The role of endothelial cell apoptosis in the effect of etanercept in psoriasis

Background Psoriasis is a chronic inflammatory skin disease associated with abnormal vascular expansion in the papillary dermis. Tumour necrosis factor (TNF)‐α is a proinflammatory cytokine that can induce antiapoptotic proteins and endothelial cell activation factors in psoriasis. Objectives The present study investigated the effect of the anti‐TNF‐α agent etanercept on the expression of endothelial nuclear factor‐κB (NF‐κB), angiogenic vascular endothelial growth factor (VEGF), endothelial cell marker CD31, antiangiogenic factor thrombospondin‐1 (TSP‐1), and antiapoptotic factors Bcl‐2 and Bcl‐xL in psoriasis. Methods Sixteen patients with moderate‐to‐severe psoriasis were included in the study and treated with etanercept 50 mg twice weekly subcutaneously for 12 weeks. Biopsies of lesional skin (baseline, weeks 3, 6 and 10) were obtained and immunohistochemically stained with antibodies for CD31, VEGF, TSP‐1, NF‐κB, Bcl‐2 and Bcl‐xL. Double immunofluorescence staining for VEGF and CD31 was evaluated with confocal laser microscopy. The terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labelling (TUNEL) assay was applied for apoptosis detection. Results Etanercept caused a statistically significant time‐dependent reduction in the number of dermal blood vessels, the number of CD31+ cells and VEGF in psoriatic lesions, with induction of endothelial cell apoptosis and statistically significant upregulation of TSP‐1 in psoriatic vessels. Immunohistochemical analysis showed significant reduction of NF‐κB, Bcl‐2 and Bcl‐xL expression in endothelial cells during treatment. These changes were accompanied by a marked clinical response. Conclusions The present findings suggest that treatment with etanercept induces apoptosis, reduces apoptosis‐inhibiting factors in psoriatic endothelial cells, and decreases angiogenesis in psoriatic skin.     

The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA,PRINS

Please cite this paper as: The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA, PRINS. Experimental Dermatology 2010; 19: 269–278. Abstract: Psoriasis Susceptibility‐Related RNA Gene Induced by Stress (PRINS) is a non‐coding RNA overexpressed in lesional and non‐lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti‐apoptotic effects in keratinocytes. siRNA‐mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon‐inducible gene with anti‐apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400‐fold upregulated in hyperproliferative lesional and ninefold upregulated in non‐lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA‐mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.     

流式细胞仪分析银屑病表皮角朊细胞凋亡及细胞周期变化

本文采用流式细胞仪分析银屑病不同阶段皮损表皮朊细胞的凋亡状况。结果发现银屑病皮损表皮角朊细胞凋亡和细胞周期呈现连续性变化。提示银屑病皮损表皮角朊细胞的病理改变是始发于角朊细胞凋亡异常增多，继而出现角朊细胞过度增殖。     

Evidence for an alternative pathway of keratinocyte maturation in psoriasis from an antigen found in psoriatic but not normal epidermis

We have isolated a murine monoclonal antibody, called psi-3, which immunolabels maturing keratinocytes in psoriatic skin but not in normal epidermis. The staining is cytoplasmic and is not extractable with 1% Triton X-100, which suggests that the psi-3 antigen is a structural component of the keratinocyte. Neither basal cells nor invading inflammatory cells are stained in psoriatic skin and the antigen appears to be associated specifically with maturing and not proliferating keratinocytes. Keratinocytes cultured in vitro from skin from nonpsoriatic individuals display the antigen in a granular pattern in differentiated cells. The antigen is also expressed after tape-stripping of normal skin and, therefore, represents an inducible product of normal keratinocytes. The antigen is destroyed by proteinase K and appears to be a protein. On discontinuous sodium dodecyl sulfate-gel electrophoresis, the antigen has been found to have a molecular weight of 135,000. The psi-3 antigen is interpreted as a new keratinocyte product expressed in psoriasis, culture, wound healing, and certain other pathologic skin conditions. The synthesis of such a new antigen would not be expected if keratinocyte maturation in psoriasis is a truncated version of the normal system and supports the hypothesis that psoriatic keratinocytes are following an alternative pathway. Results using experimental injury suggest that the psoriatic pathway is normally expressed during wound healing.     

microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris

Background Psoriasis is a chronic inflammatory skin disease characterized by intense proliferation and abnormal differentiation of keratinocytes, although the pathogenesis is still not completely clarified. Objectives We investigated the mechanism of keratinocyte proliferation seen in psoriasis, focusing on microRNA (miRNA). Materials and methods miRNAs were extracted from tissues and sera of psoriasis, atopic dermatitis and healthy control. To determine pathogenic miRNAs, we performed miRNA polymerase chain reaction (PCR) array analysis. The results were confirmed with quantitative real‐time PCR, in situ hybridization, immunohistochemistry, transient transfection of siRNA and inhibitor in cultured keratinocytes and Western blotting. Results PCR array analysis using tissue miRNA demonstrated miR‐424 level was markedly decreased in psoriasis skin in vivo. Protein expression of mitogen‐activated protein kinase kinase 1 (MEK1) or cyclin E1, predicted target genes of miR‐424, was increased in psoriatic skin, although their mRNA levels were not. The transfection of specific inhibitor of miR‐424 in normal human keratinocytes led to upregulation of MEK1 or cyclin E1 protein, and resulted in increased cell proliferation. On the other hand, cell number was significantly decreased when cells were transfected with siRNA for MEK1 or cyclin E1. Furthermore, we first investigated serum miRNA levels in psoriasis. Although not significant, serum miR‐424 concentration tended to be decreased in patients with psoriasis compared with healthy controls. Conclusions Decreased miR‐424 expression and subsequently increased MEK1 or cyclin E1 may play a key role in the pathogenesis of psoriasis. Investigation of the regulatory mechanisms of keratinocyte proliferation by miRNA may lead to new treatments and a disease activity marker.     

MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes.     

TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation

Tumor necrosis factor (TNF)‐like weak inducer of apoptosis (TWEAK) has been reported to induce keratinocyte apoptosis in vitro by engaging its sole receptor of fibroblast growth factor‐inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the growth of psoriatic keratinocytes that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary keratinocytes were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by keratinocytes in vitro, which expressed TNF receptor 2 predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein 2 and cellular FLICE‐inhibitory protein in lesional keratinocytes. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of keratinocytes under psoriatic inflammation. The activation of nuclear factor‐κB signalling‐dependent anti‐apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in keratinocytes under psoriatic inflammation.     

Decreased growth inhibition by recombinant gamma interferon is associated with increased transforming growth factor-α production in keratinocytes cultured from psoriatic lesions

Keratinocytes from involved psoriatic plaques (PP), uninvolved, clinically symptomless skin of psoriatic patients (PN) and normal healthy skin (NN) have been cultured in a low calcium serum free system for multiple passages. In this way, the keratinocytes were removed from microenvironmental factors present in the skin. While the basal rate of proliferation of the PP, PN and NN keratinocytes was not different, the PP cells produced more transforming growth factor-alpha (TGF-alpha) than NN cells, and the antiproliferative response of PP cells to gamma interferon (IFN-gamma), a product of activated T lymphocytes, was reduced. We studied IFN-gamma because it can inhibit the proliferation of NN keratinocytes, induce their differentiation and the appearance of two immunoregulatory cell surface molecules, HLA-DR and intercellular adherence molecule-I (ICAM-I), and because in another epithelial cell system, epidermal growth factor (EGF) modulates IFN-gamma activity. The mean antiproliferative effects of IFN-gamma at 50,200, and 500 U/ml for the PP group (n = 10) was less compared to the NN group (n = 11); P less than 0.001, while the PN group (n = 5) had a less dramatic, but statistically significant, reduction in growth inhibition by IFN-gamma only at 200 and 500 U/ml compared to NN cells; P less than 0.05 and P less than 0.01, respectively. The amount of TGF-alpha produced and secreted by PP keratinocytes from five different individuals was significantly greater than by NN keratinocyte cultures. In addition, IFN-gamma induced TGF-alpha to a lesser extent in PP keratinocytes compared to NN keratinocyte cultures. Keratinocytes isolated from atopic dermatitis and Sézary syndrome patients were similar to NN keratinocytes. In contrast to its differential effects on TGF-alpha production and proliferation, IFN-gamma induced similar amounts of HLA-DR and ICAM-I on PP, PN and NN keratinocytes. Thus, for the PP keratinocytes, there was a dissociation between the antiproliferative and immunomodulatory effects of IFN-gamma. These results support our previous hypothesis that the hyperproliferation and altered differentiation of keratinocytes in psoriatic plaques is linked to an altered responsiveness of the keratinocytes to IFN-gamma. Moreover, these results provide an in vitro correlate of our in vivo observation of increased TGF-alpha levels in psoriatic plaques. A new pathophysiological model to understand psoriasis is proposed which integrates these observations involving IFN-gamma and TGF-alpha. This experimental approach also provides a system to dissect biochemical pathways of pathophysiological importance for keratinocyte hyperproliferation in psoriasis.     

