HYPOXIA INDUCIBLE FACTOR-1 (HIF-1) ALPHA IN PSORIATIC PATIENTS

Transcription

1 HYPOXIA INDUCIBLE FACTOR-1 (HIF-1) ALPHA IN PSORIATIC PATIENTS Ahmed W. Amer; Sameh Fawzy Fahmy; Mohammed Al Said Faheem and Osama Abdel Azeem Ibrahim Dermatology department; Al-Azhar Faculty of Medicine ABSTRACT Background: Psoriasis is one of the commonest skin disorders, affecting 2-3%. However, its exact pathogenesis is not yet known. Hypoxia inducible factors proposed to play a role in psoriasis pathogenesis. Aim of the study: The aim of this study is the evaluation of hypoxia inducible factor (HIF-1) alpha in tissue samples of patients with psoriasis. Patients and methods: The current study is a cross sectional study that included forty patients suffering from any type of psoriasis). The diagnosis of psoriasis was based on the typical appearance of the skin lesions and confirmed by histopathological examination. The studied persons were collected from the Outpatient Clinic of Dermatology and Venereology Department, Al-Azhar University Hospital (Damietta) during the period from January 2011 to January All cases were submitted to detailed history, thorough general and dermatological examination; routine laboratory investigation (such as hepatic and renal function tests, fasting and post-prandial blood glucose level). In addition, skin biopsies (after 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 during surgical operations; after removal, the biopsies were immediately fixed in formalin). Hematoxylin & Eosin (H&E) staining was done to confirm the histopathological changes of psoriasis and to use it as guide when compared with HIF-1 Alph expression and finally immunohistochemical staining was done to detect HIF-1 Alpha expression. Results: Twenty six (26) of cases (65%) were 267

2 Ahmed W. Amer et al males and 14 (35%) were females with male to female ratio of 1.85:1; their age ranged from 24 to 70 years with a mean of 47.85±13.06 years. Family history of similar disease was reported in 6 cases (15%); the clinical type of psoriasis was localized in 60% of cases and generalized in 40% of cases; PASI score ranged from 2.1 to 22.7 with a mean of 7.24±4.81; hypoxia inducible factor score ranged from 1 to 9 with a mean of 4.55±2.43. Skin localization of HIF-1 in psoriatic cases was supra basal in 26 cases (65.0%), prickle in 38 cases (95%), papillary in 16 cases (40%) and supra papillary in 38 cases (95.0%). Thus, the major concentration was found in prickle and supra-papillary areas and the least at the papillary area (table 1). In the present study, there was no significant difference between males and females; cases with positive and negative family history; localized and generalized types; positive and negative areas in all locations except in papillary area, where there was significant increase in positive area when compared to negative area (6.25±1.90 vs 3.41±2.10 respectively). Conclusion: The present study demonstrated a marked increase of HIF- 1 alpha immunoreactivity in psoriatic skin. In addition, our results indicated that the immunohistochemical pattern of this overexpression could be used as a diagnostic adjunct in distinguishing psoriasis from its histopathologic mimics. Keywords: hypoxia inducible factor; psoriasis INTRODUCTION Psoriasis is a chronic immune-mediated inflammatory disorder with an estimated population prevalence of 2% to 3%, with approximately 17% of these patients having moderate to- severe plaque psoriasis. Up to 30% of psoriasis patients have psoriatic arthritis (Kurd and Gelfand, 2009). It has a major impact on patient psychosocial status and quality of life. The disease also carries an increased risk of comorbidities (including psoriatic arthritis, cardiovascular disease, metabolic syndrome, and inflammatory bowel disease), which can, in 268

