Dill Extract Preserves Dermal Elastic Fiber Network and Functionality: Implication of Elafin.

INTRODUCTION: Elastic skin fibers lose their mechanical properties during aging due to enzymatic degradation, lack of maturation, or posttranslational modifications. Dill extract has been observed to increase elastin protein expression and maturation in a 3D skin model, to improve mechanical properties of the skin, to increase elastin protein expression in vascular smooth muscle cells, to preserve aortic elastic lamella, and to prevent glycation. OBJECTIVE: The aim of the study was to highlight dill actions on elastin fibers during aging thanks to elastase digestion model and the underlying mechanism. METHODS: In this study, elastic fibers produced by dermal fibroblasts in 2D culture model were injured by elastase, and we observed the action of dill extract on elastic network by elastin immunofluorescence. Then action of dill extract was examined on mice skin by injuring elastin fibers by intradermal injection of elastase. Then elastin fibers were observed by second harmonic generation microscopy, and their functionality was evaluated by oscillatory shear stress tests. In order to understand mechanism by which dill acted on elastin fibers, enzymatic tests and real-time qPCR on cultured fibroblasts were performed. RESULTS: We evidence in vitro that dill extract is able to prevent elastin from elastase digestion. And we confirm in vivo that dill extract treatment prevents elastase digestion, allowing preservation of the cutaneous elastic network in mice and preservation of the cutaneous elastic properties. Although dill extract does not directly inhibit elastase activity, our results show that dill extract treatment increases mRNA expression of the endogenous inhibitor of elastase, elafin. CONCLUSION: Dill extract can thus be used to counteract the negative effects of elastase on the cutaneous elastic fiber network through modulation of PI3 gene expression.

FBN2 Silencing Recapitulates Hypoxic Conditions and Induces Elastic Fiber Impairment in Human Dermal Fibroblasts.

Most chronic wounds are characterized by varying degrees of hypoxia and low partial pressures of O2 that may favor the development of the wound and/or delay healing. However, most studies regarding extracellular matrix remodeling in wound healing are conducted under normoxic conditions. Here, we investigated the consequences of hypoxia on elastic network formation, both in a mouse model of pressure-induced hypoxic ulcer and in human primary fibroblasts cultured under hypoxic conditions. In vitro, hypoxia inhibited elastic fiber synthesis with a reduction in fibrillin-2 expression at the mRNA and protein levels. Lysyl oxidase maturation was reduced, concomitant with lower enzymatic activity. Fibrillin-2 and lysyl oxidase could interact directly, whereas the downregulation of fibrillin-2 was associated with deficient lysyl oxidase maturation. Elastic fibers were not synthesized in the hypoxic inflammatory tissues resulting from in vivo pressure-induced ulcer. Tropoelastin and fibrillin-2 were expressed sparsely in hypoxic tissues stained with carbonic anhydrase IX. Different hypoxic conditions in culture resulted in the arrest of elastic fiber synthesis. The present study demonstrated the involvement of FBN2 in regulating elastin deposition in adult skin models and described the specific impact of hypoxia on the elastin network without consequences on collagen and fibronectin networks.

Dill Extract Induces Elastic Fiber Neosynthesis and Functional Improvement in the Ascending Aorta of Aged Mice with Reversal of Age-Dependent Cardiac Hypertrophy and Involvement of Lysyl Oxidase-Like-1.

Elastic fibers (90% elastin, 10% fibrillin-rich microfibrils) are synthesized only in early life and adolescence mainly by the vascular smooth muscle cells through the cross-linking of its soluble precursor, tropoelastin. Elastic fibers endow the large elastic arteries with resilience and elasticity. Normal vascular aging is associated with arterial remodeling and stiffening, especially due to the end of production and degradation of elastic fibers, leading to altered cardiovascular function. Several pharmacological treatments stimulate the production of elastin and elastic fibers. In particular, dill extract (DE) has been demonstrated to stimulate elastin production in vitro in dermal equivalent models and in skin fibroblasts to increase lysyl oxidase-like-1 (LOXL-1) gene expression, an enzyme contributing to tropoelastin crosslinking and elastin formation. Here, we have investigated the effects of a chronic treatment (three months) of aged male mice with DE (5% or 10% v/v, in drinking water) on the structure and function of the ascending aorta. DE treatment, especially at 10%, of aged mice protected pre-existing elastic lamellae, reactivated tropoelastin and LOXL-1 expressions, induced elastic fiber neo-synthesis, and decreased the stiffness of the aging aortic wall, probably explaining the reversal of the age-related cardiac hypertrophy also observed following the treatment. DE could thus be considered as an anti-aging product for the cardiovascular system.

