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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 40  |  Issue : 2  |  Page : 76-83

Immunohistochemical expression of hypoxia-inducible factor-1 alpha in psoriatic patients


1 Lecturer of Dermatology and Venereology, Tanta University, Egypt
2 Proffesor Pathology Department, Faculty of Medicine, Tanta University, Egypt

Date of Submission30-Jun-2019
Date of Acceptance18-Dec-2019
Date of Web Publication09-Jun-2020

Correspondence Address:
Soha Abdalla Hawwam
Lecturer of Dermatology and Venereology Department, Faculty of Medicine, Tanta University, El Geish Street, Tanta 31111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejdv.ejdv_32_19

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  Abstract 


Introduction Psoriasis is a chronic inflammatory skin disease characterized by aberrant differentiation and excessive growth of keratinocytes, with dermal and epidermal mixed leukocytic infiltrate and angiogenesis in the dermis. Oxygen consumption could be increased by the proliferation of cells, and impaired oxygen supply might occur from the thickening of the epidermis. A primary role in the maintenance of oxygen and energy homeostasis is hypoxia-inducible factor-1 (HIF-1). Several gene expression-encoding inflammatory proteins are stimulated by HIF-1. The pathogenesis of various forms of inflammation shows the involvement of hypoxia.
Aim This immunohistochemical study was done to evaluate the role of HIF-1α in the pathogenesis of psoriasis.
Patients and methods This study was conducted on 20 patients with chronic plaque psoriasis, and 10 healthy individuals as a control group. They underwent hematoxylin and eosin staining and immunohistochemical staining detection of HIF-1α expression.
Results Regarding the correlation between HIF-1α expression and clinical parameters of the patients, there was a statistically significant positive correlation with the Psoriasis Area and Severity Index score.
Conclusion In this study, there was significantly increased HIF-1α expression in psoriatic patients’ skin in comparison with normal control, and also its increase with elevated Psoriasis Area and Severity Index score might represent new promising therapeutic approaches and pharmacological research target for psoriasis treatment.

Keywords: hypoxia-inducible factor-1 alpha, immunohistochemical, psoriasis


How to cite this article:
Hawwam SA, Shareef MM. Immunohistochemical expression of hypoxia-inducible factor-1 alpha in psoriatic patients. Egypt J Dermatol Venerol 2020;40:76-83

How to cite this URL:
Hawwam SA, Shareef MM. Immunohistochemical expression of hypoxia-inducible factor-1 alpha in psoriatic patients. Egypt J Dermatol Venerol [serial online] 2020 [cited 2020 Jul 6];40:76-83. Available from: http://www.ejdv.eg.net/text.asp?2020/40/2/76/286284




  Introduction Top


Psoriasis is a chronic inflammatory skin disease characterized by aberrant differentiation and excessive growth of keratinocytes, with dermal and epidermal mixed leukocytic infiltrate and angiogenesis in the dermis. The pathogenesis of psoriasis has many factors such as genetic factors, environmental factors, and activation of the immune system, adaptive and innate [1].

Angiogenesis is a hallmark of cancer. Papillary dermis microvessels are proliferated, dilated, elongated and tortuous, with increased endothelial cell proliferation. Cell proliferation causes increased oxygen consumption, and impaired oxygen supply could be owing to epidermal thickening [2].

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcriptional complex of HIF-1α and HIF-1β subunit. It has an important role in oxygen maintenance and energy homeostasis. HIF-1 can be activated by several inflammatory mediators and several genes expression-encoding inflammatory proteins stimulated by HIF-1 [3].

Various forms of inflammation have been caused by hypoxia and have a role in diseases such as colitis [4]. Moreover, the role of hypoxia and the development of angiogenesis following HIF activation in the psoriatic skin have been studied for searching the potential therapeutic intervention opportunity [5].


  Aim Top


The aim of this study was immunohistochemical evaluation of HIF-1α in tissue samples of patients with psoriasis to provide an insight into its role in the pathogenesis of psoriasis.