3 severe disease cases, reduce life expectancy (Nestle et al., 2009). Its exact pathogenesis which likely combines genetic, environmental, and immunologic factors remains to be fully elucidated. Angiogenesis is a hallmark of psoriasis. Microvessels of the papillary dermis are elongated tortuous, dilated and show increased endothelial cell proliferation. In psoriasis, cell proliferation could increase oxygen consumption, and epidermal thickeneing could lead to impaired oxygen supply (Rosenberger et al., 2007). Hypoxia inducible factor-1 (HIF-1) is heterodimeric transcriptional complex of HIF-1 alpha and HIF-1 beta subunit that has been recognized primarily for its role in the maintenace of oxygen and energy homeostaisis (Lee et al., 2004). Some studies have shown that HIF-1 plays a central role in the stress response beyond hypoxia. A number of mediators of inflammation can activate HIF-1 and HIF-1 stimulates the expression of several genes encoding proteins that promote inflammatory reactions (Hellwig-Buergel et al., 2005). In addition, hypoxia has been implicated in the pathogenesis of various forms of inflammation. The role of hypoxia in colitis has been studied in humans providing an opportunity to search for potential therapeutic interventions (Karhausen et al., 2005). AIM OF THE WORK The aim of this study is the evaluation of hypoxia inducible factor (HIF-1) alpha in tissue samples of patients with psoriasis. PATIENTS AND METHODS The current study is a cross sectional study that included forty patients suffering from any type of psoriasis). The diagnosis of psoriasis was based on the typical appearance of the skin lesions and confirmed by histopathological examination. The studied persons were collected from the Outpatient Clinic of 269

4 Ahmed W. Amer et al Dermatology and Venereology Department, Al-Azhar University Hospital (Damietta) during the period from January 2011 to January Inclusion criteria included diagnosed cases who did not receive any systemic treatment for psoriasis or phototherapy in the last 6 months; patients did not receive any topical preparations other than emollients in the past 6 weeks; patients did not receive any other medications and did not have any other systemic or skin diseases; non pregnant females or females not receive any hormonal contraceptives and finally patients who give consent to participate in the study. Exclusion criteria included patients who applied any topical preparations for psoriasis in the past 6 weeks; patients who received other systemic medications during the past 6 weeks; pregnant and lactating females; and finally patients who have other systemic or skin diseases. Studied subjects were submitted to detailed history, thorough general and dermatological examination; routine laboratory investigation (such as hepatic and renal function tests, fasting and post-prandial blood glucose level). In addition, skin biopsies (after consent, punch biopsies of 4 mm were taken under local anesthesia from lesional skin; after removal, the biopsies were immediately fixed in formalin). Hematoxylin & Eosin (H&E) staining was done to confirm the histopathological changes of psoriasis and to use it as guide when compared with HIF-1 Alph expression and finally immunohistochemical staining was done to detect HIF-1 Alpha expression. Immunohistochemical staining was done as the following: sections from paraffin blocks were cut on 4 micron thickness on adhesive positively charged slides.the slides were placed in xylene bath overnight to remove the paraffin. The sections were then rehydrated by placing them in descending grades of ethanol, followed by rinsing with distilled water. After washing, the slides were dried around the tissue sections. Endogenous peroxidase activity 270

5 was blocked by adding 1to 3 drops of 3 hydrogen peroxide in methanol solution to cover the sections. It was left for 30 minutes in room temperature. The slides were then rinsed by phosphate buffered saline (PBS) for 5 minutes and dried around the tissue sections. Antigen retrival was done by immersing the slides in 1Mm EDTA ph 8 then microwaved at a power of 800 for 20 minutes. Allow them to cool, the sections were then rinsed three times by PBS buffer and dried. Slides were then incubated overnight at 4 C with the primary antibody: Purified Mouse Anti-Human HIF-1 alpha (clone 54/HIF-1 alpha aa available from BD Transduction Laboratories, San Deigo,USA ).The slides then were rinsed three times with PBS and dried. The slides were then incubated with secondary antibody: Polyclonal FITC Goat Anti-mouse Ig (cataloge number ) available from BD Transduction Laboratories, San Deigo, USA. Then washing the slides three times with PBS buffer and dried. The enzyme labeled streptavidin-biotin complex was applied and incubated in a humid chamber at room temperature for 30 minutes and the sections were rinsed with PBS three times and dried. The chromogen used was 3,3 diaminobenzidine (DAB). One drop of DAB was added to each ml of buffered substrate. The component were mixed well and kept in a dark place. The working color reagent was applied for 15 minutes then the sections were rinsed well with distilled water. The slides were then counterstained with hematoxylin and washed in running water. Afterwards, the sections were dehydrated in ascending grades of alcohol, cleared twice in xylene then cover slipped with DPX. The internal positive control is the epithelial cells of hair follicle, sweat glands and sebaceous glands. The negative controls were prepared by substitution of primary antibody by PBS. Evaluation of immunostaining: HIF-1alpha positivity was detected in the nuclei of epidermal keratinocytes. Immunoexpression was graded according to the proportion and intensity of staining of positive epidermal cells (no staining 271

