Expression of Vascular Endothelial Growth Factor and Microvessel Density in Psoriatic Skin Lesions.

BACKGROUND: Psoriasis is a common chronic and immune-mediated skin disorder having a significant impact on the patient's quality of life. An analysis of the role of angiogenic vascular endothelial growth factor (VEGF) expression and microvessel density in psoriatic skin lesions may help in better understanding of the disease pathogenesis. The aim of this study was to examine the expression of vascular endothelial growth factor (VEGF) and microvessel density using CD34 antibodies in psoriatic skin lesions by immunohistochemical examination using normal skin of healthy individuals as controls.
MATERIALS AND METHODS: Patients with clinical diagnosis of plaque type of psoriasis (e.g., chronic plaque) (n = 49) were included in the study. 5-mm punch biopsies were taken from the psoriatic skin lesions in these patients. A total of 20 punch biopsies were taken from the control group comprising of 20 healthy volunteers. The biopsies were subjected to histopathological examination for confirmation of diagnosis and grading. Immunohistochemical evaluation was done for the expression of VEGF, and microvessel density was assessed using CD34 and compared with the controls.
RESULTS: An increased VEGF expression by keratinocytes (49.80% ± 21.16%) and microvessel density in the papillary dermis (15.302% ± 3.8061%) was observed in patients with psoriasis, which was significantly higher as compared to controls (P < 0.0001). A significant positive correlation was observed between VEGF expression by keratinocytes and the microvessel density in the dermis (r = 0.664, P = 0.01). No significant correlation was observedbetween the histopathological grade of psoriasis and microvessel density, or with the VEGF expression.
CONCLUSION: VEGF expression ascertained to be a significant factor in the pathogenesis of psoriasis.

Introduction

Psoriasis vulgaris is an inflammatory and chronic proliferative dermatological disorder characterized clinically by the appearance of red, scaly, and raised skin lesions[1] as a result of abnormal differentiation, hyperproliferation, and inflammatory infiltration in the dermis and epidermis.[1] Long-term treatment is often required, and refractoriness to treatment modalities and relapses are common reinforcing the need of newer treatment modalities. A study by Folkman et al. reported that increased angiogenesis is a contributory factor in psoriasis pathogenesis.[2]

Vascular endothelial growth factor (VEGF), CD31, CD34, and nerve growth factor (NGF) have been established as prognostic marker of angiogenesis.[3] The keratinocytes in the psoriatic lesions are considered as the main source of proangiogenic cytokines, which induce angiogenesis through VEGF.[4] VEGF, a 40- to 45-kDa dimeric glycoprotein, regulates neovascularization during skeletal growth, reproductive functions, embryogenesis, and pathological processes.[5] Therefore, overexpression of VEGF plays a predictive marker in psoriasis. Detection of CD34 antigen, in the endothelial cells of capillaries (approximately 1% of human thymocytes), acts as a marker of angiogenesis to measure the microvascular density (MVD).[6]

Currently, anti-VEGF therapy is under developmental stage for the treatment of numerous malignancies. Evaluation of the expression of VEGF and CD34 in psoriatic plaques and comparing the same with normal skin may provide some insights to the pathogenesis of psoriasis thus helping the development of targeted therapy. Hence, we aimed to study the expression of VEGF and MVD using CD34 antibodies in psoriatic skin lesions by immunohistochemical examination and tried to correlate it with the histological grade.

Materials and Methods

The psoriasis cases were chosen randomly from the archives of Department of Pathology from January 2007 to July 2014. 49 (men: 29; women: 20) patients who were recently diagnosed with psoriasis vulgaris between 15 and 70 years of age were recruited.

A 5-mm punch biopsy was taken from each patient of psoriasis including the full-thickness skin and subcutaneous fat. The control group included biopsies from 20 volunteers in which 10 were healthy volunteers and 10 were autopsy specimens (within 12 h of death). The control biopsies were matched with psoriatic patient specimens with respect to age group.

Each tissue sample was fixed in 10% buffered neutral formalin for 24 h and routinely processed and stained with hematoxylin–eosin. Histopathological grading was done based on Trojak's grading system for psoriatic lesions.[7]

Immunohistochemistry

Immunohistochemistry (IHC) was performed on 4-μm-thick section from 10% formalin-fixed paraffin-embedded tissues using a nonbiotin polymer-based horseradish peroxidase (HRP) detection system for antibodies VEGF165 (Biogenix, USA; catalogue No. AM236-5M ready to use) and CD34 (Biogenix; catalogue No. AR483-5R ready to use).

The MVD index for the quantification of angiogenesis was calculated using the method described by Behrem et al.[8] The slide was screened at low power magnification (×40) to identify the areas with highest vascularization in papillary dermis. Number of vessels were counted in three such high-density areas at highermagnification (×400). Areas with dense hemorrhagic infiltrates and vessels with caliber larger than eight red blood cells were excluded from the count. Single endothelial cells or a cluster of endothelial cells positive for CD34 in membrane/cytoplasm were regarded as a distinct single countable microvessel. The values were finalized after mutual consensus was obtained between two observers and was entered as the MVD/high-power field (HPF) for each individual lesion. Immunostaining for VEGF was semiquantitatively scored using the modified scoring system protocol by Soo et al.[9]

Suprabasilar layers of the epidermis were assessed for VEGF expression. Cell showing granular positivity within the cytoplasm was considered positive for VEGF. The mean percentage of positive cells for the expression was determined after examining a minimum of five areas at ×400 magnification. Biopsies with less than 10% cells which were stained positive were defined as negative.