A single-chip integrated transceiver for high field NMR magnetometry.

We present the design and performance of a broad-band single-chip integrated transceiver specifically conceived for nuclear magnetic resonance magnetometry. The single-chip transceiver is realized using a standard silicon complementary metal-oxide-semiconductor integrated circuit technology. A radio-frequency (RF) transmit amplifier, a transmit/receive switch, a low noise RF receive amplifier, a quadrature (IQ)-mixer, and two intermediate frequency amplifiers are integrated on a single silicon chip of 1.8 mm2. The advantages and problematic aspects with respect to conventional discrete electronic approaches are discussed. We show the results of magnetic field measurements performed at 1.4 and 7.05 T, using solid and liquid samples having volumes from 40 µl down to 100 pl. Particular attention is devoted to the comparison of the experimentally measured magnetic field standard deviation with respect to the Cramer-Rao lower bound value. With a sample of distilled water (T1 ≅ T2 ≅ 3 s, T2 *≅ 20 ms) having a volume of 40 µl, a standard deviation of 2.5 nT at 7.05 T (i.e., 0.5 ppb) in 1 s of averaging time is achieved, with a projected Cramer-Rao lower bond of 8 pT (i.e., 1.1 ppt).

In vitro epidermis model mimicking IGF-1-specific age-related decline.

Ageing is a complex multifaceted process affecting skin functionality and structure. Several 3D organotypic skin culture models have reproduced ageing by inducing replicative senescence, glycation or oxidative stress. Yet, very few models have focused on hormonal ageing and especially the insulin-like growth factor 1 (IGF-1) signalling pathway, which has been associated with longevity in animal studies and is necessary for the early stages of skin development. In this study, we built an organotypic epidermis model with targeted IGF-1 receptor knockdown to reproduce some aspects of hormonal ageing on skin. Our model displayed morphological and functional features of aged epidermis, which were mostly attributed to a loss of function of the Stratum basale. IGF-1 receptor knockdown keratinocytes depicted an extended cell cycle, reduced proliferation potential and reduced adhesion capacities and greater sensitivity to oxidative stress than control cells. Altogether, this model represents an essential tool for further investigations into the mechanisms linked to some aspects of hormonal decline or when screening for potent anti-ageing compounds.

Methylation of LOXL1 Promoter by DNMT3A in Aged Human Skin Fibroblasts.

Lysyl oxidase-like 1 (LOXL1) is an amino-oxidase involved in maturation of elastic fibers. Its downregulation has been associated with elastic fibers repair loss in aging aorta, lung, ligament, and skin. Several evidences of LOXL1 epigenetic silencing by promoter methylation were reported in cancer and cutis laxa syndrome. We hypothesized that this mechanism could be implicated in skin aging process, as far as elastic fibers are also concerned. Anti-DNMT3A chromatin immunoprecipitation was conducted with nuclear extracts from skin fibroblasts isolated from young and elderly individuals, and showed a higher level of DNMT3A protein binding to the LOXL1 promoter in older cells concomitantly to the decrease of LOXL1 mRNA expression and the increase of LOXL1 promoter methylation. Using luciferase reporter assay driven by LOXL1 promoter in HEK293 cells, we demonstrated that LOXL1 transcriptional activity was dramatically reduced when a recombinant DNMT3A was concomitantly overexpressed. LOXL1 promoter transcriptional activity was restored in the presence of a broad-spectrum inhibitor of DNMT activity, 5-aza-2'-deoxycytidine. Finally, to assess whether the interplay between DNMT3A and LOXL1 promoter could be targeted to increase LOXL1 mRNA expression level, an Origanum majorana extract was selected among 43 plant extracts as a new inhibitor of human DNMT3A activity to restore LOXL1 secretion without cytotoxicity in aged skin fibroblasts.

Biosynthetic support based on dendritic poly(L-lysine) improves human skin fibroblasts attachment.