  Patients and methods Top


Patients

The current study was conducted on 20 patients with chronic plaque psoriasis who were attending the outpatient clinic of Dermatology and Venereology Department, Tanta University Hospital, in addition to ten healthy individuals who were selected as a control group, with no history of any systemic or skin disease. The patients were diagnosed clinically by the typical appearance of the skin lesions and histopathologically confirmed. The study was approved by the medical ethics committee of the Faculty of Medicine, Tanta University.

The studied persons were divided into the following groups:
  1. Group I: 20 patients with psoriasis.
  2. Group II: 10 healthy persons as a control group.


Inclusion criteria were newly diagnosed cases with no previous topical treatment in the past 6 weeks or any systemic treatment for psoriasis or phototherapy in the past 6 months. Patients did not receive any other medications and did not have any other systemic or skin diseases. Female patients were not pregnant or receiving any hormonal contraceptives. Patients agreed to participate in the study were informed about the aim of the study and the procedures and signed written consent.

Exclusion criteria were patients who applied any topical preparations for psoriasis in the past 6 weeks, patients who received other systemic medications, pregnant and lactating women, and patients who have other systemic or skin diseases.

Complete medical history taking in the studied groups, to exclude any systemic diseases like diabetes mellitus, obesity, and family history of the same conditions, and routine investigations were done. The clinical and dermatological examination was done for the patients who were subjected to psoriasis severity assessment by estimating [Psoriasis Area and Severity Index (PASI)] score for measuring erythema intensity, induration, and desquamation in four main body areas, including the head, upper limbs, trunk, and lower limbs [6].

PASI score is then calculated according to the following equation:



The highest PASI score is 72, denoting 100% affection with maximum erythema, infiltration, and desquamation, whereas the lowest is 0, denoting no lesions at all.

Methods

Skin biopsies were taken after informed written consent. Punch biopsies of 4 mm were taken under local anesthesia from lesional skin. Control biopsies were also obtained from similar sites of healthy individuals. After removal, the biopsies were immediately fixed in formalin 10% and embedded in paraffin.
  1. Hematoxylin and eosin staining was done to confirm the histopathological changes of psoriasis.
  2. Immunohistochemical staining was done by primary antibody: Purified Mouse Anti-Human HIF-1α (clone 54/HIF-1α was available from BD Transduction Laboratories, San Diego, California, USA). The slides then were rinsed three times with PBS and dried then exposed to secondary antibody, and the slides were then incubated with secondary antibody: polyclonal FITC Goat Anti-mouse Ig (catalog number 55-4001) available from BD Transduction Laboratories, to detect HIF-1α expression.


Evaluation of immunostaining [5]

The HIF-1α positivity was detected in the nuclei of epidermal keratinocytes. Immunoexpression was graded according to the proportion and intensity of the staining of positive epidermal cells.

The proportion of stained cells was graded as follows:

No staining =0 1–25% (+1).

26–50% (+2) >50% (+3).

The intensity of the reactivity was scored compared to the intensity of staining of the nuclei of hair follicle epithelium.

Staining intensity was rated on a scale of 0 to +3:

0=Negative +1=Mild.

+2=Moderate +3=Marked.

The final score used in the statistical analysis was derived by multiplying the extent of positive epidermal cells and the intensity of staining resulting in the following score [5]: 0=negative; 1, 2, and 3=mild; 4=moderate, and 6 and 9=marked.

The location of the positivity in the epidermis was recorded in relation to the epidermal papilla (papillary, suprapapillary, or both) and in relation to the affected layer of the epidermis (basal, suprabasal, or prickle cell layer) [5].

Statistical analysis

Statistical presentation and analysis of the present study were conducted, using the mean, SD, and χ2 test by SPSS, V.16 Statistical software package (SPSS for windows version 11.0; SPSS Inc., Chicago, IL, USA).


