Inverse Correlation between Caveolin-1 Expression and Clinical Severity in Psoriasis Vulgaris

Abstract

Objective:

This study investigated the clinical significance of expression of caveolin-1 - a plasma membrane protein involved in caveola formation, endocytosis, signal transduction and angiogenesis - inthe pathogenesis of psoriasis vulgaris.

Methods:

A total of 20 patients with psoriasis vulgaris and 20 healthy volunteers were recruited. The expressions of caveolin-1, Ki-67 (marker of cell proliferation) and CD34 (marker of angiogenesis) in skin biopsies were detected by immunohistochemistry, and the level of caveolin-1 protein was quantified by Western blotting. Clinical severity was assessed using the Psoriasis Area and Severity Index (PASI) score. Correlations between caveolin-1 expression and psoriasis severity, cell proliferation and angiogenesis were analysed using the Spearman rank correlation test.

Results:

Expression of caveolin-1 was significantly lower in psoriasis samples than in healthy skin samples. In psoriasis lesions, the level of caveolin-1 expression was inversely correlated with the severity of psoriasis, cell proliferation and angiogenesis.

Conclusions:

The level of caveolin-1 expression seems to be related to the clinical severity of psoriasis, and may play a role in the abnormal keratinocyte hyperplasia and angiogenesis seen in this condition.

Introduction

Psoriasis vulgaris is a chronic inflammatory skin disease, the exact cause of which is unclear. It is associated with various immunological abnormalities and has a polygenic inheritance pattern. Abnormal keratinocyte hyperplasia and angiogenesis in the dermal papilla layer are the main pathological features of psoriasis and are closely related to the course and recurrence of this disease. ¹ Ki-67, a nonhistone nuclear protein, is a reliable marker that can be used to determine the cell proliferation index (PI) and occurs in all phases of the cell cycle except G₀; Ki-67 expression in psoriasis has been shown to reflect the degree of abnormal keratinocyte hyperplasia. ² CD34, a single-chain transmembrane glycoprotein with a molecular weight of 110 kDa, is widely recognized as a highly sensitive and specific marker for vascular endothelial cells; ³ CD34 can be used to calculate microvessel density (MVD) in the dermal papilla, which reflects the level of angiogenesis in psoriasis. ⁴

Caveolae, first identified in the 1950s, are invaginations of the plasma membrane that are rich in cholesterol, sphingomyelin and glycosphingolipids. In the 1990s, the protein caveolin was isolated from caveolae in chicken fibroblasts transformed by Rous sarcoma virus. ⁵ The presence of this family of proteins, which consists of caveolin-1, -2 and -3, differentiates caveolae from other lipid raft structures. Caveolin-1 (cav-1), the most important molecule in this family, is involved in the formation of caveolae, and in endocytosis, signal transduction, angiogenesis and other physiological processes. ⁶ It has been shown to have an inhibitory role in cell proliferation, angiogenesis and lymph node metastasis in breast cancer ⁷ and colon cancer. ⁸ Reduced levels of cav-1 protein have been reported in psoriasis and may be related to the development and/or progression of this disease. ⁹ The exact clinical significance and pathogenic role of cav-1 in psoriasis have not yet been clarified, and the relationships between cav-1 and clinical severity, abnormal keratinocyte hyperplasia and angiogenesis in psoriasis are uncertain.

In the present study, expression of cav-1 was compared between psoriasis vulgaris lesions and normal skin, and the relationships between the level of expression of cav-1 and the severity of psoriasis, PI and MVD were investigated, in order to help clarify the clinical significance of cav-1 in the pathogenesis of psoriasis vulgaris.

Patients and methods

Patients

Patients with mild to moderate psoriasis vulgaris who attended the Department of Dermatology, Qilu Hospital of Shandong University, Jinan, China, were considered for recruitment to the study. Diagnosis was made on the basis of clinical and pathological assessments. Those patients who were recruited were required to have a Psoriasis Area and Severity Index (PASI) ¹⁰ score of < 30 out of a possible maximum of 72. Patients with evidence of any other systemic disease or who had received systemic or topical treatment within the previous month were excluded from the study. Recruitment began in September 2010 and ended when 20 patients had been recruited (March 2011).

Skin biopsies were cut from psoriatic lesions of the patients, and control samples of healthy skin were obtained from tissue removed from other patients during plastic surgery at the same hospital, with each patient's permission. The control samples were matched with the psoriasis samples for body location, and patient age and sex. Samples were divided into two parts: one half was fixed in 10% neutral formaldehyde solution and embedded in paraffin wax for immunohistochemistry; the other half was frozen in liquid nitrogen for subsequent examination by Western blotting. All samples were tested within 1 month of being taken.

