Shingo Tajima

Photodynamic therapy of acne vulgaris with topical δ-aminolaevulinic acid and incoherent light in Japanese patients

Background  Photodynamic therapy (PDT) is useful for treatment of epidermal neoplasia but may also have a role in the treatment of inflammatory dermatoses.

Collagen-derived dipeptide, proline-hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fibroblasts.

Orally ingested collagen undergoes degradation to small di‐ or tripeptides, which are detected in circulating blood 2 h after ingestion. The influence of collagen‐derived peptides on dermal extracellular matrix components and cell proliferation was studied using cultured human dermal fibroblasts. Of the various collagenous peptides tested here, the dipeptide proline‐hydroxyproline (Pro‐Hyp) enhanced cell proliferation (1.5‐fold) and hyaluronic acid synthesis (3.8‐fold) at a dose of 200 nmol/mL. This was concomitant with a 2.3‐fold elevation of hyaluronan synthase 2 (HAS2) mRNA levels. Small interfering RNA (siRNA)‐mediated knockdown of the HAS2 gene in human dermal fibroblasts inhibited Pro‐Hyp‐induced HAS2 mRNA transcription and cell mitotic activity. Addition of genistein or H7, a protein kinase inhibitor, abolished the Pro‐Hyp‐induced HAS2 mRNA stimulation. Pro‐Hyp elevated phosphorylation of signal transducer and activator of transcription 3 (STAT3) within a short time period (60 min). These results suggest that Pro‐Hyp stimulates both cell mitotic activity and hyaluronic acid synthesis, which is mediated by activation of HAS2 transcription.

Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells

Abstract Alginate oligosaccharides cleaved from alginic acid polysaccharides of seaweed were tested to determine their ability to enhance proliferation and migration of human umbilical vein endothelial cells. A mixture of alginate oligosaccharides (5 μg/ml in culture broth) stimulated endothelial cell growth, [ 3 H]thymidine uptake and migration in the presence of recombinant vascular endothelial growth factor 165 (VEGF 165 ). In contrast, a high concentration mixture of the oligosaccharides (∼100 μg/ml) suppressed cell growth. The stimulatory activity was comparable to that of heparin, with affinity to VEGF 165 , and decreased on heparin-induced stimulation. Each effective oligosaccharide had guluronic acid at the reducing end. A mixture of alginate oligosaccharides (5 μg/ml) and the most ¶effective fraction (1 μg/ml) stimulated endothelial cell migration. In the presence of VEGF and heparin, some alginate oligosaccharides with the peripheral guluronic acid demonstrated marked stimulatory effects, and one fraction also showed a migratory effect. These findings indicate novel activities of alginate oligosaccharide(s) in endothelial cell growth and migration and suggest synergistic and/or stabilizing effects on VEGF 165 -dependent stimulation of endothelial cells.

Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture

Oligosaccharides, involved in regulation of plant developmental and defensive processes, were tested to determine their ability to enhance proliferation of human keratinocytes. A mixture of alginate oligosaccharides remarkably stimulated keratinocyte growth and [3H]thymidine uptake in the presence of epidermal growth factor (EGF). The activity was comparable to bovine pituitary extract, a common complement in keratinocyte culture, and additive on BPE‐induced stimulation. The most effective oligosaccharide in the mixture was identified and its chemical structure was determined. These findings demonstrate a novel activity of alginate oligosaccharide(s) in keratinocyte growth and suggest a possible co‐factor for EGF‐dependent stimulation in medium for keratinocytes.

The presence of D-β-aspartic acid-containing peptides in elastic fibers of sun-damaged skin : A potent marker for ultraviolet-induced skin aging

Biologically uncommon d-aspartyl residues have been reported in proteins of various elderly tissues. We prepared a polyclonal antibody against d-beta-Asp-containing peptide and examined its immunoreactivity in the skin. The antibody recognized integrated or disintegrated elastic fibers in the sun-exposed skin but not in the sun-protected skin of the elderly donors. Western blot analysis of the proteins isolated from sun-damaged skin demonstrated that the 50 kDa protein was immunoreactive with both antibodies for d-beta-Asp-containing peptide and elastin. Ultraviolet (UV) irradiation on normal skin caused the appearance of d-beta-Asp-containing peptide-immunoreactive fibers in the dermis. These results suggest that UV irradiation is closely related to the formation of d-beta-Asp in the elastic fibers of skin. We propose that the antibody could be a useful indicator for sun damage of the skin.

Immunohistochemical detection of lipid peroxidation products, protein-bound acrolein and 4-hydroxynonenal protein adducts, in actinic elastosis of photodamaged skin

Abstract Acrolein and 4-hydroxy-2-nonenal (HNE) are both byproducts of a lipid peroxidation reaction. Actinic elastosis in photodamaged skin of aged individuals is characterized by the accumulation of fragmented elastic fibers in the sun-exposed areas. To study whether a lipid peroxidation reaction is involved in the accumulation of altered elastic fibers in actinic elastosis, skin specimens obtained from sun-damaged areas were immunohistochemically examined using the antibodies against acrolein and HNE. Both antibodies were found to react with the accumulations of elastic material. Double immunofluorescence labeling demonstrated that acrolein/elastin and HNE/elastin were colocalized in the actinic elastosis. Western blot analysis showed that the polypeptide with a molecular weight of 62 kDa reacted with anti-acrolein, anti-HNE and anti-elastin antibodies. The results suggest that acrolein and HNE may be associated with actinic elastosis.

