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Five hydrodynamic systems are presented in this short review: the parallel plate flow cell, the impinging jet, the radial flow cell, the rotating disk and fluid dynamic gauging. These systems are of particular relevance to study surface fouling, surface cleaning or adhesion onto solid surfaces in laboratory environment. The key features of their hydrodynamics are given as well as their practical advantages and drawbacks. Examples of applications fields are also listed.

In space, cells sustain strong modifications of their mechanical environment. Mechanosensitive molecules at the cell membrane regulate mechanotransduction pathways that induce adaptive responses through the regulation of gene expression, post-translational modifications, protein interactions or intracellular trafficking, among others. In the current study, human osteoblastic cells were cultured on the ISS in microgravity and at 1 g in a centrifuge, as onboard controls. RNAseq analyses showed that microgravity inhibits cell proliferation and DNA repair, stimulates inflammatory pathways and induces ferroptosis and senescence, two pathways related to ageing. Morphological hallmarks of senescence, such as reduced nuclear size and changes in chromatin architecture, proliferation marker distribution, tubulin acetylation and lysosomal transport were identified by immunofluorescence microscopy, reinforcing the hypothesis of induction of cell senescence in microgravity during space flight. These processes could be attributed, at least in part, to the regulation of YAP1 and its downstream effectors NUPR1 and CKAP2L., (© 2023. The Author(s).)

The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-β signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo , by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leduc, Dupont, Joannes, Monseur, Baiwir, Mazzucchelli, Deroanne, Colige and Bekhouche.)

The mitral valve is a complex multilayered structure populated by fibroblast-like cells, valvular interstitial cells (VIC) which are embedded in an extracellular matrix (ECM) scaffold and are submitted to the mechanical deformations affecting valve at each heartbeat, for an average of 40 million times per year. Myxomatous mitral valve (MMV) is the most frequent heart valve disease characterized by disruption of several valvular structures due to alterations of their ECM preventing the complete closure of the valve resulting in symptoms of prolapse and regurgitation. VIC and their ECM exhibit reciprocal dynamic processes between the mechanical signals issued from the ECM and the modulation of VIC phenotype responsible for ECM homeostasis of the valve. Abnormal perception and responsiveness of VIC to mechanical stress may induce an inappropriate adaptative remodeling of the valve progressively leading to MMV. To investigate the response of human VIC to mechanical strain and identify the molecular mechanisms of mechano-transduction in these cells, a cyclic equibiaxial elongation of 14% at the cardiac frequency of 1.16 Hz was applied to VIC by using a Flexercell-4000 T™ apparatus for increasing time (from 1 h to 8 h). We showed that cyclic stretch induces an early (1 h) and transient over-expression of TGFβ2 and αSMA. CTGF, a profibrotic growth factor promoting the synthesis of ECM components, was strongly induced after 1 and 2 h of stretching and still upregulated at 8 h. The mechanical stress-induced CTGF up-regulation was dependent on RhoC, but not RhoA, as demonstrated by siRNA-mediated silencing approaches, and further supported by evidencing RhoC activation upon cell stretching and suppression of cell response by pharmacological inhibition of the effector ROCK1/2. It was also dependent on the MEK/Erk1/2 pathway which was activated by mechanical stress independently of RhoC and ROCK. Finally, mechanical stretching induced the nuclear translocation of myocardin related transcription factor-A (MRTF-A) which forms a transcriptional complex with SRF to promote the expression of target genes, notably CTGF. Treatment of stretched cultures with inhibitors of the identified pathways (ROCK1/2, MEK/Erk1/2, MRTF-A translocation) blocked CTGF overexpression and abrogated the increased MRTF-A nuclear translocation. CTGF is up-regulated in many pathological processes involving mechanically challenged organs, promotes ECM accumulation and is considered as a hallmark of fibrotic diseases. Pharmacological targeting of MRTF-A by newly developed inhibitors may represent a relevant therapy for MMV., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Muscle formation is controlled by a number of key myogenic transcriptional regulators that govern stage-specific gene expression programs and act as terminal effectors of intracellular signaling pathways. To date, the role of phosphatases in the signaling cascades instructing muscle development remains poorly understood. Here, we show that a specific PP2A-B55δ holoenzyme is necessary for skeletal myogenesis. The primary role of PP2A-B55δ is to dephosphorylate histone deacetylase 4 (HDAC4) following myocyte differentiation and ensure repression of Myocyte enhancer factor 2D (MEF2D)-dependent gene expression programs during myogenic fusion. As a crucial HDAC4/MEF2D target gene that governs myocyte fusion, we identify ArgBP2, an upstream inhibitor of Abl, which itself is a repressor of CrkII signaling. Consequently, cells lacking PP2A-B55δ show upregulation of ArgBP2 and hyperactivation of CrkII downstream effectors, including Rac1 and FAK, precluding cytoskeletal and membrane rearrangements associated with myoblast fusion. Both in vitro and in zebrafish, loss-of-function of PP2A-B55δ severely impairs fusion of myocytes and formation of multinucleated muscle fibers, without affecting myoblast differentiation. Taken together, our results establish PP2A-B55δ as the first protein phosphatase to be involved in myoblast fusion and suggest that reversible phosphorylation of HDAC4 may coordinate differentiation and fusion events during myogenesis.

