Immunohistochemical analysis of vascular endothelial growth factor (VEGF) and p53 expression in pterygium from Tunisian patients.

A pterygium is characterized by abnormal fibrovascular corneoconjunctival tissue. A number of investigations have attempted to elucidate this incompletely understood pathology. Since vascular endothelial growth factor (VEGF) and p53 are known to participate in tumor vascularization, our purpose was to study VEGF and p53 expression in active primary and recurrent pterygium from Tunisian patients. To this end, 15 cases of active primary pterygium and five cases of recurrent pterygium from Tunisia were studied by immunohistochemistry. Antibodies raised against VEGF and p53 were used to analyze the distribution and expression of these markers in pterygium and normal human conjunctiva were used as negative control. VEGF and p53 proteins were found in all cases of primary pterygium in epithelial, fibroblast and vascular endothelial cells. Active primary and recurrent pterygium have different patterns of expression. In primary pterygium, an important variability of p53 and VEGF expression was observed. However, in recurrent pterygium, p53 immunoreactivity was weak to moderate, whereas VEGF immunoreactivity was strong. In normal human conjunctiva, VEGF and p53 expression was weak to negative. The overexpression of VEGF in active primary and recurrent pterygium suggests that angiogenesis may play a role in pterygium pathogenesis and the expression of p53 in active primary pterygium, which might be associated with its mutated form, supports the hypothesis that actinic radiation may be involved in the genesis of pterygium. Thus, VEGF and p53 may be useful biomarkers for understanding the physiopathology of pterygium.

Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol.

BACKGROUND AND AIMS: Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7ß-hydroxycholesterol (7ß-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 µM), a polyphenol from red wine. METHODS: ARPE-19 cells were treated with 7ß-OH, 7KC, or 25-OH (5-40 µg/mL; 24-48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1ß, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF. RESULTS: 7ß-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7ß-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion. CONCLUSION: 7ß-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.

Immunohistochemical analysis of cellular adhesion molecules (ICAM-1, VCAM-1) and VEGF in fibrovascular membranes of patients with proliferative diabetic retinopathy: preliminary study.

PURPOSE: Diabetic fibrovascular membranes are the main pathological changes of proliferative diabetic retinopathy that can cause serious complications leading to blindness. Since the mechanism of fibrovascular membrane development is still unknown, the aim of our study was to identify potential biomarkers for this pathology. To this end, we analyzed the simultaneous expression of ICAM-1, VCAM-1 and VEGF within tissues of diabetic fibrovascular membranes. PATIENTS AND METHODS: Fibrovascular membranes were taken from nine diabetic patients with proliferative diabetic retinopathy. The fibrovascular membrane specimens were analyzed by immunohistochemistry to determine ICAM-1, VCAM-1 and VEGF expression. Controls were collected on nine normal conjunctivas removed during senile cataract surgery. RESULTS: Coexpression of ICAM-1, VCAM-1 and VEGF was found in most of the diabetic fibrovascular membranes studied. Thus, ICAM-1 was positive in eight of nine membranes (82%), VCAM-1 in seven of nine membranes (78%) and VEGF in all the membranes. CONCLUSIONS: The substantial overexpression of adhesion molecules ICAM-1, VCAM-1 and of VEGF suggests that these molecules might contribute to the development of fibrovascular membranes in patients with proliferative diabetic retinopathy, and that they could constitute suitable markers of this pathology.

[Expression of Bax and Bcl-2 apoptotic factors in the conjunctiva of diabetic patients: a preliminary study]. / Expression des facteurs apoptotiques Bax et Bcl-2 dans la conjonctive des patients diabétiques: étude préliminaire.

INTRODUCTION: Apoptosis, or programmed cell death, is necessary to multicellular organism survival, in contrast to involuntary necrosis that is devastating for tissue. It is positively or negatively regulated by proteins of the Bcl-2 family. The aim of our study was to analyze the expression of apoptotic factors Bax and Bcl-2 in the bulbar conjunctiva of diabetic patients without retinopathy and to compare it to the expression of these factors in nondiabetic patients. MATERIAL AND METHODS: Twenty-five conjunctival biopsies were obtained from diabetic patients without retinopathy. The ocular fundus and retinal fluorescein angiography results were normal. Normal human conjunctiva was taken from 15 patients undergoing senile cataract surgery. Immunohistochemical analysis was performed using indirect immunoperoxidase with antibodies against Bax and Bcl-2. RESULTS: In the human diabetic conjunctiva, The Bax protein was highly expressed in all specimens (100%). It was distributed in epithelial cells, vascular endothelial cells, fibroblasts, and inflammatory cells. The Bcl-2 protein was always at a low level or absent. In normal conjunctiva, Bax showed a significant level, whereas Bcl-2 showed no trace of positivity. CONCLUSION: Bax is often localized in tissues characterized by an elevated rate of apoptosis; in contrast, Bcl-2 is absent in these places. Our results suggest that diabetic human conjunctiva, with its inflammatory and cicatricial phenomena, is a privileged target for apoptotic cell death.

[Expression of intercellular adhesion molecule type 1 in the conjunctiva of diabetic patients: a preliminary study]. / Expression de la molécule d'adhésion intercellulaire de type 1 au niveau de la conjonctive des patients diabétiques: une étude préliminaire.

PURPOSE: Recent research has incriminated adhesion molecules in the pathogenesis of diabetic retinopathy. These molecules have been found to be expressed in many cells participating in inflammatory processes and neovascularization. The purpose of our investigation was to study the expression of intercellular adhesion molecule type 1 (ICAM-1) in the conjunctiva of diabetic patients without retinopathy in comparison with normal human conjunctiva. PATIENTS AND METHODS: Fifteen conjunctival biopsies were obtained from diabetic patients without retinopathy. The ocular fundus examination and retinal fluorescein angiography were normal. The normal human conjunctiva were taken from five patients undergoing senile cataract surgery. Immunohistochemical analysis consisted of indirect immunoperoxidase using the monoclonal antibody ICAM-1. RESULTS: The adhesion molecule ICAM-1 was immunolocalized in epithelial, vascular endothelial, and inflammatory cells. The expression of this molecule was different in diabetic patients for the same duration. In the normal human conjunctiva, the expression of ICAM-1 was very low. CONCLUSION: This preliminary study shows that ICAM-1 is present in the conjunctiva of diabetic patients without retinopathy and thus may add new insights into the pathogenesis of diabetic retinopathy.