Elafin is induced in epidermis in skin disorders with dermal neutrophilic infiltration: interleukin-1β and tumour necrosis factor-α stimulate its secretion in vitro

Background Elafin, an elastase inhibitor produced by keratinocytes, is overexpressed in the subcorneal region of skin affected by psoriasis, a major feature of which is epidermal infiltration by neutrophil leucocytes. Objectives We studied the expression of elafin in the epidermis in other skin disorders characterized by dermal neutrophil infiltration and in skin disorders with dermal lymphocyte infiltration. Patients/methods We examined biopsies from the lesional skin of patients with Behçet’s syndrome, Sweet’s syndrome, pyoderma gangrenosum, cutaneous allergic vasculitis and acute bacterial infection (cellulitis), and from the skin of patients with chronic prurigo, discoid lupus erythematosus and psoriasis. We performed in vitro experiments using cultured keratinocytes treated with mediators such as interleukin (IL) ‐1β, tumour necrosis factor (TNF) ‐α, IL‐6, neutrophil elastase and interferon (IFN) ‐γ. Results Anti‐elafin antibody showed a strong reaction with the subcorneal region of the epidermis in patients with Behçet’s syndrome, Sweet’s syndrome, pyoderma gangrenosum, cutaneous allergic vasculitis and acute bacterial infection (cellulitis), but showed no reaction in skin from patients with dermal lymphocyte infiltration such as is seen in chronic prurigo and discoid lupus erythematosus. The in vitro experiments demonstrated that treatment with IL‐1β and TNF‐α resulted in 2·6‐fold and 4‐fold stimulation of elafin secretion, respectively, whereas IL‐6, neutrophil elastase and IFN‐γ caused no significant changes in elafin release. Conclusions These results suggest that inflammatory mediators such as IL‐1β or TNF‐α secreted by dermal neutrophils may be involved in overexpression of elafin in keratinocytes; this could protect the epidermis from degradation by dermal neutrophil infiltration.

Expression of elastin-related proteins and matrix metalloproteinases in actinic elastosis of sun-damaged skin.

Abstract Actinic elastosis is characterized by an accumulation of elastotic material in the upper dermis and is considered to be a manifestation of ultraviolet-induced skin aging. To compare the structural components of the elastotic material in actinic elastosis with those in normal skin, skin specimens were stained with antibodies raised against various elastin-related proteins. Elastotic materials exhibited a strong reaction to the antibodies for elastin, microfibril-associated glycoprotein-1 (MAGP-1), MAGP-4, matrix metalloproteinase 1 (MMP-1), MMP-2 and MMP-3, but a diminished reaction to anti-MMP-9 antibody. Fibroblast cell lines from the upper dermis of affected and unaffected skin were established, and the mRNA levels of MMPs were determined. MMP-1 and -2 mRNA levels were found to be elevated approximately twofold in the fibroblasts from actinic elastosis. Since MMP-1 and -2 are considered to be major enzymes involved in the degradation of matrix components, the accumulation of elastotic materials in actinic elastosis may be related to the degradation process.

Modulation by elastin peptide VGVAPG of cell proliferation and elastin expression in human skin fibroblasts

Elastin is a major component of dermal connective tissue and confers elasticity to the tissues [1]. Elastin has unique repeating sequences in the hydrophobic region: the pentapeptide VPGVG and the hexapeptide XPGVGV (X = 1 or V). The pentapeptide VPGVG is the only repeating sequence present in the elastin molecules of all animal species so far analyzed including human, bovine, porcine and chicken elastin [2–6]. VGVAPG is a hexapeptide repeated many times in human, bovine and porcine elastin molecules, but not present in the chicken elastin molecule [2, 7]. The hydrophobic repeating peptides of elastin VGVAPG are able to generate directed cell movements of human monocytes, bovine ligamentum nuchae fibroblasts [8], aortic endothelial cells [9] and murine lung carcinoma cells [10]. The interaction of the elastin peptides with these cells is thought to be mediated by a high-affinity cell receptor for VGVAPG sequences, which is related to membrane-bound protein kinase C (PKC) activity [10, 11]. We have previously reported that synthetic elastin peptide VPGVG stimulates the proliferation of chick vascular smooth muscle cells and downregulates elastin expression [12]. In this study, we evaluated the effects of two synthetic elastin peptides VPGVG and VGVAPG, both of which are present in human elastin molecules, on the biosynthetic phenotype of extracellular matrix proteins and on the proliferation of human dermal fibroblasts. The following chemicals were purchased from Seikagaku Corporation, Tokyo: the inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) [13]; the inhibitor for cAMPand cGMP-dependent protein kinase, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004) [14]; the calcium-calmodulin binding inhibitor, N-(6-amino-hexyl)-5-chloro1naphthalenesulfonamide (W-7) [15], and the inhibitor for cAMP-dependent protein kinase, (N-[2-(P-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) [16]. Heparin and 2-O-tetradecanoylphorbol 13-acetate (TPA) were purchased from Sigma. The peptides, VPGVG and VGVAPG were synthesized by a solid-phase method. Polymers of VPGVG and VGVAPG [(VPGVG)n, and (VGVAPG)n, respectively] were synthesized as previously deShingo Tajima · Hiroshi Wachi · Yuko Uemura · Kouji Okamoto

Facial lichen striatus: successful treatment with tacrolimus ointment.

Summary We report a 22‐year‐old Japanese woman with facial lichen striatus (LS). The distribution of the lesions corresponded to that of Blaschkos lines. Histology of the lesional skin showed an inflammatory cell infiltrate around hair follicles and eccrine glands. Treatment of the linear lesions with tacrolimus ointment once or twice daily resulted in a dramatical improvement in a short time. LS is a T‐cell‐mediated inflammatory disease and tacrolimus ointment may be an effective alternative treatment for this disease especially when the lesions are located on the face.