Poly(L-lysine) (PLL) dendrigrafts (DGLs) are arborescent biosynthetic polymers of regular and controlled structures. They have specific properties such as biocompatibility and non-immunogenicity, and their surface density of NH2 functions can be easily modified and therefore appears as a powerful tool for the functionalization of hydrophobic polymers used in the context of tissue engineering. In this study, we evaluated several criteria of human skin fibroblasts when cultured with DGL of generations 2, 3 and 4, with linear PLL polymer as reference. In aqueous phase, DGLs and PLL displayed a similar cytotoxicity towards fibroblasts. Plastic culture plates grafted with DGLs were further characterized as homogeneous surfaces by atomic force microscopy and surface characterization by amino density estimation by colorimetric assay. Proliferation of fibroblasts was increased when cultured onto PLL and DGLs monolayers when compared with crude plates. Cellular adhesion was increased by 20% on DGLs in comparison to PLL. Integrin α5 subunit protein expression level was increased after 48 h of culture on DGLs, in comparison to control or PLL-coated surfaces. The presence of DGLs did not lead to overexpression or activation of matrix metalloproteinases 2 and 9. Finally, fibroblasts adhesion was increased by 40% on poly-(lactic-co-glycolic acid) matrices functionalized with DGLs when compared to PLL. Overall, these features make DGL promising candidates for the surface engineering of biomaterials in tissue engineering.

Effect of ageing on tactile transduction processes.

With advancing age, a decline in the main sensory modalities including touch sensation and perception is well reported to occur. This review mainly outlines the peripheral components of touch perception highlighting ageing influences on morphological and functional features of cutaneous mechanical transducers and mechanosensitive ion channels, sensory innervation, neurotransmitters and even vascular system required to ensure efferent function of the afferent nerve fibres in the skin. This, in conjunction with effect of ageing on the skin per se and central nervous system, could explain the tactile deficit seen among the ageing population. We also discuss appropriate tools and experimental models available to study the age-related tactile decline.

Skin microvascular response to pressure load in obese mice.

PURPOSE: The role of obesity in the appearance of skin pressure ulcers remains controversial. The aim of the present study was to evaluate blood perfusion and related lesions after skin compression in obese mice. METHODS: Sixty C57BL6 male mice were randomly assigned to a control or hypercalorific diet (HCD) for 2, 4 and 12weeks. Skin compression was induced by a magnetic force of 11 kPa overlying a subcutaneous metal plate and applied for 4h. Skin perfusion was examined using laser Doppler imaging before skin compression, immediately after compression release and 24h later. 24h after magnet removal, skin injuries were determined by photography. RESULTS: A heterogeneous distribution of blood perfusion was observed using the colour-coded map of the skin perfusion on the compressed area. At 24-h post-compression release, 60% to 75% of the compressed area was ischaemic in the 2-week HCD group and in all the control groups compared to 35% in the 4- and 15% in the 12-week HCD groups. The lowest occurrence of skin lesion seen as skin redness or pressure-sores was observed in the 12- week HCD group (4%) compared to about 12% in either the control or the 2- and 4-week HCD groups. CONCLUSIONS: This study suggests that there was no clear relationship between the extent of ischaemia and skin lesion occurrence after skin compression in short-duration obese mice. In contrast, it appears that long-duration obesity could reduce both ischaemia and skin lesions in response to skin compression through changes in skin structure.

Heparan sulfate affects elastin deposition in fibroblasts cultured from donors of different ages.

Heparan sulfate (HS), due to its presence on the cell surface and in the extracellular milieu and its ability to modulate cell signaling, has a fundamental role in both physiological and pathological conditions. For decades we have demonstrated the occurrence of interactions between glycosaminoglycans (GAGs) and elastic fibers. In particular, we have recently shown that HS is present inside elastic fibers and plays a role in the assembly and stability of elastin coacervates. Elastin represents, within the extracellular matrix, the component most severely affected during aging, and changes in the synthesis and posttranslational modifications of HS have been described, possibly influencing cellular behavior and protein interactions. Thus, the present study has investigated, in two different in vitro experimental models, the role of HS on elastin deposition and assembly. Results demonstrate that: (1) Biological effects of HS are partly dependent on the physicochemical characteristics of the GAGs; (2) HS does not affect attachment, viability, and growth of human dermal fibroblasts; (3) HS does not modify elastin gene expression nor elastin synthesis, but favors α-elastin aggregation and, independently from the age of donors, elastin assembly; (4) HS significantly increases the expression of fibulin 5, and these effects are especially evident in fibroblasts isolated from aging donors. These data provide a better understanding of the biological role of HS and offer new perspectives regarding the possibility of restoring and/or preserving the elastic component with aging.

The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth.

