|Year : 2014 | Volume
| Issue : 1 | Page : 21-26
Role of vascular endothelial growth factor, survivin, and inducible nitric oxide synthase expression in psoriasis: an immunohistochemical study
Ahmed Salem1, Amani Nasar1, Abdalla Kandil1, Randa Farag1, Kamal El-Kasheshy2
1 Department of Dermatology and Venereology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||01-Aug-2013|
|Date of Acceptance||01-Dec-2013|
|Date of Web Publication||24-Jul-2014|
Department of Dermatology and Venerology, Faculty of Medicine, Zagazig University, Zagazig
Source of Support: None, Conflict of Interest: None
Psoriasis (PS) is a common chronic, relapsing, immune-mediated disease involving the skin and joints of genetically predisposing individuals. Despite numerous studies, the pathogenesis of PS has not been fully elucidated. However, many pathogenic theories have been suggested. There is much evidence that PS is a polygenic disease modified to expression by triggering factors. PS is characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes, lymphocyte infiltration consisting mostly of T lymphocytes, and various endothelial vascular changes in the dermal layer, such as angiogenesis, dilation, and high endothelial venule formation. Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis, causing aberrant angiogenesis and vascular leakage in the upper dermis; it may also contribute to keratinocyte proliferation and epidermal barrier homeostasis in PS. Inducible nitric oxide synthase (iNOS) was found to be expressed in the keratinocytes of psoriatic lesions; in addition, there is increased expression of iNOS-specific mRNA transcripts. Survivin upregulation in psoriatic lesion in comparison with normal skin was evident, suggesting its role in the pathogenesis of PS.
The aim of this study was to investigate the immunohistochemical expression of VEGF, survivin, and iNOS in psoriatic skin and to detect their role in the pathogenesis of PS.
Patients and methods
This study was carried out on 20 patients with PS vulgaris. Immunohistochemical reactions were carried out using the streptavidin-biotin immunoperoxidase system in this study to detect the extent of expression of VEGF, survivin, and iNOS in each specimen of the skin biopsy.
A strong VEGF expression through the epidermis (mean 46.4 19.7) was observed. VEGF was significantly upregulated in psoriatic skin in comparison with normal healthy skin (P < 0.0001). Survivin was significantly upregulated in psoriatic specimen in comparison with healthy controls (P < 0.0001). INOS expression was significantly increased in psoriatic epidermis and dermis compared with healthy skin (P < 0.0001).
VEGF, survivin, and iNOS appeared to be important factors in the pathogenesis of PS.
Keywords: Psoriasis, iNOS, VEGF, survivin, IHC, clinical study
|How to cite this article:|
Salem A, Nasar A, Kandil A, Farag R, El-Kasheshy K. Role of vascular endothelial growth factor, survivin, and inducible nitric oxide synthase expression in psoriasis: an immunohistochemical study. Egypt J Dermatol Venerol 2014;34:21-6
|How to cite this URL:|
Salem A, Nasar A, Kandil A, Farag R, El-Kasheshy K. Role of vascular endothelial growth factor, survivin, and inducible nitric oxide synthase expression in psoriasis: an immunohistochemical study. Egypt J Dermatol Venerol [serial online] 2014 [cited 2020 Jan 26];34:21-6. Available from: http://www.ejdv.eg.net/text.asp?2014/34/1/21/137262
| Introduction|| |
Psoriasis (PS) is a common chronic, relapsing, immune-mediated disease involving the skin and joints of genetically predisposing individuals, which potentially shares the disease pathways with other chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases .
Several variants of PS are well characterized, including chronic plaque PS, which is the most common variant, guttate PS, erythrodermic PS, pustular PS, and inverse PS .
Despite numerous studies, the pathogenesis of PS has not been fully elucidated. However, many pathogenic theories have been suggested . There is much evidence that PS is a polygenic disease modified to expression by triggering factors .
PS is characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes, lymphocyte infiltration consisting mostly of T lymphocytes, and various endothelial vascular changes in the dermal layer, such as angiogenesis, dilation, and high endothelial venule formation. T lymphocytes and the released cytokines and chemokines appear to be the principal driver of lesion development and persistence. In addition, endothelial cells (EC), neutrophils, and natural killer T cells may play an adjunctive role along with other cytokines such as intercellular adhesion molecule-1 .