The development of new lymphatic vessels occurs in many cancerous and inflammatory diseases through the binding of VEGF-C to its receptors, VEGFR-2 and VEGFR-3. The regulation of VEGFR-2/VEGFR-3 heterodimerisation and its downstream signaling in lymphatic endothelial cells (LECs) remain poorly understood. Here, we identify the endocytic receptor, uPARAP, as a partner of VEGFR-2 and VEGFR-3 that regulates their heterodimerisation. Genetic ablation of uPARAP leads to hyperbranched lymphatic vasculatures in pathological conditions without affecting concomitant angiogenesis. In vitro, uPARAP controls LEC migration in response to VEGF-C but not VEGF-A or VEGF-CCys156Ser. uPARAP restricts VEGFR-2/VEGFR-3 heterodimerisation and subsequent VEGFR-2-mediated phosphorylation and inactivation of Crk-II adaptor. uPARAP promotes VEGFR-3 signaling through the Crk-II/JNK/paxillin/Rac1 pathway. Pharmacological Rac1 inhibition in uPARAP knockout mice restores the wild-type phenotype. In summary, our study identifies a molecular regulator of lymphangiogenesis, and uncovers novel molecular features of VEGFR-2/VEGFR-3 crosstalk and downstream signaling during VEGF-C-driven LEC sprouting in pathological conditions.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18 F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.

Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine612. Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite "pro-tumoral" effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies., Competing Interests: The authors have declared that no competing interests exist.

Membrane type 4 matrix metalloproteinase (MT4-MMP) [matrix metalloproteinase (MMP) 17] is a GPI-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple-negative breast cancer cells, MT4-MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG-tagged or Myc-tagged MT4-MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4-MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the clathrin-independent carriers/GPI-enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane-type MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer., (© 2015 FEBS.)

A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions.

Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects., (© FASEB.)

Introduction: Myxomatous mitral valve is one of the most common heart valves diseases in human and has been well characterized at a functional and morphological level. Diseased valves are thickened as a result of extracellular matrix remodeling and proteoglycans accumulation accompanied by the disruption of the stratified structures of the leaflets., Methods: Global transcriptomic analysis was used as a start-up to investigate potential pathogenic mechanisms involved in the development of the human idiopathic myxomatous mitral valve, which have been elusive for many years., Results: These prospective analyses have highlighted the potential role of apparently unrelated molecules in myxomatous mitral valve such as members of the transforming growth factor-β superfamily, aggrecanases of the "a disintegrin and metalloprotease with thrombospondin repeats I" family, and a weakening of the protection against oxidative stress. We have integrated, in this review, recent transcriptomic data from our laboratory [A. Hulin, C.F. Deroanne, C.A. Lambert, B. Dumont, V. Castronovo, J.O. Defraigne, et al. Metallothionein-dependent up-regulation of TGF-beta2 participates in the remodelling of the myxomatous mitral valve. Cardiovasc Res 2012;93:480-489] and from the publication of Sainger et al. [R. Sainger, J.B. Grau, E. Branchetti, P. Poggio, W.F. Seefried, B.C. Field, et al. Human myxomatous mitral valve prolapse: role of bone morphogenetic protein 4 in valvular interstitial cell activation. J Cell Physiol 2012;227:2595-2604] with existing literature and information issued from the study of monogenic syndromes and animal models., Conclusion: Understanding cellular alterations and molecular mechanisms involved in myxomatous mitral valve should help at identifying relevant targets for future effective pharmacological therapy to prevent or reduce its progression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies have shown that specific inhibition of HDAC7 blocks angiogenesis both in vitro and in vivo. However, the underlying molecular mechanisms are not fully understood and hence preclude any meaningful development of suitable therapeutic modalities. The goal of the present study was to further the understanding of HDAC7 epigenetic control of angiogenesis in human endothelial cells using the proteomic approach. The underlying problem was approached through siRNA-mediated gene-expression silencing of HDAC7 in human umbilical vein endothelial cells (HUVECs). To this end, HUVEC proteins were extracted and proteomically analyzed. The emphasis was placed on up-regulated proteins, as these may represent potential direct epigenetic targets of HDAC7. Among several proteins, A-kinase anchor protein 12 (AKAP12) was the most reproducibly up-regulated protein following HDAC7 depletion. This overexpression of AKAP12 was responsible for the inhibition of migration and tube formation in HDAC7-depleted HUVEC. Mechanistically, H3 histones associated with AKAP12 promoter were acetylated following the removal of HDAC7, leading to an increase in its mRNA and protein levels. AKAP12 is responsible for protein kinase C mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 increasingly binds to the chromatin and AKAP12 promoter and is necessary for maintaining the elevated levels of AKAP12 following HDAC7 knockdown. We demonstrated for the first time that AKAP12 tumor/angiogenesis suppressor gene is an epigenetic target of HDAC7, whose elevated levels lead to a negative regulation of HUVEC migration and inhibit formation of tube-like structures.

