IMMUNOLOGY, AUTOIMMUNITY, AND GRAVES’ OPHTHALMOPATHY Enalapril Reduces Proliferation and Hyaluronic Acid Release in Orbital Fibroblasts Roberta Botta, 1 Simonetta Lisi, 1 Claudio Marcocci, 1 Stefano Sellari-Franceschini, 2 Roberto Rocchi, 1 Francesco Latrofa, 1 Francesca Menconi, 1 Maria Antonietta Altea, 1 Marenza Leo, 1 Eleonora Sisti, 1 Giamberto Casini, 3 Marco Nardi, 3 Aldo Pinchera, 1,{ Paolo Vitti, 1 and Michele Marino ` 1 Background: Orbital fibroblast proliferation and hyaluronic acid (HA) release are responsible for some of the clinical features of Graves’ ophthalmopathy (GO). Thus, inhibition of these processes may be a possible thera- peutic approach to this syndrome. Enalapril, a widely used antihypertensive drug, was found to have some inhibitory actions on fibroblast proliferation in cheloid scars in vivo, based on which we investigated its effects in primary cultures of orbital fibroblasts from GO patients and control subjects. Methods: Primary cultures of GO and control fibroblasts were treated with enalapril or with a control compound (lisinopril). Cell proliferation assays, lactate dehydrogenase release assays (as a measure of cell necrosis), apo- ptosis assays, and measurement of HA in the cell media were performed. Results: Enalapril significantly reduced cell proliferation in both GO and control fibroblasts. Because enalapril did not affect cell necrosis and apoptosis, we concluded that its effects on proliferation reflected an inhibition of cell growth and/or a delay in cell cycle. Enalapril significantly reduced HA concentrations in the media from both GO and control fibroblasts. Conclusions: Enalapril has antiproliferative and HA suppressing actions in both GO and control fibroblasts. Clinical studies are needed to investigate whether enalapril has any effects in vivo in patients with GO. Introduction G raves’ ophthalmopathy (GO) is the most common extrathyroidal manifestation of Graves’ disease, as it occurs with various degrees of severity in about 50% of patients (1– 3). Because the pathogenesis of GO is poorly understood, treat- ment is difficult, and symptomatic rather than causal measures are mostly employed (1–6). Although the initiating events leading to the syndrome are still not known with certainty, the pathophysi- ological events responsible for the clinical features of GO are well established (1). Proliferation of orbital fibroblasts and increased secretion of glycosaminoglycans (GAG), especially hyaluronic acid (HA), by these cells, result in swelling of eye tissues, protru- sion of the eye globe, compensatory enlargement of eye muscles, and, in extreme cases, compression of the optic nerves (1). High doses of glucocorticoids (GC), either alone or in associ- ation with orbital irradiation, is the most common therapeutic approach in patients with moderate-severe and active forms of GO (2–6). In contrast, patients with mild and inactive GO are generally given only local, supportive measures, because these forms of GO are scarcely responsive to GC and because of the risks that high doses of GC carry (7–9). Nevertheless, patients with mild GO still suffer from the syndrome, because of their symptoms, and also because of the changes in their physical appearance, which influence their quality of life profoundly (2–5). Thus, efforts in the last few years have been aimed at identifying drugs that may be helpful to patients with mild forms of GO, possibly being devoid of the side effects that GC carry. On the clinical side, in a recent study, selenium was found to be relatively effective (10). On the laboratory side, quercetin was found to reduce proliferation and HA release in cultured orbital fibroblasts from GO patients, but also from subjects without GO (11). Here we investigated the effects of the angiotensin- converting enzyme (ACE) inhibitor enalapril in primary cul- tures of orbital fibroblasts. Our study is based on the previous observations of a possible healing effect of enalapril in cheloid scars, in subjects who were given the drug for high blood pressure (12), and also of an inhibitory effect of enalapril on the transforming growth factor (TGF)-b (13), a cytokine in- volved in HA synthesis in fibroblasts (14). Materials and Methods Primary cultures of fibroblasts Orbital adipose tissue samples were collected from five GO patients who underwent orbital decompression. Normal 1 Endocrinology, 2 Ear–Nose–Throat, and 3 Ophthalmology Units, University of Pisa, Pisa, Italy. { Dr. Aldo Pinchera passed away on October 11, 2012; see Bartalena et al. 2013 In memoriam: Aldo Pinchera, MD, PhD (1934–2012). Thyroid 23:1–4. THYROID Volume 23, Number 1, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/thy.2012.0373 92