Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells.

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Pectoralis major turnover flap based on thoracoacromial vessels for sternal dehiscence.

BACKGROUND: Reconstruction of long and deep sternal defects has been challenging. The pectoralis major can be used in the conventional turnover method that requires the internal thoracic vessel. We developed a new turnover pectoralis major flap based on thoracoacromial vessels. The purpose of this report is to present results from 14 patients. METHODS: Fourteen patients with a mean age of 73.6 years (range, 53-83 years) who had sternal defects underwent reconstruction via this procedure. The defects were caused by mediastinitis and sternal osteomyelitis in six and eight patients, respectively. The internal thoracic artery (ITA) was harvested in two patients. The mean defect size was 2.4 × 15.5 cm (ranging 1-4.3 × 13-18 cm). After elevation of the lateral border of the muscle and ligation of the third to fifth perforators from ITA, the lateral side was turned over and the medial lower portion of the flap was additionally transplanted to the defect. RESULTS: The mean flap size was 10.7 × 18 cm (ranging 9-13 × 15-21 cm). For 11 patients, defects healed without any complications. Discharge after flap reconstruction was observed in three patients, two of whom were managed using conservative treatments. Only one patient who needed additional debridement required transplantation of the contralateral pectoral major flap. CONCLUSIONS: This muscle flap is nourished primarily by the thoracoacromial vessel. The long length and large volume of the muscle flap could be successfully turned over with this procedure even in patients that had their internal thoracic artery sacrificed.

Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated monocytes/macrophages.

BACKGROUND: Serum levels of S100A8/A9 may correlate with disease activity in psoriasis. OBJECTIVE: We sought to elucidate the association of serum levels of S100A8/A9 heterodimers with the clinical subtypes of psoriasis and the major cell source. METHODS: Serum samples were collected from patients with psoriasis vulgaris (n = 30), psoriatic arthritis (PA) (n = 16), pustular psoriasis (n = 24), and atopic dermatitis (n = 14) and from healthy control subjects (n = 21). Serum concentrations of S100A8/A9 were measured, and the expression levels were examined in psoriatic lesions. The messenger RNA levels were quantified in circulating monocytes and neutrophils. RESULTS: Serum levels of S100A8/A9 were significantly increased in all subtypes of psoriasis as compared with healthy controls and atopic dermatitis. Among the psoriatic subtypes, PA and pustular psoriasis showed remarkably high concentrations of S100A8/A9 heterodimers. The higher serum levels were associated with the presence of articular symptoms, but not significantly correlated with body surface areas of psoriatic lesions. S100A8 was expressed by both keratinocytes and infiltrating mononuclear cells, whereas S100A9 was predominantly expressed by neutrophils. The expression levels of S100A8 and S100A9 messenger RNA in monocytes were increased by approximately 2.25- and 1.91-fold in PA, respectively, whereas no significant increase was observed in psoriasis vulgaris and pustular psoriasis. LIMITATIONS: Difficulty in acquisition of clinical and laboratory samples in untreated patients, and of a sufficient number of subjects, were limitations. CONCLUSIONS: Although serum levels of S100A8/A9 were increased in all types of psoriasis examined, patients with PA had higher levels of S100A8/A9, probably because of an activated monocyte/macrophage system.

Tetracyclines modulate protease-activated receptor 2-mediated proinflammatory reactions in epidermal keratinocytes.

In addition to their antibiotic effects, tetracyclines have anti-inflammatory action that is often beneficial in the control of inflammatory skin disorders. In this study, we examined the effects of tetracycline (TET) and two of its derivatives, doxycycline (DOX) and minocycline (MIN), on the production of interleukin-8 (IL-8) elicited by the activation of protease-activated receptor 2 (PAR2) in normal human epidermal keratinocytes (NHEK). In NHEK, the production of IL-8 stimulated by an agonist peptide of PAR2, SLIGKIV-NH(2), at 100 microM was significantly reduced by TET, DOX, or MIN at 5 and 10 microM, concentrations that are noncytotoxic. The tumor necrosis factor alpha (TNF-alpha)-induced production of IL-8 was synergistically augmented by SLIGKIV-NH(2), and that synergistic increase in the production of IL-8 was suppressed by 100 nM PAR2-specific small interfering RNA. It was also suppressed by TET, DOX, or MIN but not by the 14-membered-ring macrolide antibiotics erythromycin, roxithromycin, and clarithromycin, which also have anti-inflammatory activities, at 10 microM. These results suggest that tetracyclines attenuate the PAR2-IL-8 axis in keratinocytes and thereby effectively modulate proinflammatory responses in the skin.

Adult cutaneous alveolar rhabdomyosarcoma on the face diagnosed by the expression of PAX3-FKHR gene fusion transcripts.