Infliximab restores the balance between pro- and anti-apoptotic proteins in regressing psoriatic lesions

Background Psoriasis and psoriatic arthritis are treated very efficaciously with infliximab, a chimaeric human–murine antitumour necrosis factor (TNF)‐α antibody. As we reported earlier, infliximab, besides its anti‐inflammatory properties, induces a caspase‐independent programmed cell death of psoriatic keratinocytes. Objectives To elucidate this finding further, we investigated the epidermal expression of proteins involved in the mitochondria‐dependent (intrinsic) pathway of cell death. Methods Quantification of proteins with pro‐ (p53, AIF, Bax) and anti‐apoptotic functions (Bcl‐2, Bcl‐XL) and of NF‐κB was performed by means of immunohistochemistry and digital image analysis of the staining of nonlesional skin and lesional psoriatic skin from patients treated with infliximab at weeks 0, 2 and 6. Results Serial biopsies from psoriatic plaques of samples taken at days 0, 5, 14 and 21 of therapy demonstrated a significant downregulation of anti‐apoptotic proteins Bcl‐2, Bcl‐XL and NF‐κB during treatment and, in parallel, a significant upregulation of pro‐apoptotic proteins p53, Bax and AIF. These differences in expression correlated with decreases in epidermal thickness and clinical outcome (Psoriasis Area and Severity Index). At day 21, expression levels of apoptosis‐related proteins in lesional skin approximated those found in nonlesional skin. Conclusions Our data therefore suggest that TNF‐targeting agents may induce the regression of psoriasis at least in part by normalizing the expression of apoptosis‐related proteins in lesional keratinocytes.     

Cyclosporine in psoriasis treatment. Inhibition of keratinocyte cell-cycle progression in G1 independent of effects on transforming growth factor alpha/epidermal growth factor receptor pathways.

Cyclosporine, an immunosuppressive drug, is effective in the treatment of recalcitrant psoriasis. Previous work suggested that keratinocyte hyperproliferation and inflammation are linked in psoriasis and that immune mechanisms participate in the pathogenesis of psoriasis. Transforming growth factor (TGF) alpha may be an important regulatory factor of epidermal growth as overproduction of TGF-alpha is associated with epidermal hyperplasia in psoriatic plaques and epidermal growth factor receptors are overexpressed in psoriatic epithelium. In this study, the effects of cyclosporine on cultured human keratinocytes were examined. Cyclosporine specifically inhibited keratinocyte cell-cycle progression in the G1 phase without causing keratinocytes to terminally differentiate. Cyclosporine did not decrease the expression of TGF-alpha or epidermal growth factor receptors. These results suggest that the effects of cyclosporine on psoriatic skin are unrelated to direct effects on autocrine growth regulation of keratinocytes via TGF-alpha production or of epidermal growth factor receptor modulation.     

