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• All-ceramic composite thick films seamlessly integrated with a polymeric substrate. • Simple method for manufacture all-ceramic thick films. • Printable ceramic pastes realized without the use of organic additives or vehicles. • High temperature processing not needed. Direct integration of all-ceramic thick films and a polymer substrate has been realized for the first time without high temperature processing using the Room Temperature Fabrication method. Printable Li 2 MoO 4 -BaTiO 3 composite pastes with 0, 10, and 20 vol.% of BaTiO 3 were fabricated from the respective ceramic powders and water without organic additives or vehicles. The pastes were stencil printed on a polyimide substrate and dried at 120 °C without pressing or lamination. Using scanning electron microscopy, the films were observed to be in seamless contact with the substrate and to have a uniform microstructure. Relative permittivities of the ceramic films increased from 4.2 to 7.2 (at 2.5 GHz) and 4.5 to 7.5 (at 9.9 GHz) according to the vol.% content of the added BaTiO3, with corresponding dielectric losses from 10−3 to 10-2. The results show that the room temperature fabrication method enables 2D printing of all-ceramic thick films on temperature-sensitive substrates. [ABSTRACT FROM AUTHOR]

This paper presents the incorporation of large amounts of insoluble additives into a Li 2 MoO 4 ceramic matrix fabricated with a room-temperature densification method. Li 2 MoO 4 –TiO 2 composite ceramics were fabricated by a method based on the water-solubility of Li 2 MoO 4 , where the densification occurs at room temperature during sample pressing and is followed by further post-processing at 120 °C to remove residual water. As the amount of added rutile TiO 2 rose from 10 to 30 vol%, relative permittivity and loss tangent values at ~9 GHz increased from 6.9 to 10.1 and from 1.1×10 −3 to 3.8×10 −3 , respectively, and thermal coefficient of permittivity decreased from 180 ppm/°C to −170 ppm/°C. The Lichtenecker equation used in the analysis of relative permittivity was applicable in this study for Li 2 MoO 4 –TiO 2 composite ceramics with up to 20 vol% of TiO 2 . However, with 25 and 30 vol% additions of TiO 2 , correspondence decreased due to increased porosity in the composites. The low post-processing temperature made this material feasible for Ag electrode integration without formation of unwanted extra phases. [ABSTRACT FROM AUTHOR]

Abstract: Background: Matrix metalloproteinases (MMPs) are a family of ubiquitously expressed zinc-dependent endopeptidases with broad substrate specificity and strictly regulated tissue specific expression. They are expressed in physiological situations and pathological conditions involving inflammation. MMPs regulate several functions related to inflammation including bioavailability and activity of inflammatory cytokines and chemokines. There is also evidence that MMPs regulate inflammation in tumor microenvironment, which plays an important role in cancer progression. Scope of review: Here, we discuss the current view on the role of MMPs in the regulation of inflammation. Major conclusions: MMPs modulate inflammation by regulating bioavailability and activity of cytokines, chemokines, and growth factors, as well as integrity of physical tissue barriers. MMPs are also involved in immune evasion of tumor cells and in regulation of inflammation in tumor microenvironment. General significance: There is increasing evidence for non-matrix substrates of MMPs that are related to regulation of inflammatory processes. New methods have been employed for identification of the substrates of MMPs in inflammatory processes in vivo. Detailed information on the substrates of MMPs may offer more specific and effective ways of inhibiting MMP function by blocking the cleavage site in substrate or by inhibition of the bioactivity of the substrate. It is expected, that more precise information on the MMP–substrate interaction may offer novel strategies for therapeutic intervention in inflammatory diseases and cancer without blocking beneficial actions of MMPs. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. [Copyright &y& Elsevier]

Abstract: Three mammalian collagenases (MMP-1, MMP-8, and MMP-13) belong to family of matrix metalloproteinases and are the principal secreted endopeptidases capable of cleaving collagenous extracellular matrix. In addition to fibrillar collagens, collagenases can cleave several other matrix and non-matrix proteins including growth factors, and this way regulate cell growth and survival. Collagenases are important proteolytic tools for extracellular matrix remodeling during organ development and tissue regeneration, but they also apparently play important roles in many pathological situations and tumor progression and metastasis. Because of their potentially destructive characteristics the expression and activity of collagenases are strictly controlled. Synthesis of collagenases is regulated by extracellular signals via cellular signal transduction pathways at transcriptional and post-transcriptional level. Collagenases are synthesized as inactive pro-forms, and once activated, their activity is inhibited by specific tissue inhibitors of metalloproteinases, TIMPs, as well as by non-specific proteinase inhibitors. In this review we discuss the current view on the role of collagenases in tumor growth, invasion, and metastasis, as a basis for their feasibility in diagnosis and prognostication, as well as therapeutic targets in cancer patients. [Copyright &y& Elsevier]