Adaptation to hypoxia is a driving force for tumor progression that leads to therapy resistance and poor clinical outcome. Hypoxic responses are mainly mediated by hypoxia-inducible transcription factor-1 (HIF-1). One critical HIF-1 target mediating tumor progression is lysyl oxidase (LOX), which catalyzes cross-linking of collagens and elastin in the extracellular matrix, thereby regulating tissue tensile strength. Paradoxically, LOX has been reported to be both upregulated and downregulated in cancer cells, especially in colorectal cancer. Thus, we hypothesized that LOX might regulate expression of HIF-1 to create a self-timing regulatory circuit. Using human colorectal carcinoma cell lines in which HIF-1 and LOX expression could be modulated, we showed that LOX induction enhanced HIF-1 expression, whereas LOX silencing reduced it. Mechanistic investigations revealed that LOX activated the PI3K (phosphoinositide 3-kinase)-Akt signaling pathway, thereby upregulating HIF-1α protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Consistent with these results, cancer cell proliferation was stimulated by secreted and active LOX in an HIF-1α-dependent fashion. Furthermore, nude mice xenograft assays established that HIF-1 potentiated LOX action on tumor growth in vivo. Taken together, these findings provide compelling evidence that LOX and HIF-1 act in synergy to foster tumor formation, and they suggest that HIF-1/LOX mutual regulation is a pivotal mechanism in the adaptation of tumor cells to hypoxia.

Lysyl oxidase silencing impairs keratinocyte differentiation in a reconstructed-epidermis model.

Lysyl Oxidase (LOX) is an extracellular enzyme involved in the maturation of connective tissues. It also acts in many cell types as a regulator of cell behaviour and phenotype through intracellular signalling pathways. Recently, LOX was shown to be present in human epidermis where its precise functions remain unclear. We showed here that in confluent monolayer cultures of normal human keratinocytes (KCs) and N/TERT-1-immortalized KCs, LOX expression was induced during the first differentiation steps. Moreover, the silencing of LOX by stable RNA interference disrupted the expression of early differentiation markers. In a reconstructed-epidermis model, LOX silencing did not impair the stratification process nor the formation of the first differentiated layers. However, terminal differentiation was strongly impaired, as shown by a decreased expression of late differentiation proteins and by the absence of stratum corneum. Nonetheless, inhibition of LOX enzymatic activity by ß-aminopropionitrile did not affect the differentiation process. Therefore, LOX protein acts during the first steps of KC differentiation and is important for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is necessary for normal KC differentiation and thus for maintenance of epidermal homeostasis.

Epigenetic silencing of lysyl oxidase-like-1 through DNA hypermethylation in an autosomal recessive cutis laxa case.

We have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation.

European silver paper on the future of health promotion and preventive actions, basic research and clinical aspects of age-related disease.

The current article is a statement of the meeting with international and multidisciplinary participation, held in Wroclaw, Poland on September 11-13, 2008. The meeting was devoted to working out a position focusing on the challenge for individuals, health care systems, biological, psychosocial, epidemiological, medical, and public health sciences in the ageing populations of the twenty-first century. The statement is presented as an overview, in tabular format, of the current European situation regarding basic biological research on ageing, health promotion and preventive action, clinical care for older people, and recommendations for future actions.

Silver paper: the future of health promotion and preventive actions, basic research, and clinical aspects of age-related disease--a report of the European Summit on Age-Related Disease.