All study participants provided written informed consent and the study protocol was approved by the Ethics Committee of Qilu Hospital of Shandong University, Jinan, China.

Clinical Severity

Clinical severity was measured using the PASI score, a globally recognized system for the assessment of psoriasis.^11,12 Lesion locations were classified as head (h), upper extremities (u), trunk (t) and lower extremities (l). For each region, the levels of erythema (E), desquamation (D) and infiltration (I) were scored from 0 to 4 (0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, extremely severe). The percentage of the skin area (A) affected by lesions in each region was scored between 0 and 6 (0, 0%; 1, < 10%; 2, 10 – 29%; 3, 30 – 49%; 4, 50 – 69%; 5, 70 – 89%; 6, 90 – 100%). The overall PASI score was then calculated as follows: 0.1 × (E_h + D_h + I_h) × A_h + 0.2 × (E_u + D_u + I_u) × A_u + 0.3 × (E_t + D_t + I_t) × A_t + 0.4 × (E_l + D_l + I_l) × A_l.

Immunohistochemistry

Specimens embedded in paraffin wax were cut into 3-μm thick sections, deparaffinized with xylene and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 5 min at room temperature after microwave antigen retrieval. Rabbit polyclonal anti-cav-1 antibody, mouse polyclonal anti-CD34 antibody or mouse polyclonal anti-Ki-67 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1 : 100 was applied to the sections at 4 °C, and they were incubated overnight. After washing with 0.1 M phosphate-buffered saline (pH 7.4) three times for 5 min each time, antibodies were detected using the ChemMate™ EnVision™ Detection Kit Peroxidase/DAB for Rabbit/Mouse (Dako, Glostrup, Denmark) according to the manufacturer's instructions. After application of the chromogenic reagent 3,3′-diaminobenzidine, sections were counterstained with Harris's haematoxylin, differentiated using hydrochloric acid ethanol and blued using ammonia water. After dehydration in a graded ethanol series and clearing with dimethylbenzene, the slides were mounted with neutral gum and examined using the Image-Pro^® Plus analytical imaging system (Media Cybernetics, Bethesda, MD, USA). All samples were rated by investigators blinded to the source of the specimens.

Expression of cav-1 was indicated by the presence of light-yellow to brown granules in the cell membrane and cytoplasm. The colour and location of staining for cav-1 were recorded.

Expression of Ki-67 was indicated by the presence of yellow to brown granules in the nucleus. Ten representative views with the highest number of positive cells were selected per sample to calculate the PI. The number of Ki-67-positive cells and the total number of cells were counted under light microscopy at a magnification of × 400, and the PI was the percentage of Ki-67-positive cells. ¹³

Specimens were examined at a magnification of × 100 for CD34 staining in the superficial microvascular region of the dermis, which includes capillaries, small arteries and small veins. Expression of CD34 was indicated by the presence of yellow to brown granules in the plasma of vascular endothelial cells. The most vascularized area was selected for observation at a magnification of × 400. Endothelial cells or endothelial cell clusters that were stained brown by the CD34 antibody were counted as microvessels, regardless of whether red blood cells were seen within the lumen. The number of microvessels was counted in 10 visual fields at a magnification of × 400 and the mean calculated to give the MVD. ¹³

Western Blotting

Frozen samples were homogenized and 10 g of lysis buffer (Cell Lysis Buffer for Western and IP; Beyotime, Shanghai, China) was added to 2 g of homogenate; the mixture was incubated at 4 °C for 24 h. The supernatant containing total protein from the tissue homogenate was separated by centrifuging for 5 min at 2500 g . The concentration of total protein was measured using the Bradford method (Protein Assay Kit; Beyotime). The same weight of protein (20 μg) from each sample was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane by electrophoresis at 40 V for 2 h. The membranes were then blocked with 5% skimmed milk at room temperature for 1 h and incubated at 4 °C overnight with polyclonal antihuman cav-1 antibody (Santa Cruz Biotechnology) at a dilution of 1 : 400, or with anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1 : 400. After washing with Tris-buffered saline (TBS; 10 mmol/l, pH 7.4) and TBS-T (TBS containing 0.1% Tween 20; 10 mmol/l, pH 7.4) for 1 h, the membranes were incubated with horseradish peroxidase-labelled goat antirabbit immunoglobulin G (heavy and light) antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1 : 50 at room temperature for 1 h. The membranes were then washed with TBS and TBS-T for 1 h and visualized using 3,3′-diaminobenzidine. Band intensity was determined using an automatic electrophoresis gel image analysis system. An image of the gel was obtained (ChemiImager™ 5500; Alpha Innotech, San Leandro, CA, USA), and scanned with a gel documentation system (Vilber Lourmat, Marne-la-Vallée, France). Image-Pro^® Plus software was then used to calculate the ‘grey value' (absorbance ratio of the band of interest to the β-actin band).

