Psoriasis is a chronic multifactorial autoimmune skin disease with altered regulation in angiogenesis, inflammation, and proliferation of keratinocytes (Lowes et al., 2014).
The key histopathological features characteristic of psoriasis are hyperkeratosis, parakeratosis, keratinocyte hyperproliferation (acanthosis) with loss of the granular cell layer, and regular elongation of the rete ridges. The dermis shows leukocyte infiltration and increased vascularity (Wolk et al., 2009).
A vast array of growth factors, cytokines, and inflammatory mediators are involved in psoriasis development. Among them are basic fibroblast growth factor, vascular endothelial growth factor (VEGF), tumor necrosis factor, transforming growth factor-α, and interleukins (IL-17 and IL-18) (Torales-Cardeña et al., 2015).
Signal transducer and activator of transcription 3 (STAT3) behaves as a signal transducer in the cytoplasm and as a transcription factor in the nucleus and is involved in the JAK-STAT signaling pathway (Jarnicki et al., 2010). Some studies have reported the role of STAT3 in regulating basic cellular biological processes such as cell differentiation, proliferation, survival, and apoptosis (Kim et al., 2009).
The reported roles of STAT3 in promoting cell differentiation and proliferation is mediated primarily by stimulating transcription of cell cycle regulators, such as cyclin D1, cyclin E, and c-myc, and inhibition of cell cycle inhibitors p21 and p53 (Calo et al., 2003; Wolk et al., 2009).
It has been proposed that the biological activity of many growth factors and cytokines is mediated by activation of STAT3. In addition, some studies have reported that several growth factors and cytokines that are upregulated in psoriasis, such as IL-6, IL-10, and IL-20 family cytokines, are able to induce STAT3 activation. Moreover, STAT3 has been shown to play a role in psoriasis-associated IL-23 signaling pathway (Sano et al., 2005; Wolk et al., 2009). STAT3 is a key intracellular signaling molecule that mediates IL-22-induced keratinocyte hyperproliferation and is important for Th17 development (Wolk et al., 2009).
VEGF was identified to play a critical role in the process of angiogenesis by stimulating the proliferation and migration of vascular endothelial cells and enhancing the vascular permeability. It has been suggested that VEGF and its receptors (VEGFR-1 or VEGFR-2) were upregulated in psoriatic skin lesions (Heidenreich et al., 2009). VEGF may also contribute to keratinocyte hyperproliferation and might play a causative role in the vascular changes and inflammatory alterations seen in this disease (Bhushan et al., 1999; Zheng et al., 2014).
Among the angiogenic factors, VEGF stands out as a prominent transcriptional target for STAT3 (Torales-Cardeña et al., 2015). Several studies reported that STAT3 is required for endothelial cell survival and their arrangement into new vascular structures, whereas nuclear STAT3 correlates with VEGF overexpression and increased microvessel density in some tumors (Niu et al., 2002).
The aim of this study was to evaluate the expression of STAT3 and VEGF in psoriatic skin and compare them with normal skin, and to explore the role of STAT3 and VEGF in the pathogenesis of psoriasis.
Patients and methods
The present study included 30 skin biopsies taken from psoriatic patients presenting with classic psoriasis attending the outpatient clinic of Dermatology and Andrology, Faculty of Medicine, Zagazig University during the period from June to December 2015. Skin biopsies from 15 age-matched and sex-matched healthy participants were taken to represent the control group. All biopsies were fixed in 10% neutral formalin and submitted for histopathologic examination in the Pathology Department, Faculty of Medicine, Zagazig University to confirm the clinical diagnosis and for immunohistochemical staining for STAT3 and VEGF. This study was approved by the Institutional Review Board (IRB), Faculty of Medicine, Zagazig University.
Sections of 3–5 μm thickness, cut from formalin-fixed paraffin-embedded blocks, were deparaffinized in xylene and rehydrated in graded alcohol. The slides were boiled in 10 mmol/l citrate buffer (pH 6.0) for 20 min in a microwave for antigen retrieval and then washed in PBS (pH 7.4). The sections were treated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activation, and rinsed with distilled water. The slides were then incubated overnight with the primary rabbit polyclonal antibodies against STAT3 (sc7179; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) at a dilution of 1 : 200 and rabbit polyclonal antibodies against VEGF (sc152; Santa Cruz Biotechnology Inc.) at a dilution of 1 : 200 at 4°C. The slides were rinsed with PBS solution for 30 min. Sections were incubated with biotinylated secondary antibodies for 30 min. This is followed by incubation with streptavidin–biotin–peroxidase complex. The slides were rinsed with PBS and incubated with diaminobenzidine for 15 min. The slides were counterstained with hematoxylin, dehydrated, cleared, and mounted.
Sections of infiltrating duct carcinoma and pancreatic adenocarcinoma were used as positive control for STAT3 and VEGF, respectively. For negative control, we substituted the primary antibody with PBS.
