Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Non-pathogenic pemphigus foliaceus (PF) IgG acts synergistically with a directly pathogenic PF IgG to increase blistering by p38MAPK-dependent desmoglein 1 clustering.

BACKGROUND: Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). OBJECTIVE: To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. METHODS: Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. RESULTS: 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. CONCLUSION: These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Paraneoplastic pemphigus associated with fatal bronchiolitis obliterans and intractable mucosal erosions: Treatment with cyclosporin in addition to steroid, rituximab and intravenous immunoglobulin.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that presents as severe mucosal erosions and variable cutaneous lesions and is primarily associated with hematologically malignant or benign diseases. A 59-year-old Japanese woman presented with oral, ocular and vaginal mucosal erosions and erythema as well as blistering on her trunk and limbs. She developed bronchiolitis obliterans; lymphadenopathy in the cervical, subclavian, para-aortic and intraperitoneal regions; and splenomegaly. PNP with B-cell lymphoma was diagnosed. She was treated with two courses of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) for B-cell lymphoma, rituximab once every 3 weeks for five cycles, steroid pulse therapy, oral prednisolone, cyclosporin and high-dose i.v. immunoglobulin. The B-cell lymphoma was in remission after two courses of R-CHOP treatment. Although her skin erythema and blistering were also improved, the mucosal erosions and bronchiolitis obliterans gradually worsened. The patient died of bronchiolitis obliterans after 6 months of hospitalization. Because a cellular immune response is thought to be involved in the pathogenesis of PNP, cyclosporin therapy is expected to aid in suppressing the cellular response. In this case, however, the patient's mucosal lesions and bronchiolitis obliterans were not improved by regular administration of cyclosporin therapy.

Predictive Factors Associated With Acute Ocular Involvement in Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis.

PURPOSE: To suggest an objective score for grading the acute ocular severity of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), and to determine predictive factors for severe acute ocular involvement such as ocular surface epithelial defect and/or pseudomembrane formation. DESIGN: Retrospective cohort study. METHODS: The medical records of SJS (n = 87) and TEN (n = 48) patients between 2005 and 2007 were reviewed. An acute ocular severity score was determined on a scale from 0 to 3 (none, mild, severe, and very severe) according to the existence of hyperemia, corneal or conjunctival epithelial defect, and pseudomembrane formation. The associations between the severe acute ocular involvement and factors such as patient age, exposed drugs, systemic severity, and the prevalence of ocular sequelae were examined. RESULTS: The number of cases with score grade 0, 1, 2, and 3 was 19 (21.8%), 31 (35.6%), 22 (25.3%), and 15 (17.2%) in 87 SJS cases and 12 (25.0%), 11 (22.9%), 17 (35.4%), and 8 (16.7%) in 48 TEN cases. Multivariate logistic regression analysis revealed that patient age (odds ratio [OR], 0.98; 95% confidence interval [CI], 0.96-0.99; P = .007) and nonsteroidal anti-inflammatory drugs NSAIDs or cold remedies (OR, 2.58; 95% CI, 1.26-5.29; P = .010) were predictive factors for severe acute ocular involvement. The prevalence of visual disturbance and eye dryness increased according to the increase of acute ocular severity (P = .001 and P = .007 in SJS; P = .007 and P = .014 in TEN, respectively). CONCLUSIONS: At the onset of SJS/TEN, strict attention should be paid to ocular involvement in young patients and in patients exposed to NSAIDs or cold remedies.

Development of full-thickness human skin equivalents with blood and lymph-like capillary networks by cell coating technology.

We developed a human skin equivalent (HSE) containing blood and lymph-like capillary networks using a cell coating technique, which is a rapid fabrication technology of three-dimensional cellular constructs by cell surface coating using layer-by-layer assembled nanofilms of extracellular matrices. The thickness of dermis consisting of normal human dermal fibroblasts was easily controlled from approximately 5 to 100 µm by altering the seeded cell number. Keratinocytes as a major cell population showed homogeneous differentiation on the surface of the dermis by lifting to air-liquid interface. Histological analysis revealed four distinct layers such as basal layer, spinous layer, granular layer, and cornified cell layer in the epidermis. Interestingly, the measurement of transepithelial electrical resistance (TEER) indicated prolongation of the attainment time for maximum value by increasing the number of the dermal fibroblasts, and the HSEs with six layers of dermis revealed the longest period maintaining over 500 Ω cm(2) of TEER. The co-sandwich culture of human umbilical vein endothelial cells and normal human dermal lymphatic microvascular endothelial cells within eight-layered dermis showed in vitro co-network formation of individual blood and lymph-like capillaries inside the dermis. This is the report for homogeneous full-thickness HSEs with blood and lymph capillary networks, which will be useful for biomedical and pharmaceutical applications.

Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-ß1, TGF-ß2, and TGF-ß3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-ß family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea.

Infantile generalized pustular psoriasis: successful disease control with intermittent etretinate.

Infantile generalized pustular psoriasis is a rare form of psoriasis and the best treatment is controversial. We experienced a 2-year-old female with erythema on her neck and axilla starting at 3 months of age. She presented with recurrent annular and geographic scaly erythema with a few pustules on the neck, precordium and axilla, but no fever. The histopathology revealed subcorneal neutrophilic infiltration and microabscesses without Kogoj's spongiform pustules. The initial diagnosis was subcorneal pustular dermatosis. However, she developed widespread geographic erythema and numerous pustules over her entire body with a fever when she got a cold. A second skin biopsy revealed monolocular pustules and Kogoj's spongiform pustules in the subcorneal layer. Etretinate was administrated after a diagnosis of pustular psoriasis was made and her condition improved gradually. The choice of treatment depends on patient age, general condition and the disease severity.

Second harmonic generation reveals collagen fibril remodeling in fibroblast-populated collagen gels.

Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.

Eccrine sweat contains IL-1α, IL-1ß and IL-31 and activates epidermal keratinocytes as a danger signal.

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1ß, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1ß by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

Role of the aryl hydrocarbon receptor in tobacco smoke extract-induced matrix metalloproteinase-1 expression.

Findings from large epidemiologic studies indicate that there is a link between smoking and extrinsic skin ageing. We previously reported that matrix metalloproteinases (MMPs) mediate connective tissue damage in skin exposed to tobacco smoke extracts. Tobacco smoke contains more than 3800 constituents, including numerous water-insoluble polycyclic aromatic hydrocarbons (PAHs) that trigger aryl hydrocarbon receptor (AhR) signalling pathways. To analyse the molecular mechanisms involved in tobacco smoke-induced skin ageing, we exposed primary human fibroblasts and keratinocytes to tobacco smoke extracts. Hexane- and water-soluble tobacco smoke extracts significantly induced MMP-1 mRNA in both human cultured fibroblasts and keratinocytes in a dose-dependent manner. To clarify the involvement of the AhR pathway, we used a stable AhR-knockdown HaCaT cell line. AhR knockdown abolished the increased transcription of the AhR-dependent genes CYP1A1/CYP1B1 and MMP-1 induced by either of the tobacco smoke extracts. Furthermore, the tobacco smoke extracts induced 7-ethoxyresorufin-O-deethylase activity, which was almost completely abolished by AhR knockdown. Likewise, treating fibroblasts with AhR pathway inhibitors, that is, the flavonoids 3-methoxy-4-nitroflavone and α-naphthoflavone, blocked the expression of CYP1B1 and MMP-1. These findings suggest that the tobacco smoke extracts induce MMP-1 expression in human fibroblasts and keratinocytes via activation of the AhR pathway. Thus, the AhR pathway may be pathogenetically involved in extrinsic skin ageing.

Human antigen R-mediated mRNA stabilization is required for ultraviolet B-induced autoinduction of amphiregulin in keratinocytes.

All members of the EGF family are produced as transmembrane precursors that are proteolytically processed into soluble forms by disintegrin and metalloproteinases (ADAMs) for autocrine/paracrine pathways. In turn, the ligand-activated EGF receptor (EGFR) induces the expression of EGF family members, so-called "autoinduction." However, it is not well understood how this autoinduction occurs. In this study, we investigated the molecular mechanism of the autoinduction of amphiregulin (AREG), a member of the EGF family. We found that ultraviolet B (UVB) exposure increased the AREG mRNA level by stabilization of its mRNA in a human immortalized keratinocyte cell line, HaCaT. The 3' UTR of AREG mRNA was responsible for binding to an mRNA-binding protein, human antigen R (HuR), and the interaction between AREG mRNA and HuR was enhanced by UVB. Inducible knockdown of HuR expression significantly decreased AREG mRNA stability. Interestingly, treatment of HaCaT cells with an EGFR inhibitor, an EGFR neutralizing antibody, or an ADAM inhibitor destabilized AREG mRNA. In the case of ADAM inhibition, administration of soluble AREG restored the mRNA level, indicating that the stabilization occurs in a shedding-dependent manner of EGFR ligands. The HuR dependence of AREG mRNA and protein expression was also confirmed in human primary keratinocytes. Taken together, we propose a novel mechanism by which HuR regulates the stability of AREG mRNA in keratinocytes after UVB exposure and suggest that targeting of HuR functions might be crucial for understanding skin cancers caused by aberrant EGF family member-EGFR signaling.

Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin.

A disintegrin and metalloproteinase (ADAM) is a family of enzymes involved in ectodomain shedding of various membrane proteins. However, the molecular mechanism underlying substrate recognition by ADAMs remains unknown. In this study, we successfully captured and analyzed cell surface transient assemblies between the transmembrane amphiregulin precursor (proAREG) and ADAM17 during an early shedding phase, which enabled the identification of cell surface annexins as components of their shedding complex. Annexin family members annexin A2 (ANXA2), A8, and A9 interacted with proAREG and ADAM17 on the cell surface. Shedding of proAREG was increased when ANXA2 was knocked down but decreased with ANXA8 and A9 knockdown, because of enhanced and impaired association with ADAM17, respectively. Knockdown of ANXA2 and A8 in primary keratinocytes altered wound-induced cell migration and ultraviolet B-induced phosphorylation of epidermal growth factor receptor (EGFR), suggesting that annexins play an essential role in the ADAM-mediated ectodomain shedding of EGFR ligands. On the basis of these data, we propose that annexins on the cell surface function as "shedding platform" proteins to determine the substrate selectivity of ADAM17, with possible therapeutic potential in ADAM-related diseases.

Hair-inducing ability of human dermal papilla cells cultured under Wnt/ß-catenin signalling activation.

It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/ß-catenin activation results in maintenance of the hair-inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/ß-catenin signalling stimulation by GSK-3ß inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu-positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P-cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/ß-catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell-based therapy.

Pathogenic anti-desmoglein 3 mAbs cloned from a paraneoplastic pemphigus patient by phage display.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca(2+)-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP.

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents.

BACKGROUND: During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. OBJECTIVE: In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. METHODS: We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-ß (TGF-ß) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. RESULTS: The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-ß1 expression in the epidermis. The inhibition of TGF-ß kinase completely suppressed α-SMA expression in the dermal matrix. CONCLUSIONS: Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-ß1, which is responsible for myofibroblast differentiation.

Targeted overexpression of Angptl6/angiopoietin-related growth factor in the skin promotes angiogenesis and lymphatic vessel enlargement in response to ultraviolet B.

Angiogenesis is required for physiological tissue repair processes, such as cutaneous wound healing. However, recent studies indicate that endogenous angiogenic factors may enhance photo-induced skin alterations in response to experimental ultraviolet (UV)-B exposure. Angiopoietin-related growth factor (AGF), also known as angiopoietin-like protein 6 (Angptl6), is known to promote new blood vessel formation and vascular hyperpermeability. Importantly, epidermal overexpression of Angptl6/AGF in mice promotes wound healing in the skin. However, it remains unclear whether overexpression of Angptl6/AGF facilitates tissue repair processes in response to UV-B irradiation. To test this hypothesis, we subjected Angptl6/AGF transgenic mice to acute or chronic UV-B exposure. Surprisingly, transgenic mice showed enhanced photosensitivity to subthreshold doses of UV-B that did not induce skin alterations in wild-type littermates. Marked enlargement of blood vessels was observed after a single exposure to UV-B in Angptl6/AGF transgenic mice, although no epidermal changes were observed. Chronic UV-B exposure over 14 weeks promoted cutaneous skin damage in Angptl6/AGF transgenic mice, whereas wild-type mice showed little or no macroscopic skin alteration. In addition to pronounced angiogenesis and epidermal hyperplasia, marked enlargement of dermal lymphatic vessels was observed in UV-B-exposed Angptl6/AGF transgenic mice. Electron microscopy analysis further revealed that the number and size of collagen bundles in the dermis was markedly reduced after chronic UV-B exposure in Angptl6/AGF transgenic mice. Taken together, these results indicate that ectopic expression of Angptl6/AGF in mice likely promotes UV-B-induced skin alterations, and that angiogenesis could be a therapeutic target in prevention of skin photo-aging.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.