In this issue, Rohani et al. (2015) report on the role of macrophage-derived stromelysin-2 (matrix metalloproteinase (MMP)-10) in promoting the turnover of extracellular matrix (ECM) during cutaneous wound repair. They provide evidence that MMP-10 specifically enhances collagenolytic activity of murine MMP-13 produced by M2-like macrophages. These results emphasize the important role of macrophage-derived MMP-10 in regulating tissue remodeling and scar formation during wound healing. [ABSTRACT FROM AUTHOR]

Skin biopsies from seven patients with localized scleroderma (morphea) and from two healthy individuals were studied by in situ hybridization to localize the cells responsible for increased procollagen production. In scleroderma lesions, high levels of proα1(I) and proα1 (III) collagen mRNAs were detected in some but not all fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of proα1(I) and proα1(III) collagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens hybridized at the same time under identical conditions. Most of the scleroderma samples represented intermediate stages where the fibroblasts containing elevated levels of type I and type III procollagen mRNAs were located in the papillary and upper reticular layer of the dermis. One of the scleroderma samples from an early inflammatory stage of the disease was found to contain activated fibroblasts in all dermal layers and also in aggregates adjacent to inflammatory cell infilitrates. In situ analyses were also performed on cell cultures from affected and unaffected skin of one scleroderma patient. These experiments revealed a homogeneous population of activated fibroblasts in cultures producing high levels of collagen. The results suggest that development of fibrosis in scleroderma could evolve through activation of a certain fibroblast subpopulation. During cell culturing, however, cell selection or uncharacterized regulatory mechanisms appear to modulate the behavior of these cells with respect to collagen production. [ABSTRACT FROM AUTHOR]

Proteoglycans participate in the assembly of extracellular matrix, directly by interacting with other matrix components and indirectly by regulating cellular growth-factor responses. We have studied the regulation of gene expression of two small extracellular matrix chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, by dexamethasone and retinoic acid In cultured human skin fibroblasts. Dexamethasone increased decorin production, maximally 4,8- fold, and decorin mRNA levels up to 2.3-fold, but had no effect on biglycan production or mRNA levels. Dexamethasone also prevented transforming growth factor-β-elicited down-regulation of decorin mRNA levels and production by dermal fibroblasts. In addition, dexamethasone potently inhibited enhancement of biglycan production and mRNA levels by transforming growth factor-β. Retinoic acid dose dependently reduced decorin mRNA levels (by 51%) and production (by 72%), but had no effect on biglycan gene expression. Retinoic acid did not alter the effect of transforming growth factor-β on decorin or biglycan production or mRNA levels. These results provide evidence that tile effects of glucocorticoids and retinoids on dermal connective tissue are partially mediated via altered expression of decorin and biglycan, which both in turn regulate the activity of transforming growth factor-β, the most potent stimulator of connective tissue deposition. [ABSTRACT FROM AUTHOR]

Cyclosporin A is successfully used in the treatment of scleroderma, a condition with excessive deposition of collagen in the dermis. Cultured human dermal fibroblasts were used as a model to study the effects of cyclosporin A on metalloproteinase expression and activity. Fibroblasts were treated with collagenase inducing agents, phorbol 12-myristate 13-acetate (PMA), cytokines interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and the calcium ionophore A23187 in the presence of cyclosporin A under serum-free conditions, and alterations in metalloproteinase expression were studied by Northern hybridization and immunoblotting analyses, and assays for collagenolytic activity. Induction of collagenase expression by PMA and cytokines was enhanced severalfold by 1-10 μM cyclosporin A. Treatment of cells with cyclosporin A alone caused only a minor increase in collagenase mRNA levels. The secretion of immunoreactive collagenase protein and the level of p-aminophenylmercuric acetate activatable collagenase activity were increased by PMA and further enhanced by cyclosporin A. The expression of the other metalloproteinases stromelysin-1, 92-kD gelatinase, and 72-kD gelatinase or metalloproteinase inhibitor TIMP-1 were not affected by cyclosporin A. Time dependence analysis of the expression of the mRNAs for c-jun and junB indicated that the induction of these genes persisted significantly longer in cells treated with both PMA and cyclosporin A than in cells treated with PMA alone. Enhanced induction of collagenase mRNA may thus result from prolonged AP-1 activity. The results indicate that cyclosporin A potently enhances the expression of collagenase in dermal fibroblasts. [ABSTRACT FROM AUTHOR]

Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor β1 (TGFβ1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of proα1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGFβ1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGFβ1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGFβ1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGFβ1 in the pathogenesis of skin fibrosis in PSS. [ABSTRACT FROM AUTHOR]

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3' → 5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-hp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases. [ABSTRACT FROM AUTHOR]

A crystalline film of α-MnMoO4 has been prepared at an ultra-low temperature of 220°C on paper by a direct solution reaction and subsequent dehydration. First, MnMoO4·0.9H2O was prepared by immersing the substrate in an aqueous solution of MnCl2, followed by spraying a determined area of the substrate with an aqueous solution of Na2MoO4. The ceramic film of MnMoO4·0.9H2O was then dehydrated at 220°C to form a crystalline film of α-MnMoO4. The crystal structure of the material was confirmed by X-ray powder diffraction and an average film thickness of 60μm was measured using a profilometer. The permittivity of the film was also studied and showed a value of 12 at 1GHz. Additionally, the low loss performance of a capacitance structure was obtained. [ABSTRACT FROM AUTHOR]

In this issue, Zigrino et al. report on the role of host-derived mouse collagenase-3 (matrix metalloproteinase (MMP)-13) in melanoma growth and metastasis using a mouse model that lacks MMP-13. The authors demonstrate that vascularization of cutaneous melanomas in these mice is impaired compared with that of controls. This study emphasizes the importance of stromal murine MMP-13, a functional homologue of human MMP-1, in tumor progression. [ABSTRACT FROM AUTHOR]

Piezoelectric materials have a multi-billion dollar impact on the electromechanical transducers market. Their conventional synthesis includes a sintering step (at over 1000 °C) that often hampers direct integration into monolithic devices and confines their applications to heterostructures made using tedious multi-step assembly or to composites with poor electromechanical behavior. Here, we demonstrate a new and easy to scale method for the integration of piezoelectric ceramics at ultra-low temperatures without compromising their functionality. We show that all-ceramic "upside-down" composites with exceptionally high fractions of piezoelectric filler (˜75 vol. %) and low porosity can be achieved using aqueous dispersion of lithium molybdate as a binder. The method is based only on coating, mixing, moulding and drying sequences. The measured piezoelectric charge coefficient, d 33 ˜84 pC·N−1, outperforms any other known composite, whereas the voltage constant, g 33 ˜33 mV m·N−1, competes with bulk materials, thus paving the way for versatile applications not previously considered. [ABSTRACT FROM AUTHOR]

Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an ∼ 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and ∼ 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from -864 to -1 and from -534 to -1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone. [ABSTRACT FROM AUTHOR]

The effects of recombinant human Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), and Tumor Necrosis Factor-α (TNF-α) on collagen biosynthesis were studied in vitro using dermal fibroblast cultures. Both forms of IL-1 and TNF-α induced a dose-dependent inhibition of both types I and III collagen synthesis, as measured by radioimmunoassay, gel electrophoresis, or collagenase-sensitive material. This effect was accompanied by a significant release of postaglandin E2 into the culture medium. However, indomethacin, a potent inhibitor of prostaglandin synthesis, could not prevent the inhibitory effect of the three cytokines on collagen synthesis. Measurement of type I and type III procollagen mRNA levels in IL-1 treated cells revealed that both IL-1α and IL-1β were potent enhancers of procollagen gene expression at pre-translational level. On the other hand, TNF-α was found to reduce the steady-state levels of type I and III procollagen mRNA in a dose-dependent manner. Quantitation of IL-1β and TNF-α transcripts following TNF-α treatment of fibroblasts indicated that this cytokine can induce IL-1β gene expression in these cells. By contrast, TNF-α mRNA remained at a constant level after TNF-α exposure. These data suggest that IL-1 and TNF-α, two cytokines that share several biologic activities, modulate collagen deposition in dermal fibroblasts by mechanisms that are clearly different: TNF-α appears to act at a transcriptional level to inhibit collagen synthesis, whereas IL-1 inhibitory action involves important translational regulation, still unknown, that counterbalances its stimulatory effect on procollagen mRNA levels. Moreover, our data suggest the existence of local fibroblastic cytokine production that may be involved in the modulation of extracellular matrix deposition. [ABSTRACT FROM AUTHOR]