Human papillomavirus (HPV) infections account for more than 50% of infection-linked cancers in women worldwide. The immune system controls, at least partially, viral infection and around 90% of HPV-infected women clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and no study has evaluated the direct interaction between HPVs and NK cells, a key player in host resistance to viruses and tumors. We demonstrated an NK-cell infiltration in HPV-associated preneoplastic cervical lesions. Since HPVs cannot grow in vitro, virus-like particles (VLPs) were used as a model for studying the NK-cell response against the virus. Interestingly, NK cells displayed higher cytotoxic activity and cytokine production (TNF-α and IFN-γ) in the presence of HPV-VLPs. Using flow cytometry and microscopy, we observed that NK-cell stimulation was linked to rapid VLP entry into these cells by macropinocytosis. Using CD16(+) and CD16(-) NK-cell lines and a CD16-blocking antibody, we demonstrated that CD16 is necessary for HPV-VLP internalization, as well as for degranulation and cytokine production. Thus, we show for the first time that NK cells interact with HPVs and can participate in the immune response against HPV-induced lesions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The final goal of the present study was the development of a 3-D chitosan dressing that would shorten the healing time of skin wounds by stimulating migration, invasion, and proliferation of the relevant cutaneous resident cells. Three-dimensional chitosan nanofibrillar scaffolds produced by electrospinning were compared with evaporated films and freeze-dried sponges for their biological properties. The nanofibrillar structure strongly improved cell adhesion and proliferation in vitro. When implanted in mice, the nanofibrillar scaffold was colonized by mesenchymal cells and blood vessels. Accumulation of collagen fibrils was also observed. In contrast, sponges induced a foreign body granuloma. When used as a dressing covering full-thickness skin wounds in mice, chitosan nanofibrils induced a faster regeneration of both the epidermis and dermis compartments. Altogether our data illustrate the critical importance of the nanofibrillar structure of chitosan devices for their full biocompatibility and demonstrate the significant beneficial effect of chitosan as a wound-healing biomaterial.

This critical review article presents the current state of knowledge on isomalto-oligosaccharides, some well known functional oligosaccharides in Asia, to evaluate their potential as emergent prebiotics in the American and European functional food market. It includes first a unique inventory of the different families of compounds which have been considered as IMOs and their specific structure. A description has been given of the different production methods including the involved enzymes and their specific activities, the substrates, and the types of IMOs produced. Considering the structural complexity of IMO products, specific characterization methods are described, as well as purification methods which enable the body to get rid of digestible oligosaccharides. Finally, an extensive review of their techno-functional and nutritional properties enables placing IMOs inside the growing prebiotic market. This review is of particular interest considering that IMO commercialization in America and Europe is a topical subject due to the recent submission by Bioneutra Inc. (Canada) of a novel food file to the UK Food Standards Agency, as well as several patents for IMO production.

The factors influencing the adherence of starch were examined to improve the understanding of the mechanisms affecting soiling and cleanability. Therefore an aqueous suspension of starch granules was sprayed on four model substrates (glass, stainless steel, polystyrene and PTFE) and dried, and the substrates were cleaned using a radial-flow cell. The morphology of the soiled surfaces and the substrate chemical composition were also characterized. By influencing droplet spreading and competition between granule-substrate and granule-granule interfaces regarding the action of capillary forces, substrate wettability affected the shape and compactness of the adhering aggregates, the efficiency of shear forces upon cleaning, and finally the adherence of soiling particles. The rate of drying had an influence explained by the duration left to capillary forces for acting. X-ray photoelectron spectroscopy demonstrated the presence of macromolecules, mainly polysaccharides, which were adsorbed from the liquid phase, or carried by the retracting water film and deposited at the granule-substrate interface. These macromolecules acted as an adhesive joint, the properties of which seemed to be influenced by the detailed history of drying and subsequent exposure to humidity. In summary, the substrate surface energy affects the adherence of starch aggregates by different mechanisms which are all linked together: suspension droplet spreading, action of capillary forces, direct interaction with starch particles and interfacial macromolecules., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated., Methodology/principal Findings: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization., Conclusions/significance: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

