RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug–activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

BackgroundDysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies.ObjectiveTo analyse vascular development, angiogenesis and lymphangiogenesis markers expression in uninvolved skin in psoriatic patients (N), early psoriasis lesions or pinpoints (PP) and psoriasis plaques (PSO).MethodsSkin biopsies were taken in 17 patients in N and in PSO and/or PP. The mRNA steady-state level of angiogenesis and lymphangiogenesis markers was measured by RT-PCR. Immunohistochemistry was performed for von Willebrand factor, podoplanin, Ki-67 and VEGFR3. Blood (BV) and lymphatic (LV) vessels expansion was measured by computer-assisted morphometry.ResultsClinical and epidermal aspects indicated that PP are intermediate between N and PSO. While total BV area was already increased in PP similarly to PSO as compared to N, LV area in PP was intermediate between N and PSO. Mean LV size was identical in N and PP and increased in PSO, mean BV size in PP being intermediate between N and PSO. VEGF-A 189 variant was increased in PP as compared to N and PSO. As compared to N, angiogenesis markers (VEGF-A isoforms, PlGF, VEGFR2, NRP-1), VEGF-C and NRP-2 were similarly increased in PP and PSO. Keratin 16 and the lymphangiogenesis markers (VEGFR3, prox-1) were intermediate in PP.ConclusionThese data suggest that the expansion of lymphatic vessels occurs after blood vascular development in psoriasis. Expansion of BV in PP could be followed by vessel enlargement during progression to PSO, in parallel with a decreased VEGF-A 189/VEGF-A 121 balance in plaques.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and mono-oxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription—polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxyand amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.

RhoA plays a significant role in actin stress fibers formation. However, silencing RhoA alone or RhoA and RhoC did not completely suppress the stress fibers suggesting a residual “Rho-like” activity. RhoB, the third member of the Rho subclass, is a shortlived protein barely detectable in basal conditions. In various cell types, the silencing of RhoA induced a strong up-regulation of both total and active RhoB protein levels that were rescued by re-expressing RhoA and related to an enhanced half-life of the protein. The RhoA-dependent regulation of RhoB does not depend on the activity of RhoA but is mediated by its GDP-bound form. The stabilization of RhoB was not dependent on isoprenoid biosynthesis, Rho kinase, extracellular signal-regulated kinase, p38 mitogen-activated kinase, or phosphatidylinositol 3′-OH kinase pathways but required RhoGDIα. The forced expression of RhoGDIα increased RhoB half-life, whereas its knock-down antagonized the induction of RhoB following RhoA silencing. Moreover, a RhoA mutant (RhoAR68E) unable to bind RhoGDIα was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDIα is rate-limiting and the suppression of RhoA makes it available to stabilize RhoB. Our results highlight RhoGDIα-dependent cross-talks that regulate the stability of RhoGTPases.

This paper reports on the reliability issues encountered during the development of a dry ohmic switch fabricated in MEMS technology. Particularly, focus is made on electrical contact reliability. Contact degradation during hot switching tests has been noticed, and leads to switch lifetime limitation. In order to understand this phenomenon, characterizations have been performed on damaged switches and degradation modes have been cleary identified. Solutions have been implemented and finally the Schneider Electric MEMS switch is able to switch 5 V/1 mA loads for several millions of cycles and 14 V/10 mA loads for several 104 of cycles.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis. The complexity of mechanisms involved in dermatophytic infections makes relevant in vivo studies particularly difficult to perform. The aim of this study was to develop a new in vitro model of M. canis dermatophytosis using feline fetal keratinocytes in reconstructed interfollicular epidermis, and to investigate its relevance in studying the host-pathogen relationship. Histological analysis of reconstructed interfollicular feline epidermis (RFE) revealed a fully differentiated epidermis. A proliferation assay showed replicating cells only in the basal layer, indicating that RFE is a well-stratified living tissue, leading to the formation of a horny layer. Histopathological analysis of RFE infected by M. canis arthroconidia revealed that the fungus invades the stratum corneum and produces SUB3, a keratinase implicated in the infectious process. In view of these results, an M. canis dermatophytosis model on RFE seems to be a useful tool to investigate mechanisms involved in natural M. canis feline infections.