Increased expression of glucagon‐like peptide‐1 receptors in psoriasis plaques

Recent case reports suggest that treatment with glucagon‐like peptide‐1 (GLP‐1) agonists results in clinical improvement of psoriasis. The purpose of this study was to determine whether GLP‐1 receptors (GLP‐1Rs) are found in the skin of healthy volunteers and psoriasis patients and if so, whether GLP‐1Rs are located on keratinocytes or immune cells. Three mm‐punch skin biopsies were taken for gene expression analysis from six healthy volunteers and from affected and unaffected skin of six psoriasis patients. In addition, a blood sample was obtained from all participants. Cultured human keratinocytes were either untreated or incubated with tumor necrosis factor‐ α (TNF‐α), interferon‐γ (IFN‐γ) or a combination of TNF‐α and IFN‐γ for 48 h. Total RNA was extracted from all the samples, reversely transcribed and analysed for the expression of GLP‐1R using real‐time PCR. Gene expression analysis showed expression of GLP‐1Rs in five of six skin biopsies from psoriasis plaques, in one of six biopsies from unaffected psoriatic skin and in one of six biopsies from healthy skin. GLP‐1R expression was found in the blood of both healthy volunteers and psoriasis patients. No GLP‐1R expression was found in either stimulated or unstimulated cultured human keratinocytes. Our results show increased presence of GLP‐1Rs in psoriasis plaques and that this most likely is due to infiltration with immune cells. This offers a possible explanation for the positive effect of treatment with GLP‐1R agonists in patients with psoriasis.     

Psoriatic keratinocytes are resistant to tumor necrosis factor alpha's induction of mRNA for the NMDA‐R2C subunit

Psoriatic individuals demonstrate accelerated healing and the Koebner phenomenon, suggesting that psoriatic proliferation of keratinocytes is not inhibited appropriately after skin injury. Serial analysis of gene expression in TNFα‐exposed keratinocytes shows the greatest alteration in expression of NMDA‐R2C. Expression of the NMDA receptor is altered in diseased skin containing TNFα, and TNFα plays a prominent role in psoriasis. An abnormality in induction of NMDA‐R2C by TNFα in psoriatic keratinocytes may explain their lack of growth inhibition. We compared the capacity of TNFα to induce expression of NMDA‐R2C in normal and psoriatic (involved and uninvolved) keratinocytes in vitro. After 72 h of incubation with TNFα, normal keratinocytes demonstrated a significant induction of NMDA‐R2C mRNA compared with control cultures, whereas psoriatic keratinocytes showed no induction. In an in vitro model of wounding (scratches on monolayers), TNFα inhibited migration/proliferation of keratinocytes only at the edge of NMDA‐R2C expressing wounded monolayers of normal keratinocytes.     

Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre-coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.     

Fibroblast-keratinocyte interactions in psoriasis: failure of psoriatic fibroblasts to stimulate keratinocyte proliferation in vitro

We have tested in two ways the hypothesis that dermal fibroblasts direct the hyperproliferation of the overlying epidermis in psoriasis. First, culture medium from psoriatic and from normal skin fibroblasts was added to monolayer cultures of foreskin keratinocytes. Second, psoriatic and normal fibroblasts embedded in hydrated collagen lattices were co-cultured with monolayers of foreskin keratinocytes. There was no evidence in either study that psoriatic fibroblast products could stimulate the proliferation of the keratinocytes, or that normal fibroblast products inhibited their proliferation. A positive control for the fibroblasts was provided by leucocyte supernatants, which stimulated keratinocyte proliferation by up to 65%. Our data do not support a primary role for dermal fibroblasts in psoriasis.     

Insulin‐like growth factor‐1 in psoriatic plaques treated with PUVA and methotrexate

Background The pathogenesis of psoriasis is thought to depend on the activation of immune cells and their secreted cytokines, chemokines and growth factors like IGF‐1 which may contribute to the epidermal hyperplasia of psoriasis. Treatment of psoriasis with PUVA and methotrexate are associated with clinical improvement and decrease in epidermal hyperplasia. Objective To examine the effects of PUVA and methotrexate therapy on IGF‐1 expression in psoriatic plaques and whether this change correlates with clinical response. Methods For 24 psoriatic patients, the PASI score and levels of lesional IGF‐1 and its mRNA were determined by RT‐PCR before and after treatment with either methotrexate or PUVA. Skin biopsies from 12 healthy volunteers served as control for IGF‐1 levels in normal skin. Results Lesional skin of psoriatic patients showed a statistically significant elevation in IGF‐1 and its mRNA levels in comparison to control (P = 0.0001). Both methotrexate and PUVA treatment were associated with a significant decrease in both PASI scores and lesional IGF‐1 after 10 month treatment. Conclusion Both methotrexate and PUVA therapy for psoriasis are associated with a decrease in PASI score and IGF‐1. The IGF‐1 down‐regulation may possibly be a consequence of the decrease in cytokines and inflammatory cellular infiltrate that occur following treatment with either modalities or due to their effect on local fibroblast activity and proliferation.     

