[Contribution of « GIGA-Cancer ¼ at ULiege to the research on tumour microenvironment]. / Apport du «GIGA-Cancer¼ de l'ULiège à la recherche sur le microenvironnement tumoral.

The evolution of cancers is dictated by the intrinsic characteristics of malignant cells, but also by the multiple dynamic and reciprocal interactions that they establish with their tissue and cellular environment. This tumour microenvironment is therefore the subject of an ever-increasing part of cancer researches. These notably shed light on the plasticity of function of these non-malignant cells and on the diversity of their impact on the progression of the disease, both in primary tumours and during the formation of metastases. The improvement of the current therapy and the development of innovative treatments therefore require the identification of these cell subpopulations, either «allies¼ or «enemies¼ of aggressive cancer cells, as well as a more extensive understanding of the mechanisms modulating their phenotypes. This article summarizes some research projects carried out in two GIGA-Cancer laboratories supported by «Télévie¼ and the «Fondation Léon Frédéricq¼.

L'évolution de la pathologie cancéreuse est dictée par les caractéristiques intrinsèques des cellules malignes, mais également par les multiples interactions dynamiques et réciproques qu'elles établissent avec leur environnement tissulaire et cellulaire. Ce microenvironnement tumoral est donc l'objet d'une part sans cesse croissante des recherches en cancérologie. Celles-ci ont, notamment, mis en lumière la plasticité de fonction de ces cellules non malignes et la diversité de leurs impacts sur l'évolution de la maladie, tant dans les tumeurs primaires que lors de la formation des métastases. L'amélioration des traitements actuels et la mise au point de traitements novateurs nécessiteront donc l'identification fine de ces sous-populations cellulaires «alliées¼ ou «ennemies¼ des cellules cancéreuses agressives, ainsi qu'une compréhension accrue des mécanismes modulant leurs phénotypes. Cet article résume quelques projets de recherche menés dans deux laboratoires du GIGA-Cancer, soutenus notamment par Télévie et la Fondation Léon Fredericq.

[Benefits of positron emission tomography in the management and prognosis of abdominal aortic aneurysms]. / Intérêts de la tomographie à éimission de positons dans le suivi et le pronostic des anévrysmes de l'aorte abdominale.

Rupture of abdominal aortic aneurysm (AAA) remains a major cause of death in the elderly. Its prediction is a serious challenge for public health. Despite its regular use to identify patients requiring surgical treatment, the diameter of AAA is not a sufficiently precise and reliable parameter for discriminating aneurysms at high risk of rupture. A better targeting of high risk patients needs understanding in deep the processes and mechanisms directing wall rupture. Inflammation is a significant element in the progression ofAAA and can be visualized using medical imaging techniques such as positron emission tomography (PET) using a glucose derivative (FDG) as radiotracer. Studies conducted in our department have established a relationship between PET positivity and the presence of symptoms such as accelerated growth of the aneurysm or pain, signs generally considered as predictive of rupture. Moreover, activation of leukocytes coupled to cellular and molecular alterations of the aneurysmal wall in the sites of FDG uptake may lead to its instability and incompetence to resist blood pressure and rupture. PET therefore represents a new original exploration method to characterize the severity of AAA progression allowing to assess the need for a surgical treatment much better than does the AAA diameter.

[Chronic tendinopathies and platelet-rich plasma]. / Plasma riche en plaquettes et lésions tendineuses.

Platelets contain growth factors released during their degranulation following activation. These growth factors promote tissue remodeling, wound healing and angiogenesis. Currently, the clinical effect of Platelet-Rich Plasma (PRP) is still discussed, or even controversial. Our researches have assessed the effectiveness of PRP on the healing of animal tendons and human beings suffering from chronic jumper's knee.

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity.

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.

Altered expression of angiogenesis and lymphangiogenesis markers in the uninvolved skin of plaque-type psoriasis.