Statistical analysis

Statistical analysis was performed using SPSS v.22.0. The demographic data were analyzed using descriptive statistics, and the VEGF and MVD values were compared using independent t-test. The correlation between VEGF expression by keratinocytes and MVD among cases and controls was evaluated using Pearson's correlation. P < 0.05 was considered to be statistically significant.

Results

Most of the patients in the study were in the age group of 31–40 years. The mean age of patients included in the study was 39.67 ± 14.94 years. VEGF expression in the keratinocytes was found to be 49.80% ± 21.16% and MVD in the papillary dermis was 15.30% ± 3.80%. However, in the control skin samples, VEGF expression was 3.95% ± 3.94% and MVD was 5.52% ± 1.46% [Figures 1 and 2]. VEGF expression and MVD were significantly upregulated in psoriatic skin in comparison to normal healthy skin (P < 0.0001) [Table 1]. Among the cases, a significant positive correlation was observed between VEGF expression by keratinocytes and the MVD in the papillary dermis (r = 0.664; P = 0.01) [Figure 3]. A positive correlation was observed between VEGF expression by keratinocytes (r = 0.292) and MVD (r = 0.226) with histopathological grade of psoriasis, respectively. However, the correlation was statistically insignificant (P > 0.05).

Figure 1

Strong positive expression of VEGF by keratinocytes in a lesional biopsy on immunohistochemistry (×40)

Figure 2

Microvessel density in cases of psoriasis using CD34 antibodies (×40)

Table 1

Vascular endothelial growth factor expression by keratinocytes and microvessel density assessment in cases and control group

Figure 3

Scatter plot depicting the correlation between the microvessel density and vascular endothelial growth factor expression of the cases

Discussion

In our study, psoriasis was found to be more common in the younger age group with slight male preponderance. A study by Dogra et al. also found that the age group of 20–39 years has the highest incidence of psoriasis with male preponderance.[10]

In psoriasis patients, VEGF expression was significantly higher throughout the keratinocytes (49.8% ± 21.2%; P < 0.0001) as compared to with controls (VEGF expression was mild and focal), which is comparable to the report by Salem et al.[11] (46.4% ± 19.7%) and Simonetti et al.[12] (45.60% ± 19.84%) who observed a strong VEGF expression in psoriasis throughout the epidermis. Another study revealed that mean VEGF expression in the endothelial cells of the dermis was 30.82% ± 0.65% in psoriatic skin;[13] however, a reduction was observed in VEGF expression by 81.6% at week 10 under etanercept treatment.[20] Keratinocyte cell–derived VEGF contributes to the pathogenesis of psoriasis by the induction of permeability in the dermal vessels through its angiogenic effect and chemotactic action on the inflammatory cells and endothelial cells.[14] Another study done on 40 psoriatic patients concluded that there was overexpression of VEGF in keratinocytes of psoriatic lesions when compared with normal skin.[15]

Psoriasis begins with angiogenesis in the superficial dermal microvasculature, dilatation of dermal papillary capillaries, tortuosity, permeability, and a prominent elongation.[16] The MVD in the psoriatic lesions in our study was significantly higher as compared to normal healthy skin (P < 0.0001). The results were in agreement with the study conducted by Amin and Azim, in which CD34 expression was observed in lesional, nonlesional, and normal skin;[17] however, the expression in the lesional skin was significantly higher than the nonlesional skin and very weak in the normal skin. The other proangiogenic factors that are implicated in the pathogenesis of psoriasis include VEGF, hypoxia-inducible transcription factors (HIFs), angiopoietins, tumor necrosis factor (TNF), transforming growth factor (TGF)-a, interleukin (IL)-8, and IL-17.

In our study, we found an increased VEGF expression in psoriatic plaques, which may play an important role in the pathogenesis of the disease, vascular dilatation, increased vascular permeability, and epidermal hyperplasia. In our study, there was a positive correlation between MVD and VEGF expression in cases (r = 0. 664; P = 0.01). Similarly, a study by Chawla et al. showed that overexpression of VEGF correlated well with increased MVD which indicated an increasing trend in psoriasis when compared with the psoriasiform lesions.[18]

The expression of VEGF by keratinocytes and MVD in dermis did not correlate significantly with a histopathological grade in cases of psoriasis. Psoriasis is known to be a dynamic process, both clinically and histologically. Microscopic diversity occurs among psoriatic patients with clinically similar lesions between lesions of an individual and even within single plaques. A study conducted in severe psoriasis showed that histological grade does not correlate with disease severity.[7] Thus, the role of histopathological examination in psoriasis patients is limited to confirm the diagnosis. Furthermore, it is also difficult to differentiate it from other psoriasis forms of dermatoses, which include seborrheic dermatitis, psoriasiform drug eruption, chronic fungal infections, lichen simplex chronicus, chronic spongiotic dermatitis, secondary syphilis, pellagra, and so on.

As the sample size was small, studies with large sample sizes are required to validate the results presented in this study. In addition, further research is needed to identify newer angiogenic markers to analyze specific angiogenic pathway in psoriasis.

Conclusion

VEGF expression is higher in psoriatic lesions compared with healthy skin. A significant positive correlation was seen between the degree of expression of VEGF by the epidermis and the MVD in the dermis. When correlated with histopathological grade of psoriasis, no significant correlation was observed with VEGF expression or MVD. Altogether, VEGF plays a significant role in the pathogenesis of psoriasis by way of its angiogenic activity in the lesions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.