Angiotensin II: Immunohistochemical Study in Sardinian Pterygium

Abstract

The Angiotensin II (Ang II) is the principal effector peptide of the RAS system. It has a pleiotropic effect and, beside its physiological role, it has the property to stimulate angiogenesis and activate multiple signalling pathways related to cell proliferation. The purpose of the study was to determinate the Ang II expression and localization in Sardinian pterygium and normal conjunctiva by immunohistochemistry, and its possible involvement in the development and progression of the disease. Twenty-three pterygiums and eleven normal conjunctiva specimens obtained from Sardinian patients, were processed for paraffin embedding and assessed for the immunohistochemi-cal revelation of Ang II. Significant Ang II expression was identified in pterygium and conjunctiva. Particularly, thirteen pterygium specimens (n=13) displayed exclusively moderate to strong nuclear staining; some specimens (n=5) showed exclusively a moderate cytoplasmic immunoreactivity, and few specimens (n=2) displayed moderate to strong immunoreactivity in both cytoplasm and nucleus. Only 3 specimens were negative. Statistical significance difference in respect of nuclear and cytoplasmic localization was observed between normal conjunctiva and pterygium (P=0.020). The results showed a predominant intranuclear localization of Ang II in pterygium epithelial cells, in spite of conjunctiva that mainly showed cytoplasmic localization. These findings suggest a possible role for Ang II in the development and/or progression of pterygium mediated by the activation of local RAS system.

Introduction

The renin angiotensin system (RAS) is one of the most studied physiological systems in humans. It is well known as major actor in the regulation of cardiovascular homeostasis, maintaining electrolyte balance, body fluid volume, and arterial pressure, through the production of aldosterone.¹ The angiotensin II (Ang II) is the principal effector peptide of the RAS system. It has a pleiotropic effect and, in addition to its well-known physiological role, it is implicated in the development of cardiac, vascular and renal pathologies.² Studies showed the property by Ang II to stimulate angiogenesis³ and activate multiple signalling pathways related to proliferation, including protein kinase C (PKC),⁴ mitogen-activate protein kinase (MAPK),⁵ and JAK II-STAT3.⁶ Moreover, synergically with its receptor AT₁, it can act as trigger for the activation of phosphatidylinositol 3 (PI3)-kinase/Akt,⁷ which plays a pivotal role in a wide range of cellular processes, including cellular motility and cell-cycle progression.⁸ Furthermore, studies from Nagai and co-workers,⁹ have demonstrated that Ang II enhances the vascular endothelial growth factor (VEGF) and is implicated in recruiting inflammatory cells by inducing chemokines and adhesion molecules. Novel studies have demonstrated that Ang II is also implicated in the growth of solid tumor cells, such as gastric,¹⁰ breast, ¹¹ ovarian,¹² and pancreatic cancer cells.¹³

In these last decades has raised the hypothesis that tissue-specific RAS system may exist. It would act independently from circulating RAS in different tissues, as demonstrated in the eye.^14,16 The local system may be characterized by the presence of some RAS components inside the cell and by the synthesis of Ang II in the intracellular compartments. However, to date this hypothesis is still matter of debate.¹ Ang II and its main receptors AT₁ and AT₂ were found in distinct intracellular regions.¹⁵ Within the kidney, several groups showed a high density of Ang II in the nuclear fraction of both cortical and medullary areas in rat.^16-18 Li and Zhuo¹⁸ also demonstrated that the activation of Ang II-AT₁ receptor axis modulates the expression of different mRNA transcripts involved in some pathological panels mediated by Ang II, such as monocyte chemoattractant protein, transforming growth factor and sodium hydrogen exchange (NHE3). Other emerging studies showed the intracellular localization of some pivotal components of the RAS, particularly the three principal AT receptor subtypes in the nucleus.^1,19-21 Immunoreactive Ang II has been found in the inner membrane of the mitochondria of mouse proximal tubules and myocytes²² as well as in nuclear fraction of isolated rat cardiomyocytes.²³

In the present study, we investigated the presence and localization of Ang II in pterygium, an ocular surface lesion associated with chronic UV exposure.²⁴ For long time considered a simple degenerative lesion, pterygium is now accepted as a distorted wound-healing response and dysregulated cell proliferation disease.²⁵ It is characterized by inflammatory features, rich vasculature, and extracellular matrix remodelling.²⁶ Anti-apoptotic mechanisms,²⁷ cytokines,²⁸ growth and angiogenic factors,²⁹ and viral infections^30-32 have been proposed as causative agents in its pathogenesis. Focusing on the pterygium features, and based on our previous study where we demonstrated the presence of the angiotensin converting enzyme (ACE) in pterygium,¹⁴ we aimed to investigate the presence of Ang II in bioptic pterygium samples. We hypothesized that a different Ang II localization/expression in pterygium, compared with normal conjunctiva, may be related to tissue-specific RAS system activation.