Background: miRNAs are now recognized as key regulator elements in gene expression. Although they have been associated with a number of human diseases, their implication in acute and chronic asthma and their association with lung remodelling have never been thoroughly investigated., Methodology/principal Findings: In order to establish a miRNAs expression profile in lung tissue, mice were sensitized and challenged with ovalbumin mimicking acute, intermediate and chronic human asthma. Levels of lung miRNAs were profiled by microarray and in silico analyses were performed to identify potential mRNA targets and to point out signalling pathways and biological processes regulated by miRNA-dependent mechanisms. Fifty-eight, 66 and 75 miRNAs were found to be significantly modulated at short-, intermediate- and long-term challenge, respectively. Inverse correlation with the expression of potential mRNA targets identified mmu-miR-146b, -223, -29b, -29c, -483, -574-5p, -672 and -690 as the best candidates for an active implication in asthma pathogenesis. A functional validation assay was performed by cotransfecting in human lung fibroblasts (WI26) synthetic miRNAs and engineered expression constructs containing the coding sequence of luciferase upstream of the 3'UTR of various potential mRNA targets. The bioinformatics analysis identified miRNA-linked regulation of several signalling pathways, as matrix metalloproteinases, inflammatory response and TGF-β signalling, and biological processes, including apoptosis and inflammation., Conclusions/significance: This study highlights that specific miRNAs are likely to be involved in asthma disease and could represent a valuable resource both for biological makers identification and for unveiling mechanisms underlying the pathogenesis of asthma.

Hygiene is a permanent concern for food industries since they must commercialize high quality products in order to comply with the legislation and the expectations of the consumers. The hygienic state of a surface is thus a critical parameter with respect to the performances of the production process and to the final quality of the product. For this reason, cleaning and disinfection are essential. However, the efficiency of the cleaning process will not only depend on the optimization of the process by itself and on the equipment design but also on the characteristics of the soiled surface i.e. mainly its roughness, surface chemical composition, and surface energy. The relation between these parameters and the cleanability are presented here, mainly for food applications. Some examples of existing easy-to-clean and self-cleaning surfaces based on the modification of these surface parameters are also presented.

Background: Dysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies., Objective: To 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)., Methods: Skin 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., Results: Clinical 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., Conclusion: These 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., (2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

ATP, released at the leading edge of migrating neutrophils, amplifies chemotactic signals. The aim of our study was to investigate whether neutrophils express ATP-gated P2X(1) ion channels and whether these channels could play a role in chemotaxis. Whole-cell patch clamp experiments showed rapidly desensitizing currents in both human and mouse neutrophils stimulated with P2X(1) agonists, alphabeta-methylene ATP (alphabetaMeATP) and betagammaMeATP. These currents were strongly impaired or absent in neutrophils from P2X(1)(-/-) mice. In Boyden chamber assays, alphabetaMeATP provoked chemokinesis and enhanced formylated peptide- and IL-8-induced chemotaxis of human neutrophils. This agonist similarly increased W-peptide-induced chemotaxis of wild-type mouse neutrophils, whereas it had no effect on P2X(1)(-/-) neutrophils. In human as in mouse neutrophils, alphabetaMeATP selectively activated the small RhoGTPase RhoA that caused reversible myosin L chain phosphorylation. Moreover, the alphabetaMeATP-elicited neutrophil movements were prevented by the two Rho kinase inhibitors, Y27632 and H1152. In a gradient of W-peptide, P2X(1)(-/-) neutrophils migrated with reduced speed and displayed impaired trailing edge retraction. Finally, neutrophil recruitment in mouse peritoneum upon Escherichia coli injection was enhanced in wild-type mice treated with alphabetaMeATP, whereas it was significantly impaired in the P2X(1)(-/-) mice. Thus, activation of P2X(1) ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels may therefore play a significant role in host defense and inflammation.

Interfacial properties of surfactants are dependent on the conformation adopted by the hydrophilic headgroup or/and the hydrophobic tail at the boundary limit of two immiscible phases. Here, we demonstrate the impacts of the carbonyl group (-CO-) location of the ester bond of sugar-based surfactants by comparing some properties of two closely related esters, octyl glucuronate and glucose octanoate, at the air-water interface. The carbonyl group location influences the rate and extent of interfacial adsorption and the rheology properties of sugar esters at the air-water interface, which were evaluated by dynamic surface tension and complex surface viscoelastic measurements. Octyl glucuronate adsorbs the fastest at the air-water interface whereas glucose octanoate reduces the dynamic surface tension at the lowest value and exhibits the highest film viscoelasticity. Differences are attributed to molecular conformation constraints inducing relevant changes to the surface coverage kinetic capacity and the interaction strengths of the octyl sugar ester adsorbed films at the air-water interface. All of the results are supported by the minimum cross-sectional area values per molecule determined by both experimental and computational approaches.