The authors reflect on the achievements of the European Society for Dermatological Research (ESDR) as of 1970. They said that ESDR's success, characterized by its members' annual publications and meetings, is based upon the credit that exclusive science must be fostered and widely disseminated. They conclude that based on these facts, dermatology in Europe is strong as the strength of the foundations of ESDR nurtured dermatological research.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT /ras -HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras -HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion. • ECM component laminin-332 is produced by cancer cells located in the invasive front of cSCC tumor samples. • TGF-β signaling promotes laminin-332 synthesis in an active H-Ras-dependent manner, which increases cancer cell invasion. • Ras and TGF-β signaling pathways are coactivated in vivo in conjunction with laminin-332 accumulation. • We unveil a molecular mechanism that explains the high level of laminin-332 in human cSCC tumors. • We provide compelling evidence that fibroblasts have an integral role in the progression of metastatic cSCC. [ABSTRACT FROM AUTHOR]

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell-specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer. [ABSTRACT FROM AUTHOR]

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase-PCR, and western blotting revealed marked overexpression of complement factor I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell-specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal-regulated kinase (ERK) 1/2 activation. Knockdown of CFI expression potently inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer. [ABSTRACT FROM AUTHOR]

The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing globally. We have studied the expression of complement system components in cSCC. Expression profiling of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=5) with Affymetrix and quantitative real-time PCR (qPCR) revealed upregulation of complement factor H (CFH) and factor H-like protein-1 (FHL-1) in cSCC cell lines. The expression of CFH and FHL-1 mRNAs was also significantly higher in cSCC tumors (n=6) than in normal skin (n=11). Analysis of CFH and FHL-1 expression in vivo in invasive cSCCs (n=65), in situ cSCCs (n=38), and premalignant lesions (actinic keratoses, n=37) by immunohistochemistry showed that they were specifically expressed by tumor cells in cSCCs and the staining intensity was stronger in cSCCs than in in situ cSCCs and actinic keratoses. The expression of CFH by cSCC cells was upregulated by IFN-γ and the basal CFH and FHL-1 expression was dependent on extracellular signal-regulated kinase (ERK)1/2 and p38 signaling. Knockdown of CFH and FHL-1 expression inhibited proliferation and migration of cSCC cells and inhibited basal ERK1/2 activation. These results provide evidence for a role of CFH and FHL-1 in cSCC progression and identify them as progression markers and potential therapeutic targets in SCCs of skin. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To investigate the accuracy of velocity measurements in patients with aortic valve stenosis using phase contrast (PC) imaging accelerated with SENSE (Sensitivity Encoding) and k-t BLAST (Broad-use Linear Acquisition Speed-up Technique). Methods: Accelerated quantitative breath hold PC measurements, using SENSE and k-t BLAST, were performed in twelve patients whose aortic valve stenosis had been initially diagnosed using echocardiography. Stroke volume (SV) and peak velocity measurements were performed on each subject in three adjacent slices using both accelerating methods. Results: The peak velocities measured with PC MRI using SENSE were −8.0±9.5% lower (p <0.01) compared to the peak velocities measured with k-t BLAST and the correlation was r =0.83. The stroke volumes when using SENSE were slightly higher 0.4±17.1ml compared to the SV obtained using k-t BLAST but the difference was not significant (p >0.05). Conclusions: In this study higher peak velocities were measured in patients with aortic stenosis when combining k-t BLAST with PC MRI compared to PC MRI using SENSE. A probable explanation of this difference is the higher temporal resolution achieved in the k-t BLAST measurement. There was, however, no significant difference between calculated SV based on PC MRI using SENSE and k-t BLAST, respectively. [Copyright &y& Elsevier]

