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Year : 2019  |  Volume : 32  |  Issue : 3  |  Page : 1013-1018

Angiogenesis in involved and uninvolved skin of psoriasis highlighted by cluster of differentiation 34: an immunohistochemical study

1 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt

Date of Submission13-Nov-2017
Date of Acceptance30-Dec-2017
Date of Web Publication17-Oct-2019

Correspondence Address:
Reem A. Abd El-Aziz Hassan
2 Mahmoud Shahin Street, Shebeen El-Kom 32511, Menoufia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mmj.mmj_782_17

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The present study aimed at evaluating microvascular density (MVD) in involved and uninvolved skin of patients with psoriasis.
Psoriasis is a chronic inflammation skin disease characterized by accelerated proliferation, abnormal differentiation, and increased angiogenesis. The angiogenesis is related to many factors in the pathogenesis of psoriasis. The assessment of angiogenesis using cluster of differentiation 34 can help more understanding of psoriasis pathology.
Patients and methods
This study was carried out on biopsies of involved and uninvolved skin from 40 patients with psoriasis and 40 apparently healthy volunteers as a control group. Biopsies were processed for immunostaining procedure in the pathology department.
After assessment of the MVD highlighted by cluster of differentiation 34 immune-staining, the skin of psoriatic cases (involved and uninvolved) was of significantly higher MVD values in comparison with normal skin of control group (P < 0.001). Additionally, the involved psoriatic skin showed significantly higher MVD values compared with uninvolved skin (P < 0.001).
Angiogenesis has a role in the pathogenesis of psoriasis, as it was accelerated in both involved and uninvolved skin.

Keywords: angiogenesis, cluster of differentiation 34, psoriasis, skin

How to cite this article:
Hammam MA, Antar AG, Abdou AG, El-Aziz Hassan RA. Angiogenesis in involved and uninvolved skin of psoriasis highlighted by cluster of differentiation 34: an immunohistochemical study. Menoufia Med J 2019;32:1013-8

How to cite this URL:
Hammam MA, Antar AG, Abdou AG, El-Aziz Hassan RA. Angiogenesis in involved and uninvolved skin of psoriasis highlighted by cluster of differentiation 34: an immunohistochemical study. Menoufia Med J [serial online] 2019 [cited 2020 May 27];32:1013-8. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/1013/268846

  Introduction Top

Psoriasis is a chronic inflammatory and immunologically mediated skin disease. It has been reported in 2% of worldwide populations [1],[2]. The plaque psoriasis is the most common clinical type; the primary lesion is a sharply demarcated plaque, which appears erythematous correlating clinically with inflammation; and scaly, as a sign of accelerated epidermal proliferation and parakeratosis. The sites of predilection are over the extensor body surfaces especially over bonny prominences and the scalp [3]. The course and the duration of plaque psoriasis are highly variable [4]. The etiology of psoriasis is multifactorial with genetic, immunological, and environmental factors that result in angiogenesis, accelerated epidermal proliferation, and inflammation, resulting from both innate and cell mediated immunity alterations [5],[6]. The characteristic histopathological features of psoriatic skin lesions are epidermal acanthosis, parakeratosis, angiogenesis, epidermal and dermal inflammatory cells, especially neutrophils and T lymphocytes, with upregulation of different cytokines, growth factors, inflammatory mediators, angiogenic factors, and other biological factors including hypoxia-related factors, and some of these alterations are detected also in uninvolved skin of patients with psoriasis [7]. The angiogenesis in pathogenesis of psoriasis and its relation to hypoxia is not fully understood as epidermal hyperproliferation could increase oxygen need, and epidermal thickening could lead to decreased oxygen supply [8]. Hypoxia stimulates different angiogenic factors such as vascular endothelial growth factor (VEGF) which may be involved in psoriatic skin angiogenesis [9]. Angiogenic factors upregulation and angiogenesis help improvement of oxygenation which was satisfied with rapid proliferation of malignant cells [10]. Both of psoriasis and tumors share the features of cell excessive proliferation and relative hypoxia. The hematopoietic progenitor cell antigen cluster of differentiation 34 (CD34) is a glycosylated transmembrane protein, and it is commonly used as a marker of hematopoietic progenitor cells and endothelial cells [11]. Although the exact function of CD34 is unknown, it was suggested that CD34 may have a role in chemokine-dependent migration of T lymphocytes, eosinophils, and dendritic cells through interaction with adhesion molecules such as L selectin [12],[13].

  Aim Top

To investigate angiogenesis by evaluating microvascular density (MVD) using CD34 in involved and uninvolved skin of patients with psoriasis.

  Patients and Methods Top

This case–control study was done on 40 patients with chronic plaque psoriasis from the Outpatient Clinic of Dermatology and Venereology Department, Faculty of Medicine, Menoufia University, during the period between April 2015 and October 2016. Forty age-matched and sex-matched healthy participants seeking plastic surgery were included as a control group.