It is often described that it is difficult to really discriminate the cause of intrinsic skin aging. The aim of this study was to compare the profiles of expression of senescence-associated genes in biopsies of dermis from young and old human donors. TGF-beta1 was up-regulated in the dermis of old donors as well as the TGF-beta1-regulated genes. The anti-oxidant enzymes Selenium-dependent Glutathione peroxidase and Glutatione S-Transferase Theta 1 were also up-regulated in old dermis as well as Tumor Necrosis Factor Receptor Superfamily 1A. None of these genes had altered expression level in skin fibroblasts embedded in a collagen matrix and exposed to sublethal doses of UVB, suggesting their involvement in intrinsic aging. This study represents a proof-of-concept of larger whole transcriptome studies where all avenues should be used to subtract changes in gene expression due to extrinsic aging from changes potentially due to intrinsic aging.

Global inhibition of class I and II histone deacetylases (HDACs) impairs angiogenesis. Herein, we have undertaken the identification of the specific HDAC(s) with activity that is necessary for the development of blood vessels. Using small interfering RNAs, we observed that HDAC7 silencing in endothelial cells altered their morphology, their migration, and their capacity to form capillary tube-like structures in vitro but did not affect cell adhesion, proliferation, or apoptosis. Among several factors known to be involved in angiogenesis, platelet-derived growth factor-B (PDGF-B) and its receptor (PDGFR-beta) were the most upregulated genes following HDAC7 silencing. We demonstrated that their increased expression induced by HDAC7 silencing was partially responsible for the inhibition of endothelial cell migration. In addition, we have also shown that treatment of endothelial cells with phorbol 12-myristate 13-acetate resulted in the exportation of HDAC7 out of the nucleus through a protein kinase C/protein kinase D activation pathway and induced, similarly to HDAC7 silencing, an increase in PDGF-B expression, as well as a partial inhibition of endothelial cell migration. Collectively, these data identified HDAC7 as a key modulator of endothelial cell migration and hence angiogenesis, at least in part, by regulating PDGF-B/PDGFR-beta gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer.

In this study, inulooligosaccharides (F(n)-type inulin) resulting from the endo-inulinase hydrolysis of globe artichoke inulin were purified and characterized. The aim was to produce F(n) oligomer standards with the intention of identifying them in the complex inulin chromatogram. Inulin was extracted from globe artichoke and presented a high average degree of polymerization (DP) of about 80 as determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This inulin was hydrolyzed by a commercial endo-inulinase yielding a product with a very high F(n)/GF(n) molecule ratio, thus limiting the interference of GF(n) during the purification process. High-performance size exclusion chromatography was used to individually isolate and collect each retention peak corresponding to a specific oligomer. The purity of these fractions was checked by HPAEC-PAD and showed that relatively pure molecules were produced. Electrospray ionization mass spectrometry allowed the molecular weight determination of these purified oligomers and ascertained their DP as F2, F3 and F4. These F(2-4) standards were used with glucose, fructose, sucrose and GF(2-4) (commercially available) to spike commercial oligofructose products in order to determine the elution profile in the HPAEC-PAD chromatogram.

Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane - i.e., cortical - network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Rac1 is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.

A new sample preparation method for the water content determination of inulin by volumetric Karl Fischer (KF) titration was developed and compared to the usual method of introducing the sample directly in the methanol-based working medium, modified or not by formamide (1:3, v/v) in order to increase sample solubility. In the proposed method, inulins were externally prepared by dissolving them in pure formamide (2.5:7.5, w/w). The time of analysis of the liquid/liquid reaction of the new method between the dissolved sample and the reaction medium is about 1-2min, while the usual KF method is stopped after the 10min delay time. The developed method permits the determination of water included in the crystals of the sample, confirmed by analysing both crystalline and amorphous inulin samples. Another advantage of this new method is its applicability for the water content determination of other polysaccharides that are not readily soluble in the working medium. Moreover, water content determination can be done by any type of volumetric KF titrator, as this proposed technique is not dependent on any additional tools such as a built-in homogeniser or a heatable titration beaker.

