Kazuo Noda

Fibulin-4 conducts proper elastogenesis via interaction with cross-linking enzyme lysyl oxidase

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.

Latent TGF-β binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β–free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β–bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

Apocrine adenocarcinoma of the eyelid with aggressive biological behavior: report of a case.

A case of apocrine adenocarcinoma of the eyelid that showed unusually aggressive biological behavior is reported. The patient was a 57‐year‐old man who complained of discomfort and excessive lacrimation of the left eye. A subcutaneous tumor measuring 2.5 cm was found at the medial canthus of the upper eyelid, and a plica‐like subconjunctival spread was noted in the lacrimal caruncle. Invasion into the extraocular muscles and metastasis to the cervical lymph nodes and bone were already present at the time of initial presentation. Histopathologically, the tumor showed features of poorly differentiated adenocarcinoma, and polygonal tumor cells had large, hyperchromatic nuclei with prominent nucleoli and abundant eosinophilic cytoplasm. The formation of ductal structures was found occasionally. The differentiation of the tumor cells towards the apocrine gland was corroborated by immunohistochemistry using monoclonal antibodies GCDFP‐15 and B72.3. The histogenesis and pathological differential diagnosis are discussed briefly, and the tumor was considered to have originated in the Moll’s gland in the eyelid. This case emphasizes that apocrine adenocarcinomas of the ocular region have the potential for aggressive biological behavior, including distant metastasis.

Latent TGF-β binding protein-2 is essential for the development of ciliary zonule microfibrils

Latent TGF-β-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Latent TGF-β binding protein 2 and 4 have essential overlapping functions in microfibril development

Microfibrils are exracellular matrix components necessary for elastic fiber assembly and for suspending lenses. We previously reported that latent TGF-β binding protein 2 (LTBP-2), a microfibril-associated protein, is required for forming stable microfibril bundles in ciliary zonules. However, it was not understood why Ltbp2 null mice only showed an eye-specific phenotype, whereas LTBP-2 is abundantly expressed in other tissues containing microfibrils in wild type mice. Here, we show that LTBP-4, another microfibril-associated protein, compensates for the loss of LTBP-2 in microfibril formation. Ltbp2/4S double knockout (DKO) mice showed increased lethality due to emphysema, which was much more severe than that found in Ltbp4S null mice. Elastic fibers in the lungs of Ltbp2/4S DKO mice were severely disorganized and fragmented. Cultured mouse embryonic fibroblasts (MEFs) from Ltbp2/4S DKO embryos developed reduced microfibril meshwork in serum-free conditions, whereas the microfibril formation was restored by the addition of either recombinant LTBP-2 or -4. Finally, ectopic expression of LTBP-4 in the whole body restored ciliary zonule microfibril bundles in the eyes of Ltbp2 null mice. These data suggest that LTBP-2 and -4 have critical overlapping functions in forming the robust structure of microfibrils in vitro and in vivo.

β-Adrenergic Receptor Blockers Reduce the Occurrence of Keloids and Hypertrophic Scars after Cardiac Device Implantation: A Single-Institution Case-Control Study.

Background: Keloids and hypertrophic scars are characterized by excessive proliferation of fibroblasts; abnormal accumulation of extracellular matrix; and clinical findings of raised, red, itchy, and painful lesions. There are few sufficient interventions for keloids, and the development of new therapeutic agents is urgently needed. Several studies suggest that a therapeutic possibility is &bgr;-adrenergic receptor blocker treatment. Methods: In this single-center case-control study, patients who had undergone cardiac device implantation 7 to 23 months earlier were identified. The implantation incision scars of the patients were deemed to be normal or abnormal depending on their redness. The cases (abnormal scars) and controls (normal scars) were compared in terms of their &bgr;-blocker use rates. Results: Of the 45 eligible patients, 12 and 33 patients were cases and controls, respectively. The cases tended to be less likely to have taken blockers than the controls (25 percent versus 45.5 percent). This difference became significant when the patients whose scars were diagnosed 7 or 8 months after implantation were excluded from the analysis: the age-adjusted odds ratios of the patients who were diagnosed 8 to 23 and 9 to 23 months after implantation were 0.10 (95 percent CI, 0.00 to 0.83; p = 0.0309) and 0.11 (95 percent CI, 0.00 to 0.98; p = 0.047), respectively. Conclusions: &bgr;-Blockers may be an effective alternative modality for preventing and treating keloids and hypertrophic scars. Large-scale multicenter prospective studies that use histology to diagnose scars and diagnose the postoperative scars at the most suitable period are needed to confirm the effectiveness of blockers for abnormal scars. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Glucocorticoids suppress fibroblast apoptosis in an in vitro thermal injury model

The wounds of full- and deep partial-thickness burns result in hypertrophic scars and lead to skin contracture more severely than those of superficial partial-thickness burns. Therefore, preventing burn progression may help improve the aesthetic and functional outcomes after healing. Although a number of studies have focused on elucidating the underlying mechanisms of and preventing burn wound progression, it is still difficult to rescue burned dermis unless early tangential excision is performed. To investigate the underlying mechanisms of and prevent cell death of heat-injured fibroblasts, we developed an in vitro experimental model of heat-injured fibroblasts. We confirmed that heating at 55°C for 30s caused fibroblast necrosis immediately after heating, whereas heating at 46°C for 30s induced apoptosis 24h after heating. We also found that the supplementation of 100ng/ml betamethasone to the culture medium after heating decreased the number of apoptotic cells and increased that of live cells. Our studies suggest that glucocorticoids suppress apoptosis of heat-injured fibroblasts and may be useful for preventing burn wound progression.

The development of a novel wound healing material, silk-elastin sponge

Abstract Silk-elastin is a recombinant protein polymer with repeating units of silk and elastin blocks. This novel wound healing promoting material has the ability to self-assemble from a liquid to a gel. We have already reported that an aqueous solution of silk-elastin has the potential to accelerate wound healing; however, there are several problems in applying silk-elastin in the clinical setting. To solve these problems, we developed a silk-elastin sponge that is easy to use in the clinical setting. In the present study, we examined whether the wound healing effect of the silk-elastin sponge is equal to the aqueous solution of silk-elastin in vivo. The granulation tissue formation promoting effect of the silk-elastin sponge was equal to that of the aqueous solution the silk-elastin, as after application to the wound surface, the sponge was absorbed and dissolved by the exudate. At body temperature the silk-elastin then formed temperature gel. The silk-elastin gel that was obtained contained abundant cytokines from the exudate. We believe that silk-elastin sponge can be applied to various wounds that are difficult to treat with the aqueous solution.