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Abstract Skin pigmentation has both individual and societal implications. Focal or widespread loss of normal pigmentation not only renders individuals extraordinarily vulnerable to the harmful effects of sunlight (e.g. increased risk of skin cancer in albinism), but it can also result in severe emotional stress (e.g. vitiligo). Vitiligo is an acquired skin disorder caused by the disappearance of pigment cells from the epidermis that gives rise to well defined white patches which are often symmetrically distributed. The lack of melanin pigment makes the lesional skin more sensitive to sunburn. Loss of the functionality of melanocytes is responsible for vitiligo but the real cause of vitiligo is unknown. However, some conditions including autoimmune, genetic, neural, viral infections and oxidative stress could have an important role in pathogenesis of vitiligo, most of them acting in concert. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes, although epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. Therefore, damage to keratinocytes might have a significant effect on melanocyte survival. The fine structural changes of keratinocytes degeneration seems to be consistent with either early signs of cellular necrosis or apoptosis. Additionally, anti-keratinocyte antibodies, which have been detected in the sera of patients with vitiligo, resulting from keratinocyte death during the disease process. This will result in passive melanocyte death from reduced synthesis of keratinocyte -derived factors needed for melanocyte survival. Aquaporin 3 has been identified in multiple epithelial tissues as a transmembrane protein. AQP3 is detected in the epidermal keratinocytes below the stratum corneum and is involved in skin hydration. E-Cadherin plays a pivotal role in keratinocyte differentiation and cell-to cell adhesion. It was reported that phosphorylated phosphatidylinositol 3-kinase (PI3K) decreased in the depigmented epidermis of patients with vitiligo compared to that in normally pigmented epidermis. In fact, increased apoptosis of vitiliginous keratinocytes was found to be caused by impaired PI3K/AKT activation, which resulted in reduced activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), in the presence of increased tumour necrosis factor alpha (TNF-α) levels. Activation of PI3K depends on both E-Cadherin-dependent cell adhesion and tyrosine phosphorylation events. Therefore, a connection between AQP3 and PI3K through the E-Cadherincatenin complex was considered. Particularly, the role of AQP3 and PI3K in keratinocyte apoptosis. So, co-localization of AQP3 and E-Cadherin showed that there is a functional link between AQP3 and E-Cadherin in vitiliginous skin and they decreased at the cell-to-cell contacts in the depigmented epidermis. The present study aims at studying the possible role of keratinocytes in vitiligo by the immunolocalization of E-Cadherin and AQP3 in vitiliginous skin and hair follicles. This case-control study was carried out on 65 subjects. These included 40 patients of different clinical varieties of vitiligo and 25 age and sex matched normal subjects as a control group. The diagnosis of vitiligo was made on the basis of the patient’s history and the typical clinical features of discrete, well circumscribed, depigmented macules and patches. Every patient was subjected to: A) Personal history including: Name, age and sex. B) Present history including: Course, duration of the disease and duration of lesion from which the biopsy was taken. C) Family history of vitiligo. All the studied cases were either newly diagnosed or old patients with completely depigmented lesions. 2-Examination A) Full general examination to exclude associated systemic diseases such as thyroid disease, diabetes mellitus, and other autoimmune diseases. B) Detailed dermatological examination to identify Type and distribution of vitiligo. Assessment of disease activity was done according to VIDA score Every case and control was subjected to 1-Skin biopsies Three millimeter punch biopsies were taken under 2% lignocaine local anesthesia from vitiliginous skin of patients including perilesional skin. Biopsies were fixed in neutral formalin 10%, and submitted to routine tissue processing ending with paraffin embedded blocks formation. Haematoxylin and eosin stained sections were examined microscopically to evaluate and verify epidermal and dermal pathological changes if present. Immunohistochemical staining for E-Cadherin was done and interpretation was evaluated as a brown cytoplasmic, membranous or membrano-cytoplasmic stain was considered positive in in the studied cases (Lesional & perilesional skin) and control specimens. Immunohistochemical staining for aquaporin3 was done and interpretation was evaluated as a brown cytoplasmic, membranous or membrano-cytoplasmic stain in the cells was considered positive in the studied cases (Lesional & perilesional skin) and control specimens. The data were collected, tabulated, and analyzed by SPSS (statistical package for social science) version 17.0 on IBM compatible computer. Differences were considered as follows: Statistically significant (S) when (P<0.05). Highly significant (HS) when (P< 0.001). Not significant (NS) when (P> 0.05). In the present work, keratinocyte expression of both E- Cadherin and AQP3 is downregulated in lesional skin comapred with perilesional and control skin both in hair follicles and interfollicular epidermis. The downregulation of AQP3, in cultured normal human KCs, resulted in significantly decreased phosphorylation of PI3K-p85a and the expression of the ECadherin– catenins. Moreover, AQP3 knockdown also reduced binding of ECadherin and β-catenin to the same quantities of PI3K-p85a. AQP3 downregulation will adversely affect its downstream signalling molecules such as PI3K, E-Cadherin, and catenins which are involved in the regulation of cell survival. This may suggest AQP3 role in vitiligo, as reduce survival of KCs might lead to passive melanocyte death by loss of cell-cell contact and by reduction of KC-derived growth factors. Keratinocytes undergo apoptosis in the depigmented epidermis compared with the normally pigmented epidermis in patients with vitiligo. This may underscore the earlier observation detected in the present work which is KCs vacoulization in vitiliginous epidermis. In addition, loss of adhesion between KCs and melanocytes in vitiligo leads to detachment of melanocytes from basal to suprabasal layer of epidermis where they seem to be more susceptible to apoptosis. The present study shows AQP3 and E-Cadherin downregulation in perilesional skin compared with normal skin. Many studies demonstrated absence and discontinuous distributions of ECadherin in perilesional normal-appearing skin leading to altered cell–cell adhesion. Authors added that this downregulation was associated with oxidative stress in affected cells. These data are in agreement with the melanocytorrhagy hypothesis that has been proposed as the primary event in melanocytic detachment following a mechanical trauma and with the reduced number of melanocytes in the basal layer of reconstructed skin using melanocytes from subjects without vitilig Therefore, from this downregulation in perilesional skin, we can postulate that therapeutic options for vitiligo has to be extended to pigmented skin which may help to prevent early events in “silent” vitiligo melanocytes and prevent the spread of the disease. In summary, both AQP3 and E-Cadherin are downregulated in vitiliginous skin. This leads to altered cell-cell adhesion with decreased KCs survival. Loss of keratinocytes and loss of keratinocyte-melanocyte adherence leads to melanocyte loss due to decreased keratinocyte – derived growth factors. These changes begins in perilesional skin even before the appearance of clinically evident vitiliginous lesions. |