Objective: Constrictive remodeling accounts for lumen loss in postangioplasty restenosis. Matrix metalloproteinase-9 (MMP-9) has been shown to prevent constrictive remodeling in vivo. To investigate potential mechanisms for this observation, we investigated the role of MMP-9 in smooth muscle cell (SMC)-mediated collagen gel contraction, an in vitro model of constrictive remodeling., Methods: Fischer rat SMCs were stably transfected with a construct-expressing rat-MMP-9 under the control of a tetracycline (Tet)-off promoter. SMCs were seeded in type I collagen gels (2.4 mg/ml) in the presence or not of tetracycline (1 microg/ml), and gel contraction was defined as the percentage of retraction of the collagen gel. The depletion of MMP-9 was obtained by using siRNA targeting MMP-9 mRNA or a blocking antibody., Results: Gel contraction was significantly reduced at all times when MMP-9 was overexpressed (Tet-) as compared with the control condition (Tet+). However, MMP-9 depletion of control (Tet+) SMCs (using siRNA or antibody) also inhibited gel contraction. To resolve the apparent discrepancy and determine if MMP-9 exerts a dose-dependent biphasic effect on gel contraction, conditioned medium and purified rat-MMP-9 were prepared. Gel contraction was significantly increased by addition of 0.8 ng/ml of MMP-9, while high concentrations of MMP-9 (> or =100 ng/ml) inhibited contraction. The addition of BB94 and TIMP-1 did not alter the inhibitory or stimulatory effect of MMP-9., Conclusions: Our data suggest that MMP-9, independent of its proteolytic function, has a biphasic effect on SMC-mediated collagen gel contraction. Understanding the different roles of MMP-9 should allow the development of better therapeutic strategies for restenotic vascular disease.

Clinical practice reveals that osteoporotic women treated with BPs (bisphosphonates) show an increased bone mass density and a reduced risk of fractures. However, the mechanisms leading to these beneficial effects of BPs are still poorly understood. We hypothesized that ZOL (zoledronic acid), a potent third-generation BP, may induce the expression of proteins associated with the bone-forming potential of osteoblastic cells such as BSP (bone sialo-protein). Expression of BSP gene is up-regulated by hormones that promote bone formation and has been associated with de novo bone mineralization. Using real-time reverse transcriptase-PCR and Western-blot analysis, we demonstrated that ZOL increased BSP expression in Saos-2 osteoblast-like cells. Nuclear run-on and mRNA decay assays showed no effect at the transcriptional level but a stabilization of BSP transcripts in ZOL-treated cells. ZOL effect on BSP expression occurred through an interference with the mevalonate pathway since it was reversed by either mevalonate pathway intermediates or a Rho GTPase activator. We showed that ZOL impaired membrane localization of RhoA in Saos-2 cells indicating reduced prenylation of this protein. By the use of small interfering RNAs directed to RhoA and Rac1, we identified both Rho GTPases as negative regulators of BSP expression in Saos-2 cells. Our study demonstrates that ZOL induces BSP expression in osteoblast-like cells through inactivation of Rho GTPases and provides a potential mechanism to explain the favourable effects of ZOL treatment on bone mass and integrity.

Damage to the skin extracellular matrix (ECM) is the hallmark of long-term exposure to solar UV radiation. The aim of our study was to investigate the changes induced in unexposed human skin in vivo after single or repeated (five times a week for 6 weeks) exposure to 1 minimal erythemal dose (MED) of UV solar-simulated radiation. Morphological and biochemical analyses were used to evaluate the structural ECM components and the balance between the degrading enzymes and their physiologic inhibitors. A three-fold increase in matrix metalloproteinase 2 messenger RNA (mRNA) (P < 0.02, unexposed versus exposed) was observed after both single and repeated exposures. Fibrillin 1 mRNA level was increased by chronic exposure (P < 0.02) and unaltered by a single MED. On the contrary, a single MED significantly enhanced mRNA levels of interleukin-1alpha (IL-1alpha), IL-1beta (P < 0.02) and plasminogen activator inhibitor-1 (P < 0.05). Immunohistochemistry demonstrated a significant decrease in Type-I procollagen localized just below the dermal-epidermal junction in both types of exposed sites. At the same location, the immunodetected tenascin was significantly enhanced, whereas a slight increase in Type-III procollagen deposits was also observed in chronically exposed areas. Although we were unable to observe any change in elastic fibers in chronically exposed buttock skin, a significant increase in lysozyme and alpha-1 antitrypsin deposits on these fibers was observed. These results demonstrate the existence of a differential regulation, after chronic exposure compared with an acute one, of some ECM components and inflammatory mediators.

Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.