Materials and Methods

Immunohistochemistry

The study protocol for the use of human subjects in research was approved by the Human Research Ethics Committee of the Medical School, University of Cagliari, Italy. Patients with pterygium were admitted and treated at the Department of Surgical Science, Eye Clinic, University of Cagliari, Italy, while normal bulbar conjunctiva specimens were obtained from healthy donors undergoing strabismus surgery.

Twenty-three pterygium specimens and eleven conjunctiva specimens were processed for paraffin embedding. Demographic information on these patients is recorded in Table 1. After surgery, tissue fragments were fixed in formalin for at least 18 h. After fixation, tissues were washed in phosphate-buffered saline (0.1M PBS; pH 7.4) and processed. Paraffin-embedded serial sections (5 µm thick) were assessed for Ang II expression by immunohistochemistry. Briefly, sections were deparaffinized in xylene, rehydrated in a graded alcohol series and then equilibrated in PBS. Antigen retrieval was performed by citrate buffer (0.01M; pH 6) at 95ºC. Prior to incubation in the primary antibody, endogenous peroxidase activity was quenched with a solution of hydrogen peroxide 3% in 100% methanol for 20 min. Then, the sections were incubated for 45 min with 5% skim milk solution to block non-specific sites. Sections were incubated over night at 4ºC in rabbit primary antiserum polyclonal antibody to Ang II (1:100; Novus Biological, Littleton, CO, USA), and 30 min in anti-mouse peroxidase conjugated antibody (1:200, Chemicon International, Billerica, MA, USA) as secondary antiserum. 3,3’-diaminobenzidine (DAB) was used as final chromogen. Archival autoptic human kidney paraffin embedded specimen was used as positive control, while negative controls included omitting the primary antibody and isotype control. All sections were counterstained in haematoxylin and mounted in Entellan mounting medium (Merck KGaA, Darmstadt, Germany). Each section was viewed by two investigators, taking into account the location (cytoplasmatic and nuclear) and intensity of Ang II staining. Experiments were conducted in triplicate. Positive cells were obtained by counting manually in ten random fields at 400X. The cut-off was set at 60% of positive cells per field.

Table 1.

Demographic information for the pterygium and normal conjunctiva specimens.

Characteristic	Pterygium (n=23)	Normal conjunctiva (n=11)
Baseline mean age (SD)	52.9(±11.98)	49.76(±9.43)
Sex (%)
Male	12(52)	6(54)
Female	11(48)	5(46)
Type of lesion (M/F)
Primary	13 (7/6)	-
Recurrent	10 (5/5)	-
Eye (M/F)
Right	11(6/5)	-
Left	12(7/5)	-
Location of the lesion (M/F)
Nasal	13(7/6)	-
Temporal	10(5/5)	-

M, male; F, female.

Statistical analysis

Statistical analyses were performed using the SPSS Statistical Package for Social Science, version 15.0 (SPSS Inc., Chicago, IL, USA). The difference of Ang II expression between primary and recurrent or nasal and temporal specimens, and the correlation of this protein with the clinicopathological variables, such as sex or age, were assessed by Fisher’s exact test. All tests were two-tailed and P-values less than 0.05 were considered to be statistically significant.

Results

The results are summarized in Table 2. Significant Ang II expression was identified in pterygium and normal conjunctival epithelial cells (Figure 1). In conjunctiva, six specimens showed strong cytoplasmatic immunoreaction for Ang II (Figure 1A). Two samples showed moderate to strong immunoreaction in both cytoplasmatic and nuclear compartment (Figure 1B). Only one sample displayed strong nuclear immunoreactivity (data not shown). In regard to pterygium, a high number of pterygium specimens (n=13) displayed exclusively moderate to strong nuclear staining (Figure 1C). On the other hand, some specimens (n=5) showed exclusively a moderate cytoplasmatic immunoreactivity (Figure 1D). Furthermore, few specimens (n=2) displayed moderate to strong immunoreactivity in both cytoplasm and nucleus (Figure 1E). Occasionally, some sections showed immunopositive leukocytes-like cells (n=3) in the stroma and within the vessels (Figure 1F, arrows). No immunoreactivity developed in conjunctiva or in pterygium when incubated without a primary antibody and/or isotype control (Figure 1H). The kidney section showed strong immunoreactivity for Ang II in the cytoplasm and membrane of proximal renal tubules cells (Figure 1G).

Table 2.

Angiotensin II immunolocalization in pterygium and normal conjunctiva.

Localization	Specimens		P-value
	Pterygium	Conjunctiva
Cytoplasmic	5	6	0.038
Nuclear	13	1
Nuclear and cytoplasmatic	2	2
Negative	2	2
Total	22	11

Figure 1.