Evaluation of STAT3 and VEGF immunostaining
Positive immunoreactivity for STAT3 was defined as cytoplasmic and nuclear, whereas cytoplasmic staining was regarded as positive for VEGF protein expression. The scoring of STAT3 and VEGF expression in the tissue sections was based on the percentage of stained cells: negative, less than or equal to 10% stained cells; and positive, more than 10% stained cells (Kim et al., 2009). The staining intensity was graded as mild (+), moderate (++), and strong (+++). The distribution of STAT3 and VEGF expression was evaluated as diffuse or focal; diffuse expression indicated staining of all epidermal layers and focal expression was assigned if not all epidermal layers were stained.
Data were analyzed using SPSS, version 19 (SPSS Inc., Chicago, Illinois, USA). They were expressed as mean±SD for quantitative variables, and as numbers and percentage for categorical variables. χ2 or Fisher’s exact test were used for qualitative variables. t-Test and Mann–Whitney test were used for quantitative data and κ-test was used to estimate agreement. P value less than 0.05 was considered to be statistically significant.
This study included 30 psoriatic patients. The age of the patients ranged from 36 to 60 years, with a mean of 43.2±16.75 years. The psoriatic group included 17 men and 13 women. The duration of their disease ranged from 12 to 62 months, with a mean of 37±12.08 months. Out of 30 psoriatic patients, 10 patients had generalized psoriasis, whereas 20 patients had localized psoriasis.
Positive STAT3 immunoexpression was detected as cytoplasmic and nuclear staining (activated STAT3 form), which was observed in 18 (60%) out of 30 psoriatic lesions (Table 1 and Fig. 1). Diffuse STAT3 staining was detected in 10 (33.3%) psoriatic lesions, whereas focal epidermal staining was identified in eight (26.7%) psoriatic lesions. Strong to moderate staining intensity was observed in 14 (46.7%) cases (Table 2). No significant relationship was found between STAT3 expression and the clinical parameters in psoriatic patients (Table 4). In the control specimens, only weak and focal cytoplasmic epidermal staining (nonactive form of STAT3) was observed. There was a significant difference between STAT3 expression in psoriatic lesions and control group (P<0.001).
VEGF-positive expression was detected throughout the epidermis in 21/30 (70%) psoriatic lesions and in the endothelial cells of dermal blood vessels in nine (30%) out of 30 cases (Table 1 and Fig. 2). Diffuse epidermal staining of VEGF was seen in 12 (40%) psoriatic lesions. Strong to moderate VEGF staining intensity was seen in 14 (46.7%) cases of psoriasis (Table 3). No significant relationship was found between VEGF expression and the clinical parameters in psoriatic patients (Table 4). In normal skin, VEGF was detected in five (33.3%) specimens. They showed focal VEGF expression and mild to moderate staining intensity. VEGF was significantly upregulated in psoriatic lesions in comparison with the control group (P=0.02).
Agreement between STAT3 and VEGF expression in psoriasis
In psoriatic lesions, out of the 18 cases with positive STAT3 expression, 15 (83.3%) cases showed also positive VEGF immunoreactivity. There was a significant agreement between STAT3 and VEGF expression (κ=0.35, P=0.04) (Table 5).
Psoriasis is a chronic autoimmune skin disease. Its pathogenesis remains unclear, and whether psoriasis results from dysregulation of the immune system or from a primary abnormality in epidermal keratinocytes is still controversial (Nograles et al., 2010).
Over the past few years, new insights have been gained that changed the view for the pathogenesis of psoriasis. They include the knowledge about the effects of signal transduction activation, the role of immune cells in psoriasis such Th17 cells, and the role of new cytokines such as IL-22, IL-23, and IL-20 (Nickoloff and Nestle, 2004).
Several studies have reported the role of STAT3 in regulating fundamental cellular processes such as cell differentiation, proliferation, survival, and apoptosis (Aggarwal et al., 2009; Andrés et al., 2013). The dysregulation of these processes in keratinocytes is one of the key features in psoriasis (Aggarwal et al., 2009).
In the present study, there was an overexpression of nucleocytoplasmic STAT3 in 60% of psoriatic lesions. Moreover, an increase of STAT3 throughout the epidermal layers in psoriatic skin compared with normal control skin was observed. Only focal and weak cytoplasmic staining was detected in normal control specimens. This is in accordance with previous studies (Sano et al., 2005; Andrés et al., 2013; Zheng et al., 2014).
Other studies also reported increased phosphorylation of STAT3 in psoriatic skin; they also reported that many of the proinflammatory cytokines involved in the pathogenesis of psoriasis, such as IL-6 and the IL-20 subfamily, mediate their action, in part, through Jak/STAT3 signaling in epidermal keratinocytes (Aggarwal et al., 2009; Wolk et al., 2009; Miyoshi et al., 2011).
Sano et al. (2005) observed that STAT3 was overexpressed in 90% of the psoriatic lesions examined. They also studied STAT3 expression in three nonpsoriatic inflammatory skin diseases, which are characterized by epidermal hyperplasia (lichen planus, chronic dermatitis, and prurigo). They found that these lesions did not show nuclear localization of STAT3; instead, they showed cytoplasmic staining similar to the normal skin. They concluded that STAT3 activation seemed to be specific for psoriasis among nonmalignant dermatoses.