  Results Top


Clinical results

Demographic data of the studied groups are shown in [Table 1].
Table 1 Comparison between the two studied groups according to demographic data

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Regarding the severity of psoriasis and the PASI score among the patient group, the number of the patients with mild psoriasis, with PASI score less than 15, was nine (45.0%); the number of the patients with moderate psoriasis, with PASI score ranged between 15 and 25, was seven (35.0%); and the number of patients with severe psoriasis, with PASI score more than 25, was four (20.0%) ([Table 2]).
Table 2 Severity of psoriasis in the patients group

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Histopathological results

Hematoxylin and eosin staining

In the psoriasis group, according to the histopathological examination, the following results and changes were revealed:
  1. Nine cases ha mild psoriasis, showing parakeratosis, exocytosis, spongiosis, test tube acanthosis in the form of elongation and clubbing of the rete ridges, thinning of the suprapapillary epidermis, elongation and tortuous blood vessels in the papillary dermis, and superficial perivascular infiltrate.
  2. The rest of the cases were moderate to severe psoriasis showing marked histopathological features with formation of Munro microabscesses and spongiosis ([Figure 1]).
    Figure 1 Histopathological section in psoriatic skin showing (a) hyperparakeratosis, test tube acanthosis, and parakeratosis (hematoxylin and eosin, ×100) and (b) hyperkeratosis, Munro microabscesses, acanthosis, dermal perivascular inflammatory infiltrate, and club-shaped papillae (hematoxylin and eosin, ×100).

    Click here to view


Immunohistochemical staining

  1. The expression of HIF-1α in the two studied groups is illustrated in [Table 3]. There was a statistically significant difference between HIF-1α expression in the psoriasis group in comparison with the control group (P=0.001).
    Table 3 Comparison between the two studied groups according to hypoxia-inducible factor-1 alpha expression

    Click here to view
  2. The positivity of HIF-1α expression was nuclear. The differences in its expression between the lesional skin of psoriasis group compared with the normal skin of the control group are shown as follows ([Figure 2] and [Figure 3]):
    1. The HIF-1α expression in the skin of the control group showed three (30.0%) cases with mild expression and seven (70%) cases with no expression.
    2. The HIF-1α expression in the skin of the psoriatic group showed seven (35.0%) cases with a mild expression, 11 (55.0%) cases with moderate expression, and two (10.0%) cases with marked expression.
    Figure 2 Histopathological section in normal skin showing (a) hair follicle positivity (control) (immunohistochemistry for HIF-1α, ×200) and (b) mild nuclear positivity for HIF-1α (immunohistochemistry for HIF-1α, ×200). A histopathological section in psoriatic skin showing (c) mild nuclear HIF-1α positivity localization: suprapapillary and prickle cells (immunohistochemistry for HIF-1α, ×100) and (d) mild nuclear HIF-1α positivity. Localization: suprapapillary, prickle cells, and suprabasal (immunohistochemistry for HIF-1α, ×200). HIF-1, hypoxia-inducible factor-1.

    Click here to view
    Figure 3 Histopathological section in psoriatic skin showing (a) moderate nuclear HIF-1α positivity. Localization: suprapapillary and suprabasal (immunohistochemistry for HIF-1α, ×100). (b) Moderate nuclear HIF-1α positivity. Localization: suprapapillary and prickle cells (immunohistochemistry for HIF-1α, ×200). (c) Diffuse strong nuclear HIF-1α positivity in suprapapillary and prickle cells (immunohistochemistry for HIF-1α, ×100). (d) Strong nuclear HIF-1α positivity. Localization: suprapapillary, prickle cells, and suprabasal (immunohistochemistry for HIF-1α, ×200). HIF-1, hypoxia-inducible factor-1.