Mutations in the COL1A1 and COL1A2 genes, encoding the proalpha1 and 2 chains of type I collagen, cause osteogenesis imperfecta (OI) or Ehlers-Danlos syndrome (EDS) arthrochalasis type. Although the majority of missense mutations in the collagen type I triple helix affect glycine residues in the Gly-Xaa-Yaa repeat, few nonglycine substitutions have been reported. Two arginine-to-cysteine substitutions in the alpha1(I)-collagen chain are associated with classic EDS [R134C (p.R312C)] or autosomal dominant Caffey disease with mild EDS features [R836C (p.R1014C)]. Here we show alpha1(I) R-to-C substitutions in three unrelated patients who developed iliac or femoral dissection in early adulthood. In addition, manifestations of classic EDS in Patient 1 [c.1053C>T; R134C (p.R312C); X-position] or osteopenia in Patients 2 [c.1839C>T; R396C (p.R574C); Y-position] and 3 [c.3396C>T; R915C (p.R1093C); Y-position] are seen. Dermal fibroblasts from the patients produced disulfide-bonded alpha1(I)-dimers in approximately 20% of type I collagen, which were efficiently secreted into the medium in case of the R396C and R915C substitution. Theoretical stability calculations of the collagen type I heterotrimer and thermal denaturation curves of monomeric mutant alpha1(I)-collagen chains showed minor destabilization of the collagen helix. However, dimers were shown to be highly unstable. The R134C and R396C caused delayed procollagen processing by N-proteinase. Ultrastructural findings showed collagen fibrils with variable diameter and irregular interfibrillar spaces, suggesting disturbed collagen fibrillogenesis. Our findings demonstrate that R-to-C substitutions in the alpha1(I) chain may result in a phenotype with propensity to arterial rupture in early adulthood. This broadens the phenotypic range of nonglycine substitutions in collagen type I and has important implications for genetic counseling and follow-up of patients carrying this type of mutation.

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1-4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix-binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.

ADAMTS2 belongs to the "ADAM metallopeptidase with thrombospondin type I motif' (ADAMTS) family. Its primary function is to process collagen type I, II, III, and V precursors into mature molecules by excising the aminopropeptide. This process allows the correct assembly of collagen molecules into fibrils and fibers, which confers to connective tissues their architectural structure and mechanical resistance. To evaluate the impact of ADAMTS2 on the pathological accumulation of extracellular matrix proteins, mainly type I and III collagens, we evaluated carbon tetrachloride-induced liver fibrosis in ADAMTS2-deficient (TS2(-/-)) and wild-type (WT) mice. A single carbon tetrachloride injection caused a similar acute liver injury in deficient and WT mice. A chronic treatment induced collagen deposition in fibrous septa that were made of thinner and irregular fibers in TS2(-/-) mice. The rate of collagen deposition was slower in TS2(-/-) mice, and at an equivalent degree of fibrosis, the resorption of fibrous septa was slightly faster. Most of the genes involved in the development and reversion of the fibrosis were similarly regulated in TS2(-/-) and NW mice. Conclusion: These data indicate that the extent of fibrosis is reduced in TS2(-/-) mice in comparison with their WT littermates. Inhibiting the maturation of fibrillar collagens may be a beneficial therapeutic approach to interfering with the development of fibrotic lesions.