转化生长因子β及其受体Ⅰ、Ⅱ在银屑病皮损中的表达

目的　探讨转化生长因子 β(TGF β)及其受体Ⅰ、Ⅱ在银屑病角质形成细胞中的表达及其与银屑病的关系。方法　采用免疫组化方法检测TGF β1,TGF β2 ,TGF βRI,TGF βRⅡ在银屑病皮损、皮损周边正常皮肤及正常对照皮肤的原位表达。结果　与正常皮肤相比 ,TGF β1、TGF β2 、TGF βRⅠ和TGF βRⅡ在银屑病表皮基底层表达明显减弱或缺失 ;TGF βRⅡ在棘层至颗粒层的阳性表达率增加。结论　TGF β/TGF βR在银屑病皮损基底层的表达下调或缺乏 ,可能在银屑病角质形成细胞过度增殖中发挥作用 ,而且TGF β/TGF βR系统也可能与银屑病细胞异常分化有关     

Resident skin cells in psoriasis: a special look at the pathogenetic functions of keratinocytes

Psoriasis is a chronic inflammatory skin disease characterized by exaggerated keratinocyte proliferation. Current paradigm indicates that psoriasis is driven by T cell–mediated immune responses targeting keratinocytes. However, psoriasis cannot be explained solely on the basis of T-cell activation, and it is likely that intrinsic alterations in epidermal keratinocytes play a very relevant role in disease expression. In particular, keratinocytes may be important in initiating, sustaining, and amplifying the inflammatory responses by expressing molecules involved in T-cell recruitment, retention, and activation. Keratinocytes are also a relevant source of growth factors for angiogenesis. Finally, intrinsic defects in cytokine and growth factor signaling in keratinocytes may be responsible for their aberrant hyperproliferation and differentiation to T cell–derived signals. Other skin resident cells such as fibroblasts, mast cells, and endothelial cells also contribute to psoriasis pathogenesis by expressing molecules involved in T-cell recruitment and activation.     

RNA, DNA, and cell surface characteristics of lesional and nonlesional psoriatic skin

We have measured the RNA and DNA content and examined cell surface characteristics of human epidermal cells derived from normal skin, and lesional and nonlesional areas of psoriatic skin prior to and following treatment on a modified Goeckerman protocol. Our results show that cells from active psoriatic lesions contain greater numbers of basal keratinocytes when compared with either nonlesional skin from the same patients or skin from healthy volunteers and individuals with other inflammatory skin lesions. Follow-up measurements 2-3 weeks after the initiation of therapy showed that the numbers of basal keratinocytes in resolving psoriatic lesions had decreased and approached normal levels. Multiparameter RNA/DNA flow cytometric analysis on parallel samples from the same psoriasis patients revealed an increased growth fraction and proportion of cycling cells in both the nonlesional and lesional skin compared with controls. Furthermore, the cellular RNA content was elevated in lesional psoriatic skin when compared with either nonlesional or normal skin. Flow cytometric examination of nonlesional and lesional epidermal cells obtained 2-3 weeks after the commencement of therapy revealed that the growth fraction and mean RNA content of the keratinocytes from resolving psoriatic plaques decreased in response to therapy. In contrast, the proportion of keratinocytes within the S + G2 + M phases of the cell cycle remained elevated. These data indicate that "uninvolved" psoriatic skin exhibits characteristics more closely resembling lesional psoriatic skin than normal skin. The results further suggest that quantitation of cellular RNA content and basal cell number might be sensitive indicators of early treatment response in psoriasis.