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor and vasculoprotein, is an endothelial-specific growth factor. In addition, it is a potent angiogenic agent and very potent hyperpermeability factor being 50 000 times more potent than histamine .
It is suggested that PS is an angiogenesis-dependent disease, and vasoproliferation is suggested as a suitable target for the development of antipsoriatic drugs .
Several angiogenic factors have been identified in psoriatic epidermis, including IL-8, TNF-a, transforming growth factor-a, EC-stimulating angiogenesis factor, thymidine phosphorylase, and VEGF .
Besides the potential role of VEGF in causing aberrant angiogenesis and vascular leakage in the upper dermis, it may also contribute to keratinocyte proliferation and epidermal barrier homeostasis ,. In psoriatic skin, the VEGF receptors, VEGFR-1 and VEGFR-2, are detectable and functional in the keratinocytes .
Inducible nitric oxide synthase (iNOS) was found to be expressed in the keratinocytes of psoriatic lesions . In addition, there is increased expression of iNOS-specific mRNA transcripts. The use of topical betamethasone valerate markedly decreased iNOS-mRNA transcription, iNOS expression, and NO production .
NO released from the keratinocytes at high concentration may be considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of iNOS-mRNA and protein in PS lesion, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver antiproliferative NO concentrations. Recently, arginase 1, which substantially participates in the regulation of iNOS activity by competing for the common substrate l-arginine, is highly overexpressed in the hyperproliferative psoriatic epidemis and is coexpressed with iNOS .
Survivin is the smallest member of a family of potent endogenous caspase inhibitors called the inhibitor of apoptosis proteins . It is a multifunctional protein that controls cell proliferation, inhibition of apoptosis, and the promotion of angiogenesis. Gene encoding survivin is among the top five most tumor-specific genes in human genome . Although survivin is expressed at high levels during fetal development, it is rarely expressed in normal healthy adult tissues. It is however upregulated in the majority of cancers .
PS may not only develop as a consequence of excessive cell proliferation, but also may be the result of prolonged cell survival, upregulation of cell survival pathways, and suppression of apoptosis, which are implicated in blood vessel maintenance and tissue inflammation. Skin ECs and epidermal keratinocytes critically depend on survival machinery to preserve newly formed vessel integrity and epidermal hyperplasia, key pathogenic features of PS .
| Materials and methods|| |
This study was carried out on 20 patients with PS vulgaris. There were 10 male patients and 10 female patients and their ages ranged between 10 and 68 years. The participants included in this study were selected randomly from the outpatient clinic of the Dermatology and Venereology Department, Zagazig University Hospitals during the period from June 2010 to February 2011. Ten healthy volunteers were also included in this study as a control group.
A 6-mm punch biopsy was taken from each patient under local anesthesia (2% xylocaine). The punch biopsy specimen was obtained from a fully developed psoriatic plaque, near the margin. Each specimen was fixed in 10% buffered neutral formalin solution (not more than 24 h) and embedded in paraffin to form paraffin blocks, and serial sections of 5 μm thickness were obtained from each block. None of the patients was receiving systemic or phototherapy. All topical therapies, apart from emollients, were discontinued 3 weeks before entry into the study. All biopsies were submitted to histopathologic evaluation to confirm the diagnosis of PS and to immunohistochemical staining for anti-VEGF (dilution 1:200; Santa Cruz Biotechnology, California, USA), antisurvivin (dilution 1:10; Santa Cruz Biotechnology), and polyclonal antibodies - anti-iNOS (dilution 1 : 50; Santa Cruz Biotechnology) - in Pathology Department, Faculty of Medicine, Zagazig University (Egypt).
Immunohistochemical reactions were carried out using the streptavidin-biotin immunoperoxidase system in this study to detect the extent of expression of VEGF, survivin, and iNOS in each specimen of the skin biopsy.
Evaluation of immunostaining
Immunostaining for VEGF and iNOs was semiquantitatively scored as follows:
1, Weak (<10% expression of cells).
2, Moderate (10-20% expression of cells).
3, Strong (expression in >50%).
A score of 2 or higher was considered positive .
Immunostaining for survivin was semiquantitatively scored as follows:
The mean percentage of positive cells for the expression of survivin was determined in at least five areas at 400-fold magnification, and patients with less than 10% positively stained cells were defined as negative. Patients with 10-29% positively stained cells were defined as +, 30-59% as ++, and 60% or more as +++ .
