Enalapril Reduces Proliferation and Hyaluronic Acid Release in Orbital Fibroblasts

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, treatment 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 pathophysiological 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, protrusion 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 association 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 cultures 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)-β (13), a cytokine involved in HA synthesis in fibroblasts (14).

Materials and Methods

Results

Enalapril inhibits orbital fibroblasts proliferation

Fibroblasts were treated with enalapril 2 or 5 mM, for 3 or 5 days. The concentrations of enalapril were well below the cytotoxic dose, which was found to be >5 mM on cell viability counts at 72 hours (not shown). Proliferation of orbital fibroblasts was reduced in both GO and control fibroblasts, both at 3 and 5 days (Fig. 1a). The decrease in proliferation was statistically significant in both groups (p=0.0008 in GO fibroblasts and p=0.0010 in control fibroblasts). The effect of enalapril was specific, as lisinopril, another ACE-inhibitor, had negligible effects (Fig. 1b).

OPEN IN VIEWER

FIG. 1.

(a) Cell proliferation after treatment with enalapril for 3 or 5 days. (b) Proliferation after treatment with lisinopril 5 mM for 3 or 5 days. Values are expressed as median±interquartile range (IQR).

Enalapril does not affect cell toxicity in orbital fibroblasts

To understand the mechanisms responsible for inhibition of cell proliferation, we determined LDH release from cells, as a measure of cell toxicity (necrosis) (15). Fibroblasts were treated for 3 or 5 days with 5 mM enalapril. As shown in Figure 2a, enalapril did not cause any significant increase in cell toxicity in either of the fibroblast groups, and also lisinopril had no effect (Fig. 2b).

OPEN IN VIEWER

FIG. 2.

(a) Lactate dehydrogenase (LDH) release after treatment for 3 or 5 days with enalapril. (b) LDH release after treatment with lisinopril for 5 days. LDH is expressed as an index obtained by dividing the observed LDH release by the maximum LDH release. Values are expressed as median±IQR.

Enalapril does not induce apoptosis in orbital fibroblasts

We then investigated whether enalapril has any effects on apoptosis. As shown in Figure 3a and b, neither enalapril nor lisinopril affected apoptosis in both GO and control fibroblasts.

OPEN IN VIEWER

FIG. 3.

Apoptosis measurement after treatment with enalapril (a) or lisinopril (b) for 5 days. Values are expressed as median±IQR.

Enalapril reduces HA release

To evaluate the effects of enalapril on HA release, we measured HA in the cell media following treatment of orbital fibroblasts with 5 mM enalapril for 5 days. As shown in Figure 4a, HA concentrations were reduced upon enalapril treatment in both groups, in a statistically significant manner (p=0.0431 in both GO and control fibroblasts). Lisinopril treatment had negligible effects (Fig. 4b).

OPEN IN VIEWER

FIG. 4.

(a) Hyaluronic acid (HA) release in the cell media after treatment with enalapril (a) or lisinopril (b). Values are expressed as median±IQR.

Discussion

In the present study, we provide evidence for an inhibitory effect of enalapril on orbital fibroblast proliferation and HA production. The possibility of an inhibitory effect was considered because of previous studies suggesting that enalapril may have some inhibitory actions on fibroblast growth and HA synthesis (12 –14). Enalapril belongs to the family of ACE-inhibitor drugs and is commonly used in patients with high blood pressure (16). In addition to their effects on blood pressure, ACE-inhibitors are known to reduce the left ventricular collagen content and attenuate collagen remodeling in the postinfarctual period (16). Furthermore, they have been shown to exert a pulmonary antifibrotic effect (16). These apparent inhibitory effects on the extracellular matrix may involve an action of ACE-inhibitors on GAG production by fibroblasts. Thus, enalapril has been shown to have an inhibitory action on TGF-β, a cytokine involved in HA synthesis (13,14). Finally, recently Iannello et al. reported two cases of patients given enalapril for high blood pressure, in whom they observed healing of cheloid scars, suggesting a possible inhibitory effect of enalapril in fibroblast proliferation (12).

In the present study, we investigated the effects of enalapril in primary cultures of orbital fibroblasts taken from patients with GO or from control subjects; namely, patients who had undergone orbital surgery for reasons unrelated to GO. Enalapril markedly reduced cell proliferation in both cell groups, but it did not influence cell toxicity or apoptosis. Therefore, we concluded that its effects on proliferation reflect an inhibition of cell growth and/or a delay in cell cycle. Nevertheless, further studies are required to investigate, in detail, the molecular and cellular mechanisms involved in the action of enalapril in orbital fibroblasts. In addition to its effects on cell proliferation, enalapril markedly reduced HA release by orbital fibroblast, which, as discussed below, is quite relevant in view of the role of HA in GO (1).

As mentioned above, orbital fibroblast proliferation and increased GAG secretion are among the major mechanisms responsible for the pathological changes of orbital tissues in GO (1). Inhibition of these processes may be a reasonable therapeutic approach to GO, especially in the early stage of the syndrome, when these processes occur mostly (1 –6). Our findings raise the possibility that enalapril may be tested in patients with GO. The fact that its actions in vitro were exerted in both GO and control fibroblast should not be a limitation for a possible clinical use of enalapril, considering that, in any case, enalapril is largely employed in the clinical practice for high blood pressure, and if it had an effect on fibroblast proliferation in subjects without GO, this would be clinically irrelevant, as it has never been reported among the side effects of this drug (16). On the other hand, orbital fibroblast proliferation and HA production are known to be much greater in GO patients than in patients without GO, because of which enalapril may have a detectable effect in GO, as it seems to do in cheloid scars (12). Further studies are needed to investigate this issue.