⴙ1

ⴙ2

ⴙ3

ⴙ4

0 0

10 0

0 3

0 7

0 0

0 0

0 0

10 10

0 0

0 0

0 0

6 0

4 5

0 5

0 0

ET ⫽ endothelin; DAB ⫽ diaminobenzidine; GCA ⫽ giant cell arteritis; N ⫽ number. Staining of cells: 0 ⫽ none, ⫹1 ⫽ cells in 1/4 of the specimen or less stained, ⫹2 ⫽ cells in 1/4 to 1/2 of the specimen stained, ⫹3 ⫽ cells in 1/2 to 3/4 specimen stained, ⫹4 ⫽ 3/4 to 1 specimen stained.

Calculations and Statistical Analysis Experiments were performed using arteries from 10 patients with positive biopsies and 10 patients with negative biopsies for GCA. The immunoreaction intensity was measured using ImageJ software (available at: http://rsb.info.nih.gov/ij/; accessed May 6, 2009). Measurements were performed on multiple randomly selected regions of a minimum of 3 sections, and the mean values were calculated. Immunoreactivity was also graded according to how much of the specimen was stained: 0 ⫽ none, ⫹1 ⫽ cells in 1/4 of the specimen or less stained, ⫹2 ⫽ cells in 1/4 to 1/2 of the specimen stained, ⫹3 ⫽ cells in 1/2 to 3/4 specimen stained, and ⫹4 ⫽ 3/4 to 1 specimen stained (Table 3). Statistical analysis was performed using the Student t test. To determine the correlations between the inflammation marker C-reactive protein (CRP) or ESR and ET-1, ETA, and ETB receptor immunoreactivity, Pearson’s coefficient of correlation was used and linear regression relationships were created. Significance was defined as P⬍0.05. Values are presented as means ⫾ standard deviation.

Results Hematoxylin-Eosin Histopathology

⫺7

using blocking peptide ET-1 (10 mol/L) before the run of the staining protocol of 2 identical samples. Blocked antibody was used for 1 of the 2 identical samples, and the control was used for the other. Endothelin-1 was purchased from Sigma Chemical Company (St. Louis, MO). Samples were examined using an Olympus BX60 microscope (Tokyo, Japan) and photographed using a Pixera Pro 600ES digital camera (Los Gatos, CA).

Hematoxylin-eosin–stained sections of temporal arteries from the patients with GCA showed band-shaped infiltrates of inflammatory cells that include lymphocytes, histocytes, and multinucleated giant cells. In addition, concentric intimal hyperplasia with reduced cross-sectional area of the lumen and internal elastic lamina degeneration was observed (Table 2; Fig 1). Immunostaining for CD68 was particularly strong at sites of inflammatory cell infil-

Figure 1. Representative example of hematoxylin-eosin–stained sections of a temporal artery from (A) control patient without arteritis and (B) patient with GCA. Note the intimal hyperplasia with reduction of the lumen, internal elastic lamina degeneration, band-shaped inflammatory infiltrates of immune cells, and focal necrosis. GCA ⫽ giant cell arteritis.

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Dimitrijevic et al 䡠 Endothelin and Giant Cell Arteritis tration. CD45 labeling was also present, but not as prominent (data not shown). In more severe cases of GCA, transmural inflammatory infiltrates and necrosis in portions of the vascular wall were observed. Cellular changes such as those described above were not observed in the control samples, which were without signs of vasculitis (Table 2).

0.05) and endothelial cells (⫹39⫾4%; P⬍0.05). In addition, multinucleated giant cells showed mainly ⫹3 immunostaining for ET-1. Immunoreaction for ET-1 was also observed in SMCs and CD68-positive cells in the neointima.

Endothelin-A Receptor Immunoreactivity Endothelin-1 Immunoreaction In the temporal arteries of patients without vasculitis, low immunolabeling for ET-1 was observed in the SMC layer, whereas marginally increased levels of immunostaining were observed in the endothelial cell layer (Fig 2). Patients with GCA showed increased immunoreactivity of ET-1 in SMCs (⫹97⫾16%; P⬍

Immunostaining for ETA receptors was detected in SMCs and CD68-positive cells. Endothelin-A receptor is normally expressed in neointimal SMCs and CD68-positive cells in all blood vessels. No differences were observed in the expression of ETA receptors in SMCs or endothelial cells in samples from patients with GCA compared with controls (Fig 3).