Immunohistochemical expression of angiotensin II (Ang II) in normal conjunctiva and pterygium. A) Positive immunoreactive staining is visible in the epithelial cells of normal conjunctiva in cytoplasm. B) Positive immunoreactive staining is visible in both cytoplasm and nuclear compartment. C,D,E) Nuclear, cytoplasmatic and both nuclear and cytoplasmatic positive immunostain to Ang II in pterygium epithelial cells, respectively. F) Ang II positive leukocytes-like cells are notable in blood vessels and in the stroma (arrows). G) Pterygium negative control does not display immunoreaction. H) Kidney shows specific immunostaining to Ang II.

Statistical analysis

Statistical significance difference in respect of nuclear and cytoplasmatic localization was observed between normal conjunctiva and pterygium (P=0.038), while no significant association was obtained between Ang II expression and nasal or temporal eye localization, primary or recurrent pterygium specimens, and clinical features such as age and sex (P>0.05). Moreover, no correlation was found in temporal or nasal localization, clinical features, and between primary or recurrent pterygium and clinical features (P>0.05).

Discussion

The present study shows the expression and immunolocalization of Ang II in pterygium and normal conjunctiva. Significative immunolocalization was noted in normal and pathological sections. It is widely known that Ang II is a multifunctional bioactive octapeptide of the renin-angiotensin system that plays a fundamental role in vasoconstriction, controlling cardiovascular function and renal homeostasis.³³ In our study we were able to detect Ang II in different cellular compartments, with notable differences between normal conjunctiva and pterygium. Our findings show a predominant nuclear immunolocalization of Ang II in most of the pterygium samples. Solely few pterygium specimens show a moderate cytoplasmatic immunostaining. Over the past two decades, has raised the hypothesis of local, tissue-specific, RAS system, that could act independently from circulating RAS. On this way, many authors, with different approaches, demonstrated local RAS systems.^34-38 In 1971, Robertson and Khairallah³⁹ demonstrated nuclear localization of Ang II in smooth and cardiac muscle cells. Later, other authors correlated the presence of intracellular/nuclear Ang II to different cellular mechanisms implicated in cell growth and proliferation^2,40 and tumor growth, invasion, and angiogenesis.^41-43

In view of these studies, we found a similar pattern in pterygium. Thirteen samples showed nuclear localization versus five samples that showed positive cytoplasmatic stain. This evidence led us to hypothesize a possible response by Ang II in the inflammatory/proliferative processes in pterygium mediated in the nuclear compartment. In fact, some studies reported the ability of Ang II to bind the chromatin,^44,45 to modulate gene expression, and to mediate different mRNA transcripts.¹⁶ Recent discoveries showed Ang II inducing rapid synthesis of VEGF in proximal tubular epithelial cells (MCT), by stimulation of its mRNA translation.⁴⁶ Moreover, it has been documented that Ang II regulates activity of several key mediators of injury, such as transforming growth factor-ß (TGF-ß).^47-49 This intranuclear Ang II immunoexpression lead us to hypothe-size a similar pathway in pterygium. Moreover, in the present study we were able to detect Ang II staining also in leukocytes-like cells; previous studies on human monocytes revealed that they contain high amount of Ang II, which may be potentially released under different stimuli.⁵⁰ A well-known pterygium feature is the leukocytes infiltration consisting predominantly of lymphocyte T cells.⁵¹ In consideration of this statement, it is reasonable to think that a potential release of Ang II from leukocites, in a chronic inflammation condition, could contribute to worsen the disease stage. All these evidences are reinforced by the results obtained from normal conjunctiva. Here we found a predominant epithelial cytoplasmatic staining. This leads to believe that in physiological conditions Ang II may basically play a role in the cytosol. Ang II microinjections on rat vascular smooth muscle cells have demostrated an increase cytosolic Ca²⁺ levels inducing both intracellular stores and Ca²⁺ influx.⁵² This suggests a possible support function by Ang II to normal, physiological, cellular mechanism,¹ postulated that actually intracellular RAS system, and particularly Ang II, may work in different ways under different conditions. In physiological condition the intracellular RAS may support the circulating RAS and, as proposed by Eggena et al., it may increase the local Ang II concentration by transcription rate of some RAS components when the Ang II concentration is low, or suppressing transcription when Ang II levels are high.⁵³ To date we can not assert if the intracellular RAS is an independent regulation system, however our feeling is that it could represent a more accurate control mechanism to support local and circulating RAS. This hypothesis is supported by a study from Kalinyak and Perlman⁵⁴ which stated that circulating Ang II concentration, in normal physiological conditions, is insufficient to induce the multiple responses attributed to Ang II.

In conclusion, in this report we showed that pterygium epithelial cells exhibit Ang II in the nuclear compartment. Intranuclear localization could be attributable to the gene expression modulator role played by Ang II in pathological condition. Moreover, positive Ang II leukocytes-like cells in pterygium suggest a possible role due to the release of Ang II in pterygium. This study represents a tip of the iceberg and further studies will be needed to better clarify the intracellular role of Ang II in pterygium.