Statistical Analyses

Data were recorded and the mean ± SD was calculated. Student's t-test was used to compare the mean cav-1 protein level, PI and MVD in samples from patients with psoriasis and samples from healthy skin. Correlations between the level of cav-1 expression and PASI score, PI and MVD in samples from patients with psoriasis were analysed using the Spearman rank correlation test. A P-value of < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS^® software version 16.0 for Windows^® (SPSS Inc., Chicago, IL, USA).

Results

A total of 20 patients with psoriasis vulgaris were recruited to the study (12 males, eight females; age 24 – 66 years, mean 45.7 years). Lesions were located on the back (n = 6), thorax (n = 5), lower limbs (n = 3), abdomen (n = 2), scalp (n = 1), face (n = 1), upper limbs (n = 1) and palm of the hand (n = 1). The 20 healthy control volunteers (13 males, seven females) were aged 20 – 69 years (mean 43.2 years). For personal reasons, three of the psoriasis patients withdrew permission to use their samples after the study had started, leaving 17 samples for analysis. Samples from three control subjects were withdrawn from the analysis, retaining the 17 samples that matched the psoriasis samples most closely in position on the body and the participant's sex and age.

Expression of CAV-1

In samples from patients with psoriasis and healthy volunteers, cav-1 expression was located immunohistochemically in the membrane and cytoplasm of cells within the basal and spinous layers of the epidermis. In samples from healthy volunteers, mainly brown or brown-yellow granules were seen, whereas in samples from patients with psoriasis, the granules were mainly yellow or light yellow. The control group did not show staining for cav-1.

On Western blotting, bands at 22 kDa, corresponding to cav-1 protein, were seen in both healthy and psoriasis samples. The ‘grey value' (absorbance ratio of the band of interest to the β-actin band) for cav-1 was 1.32 ± 0.27 in healthy samples and 0.71 ± 0.29 in psoriasis samples; this difference was statistically significant (t = 2.03, P < 0.05).

Pasi Score

The PASI score ranged from 2.6 to 14.6 for the psoriatic lesions (mean ± SD of 10.17 ± 3.77). There was a significant inverse correlation between the level of cav-1 expression and PASI score for the psoriasis samples (r = -0.82, P < 0.05).

Proliferation Index

Positive staining for Ki-67 was seen in only a small number of basal cells in healthy control samples, but was widely observed in the basal layer and the middle to lower portion of the spinous layer in psoriasis samples. The PI was 26.47 ± 3.66% in psoriasis samples and 1.23 ± 0.38% in healthy control samples; this difference was statistically significant (t = 2.12, P < 0.01). There was a significant inverse correlation between the level of cav-1 expression and PI for the psoriasis samples (r = -0.84, P < 0.05).

Microvessel Density

Expression of CD34 was detected in the dermal vascular endothelial cells of both healthy controls and patients with psoriasis. The MVD was 18.26 ± 7.33 per × 400 visual field in psoriasis samples and 4.51 ± 2.04 per × 400 visual field in healthy control samples; this difference was statistically significant (t = 2.10, P < 0.01). There was a significant negative correlation between the level of cav-1 expression and MVD in the psoriasis samples (r = -0.87, P < 0.05).