Figure 2. Immunostaining for ET-1 in temporal arteries from patient with GCA compared with healthy control. (A) Panels are representative examples of immunohistologic images, and (B) panels are measurements of ET-1 immunostaining intensities. Results are shown as mean values ⫾ standard deviation. Statistical analyses were performed using the Student t test. *Significance was defined as P⬍0.05. Note that the ET-1 expression is higher in the SMC layer, endothelial cells, and giant cells in the temporal artery from patient with GCA. Immunostaining for ET-1 is shown in the neointima, infiltrating macrophages, and SMCs. ET ⫽ endothelin; GCA ⫽ giant cell arteritis; SMC ⫽ smooth muscle cells.

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Figure 3. Immunostaining for ETA receptors in temporal arteries from patient with GCA compared with healthy control. (A) Panels are representative examples of immunohistologic images, and (B) panels are measurements of ETA receptor immunostaining intensities. Results are shown as mean values ⫾ standard deviation. Statistical analyses were performed using the Student t test. *Significance was defined as P⬍0.05. Note that the ETA expression is weak, and no difference in the SMC layer is observed. Expression of ETA receptors in the neointima was also observed, located in SMC and macrophages. ET ⫽ endothelin; GCA ⫽ giant cell arteritis; NS ⫽ not significant; SMC ⫽ smooth muscle cells.

Endothelin-B Receptor Immunoreactivity As expected, immunostaining of ETB receptors was observed in the endothelial cell layer of tissue sections taken from control subjects. However, in patients with GCA there was an additional positive ETB receptor expression in the SMC layer (Fig 4) of the temporal arteries. In patients with GCA, ETB receptor immunostaining was increased (⫹75⫾18%; P⬍0.05) in SMC and the endothelium (⫹49⫾8%; P⬍0.05) compared with controls. Multinucleated giant cells exhibited distinct ETB receptor immunostaining in GCA.

Colocalization Experiments Counterstaining with alpha-gamma actin (for SMC) and CD31 (for endothelial cells) verified that ET-1, ETA, and ETB receptors were

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localized in SMCs and endothelial cells, respectively (data not shown).

Relation of C-reactive Protein to Endothelin-1, Endothelin-A, and Endothelin-B Receptor Expression in Giant Cell Arteritis The association among the ET-1, ETA, and ETB receptors’ immunoreactivity and the degree of systemic inflammation, characterized by circulating CRP levels in patients with GCA, was analyzed using Pearson’s coefficient of correlation. The quantitative immunohistochemical data allowed the analysis of a putative correlation to CRP or ESR levels. C-reactive protein levels were found to correlate to the degree of ET-1 (r⫽0.75, P⬍0.05) and ETB (r⫽0.65, P⬍0.05) receptor expression in temporal arteries from

Dimitrijevic et al 䡠 Endothelin and Giant Cell Arteritis

Figure 4. Immunostaining for ETB receptors in temporal arteries from patient with GCA compared with healthy control. (A) Panels are representative examples of immunohistologic images, and (B) panels are measurements of ETB receptor immunostaining intensities. Results are shown as mean values ⫾ standard deviation. Statistical analyses were performed using the Student t test. *Significance was defined as P⬍0.05. Note that the ETB expression is higher in the SMC layer and endothelial cells. Immunostaining for ETB receptors was observed in the neointima, SMCs, and macrophages. ET ⫽ endothelin; GCA ⫽ giant cell arteritis; SMC ⫽ smooth muscle cells.

patients with GCA, whereas no significant correlation with ESR was found (Fig 5 and data not shown).

Discussion Major Findings The present study was designed to examine the involvement ET-1 and its receptors ETA and ETB in GCA. Temporal arteries from patients diagnosed with GCA were compared with temporal arteries from age- and gender-matched morphologically GCA-negative individuals. The clinical evaluation showed that the controls had near normal temporal arteries without arteritis but remitted to temporal artery biopsy

because of clinical suspicion of GCA. The major findings from this study were (1) increased expression of the ET-1 receptor on endothelial cells and SMCs of temporal arteries from patients with GCA; and (2) increased expression of ETB receptor, normally expressed in endothelial cells, in SMCs, multinucleated giant cells, and endothelial cells in temporal arteries from patients with GCA. The ET-1 and ETB receptor expression correlated with the degree of inflammation.

Giant Cell Arteritis Diagnosis Patients were diagnosed as having GCA according to the criteria outlined by the American College of Rheumatology for the classification of GCA.1 Patients presenting with 3 or more of the following criteria were diagnosed as having

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Figure 5. Association among ET-1, ETA, and ETB receptor immunostaining and levels of CRP. The linear regression line (solid) equation is for non-zero slope. Statistical analyses were performed using Pearson’s coefficient of correlation. r ⫽ correlation coefficient; ns ⫽ not significant. *Significance was defined as P⬍0.05. Note the ET-1 and ETB receptor levels significantly correlate to CRP levels. CRP ⫽ C-reactive protein; ET ⫽ endothelin; NS ⫽ not significant.