BACKGROUND. In September 2008, under the French Presidency of the European Union and with the support of the Polish Minister of Health, a European Summit on Age-Related Disease was organised inWroclaw (Poland). At this meeting, European politicians, gerontologists and geriatricians gathered to discuss a common approach to future challenges related to age-related disease. Politicians and decision-makers from the European Union and Ministers of Health and their deputies from many European countries raised the problems and difficulties to be tackled in a growing population with a high burden of disease, and asked scientists to write a consensus document with recommendations for future actions and decisions. Scientists and clinicians worked in parallel in three different groups, on health promotion and preventive actions, basic research in age-related disease, and clinical aspects of disease in older people. Beforehand, the format of the paper with recommendations was discussed, and it was finally agreed that, for a better understanding by decision- makers, it would be divided in two different columns: one with facts that were considered settled and agreed by most experts (under the heading We know), and a second with recommendations related to each fact (We recommend). No limit on the number of topics to be discussed was settled. After careful and detailed discussion in each group, which in most cases included the exact wording of each statement, chairpersons presented the results in a plenary session, and new input from all participants was received, until each of the statements and recommendations were accepted by a large majority. Areas with no consensus were excluded from the document. Immediately after the Summit, the chairpersons sent the document both to the main authors and to a list of experts (see footnote) who had made presentations at the summit and agreed to review and critically comment on the final document, which is presented below. As regards the scientific aspects of the planning of the Summit, several organisations, under the leadership of the EUGMS, were asked both to review the program and to suggest names of speakers and participants. After the Summit, the Boards of these organizations (European Union Geriatric Medicine Society (EUGMS), International Association of Gerontology and Geriatrics-European Region (IAGGER), European Association of Geriatric Psychiatry (EAGP), International Society of Gerontechnology (ISG) and International Society for the Study of the Aging Male (ISSAM) agreed to consider the document as an official paper, and help with its dissemination. The name Silver Paper was used, recalling the grey or silvery hair of our older citizens, as an easy reference. It has been sent officially to several bodies of the European Union and to Health Ministers of most European countries; and will be published in other languages in local journals. Its declared intention is to foster changes in policies which may, in the future, reduce the burden of disease in old age.

Genotype-correlated expression of lysyl oxidase-like 1 in ocular tissues of patients with pseudoexfoliation syndrome/glaucoma and normal patients.

Pseudoexfoliation (PEX) syndrome is a generalized disease of the extracellular matrix and the most common identifiable cause of open-angle glaucoma. Two single nucleotide polymorphisms in the lysyl oxidase-like 1 (LOXL1) gene (rs1048661 and rs3825942) have been recently identified as strong genetic risk factors for both PEX syndrome and PEX glaucoma. Here we investigated the expression and localization of LOXL1, LOXL2, and lysyl oxidase (LOX) in tissues of PEX syndrome/glaucoma patients and controls in correlation with their individual single nucleotide polymorphism genotypes and stages of disease. LOXL1 ocular expression was reduced by approximately 20% per risk allele of rs1048661, whereas risk alleles of rs3825942, which were highly overrepresented in PEX cases, did not affect LOXL1 expression levels. Irrespective of the individual genotype, LOXL1 expression was significantly increased in early PEX stages but was decreased in advanced stages both with and without glaucoma compared with controls, whereas LOX and LOXL2 showed no differences between groups. LOXL1 was also found to be a major component of fibrillar PEX aggregates in both intra- and extraocular locations and to co-localize with various elastic fiber components. These findings provide evidence for LOXL1 involvement in the initial stages of abnormal fibrogenesis in PEX tissues. Alterations of LOXL1 activation, processing, and/or substrate specificity may contribute to the abnormal aggregation of elastic fiber components into characteristic PEX fibrils.

Differential expression of lysyl oxidases LOXL1 and LOX during growth and aging suggests specific roles in elastin and collagen fiber remodeling in rat aorta.

The extracellular matrix (ECM) plays an important role in vascular tissue structure, maintenance, and function. Lysyl oxidases catalyze a key step in the posttranslational cross-linking of elastin and collagens in the ECM. Gene knockout studies in mice suggested a role for lysyl oxidase-like (LOXL1) in adult elastin synthesis and a role for its isoform, lysyl oxidase (LOX), in the synthesis of both collagens and elastin during development. However, the relative expression of both isoforms as a function of age is not known and was therefore investigated here. LOX and LOXL1 immunohistochemistry and real-time RT-PCR were performed during development, growth and aging in the aorta of LOU and Brown-Norway (BN) rats, two inbred strains with different susceptibilities to arterial fragility. In addition, expression of genes encoding for elastic fiber proteins and type I collagen, together with elastin and collagen contents, was measured in adult and old rat aortas. High aortic LOX expression was observed early in the development (embryonic day 15), followed by a drastic reduction in adulthood, whereas LOXL1 was mainly detectable in the intima and media; its expression was maintained throughout life in the LOU rat. Expression of tropoelastin, type-I collagen, and LOXL1 genes was reduced in the aorta of 6-week-old BN rats. Aging is characterized by a decreased elastin/collagen ratio and a greatly decreased expression of LOX, tropoelastin, and type-I collagen. These findings indicate a different spatial and temporal expression of LOX and LOXL1 during growth and aging in the rat aorta and suggest specific roles for LOX and LOXL1 in the synthesis and remodeling of elastic and collagen fibers.

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders.

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior-posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.