6 Ahmed W. Amer et al [0]; 1-25% [+1]; 26-50% [+2]; and > 50% [+3]. Staining intensity was rated on a scale of 0 to (+3): (0= Negative; +1 = Mild; +2 = Moderate; +3 = Marked). The final score used in statistical analysis was derived by multiplying the extent of positive epidermal cells and the intensity of staining. 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 epidermis (basal, suprabasal or prickle cell layer). Statistical analysis of data: statistical presentation and analysis of the present study was conducted, using the mean, standard deviation by SPSS V.16. Quantitative data were presented as mean ± standard deviation (SD); while qualitative data represented as frequency and percent distribution. Student (t) test and Chi square test were used for comparison between groups in quantitative and qualitative data respectively. RESULTS The present study included 40 cases; 26 of them (65%) were males and 14 (35%) were females with male to female ratio of 1.85:1; their age ranged from 24 to 70 years with a mean of 47.85±13.06 years. Family history of similar disease was reported in 6 cases (15%); the clinical type of psoriasis was localized in 60% of cases and generalized in 40% of cases; PASI score ranged from 2.1 to 22.7 with a mean of 7.24±4.81; hypoxia inducible factor score ranged from 1 to 9 with a mean of 4.55±2.43. Skin localization of HIF-1 in psoriatic cases was supra basal in 26 cases (65.0%), prickle in 38 cases (95%), papillary in 16 cases (40%) and supra papillary in 38 cases (95.0%). Thus, the major concentration was found in prickle and supra-papillary areas and the least at the papillary area (table 1). In the present study, there was no significant difference between males and females; cases with positive and negative family history; localized and generalized types; positive and negative areas in all locations except in papillary area, where there was significant increase in 272

7 positive area when compared to negative area (6.25±1.90 vs 3.41±2.10 respectively) (table 2). RESULTS Table (1): General characteristics of studied cases Variable Statistics Age (years) (mean±sd; range) 47.85±13.06; Disease duration (mean±sd; range) 5.10±4.32; 1-15 Gender (male/female) (n,%) 26/ 14 (65.0%/35.0%) Positive family history 6(15.0%) Clinical type (localized/ generalized) 24/16 (60.0%/40.0%) (n,%) PASI score (mean±sd; range) 7.24±4.81; HIF-1 Score (mean±sd; range) 4.55±2.43; Skin localization of HIF-1 Supra-basal Prickle Papillary Suprapapillary 26(65.0%) 38(95.0%) 16(40.0%) 38(95.0%) Table (2): relation between different variables and HIF-1 percentage Variable Statistics Test P value Sex Male 5.23± Female 3.28±2.13 Family Positive 6.0± history Negative 4.11±2.31 Clinical Localized 3.83± type Generalized 5.62±2.55 Suprabasal Positive 5.0± Negative 3.71±2.21 Prickle Positive 4.47± Negative 5.50±.0.70 Papillary Positive 6.25± * Negative 3.41±2.10 Suprapapillary Positive 4.47± Negative 5.50±

8 Ahmed W. Amer et al Figure (1):HIF Signals appear brown; blue counter staining with hematoxylin Figure (2): upregulation of HIF-1 in psoriatic epidermis Figure (3): expression of HIF-1 in psoriatic dermis 274

9 DISCUSSION The present study was designed to evaluate hypoxia inducible factor (HIF-1) alpha- by immunohistochemical analysis- in tissue samples of patients with psoriasis. The hallmark findings of the present study are that, there was significant increase of HIF-1 expression in different sites of the skin of psoriatic patients and it revealed that, HIF-1 expression was supra basal in 65.0%, prickle in 95%, papillary in 40% and supra papillary in 95.0%. Thus, the major concentration was found in prickle and supra-papillary areas and the least at the papillary area. In addition, there was no relation between HIF-1 scores and patient gender, age, disease duration, disease severity, clinical type or family history. No significant difference was found between positive and negative cases in different sites except papillary site. These results are in agreement with that reported by Yongjian et al. (2010) who conduction a study on 32 Chinese patients with psoriasis and 20 healthy controls and estimated expression of hypoxia inducible factor 1 in psoriatic lesions and reported that, the expression of HIF-1 in control skin was very weak, but very strong in psoriatic lesions, which showed significant difference in between psoriasis and control group (p < 0.05). They concluded that, HIF-1α had high expression in psoriasis and might play an important role in genesis and development of psoriasis. In addition, results of the present study are in accordance with Rosenberger et al. (2007) who reported that, HIF-1 was poorly expressed in normal skin in animal and human models, while, it is strongly activated in psoriatic skin in cell types which express pivotal angiogenic factors. In a trial to explain the previous finding, Detmar (2000), reported that, the vasculature of adult skin remains normally quiescent due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. But, skin retains the capacity for brisk initiation of angiogenesis. Cyclic vascular expansion due to keratinocytederived VEGF occurs in the growth phase of hair follicles. VEGF can augment 275