    Click here to view
  3. Regarding the correlation between HIF-1α expression and clinical parameters of the patients; there was a statistically significant positive correlation only with the PASI score (P=0.001) ([Figure 4]).
    Figure 4 Relation between HIF-1α expression and a different PASI score of patients with psoriasis. HIF-1, hypoxia-inducible factor-1; PASI, Psoriasis Area and Severity Index.

    Click here to view
  4. Regarding the relation between HIF-1α expression and its localization to the papilla, all cases were positive in the suprapapillary portion ([Table 4], [Figure 5]).
    Table 4 Relation between hypoxia-inducible factor-1 alpha expression with its localization regarding the papilla

    Click here to view
    Figure 5 Comparison between the two studied groups according to papillary and suprapapillary localization.

    Click here to view



  Discussion Top


Psoriasis is a skin diseases that is autoimmune in nature, being chronic and hyperproliferative, and differs in severity. It is characterized by the presence of well-demarcated erythematous plaques of various sizes which are topped by silvery scales. It affects the scalp commonly, sacral region, and extensor surfaces of the extremities. The disease is characterized by spontaneous remissions and relapses [7].

Acute injury is characterized by hypoxia with high oxygen consumption by cells and granulation tissue. Keratinocyte migration and laminin-332 deposition is required for wound repair. Hypoxia promotes in vitro KC motility. The hypoxic situation induces the secretion and synthesis of growth factors induced by hypoxia, which could be HIF-dependent pathway mediated [8].

This study examined the expression of HIF-1α in both psoriatic lesional skin and control normal skin for detection of the correlation between HIF-1α expression and disease severity.

Regarding HIF-1α expression in normal skin of control group, the present study demonstrated that HIF-1α is poorly expressed in epidermal KC, whereas it is abundantly expressed in hair follicles. This agreed with Rosenberger et al. [2] who reported higher HIF-1α expression in hair follicles, sebaceous glands, and sweat glands in normal human skin, than in epidermis, which showed low and focal expression. Moreover, by using immunohistochemistry, they demonstrated that HIF-1α staining is increased in psoriatic versus normal skin throughout the epidermis. In addition, by using in-situ hybridization, they showed an increased number of HIF-1α messenger RNA signals in psoriatic skin. They also added pronounced HIF-1α expression in perivascular areas where least hypoxia is expected. However, as oxygen level is a balance between supply and consumption, in these perivascular areas, relative hypoxia may be owing to oxygen consumption (e.g. by inflammatory cells, stromal cells, and endothelial cells) exceeding oxygen supply.

In a trial by Detmar [9], it was reported that dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli keeps the vasculature of adult skin normal, but the skin retains the capacity for brisk initiation of angiogenesis. In the hair follicle growth phase, KC-derived VEGF causes cyclic vascular expansion. HIF-1α messenger RNA translation into protein via phosphoinositol-3 kinase and Akt can be augmented by VEGF.

The study of Yongjian et al. [10] agreed with these results. The study was done on 32 Chinese patients with psoriasis and 20 healthy controls and estimated HIF-1α expression in psoriatic lesions. It reported that the expression of HIF-1α in control skin was very weak, but in psoriatic lesions expression was very strong, and this might play an important role in psoriasis development and genesis.

Tovar-Castillo et al. [11], using reverse transcriptase-PCR, showed significant overexpression of HIF-1α, which was significantly overexpressed in psoriasis more than adjacent nonpsoriatic skin and healthy skin of the control group.Reports indicate that at least eight common cytokines can induce HIF-1α accumulation. Most of these cytokines are known to be ‘active’ in psoriasis, and they could be incriminated in the observed increase of HIF-1α immunoreactivity in the psoriatic keratinocytes. It is well known that HIF-1α may stimulate VEGF and enhance angiogenesis, and also HIF-1α serves as a proinflammatory cytokine by stimulating the production of cyclooxygenase-2, iNOS, and human homeo box-2. Therefore, keratinocytes overexpressing HIF-1α enhance adjacent papilla angiogenesis and inflammation, and HIF-1α overexpression is further stimulated in these KCs by sustained inflammation in a vicious circle [12],[13].