Inclusion and exclusions criteria

Patients with chronic plaque psoriasis, the most common type of psoriasis, with exclusion of other types of psoriasis and any other dermatological diseases, and having had any treatment stopped for at least 2 weeks before taking skin biopsies were included in the study [14].

Each of the patients with psoriasis was subjected to the following: full history taking, general examination, and assessment of psoriasis severity and extent using psoriasis area severity index score (PASI) [15].

Skin biopsies

After taking a written consent from the patients and control groups, two punch biopsies 3 mm each from involved and uninvolved skin of patients with psoriasis were taken under local anesthesia. One 3-mm punch biopsy was taken from healthy skin of control group. The biopsies were fixed in 10% neutral formalin and submitted to routine tissue processing ending with the formation of paraffin-embedded blocks, in Pathology Department, Faculty of Medicine, Menoufia University. Two paraffin sections, each 4-mm thick, were cut from each block. One of them was stained by hematoxylin and eosin to evaluate the pathological changes, and the other sections were cut on poly-l-lysine–coated slides for CD34 immunostaining procedure.

Histopathological evaluation

Hematoxylin and eosin stained sections were examined by light microscope to evaluate the pathological changes. The degrees of parakeratosis, acanthosis, angiogenesis, and inflammation were assigned as mild, moderate, and marked for each parameter.

Immunohistochemical staining

The method used for immunostaining was a streptavidin-biotin–amplified system. The primary antibodies used were mouse monoclonal antihuman CD34 class II antibodies (Cat. #MS-363-R7, 7.0 ml ready to use; LabVision/NeoMarkers, Fremont, California USA). The slides were subjected to subsequent steps of deparaffinization and rehydration. Antigen retrieval was performed by boiling in citrate buffer saline (pH 6) followed by cooling at room temperature. The primary antibodies were incubated overnight at room temperature, and then the secondary antibody (Ultravision detection system antipolyvalent horseradish peroxidase/diaminobenzidine, ready-to-use; NeoMarker) was applied with diaminobenzidine as a chromogenic substrate and Mayer's hematoxylin as a counter stain. Placenta tissue was used as a positive control for CD34. Replacement of the primary antibody step with a blocking buffer was included in the staining procedure as a negative control.

Evaluation of mean microvascular density using CD34 immunostaining

All specimens were analyzed in a semiquantitative way through examination of the whole immune-stained slide. Positive CD34 expression was assigned when the cell showed cytoplasmic and membranous staining. For the evaluation of MVD, slides were scanned in the light microscope at ×40 magnification and three areas of maximal MVD, so-called 'hot spots', were identified. In each hot spot, microvessels (endothelial cells with or without lumen, capillaries, and small venules) were counted at ×400 magnification (each field representing an area of 0.375 mm 2). For each slide, the mean number of microvessels from these three areas was calculated. The calculated figures were expressed as mean, median, and range [16]. The median value of MVD was used as a reference point, and then the cases were classified into the following:

  1. Cases with low MVD up to median
  2. Cases with high MVD more than median.

Ethical consideration

The study was approved by Ethical Committee of Menoufia Faculty of Medicine, and an informed consent obtained from all participants before the study was commenced.

Statistical analysis

Data were collected, tabulated, and statistically analyzed using IBM SPSS (version 20.0; IBM Corp., Armonk, New York, USA). χ2 and Fisher exact tests were used for comparison between qualitative variables. Mann–Whitney (U) and Kruskal–Wallis (K) tests were used for comparison between quantitative variables. P value up to 0.05 was considered significant.

  Results Top

The clinical data of the studied cases are demonstrated in [Table 1].
Table 1: The clinical data of patients with psoriasis

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The histopathological data of the studied cases are demonstrated in [Table 2].
Table 2: Descriptive data of the histopathological characteristics of involved psoriatic skin

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Based on CD34 immune-staining, the MVD was evaluated in normal skin of control [Figure 1]a, uninvolved psoriatic skin lesions [Figure 1]b, and involved psoriatic skin lesions [Figure 1]c and [Figure 1]d.
Figure 1: Cluster of differentiation 34 (CD34) highlighting dermal blood vessels of normal skin (a). CD34 highlighting dermal blood vessels and endothelial cells in uninvolved skin of psoriasis (b). CD34 highlighting pathological changes (angiogenesis) in involved skin of psoriasis (c). CD34 highlighting angiogenesis in involved skin of psoriasis (d). CD34 highlighting angiogenesis in involved skin of psoriasis (e). CD34 cytoplasmic staining of the endothelial cells (f) (immunohistochemical staining ×200 for a, b, d and e, ×100 for c and ×400 for f).