The extracellular tumor microenvironment is acidified, whereas the intracellular pH of tumor and stromal cells is neutral. cis-Urocanic acid (cis-UCA), an endogenous compound of the skin, can acidify the cytosol by transporting protons into the cells. This phenomenon, termed the protodynamic concept, was studied here in human cancer cells. cis-UCA dose-dependently reduced the number of viable human melanoma, cervical carcinoma, and fibrosarcoma cells at weakly acidic extracellular pH. The intracellular pH decreased by up to 0.5 pH units in a concentration-dependent manner with 0.3-30 m cis-UCA at extracellular pH 6.5 but not at pH 7.4. Under the same conditions, 30 mM cis-UCA induced annexin-V binding and activation of caspase-3 in A2058 melanoma cells as signs of apoptotic cell death. Finally, growth of human melanoma xenografts in SCID mice was suppressed by 60% following intratumoral injection of cis-UCA. Accordingly, the percentage of tumor necrosis and active caspase-3-immunopositive cells increased, whereas proliferation activity decreased. These results identify cis-UCA as an anticancer agent inhibiting melanoma growth by immediate intracellular acidification followed by apoptotic cell death in vivo. [ABSTRACT FROM AUTHOR]

The transforming growth factor-β (TGF-β) maintains epithehal homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-β promotes malignancy. TGF-β activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-β-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-β receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-β-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-β on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses. [ABSTRACT FROM AUTHOR]

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.Journal of Investigative Dermatology (2007) 127, 49–59. doi:10.1038/sj.jid.5700500; published online 17 August 2006 [ABSTRACT FROM AUTHOR]

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-β1 (TGF-β1) and is hallmarked by the neo-expression of α-smooth muscle actin (α-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-β1 treatment preceding α-SMA upregulation. TGF-β1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with α-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression.Journal of Investigative Dermatology (2006) 126, 2387–2396. doi:10.1038/sj.jid.5700427; published online 22 June 2006 [ABSTRACT FROM AUTHOR]

Connective tissue growth factor (CTGF) is secreted by fibroblasts stimulated with transforming growth factor-β (TGF-β). CTGF is a potent enhancer of fibroblast proliferation, chemotaxis, and extracellular matrix deposition, and it is thought to mediate some of the fibrogenic effects of TGF-β. Here, we have elucidated signaling pathways involved in regulating the TGF-β-induced production of CTGF in primary fibroblasts. TGF-β induced the expression of CTGF messenger RNA and protein in human gingival fibroblasts after 2 h of treatment. Adenoviral overexpression of Smad3 enhanced the TGF-β-elicited expression of CTGF, whereas Smad7 and dominant-negative Smad3 suppressed the effects of TGF-β on CTGF and Cyr61 expression. Pre-treatment of cells with PD98059, an inhibitor for extracellular signal-regulated kinase (ERK)1/2-activator mitogen-activated protein kinase (MAPK)/ERK kinase (MEK)1, potently inhibited the TGF-β-induced expression of CTGF. Furthermore, co-expression of Smad3 with constitutively active MEK1 resulted in potent induction of CTGF production without exogenous TGF-β stimulation. Together, these results demonstrate that Smad3 and ERK1/2 coordinately mediate TGF-β-induced release of CTGF by fibroblasts. It is conceivable that the crosstalk between Smad3 and ERK1/2 signaling cascades plays an important role in regulating CTGF expression, e.g., in wound repair and tissue fibrosis and could be exploited in therapeutic targeting of fibrotic conditions. [ABSTRACT FROM AUTHOR]

Here, we have examined the expression of matrix metalloproteinase-19 (MMP-19) in human cutaneous wounds and by human skin fibroblasts in culture. Expression of MMP-19 was detected by immunohistochemistry in fibroblasts, capillary endothelial cells, and macrophages in the dermal layer of large granulating wounds, as well as in chronic venous and decubitus ulcers. MMP-19 mRNA expression and pro-MMP-19 production by dermal fibroblasts in culture was potently enhanced by tumor necrosis factor-α (TNF-α). Induction of MMP-19 expression by TNF-α was prevented partially by blocking the activation of extracellular signal-regulated kinase (ERK)-1/2 by PD98059 and p38 activity by SB203580. Activation of ERK1/2 by adenovirus-mediated delivery of constitutively active MAPK/ERK kinase 1 resulted in the induction of MMP-19 expression. Activation of p38 alone by adenovirally delivered constitutively active MAPK kinase 3b (MKK3b) and MKK6b also enhanced MMP-19 production, and the most potent induction of MMP-19 expression was noted when ERK1/2 was activated in combination with p38. Activation of c-Jun N-terminal kinase (JNK). Abundant pro-MMP-19 production by fibroblasts was associated with proteolytic processing of secreted pro-MMP-19. These results suggest a role of MMP-19 in cutaneous wound repair and identify three distinct signaling pathways, which coordinately mediate induction of MMP-19 expression in fibroblasts: mitogen-activated ERK1/2 pathway and stress-activated JNK and p38 pathways, of which control proteolytic activity of dermal fibroblasts. [ABSTRACT FROM AUTHOR]