In the current study, the distribution of HIF-1 positivity was mostly in papillary and suprapapillary epidermis, in accordance with the result of Ionnou et al. [5], who added that they have noted increased HIF-1α staining, with specific localization, where HIF-1α preferential immunostaining in the keratinocytes of the suprapapillary and peripapillary epidermis overlying the inflamed, elongated dermal papillae that contained the characteristic tortuous blood vessels.


  Conclusion Top


In this study, there was significant increased HIF-1α expression in psoriatic patients’ skin in comparison with normal control, and also its increase with elevated PASI score represents promising therapeutic approaches and pharmacological research targets for psoriasis treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy of psoriasis. Nature 2007; 445:866–873.  Back to cited text no. 1
    
2.
Rosenberger C, Solovan C, Rosenberger AD, Jinping L, Treudler R, Frei U, Eckardt KU et al. Upregulation of hypoxia-inducible factors in normal and psoriatic skin. J Invest Dermatol 2007; 10:2445–2452.  Back to cited text no. 2
    
3.
Lee SH, Lee YJ, Han HJ, Eckardt KU, Brown LF, Krueger JG et al. Effect of arachidonic acid on hypoxia-induced IL-6 production in mouse ES cells: Involvement of MAPKs, NF-kappa B and HIF-1alpha. J Cell Physiol 2010; 222:574–585.  Back to cited text no. 3
    
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Karhausen J, Hhaasse VH, Colgan SP. Inflammatory hypoxia. Role of hypoxia-inducible factor. Cell Cycle 2005; 4: 256–264.  Back to cited text no. 4
    
5.
Ionnou M, Sourli F, Mylonis L, Barbanis S, Papamichali R, Kouvaras E et al. Increased HIF-1 alpha immumostaining in psoriasis compared to psoriasiformdermatitides. J Cutan Pathol 2009; 36:1255–1257.  Back to cited text no. 5
    
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Carlin CS, Feldman SR. A 50% reduction in the Psoriasis Area and Severity Index (PASI 50) is a clinically significant endpoint in the assessment of psoriasis. J Am Acad Dermatol 2004; 50:859–866.  Back to cited text no. 6
    
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Griffith CE, Barker JN. Psoriasis. In Burns T, Breathnach SM, Cox NH, Griffiths CE, editors. Rook’s textbook of dermatology. 8th ed. UK: Wiley Blackwell; 2010. 20–21.  Back to cited text no. 7
    
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Frank S, Kampfer H. Excisional wound healing. An experimental approach. Methods Mol Med 2003; 78:3–15.  Back to cited text no. 8
    
9.
Detmar M. The role of VEGF and thrombospondins in skin angiogenesis. J Dermatol Sci 2000; 24:78–84.  Back to cited text no. 9
    
10.
Yongjian LI, Guiying Z, Rong X, Huan C, Haiquan W, Ellison EC et al. Expression of iNOS and HIF-1α with angiogenesis in affected skin biopsies from patients with psoriasis. J Cent Sount Univ 2010; 35:952–956.  Back to cited text no. 10
    
11.
Tovar-Castillo LE, Cancino-Diaz JC, Garcia-Vazquez F, Cancino-Gómez FG, León-Dorantes G, Blancas-González F et al. Under-expression of VHL and over-expression of HDAC-1, HIF-1alpha, LL-37, and IAP-2 in affected skin biopsies of patients with psoriasis. Int J Dermatol 2007; 46:239–246.  Back to cited text no. 11
    
12.
Haddad JJ, Harb HL. Cytokines and the regulation of the hypoxia-inducible factor (HIF)-1a. Int Immunopharmacol 2005; 5:461–483.  Back to cited text no. 12
    
13.
Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS et al. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1α. Genes Dev 2001; 15:2520–2535.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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



 

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