In view of the potential impact of pituicyte morphology on neurohypophysial hormone secretion, we have studied the mechanisms involved in the shape changes induced by vasopressin (AVP) and oxytocin (OXT) in cultured rat pituicytes. Pituicytes induced to become stellate in the presence of 10 micro m adenosine revert to their nonstellate shape approximately 20 min after application of AVP or OXT. The IC50 for this effect is 0.1 nm for AVP and 36 nm for OXT. Both agonists induce Ca2+ signals in pituicytes, comprised of a transient peak and a plateau phase that is dependent on the presence of extracellular Ca2+. The EC50 values of AVP for the transient and sustained responses are 4.5 and 0.1 nm, respectively; corresponding values for OXT are 180 and 107 nm. We determined pharmacologically that these hormone-induced Ca2+ signals are mediated by the V1a subtype of vasopressin receptors, similar to what we previously observed for hormone-induced reversal of stellation. Removal of extracellular Ca2+ or chelation of intracellular Ca2+ partially prevented AVP from reversing stellation, suggesting a role for Ca2+ in this event. We previously established that adenosine-induced stellation of pituicytes occurs via RhoA inhibition. However, pharmacological experiments and pull-down assays presented here show that AVP-induced reversal of stellation does not involve RhoA activation. Rather, AVP was found to induce a time-dependent activation of Cdc42, another small GTPase involved in cytoskeletal plasticity. Activation of Cdc42 by AVP is sensitive to intra- and extracellular Ca2+ depletion, similar to AVP-induced reversal of stellation. Furthermore, AVP-induced reversal of stellation is blocked by expression of an NWASP fragment known to inhibit endogenous Cdc42.

Pituicyte stellation in vitro represents a useful model with which to study morphological changes that occur in vivo in these cells during times of high neurohypophysial hormone output. This model has helped us establish the hypothesis of a purinergic regulation of pituicyte morphological plasticity. We first show that ATP induces stellation in 37% of pituicytes, an effect that is secondary to the metabolism of ATP to adenosine. Adenosine-induced stellation of pituicytes appears to be mediated by A(1)-type receptors. The effect is independent of intracellular calcium and does not involve the mitogen-activated protein kinase pathway. The basal (nonstellate) state of pituicytes depends on tonic activation of a Rho GTPase because both C3 transferase (a Rho inhibitor) and Y-27632 (an inhibitor of p160Rho kinase) can induce stellation. Lysophosphatidic acid, a Rho activator, blocks the morphogenic effect of adenosine dose-dependently. Using a specific RhoA pull-down assay, we also show that downregulation of activated RhoA is the key event coupling A(1) receptor activation to pituicyte stellation, via F-actin depolymerization and microtubule reorganization. Finally, both vasopressin and oxytocin can prevent or reverse adenosine-induced stellation. The effects of vasopressin, and those of high concentrations of oxytocin, are mediated through V(1a) receptors. Placed within the context of the relevant literature, our data suggest the possibility of a purinergic regulation of pituicyte morphological plasticity and subsequent modulation of hormone release, with these hormones providing a negative feedback mechanism., (Copyright 2002 Wiley-Liss, Inc.)

The effect of hydrophobic chain length on surface properties of enzymatically prepared n-alkyl esters of glucuronic acid are examined. Dynamic parameters from Hua and Rosen's mathematical model and equilibrium surface tension are presented for esters with octyl, decyl, dodecyl, and tetradecyl alkyl segments. Increasing the alkyl chain length has a significant influence on the surface activity. Decyl and dodecyl glucuronate exhibit an interesting adsorption speed associated with foaming capacity. Octyl glucuronate exhibits a micellar organization as its bulk concentration is over 10.68 mM.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.

The transforming properties of fibroblast growth factor 3 (FGF-3) were investigated in MCF7 breast cancer cells and compared to those of FGF-4, a known oncogenic product. The short form of fgf-3 and the fgf-4 sequences were each introduced with retroviral vectors and the proteins were only detected in the cytoplasm of the infected cells, as expected. In vitro, cells producing FGF-3 (MCF7.fgf-3) and FGF-4 (MCF7.fgf-4) displayed an amount of estrogen receptors decreased to around 45% of the control value. However, MCF7.fgf-3 cell proliferation remained responsive to estradiol supply. The sensitivity of the MCF7.fgf-4 cells, if existant, was masked by the important mitogenic action exerted by FGF-4. In vivo, the MCF7.fgf-3 and MCF7.fgf-4 cells gave rise to tumors under conditions in which the control cells were not tumorigenic. Supplementing the mice with estrogen had the paradoxical effect of totally suppressing the start of the FGF-3 as well as the FGF-4 tumors. Tumorigenicity in the presence of matrigel was similar for MCF7.fgf-3 and control cells and was increased by estrogen supplementation. Once started, the MCF7.fgf-4 tumors grew with a characteristic high rate. Remarkably, FGF-4 but not FGF-3, stimulated the secretion of vascular endothelial growth factor (VEGF165) without altering the steady-state level of its mRNA, suggesting a possible regulation of VEGF synthesis at the translational level in MCF7 cells. The increased VEGF secretion is probably involved in the more aggressive phenotype of the MCF7.fgf-4 cells while a decreased dependence upon micro-environmental factors might be part of the increased tumorigenic potential of the MCF7.fgf-3 cells.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