BACKGROUND: Vascular alterations are significant events in the pathomechanism of psoriasis. A disorder in the mechanisms regulating skin angiogenesis and lymphangiogenesis could participate in the pathogenesis of the disease. OBJECTIVES: To quantify differences in the expression of angiogenesis and lymphangiogenesis growth factors, receptors, coreceptors as well as their antagonists in the uninvolved skin of patients with psoriasis compared with the skin of nonpsoriatic volunteers. METHODS: Skin biopsies were collected from the involved skin of 13 patients with untreated plaque-type psoriasis, from their nonlesional skin at distance from the lesions and from the skin of 16 healthy volunteers. The mRNA steady-state level of keratins 10, 14 and 16, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vimentin, collagen I and IV, proliferating cell nuclear antigen, the various splice variants of vascular endothelial growth factor, VEGF-A, VEGF-C and VEGF-D, their receptors VEGFR1, VEGFR2 and VEGFR3, neuropilin (NRP)-1 and its soluble forms, NRP-2, semaphorin 3A and prox-1, was measured by reverse transcription-polymerase chain reaction. Immunohistochemistry was performed for Ki-67, von Willebrand factor and D2-40. Blood and lymphatic vessel density, area and distance from epidermis were estimated by morphological analysis coupled to an original computer-assisted method of quantification. RESULTS: Skin from healthy volunteers and nonlesional skin from patients with psoriasis displayed similar histological, morphometric and proliferative features. However, a significant overexpression of VEGFR3, the VEGF-A isoform VEGF121, soluble 12 NRP-1 and GAPDH was observed in the nonlesional psoriatic skin as compared with that of normal volunteers. CONCLUSIONS: These data point to significant differences in the blood and lymphatic vascular transcriptome between the clinically normal-appearing skin of patients with psoriasis and the skin of volunteers without psoriasis.

Spontaneous atopic dermatitis due to immune dysregulation in mice lacking Adamts2 and 14.

Since its first description, ADAMTS14 has been considered as an aminoprocollagen peptidase based on its high similarity with ADAMTS3 and ADAMTS2. As its importance for procollagen processing was never experimentally demonstrated in vivo, we generated Adamts14-deficient mice. They are healthy, fertile and display normal aminoprocollagen processing. They were further crossed with Adamts2-deficient mice to evaluate potential functional redundancies between these two highly related enzymes. Initial characterizations made on young Adamts2-Adamts14-deficient animals showed the same phenotype as that of Adamts2-deficient mice, with no further reduction of procollagen processing and no significant aggravation of the structural alterations of collagen fibrils. However, when evaluated at older age, Adamts2-Adamts14-deficient mice surprisingly displayed epidermal lesions, appearing in 2 month-old males and later in some females, and then worsening rapidly. Immunohistological evaluations of skin sections around the lesions revealed thickening of the epidermis, hypercellularity in the dermis and extensive infiltration by immune cells. Additional investigations, performed on young mice before the formation of the initial lesions, revealed that the primary cause of the phenotype was not related to alterations of the epidermal barrier but was rather the result of an abnormal activation and differentiation of T lymphocytes towards a Th1 profile. However, the primary molecular defect probably does not reside in the immune system itself since irradiated Adamts2-Adamts14-deficient mice grafted with WT immune cells still developed lesions. While originally created to better characterize the common and specific functions of ADAMTS2 and ADAMTS14 in extracellular matrix and connective tissues homeostasis, the Adamts2-Adamts14-deficient mice revealed an unexpected but significant role of ADAMTS in the regulation of immune system, possibly through a cross-talk involving mesenchymal cells and the TGFß pathways.

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

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.

Liquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

Preparation and characterizations of EGDE crosslinked chitosan electrospun membranes.