We have studied the effect of a newly identified tumor suppressor tissue inhibitor of metalloproteinases- 3 (TIMP-3) on the growth of human melanoma and squamous-cell carcinoma (SCC). Adenoviral delivery of the TIMP-3 gene to human melanoma (A2058) and SCC (UT-SCC-7) cells ex vivo inhibited tumorigenesis after subcutaneous (s.c.) injection of the infected cells into SCID/SCID mice. Three daily consecutive intratumoral injections of 1.4×109 plaque-forming units (pfu) of TIMP-3 adenovirus (RAdTIMP-3) inhibited the growth of preestablished melanoma and SCC xenografts in SCID/SCID mice, whereas growth of control virus-injected tumors was not affected. The antitumor effect of RAdTIMP-3 was obtained with in vivo adenoviral transduction efficiency of 8–10%, and it was more potent than that of adenovirally delivered p53. Adenovirusmediated expression of TIMP-3 potently reduced gelatinolytic activity, increased the number of apoptotic cells, and inhibited vascularization of melanomas. Escalation of the adenoviral dose to three rounds of three daily consecutive injections with 1.4×109 pfu of RAdTIMP-3 every 6 days entirely inhibited growth of injected melanomas for 32 days. Mixing RAdTIMP-3-infected A2058 cells with uninfected cells in 1:1 ratio in culture resulted in death of all cells in 96 hours. Adenovirally delivered TIMP-3 was also expressed by A2058 cells in soluble form into the culture medium, where it exerted a cytotoxic effect on uninfected A2058 cell cultures after relocating to the cell layer. These results identify TIMP-3 as a novel type of secreted tumor suppressor, which has antiinvasive, antiangiogenic, and proapoptotic effects in vivo, and which displays a potent bystander effect validating further exploration of its applicability in human cancer gene therapy. [Copyright &y& Elsevier]

Recombinant, replication-deficient adenoviruses are efficient vectors for gene transfer to a wide range of cell types, with the exception of T lymphocytes. Here, we show that primary T lymphocytes from peripheral blood, cord blood, and the Jurkat T cell line are efficiently transduced by recombinant adenovirus. Nearly 100% infection efficiency of primary T cells is obtained with high multiplicity of infection (MOI) (5000) of recombinant adenovirus coding for lacZ. Similar infection efficiency by adenovirus-mediated gene transfer was obtained at lower MOI (3000) by activating primary T cells with PHA and PMA. Addition of cationic liposomes together with RAdlacZ markedly enhanced the infection efficiency at lower MOI (1000) resulting in over 90% infection efficiency. Primary T cells express low levels of coxsackievirus and adenovirus receptor (CAR), a cell surface receptor for adenovirus fiber attachment, as well as αvβ3 and αvβ5 integrins, cellular receptors for adenovirus internalization. This suggests that adenovirus entry to T cells at high MOI is mediated by other mechanisms. In conclusion, these results demonstrate that genes can be efficiently transferred to primary lymphocytes by adenovirus vectors at high MOI or in combination with cationic liposomes. [Copyright &y& Elsevier]

SummaryMatrix metalloproteinases play an essential role in tumor growth and invasion. Different matrix metalloproteinases are often expressed in cancers with distinct patterns. To investigate the role of human macrophage metalloelastase (MMP-12) in epidermal tumors, we studied human macrophage metalloelastase mRNA and protein expression in malignant squamous cell and basal cell carcinomas, and in premalignant Bowen's disease. Human macrophage metalloelastase was detected in 11 of 17 squamous cell carcinomas in epithelial cancer cells, whereas macrophages were positive in 15 of 17 samples. In basal cell carcinomas, human macrophage metalloelastase was more often found in macrophages (seven of 19) than in cancer cells (four of 19). Human macrophage metalloelastase mRNA was also detected in three cell lines derived from squamous cell carcinomas of the head and neck and in transformed HaCaT cells, whereas premalignant tumors and primary keratinocytes were negative for human macrophage metalloelastase mRNA. Western analysis revealed human macrophage metalloelastase protein in squamous cell carcinoma cells. Our results show that human macrophage metalloelastase can be expressed in vivo and in vitro by transformed epithelial cells and indicate that the level of human macrophage metalloelastase expression correlates with epithelial dedifferentiation and histologic aggressiveness. [ABSTRACT FROM AUTHOR]

Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases‐3 (TIMP‐3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP‐3 mRNA were elevated up to 2.5‐fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP‐1 mRNA expression was elevated up to 1.8‐fold in two and TIMP‐2 mRNA expression up to 1.8‐fold in two systemic sclerosis strains. Using in situ hybridization, TIMP‐3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP‐1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP‐3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6‐ and 8.1‐fold, respectively) by transforming growth factor‐β, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor‐α. Specific activation of TIMP‐3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP‐3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP‐3 expression by transforming growth factor‐β. Keywords: fibrosis/matrix metalloproteinase. J Invest Dermatol 110:416–421 1998. [ABSTRACT FROM AUTHOR]

Co-expression of several members of the matrix metalloproteinase (MMP) family is a characteristic of human carcinomas. To investigate the role of the recently cloned collagenase-3 (MMP-13) in epidermal tumors, we studied samples representing malignant (basal and squamous cell carcinoma, Paget's disease), pre-malignant (Bowen's disease, solar keratosis), and benign (keratoacanthoma, seborrheic keratosis, linear epidermal nevus) tumors. Basal cell carcinomas expressed collagenase-3 mRNA in focal areas of keratinized cells, the squamous differentiation of which was confirmed by positive immunostaining for involucrin. Apoptosis was observed in central pans of these foci. In squamous cell carcinomas, collagenase-3 expression was detected at the epithelial tumor front and less frequently in the surrounding stromal cells. Collagenase-3 mRNA co-localized with immunostaining for laminin-5, an adhesion molecule suggested to participate in the migration of tumor cells. The pre-malignant and benign tumors were mostly negative for collagenase-3. Stromelysin-1, a potential activator of latent collagenases, was frequently expressed by stromal cells surrounding the malignant tumors, and the two MMPs occasionally co-localized in keratotic foci. Our results demonstrate that in basal cell carcinomas, expression of collagenase-3 is associated with terminal differentiation of epithelial cells. Furthermore, the gene is activated during skin carcinogenesis, and we suggest a role for collagenase-3 in degradation of the extracellular matrix associated with malignant epithelial growth. [ABSTRACT FROM AUTHOR]

Proteolysis is an intrinsic component of cutaneous wound repair and several matrix metalloproteinases have been shown to participate in various stages of this process. Therefore, we investigated the expression of a novel metalloproteinase, collagenase-3 (MMP-13), in normally healing cutaneous wounds and chronic venous ulcers. MMP-13 was expressed abundantly by fibroblasts deep in the chronic ulcer bed but was not detected in epidermis and all the acute wounds. The spatial expression of MMP-13 differed from that of collagenase-1 (MMP-1), which was prominently expressed by migrating keratinocytes and dermal cells located just beneath the wound surface. Northern blot hybridization did not reveal expression of MMP-13 by fibroblasts cultured on tissue culture plastic. In accordance with our in vivo findings, however, fibroblasts grown in a collagen gel produced MMP-13 mRNA abundantly. Our results suggest that MMP-13 can be induced in skin during wound repair after altered cell-matrix interactions. Although both MMP-1 and MMP-13 have the unique ability to degrade fibrillar collagens, their regulation and role during wound repair seem different. Collagenase-1 is critical for re-epithelialization, and MMP-13 most likely plays a role in the remodeling of collagenous matrix in chronic wounds. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α(TNF-α) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-α are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-α by using a mutant, TNF-R55-specific form of human TNF-α. This mutant TNF-α markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-α. In addition, TNF-R55-specific TNF-α suppressed type I collagen mRNA levels as potently as wild-type TNF-α (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-α was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-γ, indicating specific enhancement of TNF- R55 signaling pathway by interferon-α. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-α on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-α may prove to be feasible in the therapy of fibrotic disorders. [ABSTRACT FROM AUTHOR]

The correlation of ejection fraction to left ventricular long-axis contractions, measured from left coronary ostium to apical arterial branches, on coronary angiograms was investigated. [ABSTRACT FROM AUTHOR]

An abstract of the article "Silica Gel in Targeted and Controlled Viral Gene Therapy," by Mika K. Koskinen and colleagues is presented.