The infection of normal mouse mammary EF43 cells by a retroviral vector carrying either Fgf-3 (EF43.Fgf-3) or Fgf-4 (EF43.Fgf-4) cDNA resulted in the transformation of cells displaying different tumorigenic potentials in nude mice (A. Hajitou and C-M. Calberg-Bacq, Int. J. Cancer, 63: 702-709, 1995). EF43.Fgf-4 produced rapidly developing tumors at all sites of inoculation, whereas EF43.Fgf-3 produced slowly growing tumors only in the mammary fat pad. Cells infected with the vector carrying the selection gene alone (EF43.C) were not tumorigenic. The angiogenic properties of these cells were tested in an in vitro angiogenesis model using human umbilical vein endothelial cells (HUVECs) cultured at the surface of a type I collagen gel and their capacity to form tube-like structures on invasion of the gel. Only the conditioned medium (CM) of EF43.Fgf-4 induced an angiogenic morphotype in HUVECs. In parallel, the mRNA expression of matrix metalloproteinase 1 and c-ETS-1 was increased in the HUVECs displaying a differentiated phenotype, whereas the tissue inhibitor of matrix metalloproteinase 1 mRNA level was decreased. Recombinant human fibroblast growth factor 4 (FGF-4) did not induce an angiogenic phenotype in HUVECs by itself. By Western blot analysis, a high expression of vascular endothelial growth factor (VEGF) was detected in the EF43.Fgf-4 CM. This result was confirmed by Northern blot analysis of total RNA extracted from the three cell types; the steady-state level of VEGF mRNA was low and equivalent in EF43.C and EF43.Fgf-3, whereas it was strongly increased in EF43.Fgf-4. Culturing EF43 cells carrying only the selection gene with increasing concentrations of recombinant human FGF-4 resulted in a dose-dependent stimulation of VEGF. The induction of the angiogenic morphotype and the parallel modulations of the biosynthetic phenotype in HUVECs were completely suppressed by adding a neutralizing antibody directed against VEGF to EF43.Fgf-4 CM. Furthermore, inhibition of protein kinase C by bisindoylmaleimide suppressed the angiogenic phenotype induced by the CM of EF43.Fgf-4. Our results point to an indirect angiogenic activity of FGF-4 through the autocrine induction of VEGF secretion by EF43.Fgf-4 cells, an original signaling pathway that might be significant in tumor progression and metastasis.

A model of collagen-induced in vitro angiogenesis was used to investigate the modulation of expression and assembly of focal adhesion plaque-associated proteins during the process of differentiation. Human umbilical vein endothelial cells (HUVEC), first attached on an adhesive substratum (gelatin-, fibronectin-, or laminin-coated dish) or adherent collagen gel and then covered by an overlaying collagen get, organized within 3-4 days in tube-like structures (TLS). Removing the overlaying collagen gel from fully differentiated HUVEC induced a reversion of the process and HUVEC returned to a monolayer pattern. Modulations of focal adhesion-associated proteins occurring in HUVEC during the in vitro differentiation process and its reversal were investigated by Western blot analysis. A significant decrease of expression of vinculin, the integrin alpha2 subunit, talin, alpha-actinin, and actin was observed in TLS whereas the amount of FVIII-related antigen did not vary as compared to control monolayer cultures. During reversal, all the reduced proteins were markedly reexpressed. Human skin fibroblasts (HSF), submitted to the same experimental conditions, did not form TLS. Most of the focal adhesion proteins in HSF were similarly modulated by an overlaying collagen gel with the exception of vinculin, which was not modified. This particular protein was therefore more thoroughly investigated. In a nondifferentiated monolayer of HUVEC, a significant proportion of vinculin was organized into a detergent-resistant juxtamembranous structure (focal adhesion plaque) which disassembled early in TLS formation and reassembled during the reversal of the process. The reduction of vinculin during TLS formation was preceded by a downregulation of its mRNA while this mRNA was upregulated during reversal of the morphotype. These results suggest that the modulations of the cytoskeletal and focal adhesion proteins and more specifically of vinculin coupled to its subcellular redistribution are critical and early events in the cascade of mechanochemical signaling during in vitro angiogenesis induced by fibrillar collagen.