The results of the study were statistically analyzed using SPSS version 15 statistical program. Data were expressed as mean ± SD for quantitative variables, numbers, and percentage. For categorical variables, the Student t-test, analysis of variance, the χ2 -test, and paired t-test were used when appropriate. Correlation (r) was used to find the relationship between quantitative variables. P value less than 0.05 was considered the significant limit.
| Results|| |
The psoriatic group included 10 male patients and 10 female patients; their ages ranged from 10 to 68 years, with a mean of 43.7 ± 18.2 years. The duration of their disease varied from 1 to 120 months, with a mean of 52.15 ± 40.1 months [Table 1].
Evaluation of vascular endothelial growth factor immunostaining
In PS, we found a strong cytoplasmic VEGF expression throughout the epidermis (mean 46.4 ± 19.7) and a moderate expression in inflammatory infiltrate and blood vessels (mean 19 ± 4.9, 8.4 ± 1.66, respectively). However, in the healthy skin, VEGF expression was faint (mean 1.2 ± 0.4). VEGF was significantly upregulated in psoriatic specimen in comparison with normal healthy skin (P < 0.0001) [Table 2] and [Figure 1].
|Table 2: Comparsion between VEGFexpression in psoriatic and healthy skin:|
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Evaluation of survivin immunostaining
Sixteen of the 20 patients with psoriatic lesions (80%) showed positivity for survivin. The staining was seen either in the epidermis or endothelium of proliferating capillaries or both. Pure epidermal staining was seen in nine (56.25%) patients, whereas endothelial staining was seen in three (18.75%) patients. Moreover, four (25%) patients showed both epidermal and endothelial staining.
In normal skin, epidermal survivin expression was mostly negative, but faint stain appeared in the epithelial basal layer and in the capillary ECs. Staining of the outer root sheath of hair follicles and sweat gland was also observed in normal specimens. Survivin was significantly upregulated in psoriatic specimens in comparison with healthy controls (P < 0.0001) [Table 3] and [Figure 2].
|Table 3: Survivin immunohistichemical expression in psoriatic and healthy skin|
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Evaluation of inducible nitric oxide synthase immunostaining
iNOs expression was significantly increased in the psoriatic epidermis and dermis compared with healthy skin (P < 0.0001). There is a modest iNOs expression in the psoriatic vessel wall [Table 4] and [Figure 3]
|Table 4: iNOS immunohistochemical expression in psoriatic and healthy skin|
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The correlation between PASI score and VEGF, iNOS, and survivin was unsignificant [Table 7].
| Discussion|| |
PS is considered to be a T cell-mediated disease with active hyperproliferation of keratinocytes and abnormal vascular expansion within the superficial dermis. This expansion is mediated by angiogenesis, an active vasoproliferative process that appears to be a key inflammatory response early in the pathogenesis of PS .
VEGF is a major regulator of physiological and pathological angiogenesis. The receptors for VEGF (VEGFR-1 or VEGFR-2) are primarily expressed by vascular ECs. VEGF binds to either of these receptors, leading to receptor activation and intracellular signal transduction .
VEGF signaling often represents a critical rate-limiting step in physiological angiogenesis. Under physiological conditions, VEGF promotes growth of ECs derived from the arteries, veins, and lymphatic vessels. Its permeability enhancing activity underlies significant roles of this molecule in inflammation and other pathological circumstances .
Besides its potential role in causing aberrant angiogenesis and vascular leakage in the upper dermis, VEGF may also contribute to keratinocyte proliferation and epidermal barrier homeostasis .
In psoriatic skin, the VEGF receptors, VEGFR-1 and VEGFR-2, are detectable and functional in the keratinocytes .
As VEGF is secreted by the keratinocytes and induces VEGFR expression in the same cells, VEGF may also contribute to keratinocyte hyperproliferation in PS in an autocrine manner. This could be relevant when PS is triggered by external injury (Koebner phenomenon), and interestingly disruption of the epidermal barrier homeostasis induces VEGF expression .
In PS, we detected a strong VEGF expression through the epithelium (mean 46.4 ± 19.7). This is in agreement with the study by Simonetti et al.  who found that VEGF was strongly expressed in PS throughout the epidermis (mean 45.60 ± 19.84).