GCA: temporal artery abnormality (tender or reduced pulsation), elevated ESR (defined as ⬎50 mm/h), age of onset ⬎50 years, onset of new headache, and abnormal arterial biopsy. Characteristics of the patients’ clinical presentations are shown in Table 1, verifying that the subjects with GCA conform to accepted diagnostic criteria. Hematoxylin-eosin–stained sections of temporal arteries demonstrated features that are typical of GCA, including intimal hyperplasia with reduction of the lumen, internal elastic lamina degeneration, and band-shaped infiltrates of inflammatory cells, including lymphocytes, histocytes, and multinucleated giant cells. In the more severe cases of GCA, transmural inflammatory infiltrates and necrosis were observed in portions of the vascular wall. No such changes were observed in the controls. Detailed presentation of the vascular wall lesions in patients with GCA and controls is shown in Table 2.

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Enhanced ET-1 expression in arteries of patients with GCA was not limited to endothelial cells but was also detected in SMCs in the media and neointima layers, as well as in multinucleated giant cells. This staining pattern was not observed in controls with vessels suspected of being affected but with no proven histopathology of arteritis. Endothelin-1 has numerous proinflammatory properties,11 which involve production of cytokines12,13 and interleukins,14,15 and stimulation of reactive oxygen species,16 which is known to increase the ET-1 expression. Endothelin-1 is also capable of inducing expression of NF-␬B17 and tumor necrosis factor-␣,18 the latter being stimulated by ET-1 itself.19 Endothelin-1 expression in GCA could therefore contribute to the progression of the disease as a result of the proinflammatory effect of ET-1. The vascular SMCs are under proinflammatory conditions known to produce ET-1 as observed (Fig 2). It is known that oxidative stress can elevate the production of ET-1 both in endothelial cells and in SMCs.16 This activity coincides with the site of inflammation as in GCA,16 which is where we observed ET-1 receptor. The high level of expression of ET-1 receptor in SMCs and giant cells is novel for GCA (Table 3). Platelet-derived growth factor has been suggested to be responsible for intimal hyperplasia in GCA, and previous work has shown that ET-1 alone or together with platelet-derived growth factor potentiates SMC growth.20,21 In addition, ET-1 has been implicated in the development of neointima lesions after balloon angioplasty. These lesions could be reduced by 50% through administration of an ET-receptor antagonist.22 Progression of GCA could thus in part be driven by ET-1 expression and its potent proliferative effects on both SMCs23 and the neointima inducing vascular wall thickening. Endothelin-1 could also enhance the production of collagen, elastin, and proteoglycans in SMCs, thus contributing to intimal proliferation after the vascular injury, as seen in GCA. Furthermore, metalloproteinase-2, implicated in the degeneration of elastin in GCA, is activated by ET-1.24 We speculate that inhibition of the ET system may provide a new pharmacologic approach to modulate the progression of GCA. This is supported by experiments with inhibitors of ET-1 receptors that have been shown to beneficial in the treatment of atherosclerosis,25,26 which is a different vascular disease but with common inflammatory components as in GCA. This is supported by the facts that ET-1 is the most potent vasoconstrictor known and that the most feared complication is vascular occlusion, which may originate from both vasoconstriction and proliferation.

Endothelin-1 Receptors in the Vascular Wall Two ET receptors are present in the human genome: ETA and ETB receptors.4,27,28 Both ETA and ETB receptors are distributed heterogeneously in human tissues and blood vessels;4 ETA receptors are primarily located on SMCs, and ETB receptors are abundant on endothelial cells.4 In the present study, we demonstrated a low level of immunoreactivity for ETB receptors on SMCs of the media in the temporal arteries in control patients. The immunostaining for ETA receptors was more pronounced and localized pri-

Dimitrijevic et al 䡠 Endothelin and Giant Cell Arteritis marily to the SMCs. In the patients with GCA, an increase in immunostaining was observed for ETB receptors located on SMCs of the temporal arteries. This is in agreement with work reported in sclerodermal renal artery crisis.7 It seems that in giant cells, ETB, rather than ETA, receptor expression is present in SMCs and endothelial cells. Thus, the results suggest that in GCA there is a shift in the expression of ET receptors in SMC toward more ETB receptors, similar to what has been observed in pulmonary arterial hypertension.29 Endothelin-1 is a more potent mitogen on vascular SMC expressing ETB receptors compared with ETA.30 The increased expression of the ETB receptor was seen in the SMCs. This could result in local vasoconstriction induced by the increased ET-1 production in the pathologic vessel, leading to vasoconstriction.31 Researchers have suggested that ETB receptors have a role in the clearance of ET-1;32,33 however, they are also involved in vasodilatation through release of nitric oxide. It is tempting to speculate that the observed increase in ETB receptor expression is secondary to the increased ET-1 production observed in GCA. Similar to what is observed in atherosclerosis, elevated ET-1 levels in the blood could in conjunction with increased ETB receptor expression in SMCs result in enhanced vasoconstriction.34 Given that ETB receptors are expressed on macrophages and ET-1 activates ETB receptors through NF-␬B, it is reasonable to speculate that in GCA the activation results in discharge of free radicals and increased levels of interleukin-8 and monocyte chemoattractant protein-1,17 maintaining the inflammatory condition. Because the ETA receptor expression was unaltered, it may have an additive effect, which supports enhanced vasoconstriction, and this may participate in the disease-associated vascular obstruction.