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After assessment of the MVD values, the normal skin MVD values ranged between 10 and 30, with a median of 20 and a mean of 18.37 ± 5.92. The uninvolved skin of patients with psoriasis showed MVD ranging between 10 and 60, with a median of 30 and a mean of 29.50 ± 11.25. MVD values of the involved skin of patients with psoriasis ranged between 50 and 130, with a median of 85 and a mean of 87 ± 21.14 [Table 3].
Table 3: Comparison of the immunohistochemical expression of cluster of differentiation 34 (microvascular density) values among the studied groups

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Comparing the MVD values of the studied groups, the skin of patients with psoriasis either involved and uninvolved showed significantly higher mean and median values than that of normal skin of control group (P < 0.001). Furthermore, the involved skin of patients with psoriasis was of higher mean and median values of MVD in comparison with uninvolved skin (P < 0.001) [Table 3].

The MVD median value of all groups was 30, so the mean MVD was classified into low density (<30) and high density (≥30). According to this classification, the mean MVD of normal skin was of low density, and the involved psoriatic skin was of high density whereas the uninvolved showed 31 (77.5%) cases with low density and nine (22.5%) cases with high density. From these results, there was a highly significant difference between normal skin and involved (P < 0.001) and uninvolved skin (P = 0.004) regarding mean MVD. Furthermore, there was a highly significant difference between uninvolved and involved skin of patients with psoriasis (P < 0.001) [Table 3].

Studying the relationship between MVD of psoriatic skin and clinicopathological variables revealed a significant positive correlation between MVD values in psoriatic skin and age of the patients (P = 0.04) and duration of the disease (P = 0.01), indicated that MVD values increased with aging and duration of the disease. Furthermore, MVD values in patients with psoriasis tended to be higher in male (91.40 ± 4.36) compared with female (80.0 ± 19.32) patients. However, the relationship did not reach significant value (P = 0.08) [Table 4].
Table 4: The relationship between microvascular density of involved psoriatic skin and the studied clinicopathological variables

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  Discussion Top

Angiogenesis is one of the landmarks of psoriasis [17].Previous studies have demonstrated that angiogenesis occurs in the early progression of psoriasis, before epidermal hyperproliferation can be detected either histologically and clinically, and this meant that psoriasis is an angiogenesis-dependent disease. Many studies demonstrated the important role of endothelial cells in psoriasis through finding elevated levels of angiogenic factors such as VEGF, prostaglandins, and nitric oxide in psoriatic lesions [18],[19]. CD34 is a glycosylated transmembrane protein and a well-known marker for primitive blood-derived and bone marrow-derived progenitor cells, especially endothelial cells [11]. CD34 functions as a cell–cell adhesion factor that interacts with other adhesion molecules such as L selectin [12]. Psoriasis pathogenesis is a complex interaction between several factors, and the immunological factor is the most important implicated factor, especially T cells [20]. The adhesion molecules such as L selectin and CD34 have role in recruitment of T lymphocytes and dendritic cells to tissues such as skin and lymph nodes [13].

In the current study, the MVD highlighted by CD34 was of significantly higher values in involved and uninvolved skin of psoriasis compared with normal skin (P < 0.001). Moreover, MVD values were significantly higher in involved psoriatic skin compared with uninvolved skin (P < 0.001). These results confirmed the possible role of enhanced angiogenesis in pathogenesis of psoriasis. Such results agreed with Amin and Azim [21] study that found significant difference between involved psoriatic skin and both uninvolved skin of psoriasis and normal skin. On the contrary, the study did not agree with our study about finding significant difference between uninvolved psoriatic skin and normal skin. The current study agreed with Simonetti et al. [22] and Sankar et al. [23] who observed significant increase in both CD34 and VEGF in psoriatic skin compared with normal skin. In addition, the study by Gupta et al. [24] of dermal vasculature in psoriasis and dermatitis demonstrated significant difference between MVD in psoriasis and psoriatic dermatitis using CD34, which agreed with our study.

The present study demonstrated that there was a significant positive correlation between MVD values in involved skin of psoriatic specimens and age of the patients (P = 0.04) and psoriasis duration (P = 0.01). From these results, we can find that angiogenesis increases with age of patients with psoriasis. These findings could be related to increased atherosclerosis and tissue hypoxia in older patients, which enhance angiogenesis [25],[26]. Furthermore, many studies demonstrated the relation between psoriasis and accelerated atherosclerosis, which may justify the relation between duration of psoriasis and enhanced angiogenesis [27],[28].

The MVD values in patients with psoriasis tended to be higher in male (91.40 ± 4.36) compared with female patients (80.0 ± 19.32), with no significant value (P = 0.08). This finding agreed with a study done by Jiamton et al. [29] that found significant association between male sex and severity of psoriasis using PASI scoring. Regarding the association between MVD highlighted by CD34 and PASI score in the present study, there was no significant association, which disagreed with Sankar et al. [23] who found significant association between expression of CD34 and PASI score.

  Conclusion Top

Angiogenesis has a role in pathogenesis of psoriasis as it was accelerated in both involved and uninvolved skin.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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