10 Ahmed W. Amer et al HIFα mrna translation into protein via phosphoinositol- 3 kinase and Akt (Kilic et al., 2006). Rosenberger et al. (2007) show that HIFα colocates with VEGF in the epidermis. In addition, they demonstrated that in psoriasis HIFα, Flt-1, and activated Akt all occur in dermal capillaries. They added, HIF expression is pronounced in perivascular areas where one would expect the least hypoxia. However, as oxygen level is a balance between supply and consumption, in these perivascular areas oxygen consumption (e.g., by inflammatory cells, stromal cells, endothelial cells, etc.) might exceed oxygen supply, thus leading to relative hypoxia. Several lines of evidence suggest a crucial role for augmented VEGF response and the so-called angiogenesis predisposition in the pathophysiology of psoriasis (Detmar, 2004). However, it remains unclear, whether such predisposition also includes alterations upstream of the VEGF gene, such as the HIF system. In this regard and in animal study, Xia et al. (2003) reported VEGF upregulation of reproducing the complete psoriatic phenotype in transgenic mice. In these mice, psoriatic plaques evolve spontaneously after a period of approximately 6 months, which is prevented by VEGF inhibition. By contrast, short-time delivery of VEGF to the skin does not produce psoriatic lesions (Sundberg et al., 2001). Such data suggest that long-term cutaneous VEGF activation is necessary and sufficient to induce psoriasis. Rosenberger et al. (2007) data offer a first hint that HIFs and Akt could be important players in psoriatic angiogenesis, which theoretically would fit into the existing plot of inflammation and epithelial proliferation. Theoretically, HIF activation in T cells may contribute to psoriatic pathophysiology, given that HIF is involved in T-cell survival and function (Nakamura et al. 2005). Furthermore, our results are in agreement with Ioannou et al. (2009) who 276

11 reported that, their overall impression was that HIF-1 alpha immuno-staining was distinct in psoriasis. This could be attributed to, and explained by, quantitative as well as qualitative differences. They added, HIF-1 alpha immunoreactivity final scores were higher in the cases of psoriasis. The corresponding scores were significantly lower in the cases of psoriasiform dermatitis, Scores were practically close to zero in the samples of normal skin. The difference in HIF-1 alpha immunoreactivity scores between psoriasis and psoriasiform dermatitis was statistically significant. They also found that, in regard to the qualitative differences in psoriasis, HIF-1 alpha immunostaining was preferentially localized to keratinocytes in the suprapapillary regions overlying the inflamed, elongated dermal papillae that contained the characteristic tortuous blood vessels. The mechanism(s) underlying the increased HIF-1 alpha immunoreactivity in psoriasis could be different from those operating in hypoxia, and they could involve stimulation by proinflammatory cytokines (Ioannou et al., 2009). Reports indicate that HIF-1 alpha accumulation can be induced by at least eight common cytokines (Haddad and Harb, 2005). Most of these cytokines are known to be active in psoriasis, and they could be incriminated in the observed increase of HIF-1 alpha immunoreactivity in the psoriatic keratinocytes (Lowes et al., 2007). Finally, Ioannou et al. (2009) added that, they have noted that HIF-1 alpha staining, besides being increased, had a specific localization, where it had preferential immunostaining of HIF-1 alpha in the keratinocytes of the suprapapillary and peripapillary epidermis overlying the inflamed, elongated dermal papillae that contained the characteristic tortuous blood vessels. In short, results of the demonstrated a marked increase of HIF- 1 alpha immunoreactivity in psoriatic. In addition, in psoriasis, our results indicated that the immunohistochemical pattern of this overexpression could be exploited as a diagnostic adjunct in distinguishing psoriasis from its histopathologic mimics. 277

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