Discussion

Caveolin-1 includes a highly conserved central hydrophobic region, located at residues 102 – 134, with variable N-terminal and C-terminal regions on both sides in the form of 14 – 16 polymers. Residues 82 – 101 in the N-terminal region, known as the caveolin scaffolding domain, constitute the functional part of cav-1 and can bind to a variety of signalling molecules, such as c-Src, Gα-guanosine diphosphate, protein kinase C and cyclin D₁, to modulate their activities.^14,15 Caveolin-1 is involved in many important physiological processes, including endocytosis, cholesterol transport, cell membrane stability, angiogenesis, cell cycle regulation and cell proliferation. ¹⁶

In the present study, expression of cav-1 was seen in the membrane and cytoplasm of the basal and spinous layers of the epidermis in both healthy skin samples and psoriatic lesions. Staining intensity for cav-1 was greater in healthy samples than in psoriasis samples, which reflected the different levels of cav-1 in the two groups. Quantification by Western blotting showed that cav-1 protein expression was significantly lower in psoriasis samples than in healthy samples. Marked downregulation of cav-1 compared with healthy skin, measured using an immunohistochemical method, was reported in a retrospective analysis ⁹ and in a prospective study, ¹⁶ which is consistent with the results of the present study. Stimulation of a variety of cytokines can reduce the expression of cav-1. ¹⁷ It is generally acknowledged that regulation of immune cells is abnormal in psoriasis. T-helper 1-type cytokines secreted by local T lymphocytes alter the biological activity and immune function of keratinocytes, and activation of keratinocytes can promote the further activation of T cells, maintaining the immune disorder in lesions. ¹⁸ It is therefore suggested that abnormal expression of cytokines (such as interleukins, tumour necrosis factor-α and interferon) may be an important factor in the downregulation of cav-1 in psoriasis. In addition, an inverse correlation between cav-1 expression and the PASI score was demonstrated for the first time in the present study. As the level of cav-1 expression decreased, the clinical severity of psoriasis worsened, indicating that cav-1 may play an important role in the occurrence and progression of psoriasis.

Previous studies reporting reduced levels of cav-1 protein in psoriasis have not clearly elucidate the pathogenic mechanism involved. ¹⁰ In the present study, cav-1 expression was correlated with hyperproliferation and angiogenesis. A significant inverse correlation was seen between the PI and cav-1 expression in psoriasis lesions. The mechanisms by which the level of cav-1 expression may be involved might include: (i) negative regulation of cyclin D₁ to arrest cells in the G₀/G₁ phase; ¹⁹ (ii) inhibition of the activation of extracellular regulated kinase (ERK) to prevent signal transduction; ¹⁹ (iii) reduced expression of epidermal growth factor (EGF), fibroblast growth factor, platelet-derived growth factor and transforming growth factor; ²⁰ (iv) prevention of the tyrosine protein kinase Fyn and the Src homology and collagen protein Shc from interacting with α-integrin by isolating the Fyn–Shc complex; ¹⁹ (v) inhibition of the phosphorylation of Src tyrosine kinase to prevent signal transduction; ¹⁹ and (vi) regulation of p53, which is a negative regulator of the cell cycle – the activity of p53 increases with increasing expression of cav-1 and vice versa. ¹⁹

Angiogenesis is an extremely important factor in the pathogenesis of psoriasis. ¹ In the present study there was a significant inverse correlation between MVD and cav-1 expression in psoriatic lesions. The cav-1 protein is a major negative regulator in the proliferation signalling pathway of endothelial cells. ¹⁷ After phosphorylation of the kinase domain receptor induced by vascular epidermal growth factor (VEGF), the p42/p44 mitogen-activated protein kinase (MAPK) signalling pathway becomes activated to transmit signals into the cells. Overexpression of cav-1 inhibits the activation of p42/p44 MAPK through direct reaction with MAPK/ERK kinase or ERK. In contrast, cav-1 inhibits the activity of flk-1, the receptor of VEGF, potently inhibiting VEGF-dependent angiogenesis. ²¹ In addition, vasodilatation and increased permeability of blood vessels, mediated by nitric oxide, constitutes an important mechanism for angiogenesis. Nitric oxide synthase (NOS) in vascular endothelial cells is located within caveolae and the caveolin scaffolding domain of cav-1 can combine with NOS making it inactive and, thereby, inhibiting angiogenesis. ²²

The main pathological features of psoriasis are abnormal keratinocyte hyperplasia and angiogenesis in the dermal papilla layer, factors that are closely related to the course and recurrence of the disease. ¹ The results of the present study suggest that cav-1 may be involved in abnormal keratinocyte hyperplasia and angiogenesis in psoriasis and indicate its potential as a target protein for the treatment of psoriasis.

In conclusion, cav-1 expression in samples from psoriasis vulgaris lesions was significantly lower than that in normal skin samples. The level of cav-1 was inversely correlated with PASI, PI and MVD. These results showed that the level of cav-1 expression seems to be related to the clinical severity of psoriasis, and may play a role in the abnormal keratinocyte hyperplasia and angiogenesis seen in patients with this condition.