A 26-year-old woman presented with an indurated subcutaneous tumor on her left cheek. The histology was compatible with alveolar rhabdomyosarcoma, but immunohistochemistry showed that the tumor cells were negative for desmin, alpha-smooth muscle actin and alpha-Sr-1, but were positive for CD56, vimentin and myogenin. The diagnosis of alveolar rhabdomyosarcoma was confirmed by the detection of PAX3-FKHR fusion gene transcripts in the paraffin-embedded tumor tissue. The tumor was unresponsive to chemotherapy with pirarubicin, carboplatin and ifosfamide, and the patient died 9 months after the diagnosis. This adult case of an alveolar rhabdomyosarcoma primarily occurring on the face is very rare, and the detection of PAX3-FKHR fusion gene transcripts was useful for diagnosis of the disease.

Development of Skin Flaps for Reconstructive Surgery: Random Pattern Flap to Perforator Flap.

Flap transplantation has been an important procedure in plastic and reconstructive surgery to cover and fill various defects. Flap necrosis due to blood circulation failure leads to severe complications, especially in a patient undergoing reconstruction concerning the body cavity after tumor ablation. Surgical procedures for flap transplantation have been further improved and developed. We have reviewed from the random pattern flap to the newest procedure, the perforator flap. Perforator vessels were investigated in the process of development of the fasciocutaneous flap and have become important for blood supply of the skin flap. Blood circulation of the flap has become more stable and reliable than ever with the development and findings of the perforator vessels. Further development of a skin flap will be based on the perforasome concept, which involves the study of the territory and linking of perforator vessels. J. Med. Invest. 63: 159-162, August, 2016.

Reduction mammaplasty and mastopexy for the contralateral breast after reconstruction surgery following cancer resection: A report of 3 cases.

BACKGROUND: Breast reconstruction generally involves autologous tissue transplantation and placement of a mammary prosthesis. When the patient's breasts are extremely large and ptotic, breast reconstruction often results in significantly asymmetrical appearance. However, a good aesthetic outcome after reconstruction surgery following cancer resection is an important quality-of-life factor. We evaluated the efficacy of touch-up surgery, either reduction mammaplasty or mastopexy, performed on the contralateral breast for symmetrization. METHODS: Reduction mammaplasty was performed on the contralateral breast in 2 patients and mastopexy was performed on the contralateral breast in 1 patient after reconstruction surgery following cancer resection, between 2008 and 2014. We reviewed each patient's medical record for general clinical information and for the methods of breast cancer resection and breast reconstruction used, wait time between breast cancer resection and touch-up surgery, preservation of the sensitivity of the nipple-areola complex after the touch-up surgery, and aesthetic outcome (based on visual analog scale score). RESULTS: Wait times in the 3 cases were 4, 9, and 18 months. Nipple-areolar sensitivity was well preserved in all 3 cases. Aesthetic outcomes were judged "excellent" or "very good." CONCLUSION: Revision surgery on the contralateral breast 4 to 18 months after breast reconstruction substantially improves the aesthetic outcome. J. Med. Invest. 63: 281-285, August, 2016.

The expression of p63 during epidermal remodeling in psoriasis.

Psoriasis is a skin disorder of chronic keratinization characterized by epidermal hyperplasia, hyperkeratosis, and inflammation. However, little is known about the mechanism (s) underlying the hyperplasia with elongated rete ridges characteristic of psoriasis. The p63 transcription factor, a homologue of the p53 tumor suppressor, has been implicated in the maintenance of epidermal stem cells and the stratification of the epidermis. p63 is up-regulated in squamous cell carcinomas with anaplasia, suggesting that it is also associated with epidermal hyperplasia. In this study, we examined the expression of p63 in the remodeling of psoriatic epidermis. Lesional tissues from 17 psoriasis patients in various stages of plaque-type psoriasis and normal skin tissues from five healthy subjects were examined by immunohistochemistry using a monoclonal anti-p63 antibody. Normal epidermis stained positively for p63 in the basal cell layer and in 2 to 4 layers of the spinous cell layer. p63 was positive in the thickened rete ridges of the epidermis even in early psoriatic lesions. As the epidermis elongated, p63-positive cells moved down and were localized in the lower parts of the rete ridges where keratinocytes densely proliferated. From these results, we suggest that p63 may be involved in the early stage of the remodeling process of the psoriatic epidermis as well as in the elongation of the rete ridges.

Lamellar ichthyosis with pseudoexon activation in the transglutaminase 1 gene.

We report a case of a 12-year-old boy who was born as a collodion baby after which thick scales developed on his entire body surface. His younger brother showed a similar skin condition. Arcuate-shaped, large, brownish scales covered his face with ectropion. His lower legs were also covered with larger thick, brownish, plate-like scales, but other areas were covered with smaller scales. Next-generation sequencing for exons and splice sites detected a stop-gain TGM1 mutation leading to p.R71* in transglutaminase 1 (TG1). Another mutation identified was a cryptic mutation in intron 3 that activated a pseudoexon, which was detected by RNA-based analysis of hair bulbs. The deep intronic mutation caused another truncation mutation, p.N171Tfs(*) 45, in TG1. An in situ TG1 assay demonstrated that TG1 activity was totally lost in this case. Thus, we conclude that the severe phenotype of the patient developed due to those novel compound heterozygous null truncation mutations in TGM1.