Limitations There are several limitations of this study that need to be addressed. The first limitation is that this is a transversal study; therefore, we could not investigate the prognostic implication of the ET-1, ETA, or ETB receptor expression. In addition, this was a retrospective immunohistochemical study with limited amounts of tissue available, and there is a need to confirm the data with protein quantization with Western blot. It needs to be taken in account that the observed correlations between CRP and ET-1 and ETB receptors are based on a small patient group, which needs further exploration. In conclusion, the present study demonstrated increased expression of ET-1 and ETB receptors in SMCs and endothelial cells in temporal arteries from patients with GCA. This finding suggests that the ET-1 pathway via ETB receptors is markedly activated in GCA in an autocrine/paracrine fashion. Increased activity in the ET system may contribute to the progression of the inflammation in GCA. The inflammation in GCA may be modulated by inhibitors of the ET system. This could be a corticosteroid-sparing alternative for the treatment of GCA.

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21. Hafizi S, Allen SP, Goodwin AT, et al. Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ET(A) receptor and is co-mitogenic with growth factors. Atherosclerosis 1999;146:351–9. 22. Douglas SA, Louden C, Vickery-Clark LM, et al. A role for endogenous endothelin-1 in neointimal formation after rat carotid artery balloon angioplasty: protective effects of the novel nonpeptide endothelin receptor antagonist SB 209670. Circ Res 1994;75:190 –7. 23. Hirata Y, Takagi Y, Fukuda Y, Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989;78:225– 8. 24. Felx M, Guyot MC, Isler M, et al. Endothelin-1 (ET-1) promotes MMP-2 and MMP-9 induction involving the transcription factor NF-kappaB in human osteosarcoma. Clin Sci (Lond) 2006;110:645–54. 25. Kowala MC, Rose PM, Stein PD, et al. Selective blockade of the endothelin subtype A receptor decreases early atherosclerosis in hamsters fed cholesterol. Am J Pathol 1995;146:819 –26. 26. Barton M, Haudenschild CC, d’Uscio LV, et al. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A 1998;95:14367–72. 27. Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 1990;348: 730 –2.

28. Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 1990;348:732–5. 29. Black SM, Mata-Greenwood E, Dettman RW, et al. Emergence of smooth muscle cell endothelin B-mediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood flow. Circulation 2003;108: 1646 –54. 30. Eguchi S, Hirata Y, Imai T, et al. Phenotypic change of endothelin receptor subtype in cultured rat vascular smooth muscle cells. Endocrinology 1994;134:222– 8. 31. Barber DA, Michener SR, Ziesmer SC, Miller VM. Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle. Am J Physiol 1996;270:H65–71. 32. Fukuroda T, Fujikawa T, Ozaki S, et al. Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Commun 1994;199:1461–5. 33. Plumpton C, Ferro CJ, Haynes WG, et al. The increase in human plasma immunoreactive endothelin but not big endothelin-1 or its C-terminal fragment induced by systemic administration of the endothelin antagonist TAK-044. Br J Pharmacol 1996;119: 311– 4. 34. Pernow J, Bohm F, Johansson BL, et al. Enhanced vasoconstrictor response to endothelin-B-receptor stimulation in patients with atherosclerosis. J Cardiovasc Pharmacol 2000; 36(suppl):S418 –20.

Footnotes and Financial Disclosures Originally received: April 6, 2009. Final revision: July 1, 2009. Accepted: July 30, 2009. Available online: December 24, 2009.

Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Manuscript no. 2009-477.

1

Department of Medicine, Institute of Clinical Sciences Lund University, Lund, Sweden. 2

Department of Pathology, Institute of Clinical Sciences Lund University, Lund, Sweden.

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Supported by the Swedish Research Council grant no 5958 and the Heart and Lung Foundation (Sweden). Correspondence: Ivan Dimitrijevic, MD, Division of Experimental Vascular Research, BMC A13, SE-221 84 Lund, Sweden. E-mail: [email protected].