Astrocytic tumours are associated with dismal prognoses due to their pronounced ability to diffusely invade the brain parenchyma. Various neuropeptides, including gastrin, are able to modulate tumour astrocyte migration. While neurotensin has been shown to influence the proliferation of glioma cells and the migratory ability of a large set of other cell types, its role in glioma cell migration has never been investigated. Neurotensin-induced modifications to the motility features of human U373 glioblastoma cells therefore constitute the topic of the present study. We evidenced that three subtypes of neurotensin receptors (NTR1, NTR2 and NTR3) are expressed in U373 glioblastoma cells, at least as far as their mRNAs are concerned. Treating U373 tumour cells with 10 nM neurotensin markedly modified the morphological patterns of these cells and also profoundly altered the organization of their actin cytoskeletons. Pull-down assays revealed that neurotensin induced the activation in U373 cells of both Rac1 and Cdc42 but not RhoA. Scratch wound assays evidenced that neurotensin (0.1 and 10 nM) very significantly inhibited wound colonization by U373 cells cultured in the absence of serum. In addition, quantitative phase-contrast videomicroscopy analyses showed that neurotensin decreases the motility levels of U373 glioblastoma cells when these cells are cultured on plastic. In sharp contrast, neurotensin stimulates the motility of U373 cells when they are cultured on laminin, which is a pro-adhesive extracellular matrix component ubiquitously secreted by glioma cells. Our data thus strongly suggest that, in addition to gastrin, neurotensin is a neuropeptide capable of modulating tumour astrocyte migration into the brain parenchyma.