Upregulation of interleukin-33 in the epidermis of two Japanese patients with Netherton syndrome.

Netherton syndrome (NS) is a rare autosomal recessive disorder which is caused by mutations in the SPINK5 gene encoding the serine-protease inhibitor LEKTI. Characteristic symptoms of NS include erythroderma with diffuse desquamation, hair abnormalities and atopic manifestations. Here, we report two Japanese patients with NS, one of whom had a novel mutation in the SPINK5 gene which leads to p.C367Lfs*3. The upregulation of interleukin-33 (IL-33) was evident in basal and thickened lower spinous layers of the epidermis in those cases. This suggests that IL-33 may be involved in the pathophysiology of NS as well as in atopic dermatitis.

Interleukin-17- and protease-activated receptor 2-mediated production of CXCL1 and CXCL8 modulated by cyclosporine A, vitamin D3 and glucocorticoids in human keratinocytes.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor which mediates a variety of functions in the skin including cutaneous inflammation. SLIGKV-NH(2) , an agonist peptide for PAR2, enhanced the interleukin (IL)-17-induced production of two CXC chemokines, CXCL1 (GRO-α) and CXCL8 (IL-8), in normal human epidermal keratinocytes (NHEK) in a concentration-dependent manner. The enhanced production of those chemokines was suppressed by a PAR2-specific siRNA. The SLIGKV-NH(2) -induced production of both CXCL1 and CXCL8 was markedly reduced by cyclosporine A. The enhanced production of CXCL1 was suppressed by 1α, 24R-dihydroxyvitamin D(3) , an active form of vitamin D(3) , and weakly by glucocorticoids, dexamethasone and clobetasol propionate, whereas production of CXCL8 was not altered by any of those receptor agonists. In psoriatic skin, the thickened upper spinous layer of the epidermis was positive for PAR2 protein and the expression of the IL17A mRNA was increased. These results suggest that the IL-17-induced pro-inflammatory reaction is enhanced by the activation of PAR2 in keratinocytes, and that the effect of PAR2 is differentially modulated by cyclosporine A, the active form of vitamin D(3) and glucocorticoids.

Knocking-in the R142C mutation in transglutaminase 1 disrupts the stratum corneum barrier and postnatal survival of mice.

BACKGROUND: Mutations in the gene encoding transglutaminase 1 (TG1) are responsible for various types of autosomal recessive congenital ichthyosis (ARCI), such as lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) and some minor variants of ARCI. A point mutation of R143C in the ß-sandwich domain of TG1 has been often identified in patients with LI or CIE. OBJECTIVE: To elucidate the effect of that point mutation on skin barrier structures and functions, we generated mice with a point mutation of R142C, which corresponds to the R143C mutation in human TG1. METHODS: A mouse line with the R142C point mutation in TG1 was established using a gene targeting technique and the Cre-loxP system. The skin phenotypes were analyzed in homozygous mutant Tgm1(R142C/R142C) mice. RESULTS: In the skin of Tgm1(R142C/R142C) mice, expression of the mutant transcripts was comparable with wild-type or Tgm1(+/R142C) mice. However, the amount of mutated protein in the skin was markedly decreased in Tgm1(R142C/R142C) mice, and the TG1 activity of Tgm1(R142C/R142C) keratinocytes was almost lost. Tgm1(R142C/R142C) mice exhibited morphological and functional skin barrier defects and neonatal lethality. The stratum corneum of those mice lacked cornified envelopes, and loricrin, the major structural component, failed to assemble at the corneocyte cell periphery. Tgm1(R142C/R142C) mice showed a marked increase in transepidermal water loss and their skin was easily permeable to toluidine blue dye. The intercellular lipid lamellar structures of the stratum corneum were irregular and the 13-nm periodic X-ray diffractions from the stratum corneum lipid molecules were lost in vivo. CONCLUSION: From these results, we suggest that the R142C mutation of TG1 reduces the enzyme stability which is indispensable for development of the stratum corneum and skin barrier function and for postnatal survival of mice.

Identification of preferred substrate sequences for transglutaminase 1--development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin.

Transglutaminase 1 (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia. This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes. To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1. The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK/RpsixxxWP (where x and psi represent non-conserved and hydrophobic amino acids, respectively). Using glutathione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific. We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity. Even in peptide form, K5 appeared to have high and specific reactivity as substrate. In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1-null mouse. In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin.

Effect of 14-membered-ring macrolides on production of interleukin-8 mediated by protease-activated receptor 2 in human keratinocytes.

The production of interleukin-8 induced by the activation of protease-activated receptor 2 and its synergism with interleukin-1beta were modulated by 14-membered-ring macrolides, namely, roxithromycin, erythromycin, and clarithromycin, in cultured normal human epidermal keratinocytes. Those macrolides may attenuate the protease-activated receptor 2-interleukin-8 axis and thereby modulate proinflammatory responses in the skin.