The absence of the active phosphorylated form of STAT3 in normal skin and its upregulation in psoriatic lesions suggested that STAT3 is a main player in such an inflammatory dermatosis. Similar observations were reported in previous results (Sano et al., 2005; Jarnicki et al., 2010).
Miyoshi et al. (2011) reported that upregulation of STAT3 activity occurs during keratinocyte differentiation and proliferation. This could be partially explained by the activation of cell cycle regulators, cyclins, and c-myc, and suppression of apoptosis. Activation of STAT3 has also been reported in other autoimmune inflammatory diseases such as rheumatoid arthritis (Isomäki et al., 2015).
In addition, a transgenic mouse model that showed overexpression of activated STAT3 in the basal layer of the epidermis developed many of the characteristic features of psoriasis (Sano et al., 2005; Wolk et al., 2009).
In favor of our hypothesis regarding the important role of STAT3 in the pathogenesis of psoriasis, Miyoshi et al. (2011) reported that keratinocytes in psoriatic lesions are characterized by activated STAT3. They also observed that topical treatment with STAT3 inhibitor (STA-21) was able to inhibit the development of psoriasiform lesions in K5.STAT3C transgenic mice, and that it also improved the psoriatic lesions in six of the eight studied cases of psoriasis. They concluded that STAT3 can be used as a therapeutic target for the treatment of psoriasis.
It is well established that psoriasis is associated with accentuated dermal vascularity within the superficial dermis (Barile et al., 2006; Canavese et al., 2010). VEGF is a multifunctional cytokine produced by epidermal keratinocytes and is a key angiogenic factor in psoriasis (Wei et al., 2003).
In the present study, there was an overexpression of VEGF in the keratinocytes of 70% of psoriatic lesion as compared with the control group that showed positive VEGF expression in 33.3% of the cases. This finding is in agreement with the study of Simonetti et al. (2009). We also observed that VEGF was also expressed in the endothelial lining of dermal blood vessels in 9/30 (30%) psoriatic lesions, whereas it was not observed in normal skin. Previous studies have shown that overexpression of VEGF was associated with psoriasis (Canavese et al., 2010; Armstrong et al., 2011).
Henno et al. (2010) reported that the expression of VEGF mRNA was significantly increased in the psoriatic patients when compared with the controls. Bhushan et al. (1999) reported that VEGF was produced predominantly by keratinocytes and to a less extent by fibroblasts. According to Armstrong et al. (2011), VEGF enhances the mitogenic activity of keratinocytes in vitro and also upregulates the expression of VEGF receptor on both keratinocytes and endothelial cells. This appears to play a key role in contributing to the process of angiogenesis and the hyperplastic psoriasiform epidermis.
Further evidence supporting the role of VEGF in the pathogenesis of psoriasis comes from the observation that overexpression of VEGF in genetically modified mice results in skin inflammation with similar features of psoriasis, including the characteristic epidermal alterations, vascular changes, and inflammatory infiltrate. Moreover, administration of a potent VEGF antagonist to this model could reverse the psoriasis-like skin lesions (Xia et al., 2003).
Another interesting study observed that VEGF and the proinflammatory cytokine IL-23, which is responsible for subsequent Th17 cell response, are both strongly expressed in keratinocytes of psoriatic patients. They also reported that targeting VEGF could lead to novel anti-inflammatory treatments for this chronic disease (Canavese et al., 2011).
The current therapies for psoriasis have two target points: the immune response and the inhibition of neoangiogenesis factors (Moens et al., 2014). Jak proteins have emerged as possible new therapeutic targets for the treatment of autoimmune inflammatory diseases, including psoriasis (Miyoshi et al., 2011).
Few studies have been performed on the relationship between STAT3 and VEGF in psoriasis. The results of these previous studies have shown that the transcription factor STAT3 mediates the transcription and activation of VEGF (Niu et al., 2002; Zheng et al., 2014). We also observed that overexpression of activated STAT3 was significantly associated with VEGF positivity.
Previous studies have shown that active STAT3 might upregulate VEGF expression and angiogenesis during tumor development (Wei et al., 2003; Huang et al., 2007). They reported that STAT3 may also promote neovascularization by mediating endothelial cell response to other growth factors, including granulocyte–macrophage-stimulating factor, and that therapeutic targeting of STAT3 may inhibit neovascularization.
In light of these results, we conclude that upregulation of STAT3 in addition to VEGF might contribute to the development of psoriatic lesions.
Epidermal keratinocytes in psoriatic lesions are characterized by activated STAT3 and VEGF upregulation. The results suggest a potential role of STAT3 and VEGF in the pathogenesis of psoriasis, showing the possibility of being researched further as potential new targeted therapies for psoriasis vulgaris.
Conflicts of interest
There are no conflicts of interest.
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