We argue that adipocytes in the subcutis are there for the best, the bad and the worse! The ideal feminine silhouette has evolved with the rhythm of culture (Fig. 1). The goddesses of antiquity were carrying a significant layer of subcutaneous fat. This was also observed in thighs and hips of women up to the Renaissance. This energy reserve was considered as an asset assisting in surviving periods of starvation. The ideal of a thick layer of subcutaneous fat progressively vanished, up to the modern lean models. Average is the best as subcutaneous fat is a typical female sexual trait. Its excess or defect is a cause for concern. Without this regular layer that smoothen their shapes as observed in lipodystrophies, the female patients acquire a cachectic face and a male aspect of their body contour with apparent veins and muscle masses (Fig. 1d). AIDS patients treated with combined antiretroviral drugs also suffer a similar distressing aspect accompanied by central adiposity (1). Evolution of the subcutaneous adipose layer with time and fashion: upper left, statute of Aphroditis, right painting of P.P. Rubens, below left a famous model of the present time and below right the destressing aspect of a lady presenting a classical acquired partial lipodistrophy. Adipocytes are not different from any other cells associated in organs in that they need a support with which they exchange information. The so-called ‘cellulite’ and its orange peel aspect is mostly apparent in the gynoïd lipodystrophy. It might be related to a sex-linked architecture of the deep dermal fat lobules and the organization of the connective tissue septum of the hypodermis, as proposed by Nürnberger and Müller (2), although this concept is not completely supported by recent magnetic resonance imaging data (3). Alteration in the connective tissue septum of the subcutaneous tissue is a main cause of the aspect of aged skin and the so-called gravitational folds (4). The adipocytes remodel their supporting connective tissue to extend it upon lipid storage. Upon reduction of the size of the adipocytes, the connective tissue is irreversibly laxed by the loss of mechanical (and endocrine) control (5). Grafting fat pad from non-affected skin locations in classical partial lipodystrophies or AIDS patients is most often of inconsistent benefit by rapid resorption (6). This perhaps also results from the lack of adequate cell–cell control of the adipocytes and its associated connective tissue support (7). Besides their energy storage capacity, subcutaneous adipocytes provide a mechanical protection to the underlying structures. They also potentially participate in many other functions by their multipotentiality in terms of differentiation and secretory activity. Cells that mature into different lineages (neurones, myocytes and adipocytes and others) have been described in skin (8) and in the subcutis (9). Neonatal mouse skin adipocytes express stem cell antigen 1 (Sca 1) (10), further supporting the concept that the subcutis is a stem cell repository. Adipocytes are not identical in all locations (11). Furthermore subcutaneous adipose tissue is not homogeneous as it contains white and brown adipocytes, at least up to the neonatal period (12). In view of the plasticity of adipocytes in culture one can envision that they participate in various physiological and pathological processes, e.g. by secretion of the adipokines that they produce (13), which include proangiogenic factors (14; Figure 2). Overview of the major functions modulated by adipocyte-derived factors. ADAM, a disintegrin and metalloprotease; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; AGT, angiotensinogen; Ang-2, angiopoietin-2; Apo E, apolipoprotein E; ASP, acylation stimulating protein; IGF-I, insulin-like growth factor-I; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemotractant protein-1; MIF, macrophage migration inhibitory factor; MMP, matrix metalloprotease; PAI-1, plasminogen activator inhibitor-1; PlGF, placenta growth factor; SAA3, serum amyloid A3; TF, tissue factor; TGF-β, transforming growth factor-β; TIMP, tissue inhibitor of MMP; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor [adapted from Lafontan (23)]. Adipocytes also collaborate in tissue repair, as suggested by their promotion of epidermal regeneration in vitro (15). In the healing wound, a high number of actively dividing hypodermal cells observed rapidly after trauma (16) might be derived from the subcutaneous adipocytes or their precursors. The abundance of the Sca1+ cells in fetal skin still needs to be considered in the context of scar-free type of healing in the foetus. During the anagen phase of the hair follicle cycle, the increased size of the subcutis (17, 18) may provide a dynamic, supportive environment for maintenance of the metabolically highly active, maximally proliferating anagen hair follicle. Interestingly the secreted ‘agouti’ protein (which regulates hair pigmentation, e.g. in mice) is also involved in adipocyte regulation (19). Agouti is physiologically expressed in skin, and this mainly during the anagen phase of the hair cycle (20). In addition to fatty acids and other lipid moieties, adipocytes synthesize a host of adipokines involved in a plethora of autocrine, paracrine and endocrine functions. In subcutaneous adipose tissue, approximately 20% of its known genes encode secreted proteins (21). The diversity of these factors includes, among others, enzymes, cytokines, growth factors and hormones (12, 22-27). Some of these proteins play a role in lipid metabolism, some are inflammatory cytokines, while others are involved in vascular haemostasis, extracellular matrix remodelling or the complement system (Fig. 2). Recent observations suggest that increased oxidative stress in accumulated fat, via increased NADPH oxidase and decreased antioxidant enzyme activity, causes dysregulated production of adipokines such as PAI-1, tumor necrosis factor-α (TNF-α), resistin, leptin and adiponectin. These participate in the pathogenesis of obesity-associated metabolic syndrome. Increased production of PAI-1 and TNF-α from accumulated fat contribute to the development of thrombosis and insulin resistance respectively. In contrast, adiponectin exerts insulin-sensitizing and anti-atherogenic effects, and hence a decrease in plasma adiponectin contributes to insulin resistance and atherosclerosis in obesity. Increased reactive oxygen species production from accumulated fat also leads to increased oxidative stress in blood, affecting other organs including the liver, skeletal muscle and aorta, suggesting that increased oxidative stress in accumulated fat represents an early instigator of obesity-associated metabolic syndrome. A current paradigm stipulates that subcutaneous fat plays a minor role in the development of the metabolic syndrome (28, 29). However, new pieces of evidence demonstrating that the subcutaneous fat, by acting as a metabolic sink, protects against the metabolic syndrome of obesity, challenge this concept (30, 31). The mesenchymal cells normally present in connective tissues include several cell types, most notably fibroblasts, endothelial cells and adipocytes. It is becoming increasingly clear that these cells actively participate in tumor development (32, 33). The existence of critical cross-talks between cancer cells and adipocytes is emerging (34, 35). Owing to their capacity to secrete adipokines, adipocytes are excellent candidates to influence tumor behaviour through heterotypic signalling processes, which prove to be critical for tumor survival, angiogenesis, growth and metastasis (36, 37). For example, breast cancer cells exhibit differential response to leptin and adiponectin, two major products of subcutaneous adipocytes: leptin stimulates while adiponectin inhibits tumor cell proliferation (38, 39). Moreover, both epidemiology and experimental observations suggest that obesity-associated dysregulation of adipokines is likely to contribute significantly to the pathogenesis of several cancers (40). Accelerated tumor formation in fatless (A-ZIP/F-1) mice with type 2 diabetes and inflammation, however, seems to contradict this paradigm (41).