Avramidis et al.  found that the mean VEGF expression in the ECs and dermis was 30.82 ± 0.65 in psoriatic skin, and they reported reduction in VEGF expression by 81.6% at week 10 under etanercept treatment.
In contrast, in the healthy skin, we noted that VEGF expression was faintly diffused (mean 1.2 ± 0.4), a finding that is in agreement with the study by Simonetti et al. . VEGF was significantly upregulated in psoriatic specimen in comparison with normal healthy skin (P < 0.001).
Keratinocytes cell-derived VEGF probably actively contributes to the pathogenesis of PS by the induction of permeability in dermal vessels, its chemotactic action on inflammatory cells and ECs, and its angiogenic effect . VEGF increases keratinocytes mitogenic activity in vitro and upregulates expression of VEGF receptor 1 and 2 on both keratinocytes and ECs. This autocrine/paracrine loop appears to contribute to the cycle of angiogenesis and epidermal hyperplasia in PS .
PS is a common inflammatory skin disease showing many pathological features, including the presence of marked T-cell infiltrates, hyperplasis/altered differentiation of keratinocytes, and infiltration by a group of CD11c + /CD1a + dendritic cells that have been termed inflammatory dendritic epidermal cells .
Advanced study on PS has also identified skin infiltration by a group of dendritic cells that synthesize TNFα and iNOS. These cells are evident in both dermis and epidermis and are a major cell type in the skin in lesion of PS .
In our study, iNOS expression was significantly increased (P < 0.001) in the psoriatic epithelium and dermis compared with normal skin. We observed a moderate expression of iNOS in the psoriatic vessel walls; these findings are in accordance with Simonetti et al.  study.
Overexpression of iNOS contributes to VEGF-induced angiogenesis . Moreover, VEGF mediates a central part of its proangiogenic effect through stimulation of eNOS and possibly iNOS-derived NO .
In our study, we observed a significant positive correlation between VEGF and iNOS expression (r = 0.793) [Table 5]. This result is in agreement with the study by Simonetti et al.  who found a significant positive correlation between VEGF and iNOS expression (r = 0.733, P = 0.038).
Survivin is the smallest member of a family of potent endogenous caspase inhibitors called the inhibitor of apoptosis proteins .
We observed absence of survivin staining of the suprabasal layers in control specimens, and its confinement to the basal layer of epidermis was similar to that of Abdou and Hanout . The basal layer of epidermis represents population of stem cells that have a high capacity for self-renewal, and it is responsible for constant epidermal turnover. However, no survivin immunostaining was detected in normal skin according to Bowen et al. . Faint survivin expression in the epithelial basal layer and in the capillary ECs was detected by Simonetti et al. .
Sixteen of the 20 psoriatic lesions (80%) showed positivity for survivin, comparable with the findings of Bowen et al.  that reached 88% and the findings of Abdou and Hanout  that reached 73%. These differences may be due to different numbers of investigated psoriatic patients, which were eight patients in Bowen et al.'s  study, 30 in Abdou and Hanout study , and 20 in this study.
In our study, survivin was significantly upregulated in psoriatic specimen in comparison with normal skin (P < 0.001). We detected nuclear survivin expression mainly in the psoriatic epithelial suprabasal layer, whereas cytoplasmic survivin expression was stained in the basal and suprabasal keratinocytes. These results were similar to those of Simonetti et al. .
In this study, the endothelial pattern of staining was seen in seven of 16 (43.8%) patients with psoriatic lesions, whereas it was not observed in control specimens. This is consistent with the study of Abdou and Hanout . ECs are one of the most critical sites for control of apoptosis in many situations such as vascular injury, vascular remodeling, and new blood vessel formation (angiogenesis) . VEGF and basic fibroblast growth factor maintain ECs viability by induction of the antiapoptotic machinery system in ECs, including survivin expression .
The antiapoptotic role of survivin not only came from its expression in the epidermis, but also in the ECs of proliferating capillaries of papillary dermis. VEGF promotes survival of ECs through induction of survivin.
In our study, we observed a significant correlation between epithelial VEGF and cytoplasmic survivin expression (r = 0.83) [Table 6].
| Conclusion|| |
VEGF, survivin, and iNOS appeared to be important factors in the pathogenesis of PS. These findings form the basis for developing novel and selective therapeutic stratigeis.
| Acknowledgements|| |
Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]