Composite Crosslinked nanofibrous membranes of chitosan, ethylene glycol diglycidyl ether (EGDE) and polyethylene oxide was successfully prepared with bead free morphology via electrospinning technique followed by heat mediated chemical crosslinking. Architectural stability of nanofiber mat in aqueous medium was achieved by chemical crosslinking of only 1% EGDE, and tensile strength tests revealed that increasing EGDE content has considerably enhance the elastic modulus of nanofibers. The structure, morphology and mechanical properties of nanofibers were characterized by Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM) and Instron machine, respectively. Skin fibroblasts and endothelial cells showed good attachment, proliferation and viability on crosslinked electrospun membranes. The results indicate a good biocompatibility and non-toxic nature of the resulted membrane.

Mutations in type 1 procollagen that cause osteogenesis imperfecta: effects of the mutations on the assembly of collagen into fibrils, the basis of phenotypic variations, and potential antisense therapies.

Work by a large number of investigators over the last decade has established that over 90% of patients with osteogenesis imperfecta have mutations in one of the two genes for type I procollagen, that most unrelated probands have different mutations in the genes, and that the mutations found in most of the serious variants of the disease cause synthesis of abnormal pro alpha chains of the protein. The results have demonstrated that synthesis of structurally abnormal but partially functional pro alpha chains can interfere with folding of the central region of the protein into a triple-helical conformation, prevent processing of the N-terminal propeptides of procollagen, or produce subtle alterations in conformation that interfere with the self-assembly of the protein into collagen fibrils. One of the unsolved mysteries about the disease is why some mutations produce severe phenotypes, whereas very similar mutations produce mild phenotypes. Recent studies in transgenic mice suggest that nongenetic factors, such as stochastic events during development, may determine the severity of the disease phenotype produced by a specific mutation. Also, recent results raised the possibility that strategies of antisense gene therapy may be effective in treating the disease some time in the future. Specific inhibition of expression of a mutated collagen gene has been obtained with antisense oligonucleotides in cell culture experiments. However, there is no means of selective delivery of antisense oligonucleotides to the appropriate tissues.

Mutation analysis of coding sequences for type I procollagen in individuals with low bone density.

Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position alpha 2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88:5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at alpha 1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon alpha 2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.

Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts.

Mechanical tension governs fibroblast proliferation and survival and the homeostasis of the extracellular matrix to adapt its resistance to the mechanical requirements of the organs. To consolidate this view, we analysed the effect of tension release on the expression of molecules involved in the architecture and stabilisation of the collagen fibres, namely the procollagens type I and III, the amino- and carboxy-procollagen peptidases (N-pCP and C-pCP) and lysyl oxidase. Cells were cultured in conditions of high mechanical stress in monolayer on a collagen coat and under reduced tension by disruption of the cytoskeleton upon treatment with cytochalasin D in monolayer on a collagen coat or by integrin-mediated stress relaxation in a freely retracting collagen gel. The mRNAs were measured by quantitative RT-PCR monitored by simultaneous reverse-transcription and amplification of an original internal standard. Tension relaxation resulted in a decreased expression of the procollagens type I and III, of the two expressed forms of C-pCP, of the two forms of N-pCP and of lysyl oxidase. Type III collagen, known to control diameter of the fibres, was less down-regulated than type I collagen. Interestingly, the expression of the two alternatively spliced forms of the N-pCP was dissimilarly regulated. These data suggest that mechanical tension may modulate the stiffness of the extracellular matrix by controlling not only the level of expression of its fibrillar constituents but also that of the enzymes participating in their extracellular processing and mechanical stabilisation.

Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. 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 carboxy- and 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.

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

The aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression.

Effect of EGF on human skin fibroblasts is modulated by the extracellular matrix.

The aim of this work was to clarify the reason why a discrepancy exists between the effects of epidermal growth factor (EGF) on fibroblasts in culture repressing collagen biosynthesis and in vivo stimulating wound healing. The effect of EGF on the biosynthetic activity of fibroblasts was measured in various conditions of cultures: on plastic, on plastic coated with various macromolecules of the extracellular matrix, on top of a type I collagen gel, and within a three-dimensional collagen lattice. While the noncollagen protein (NCP) synthesis was not affected by the interactions of the cell with the various coated matrices, collagen synthesis was inhibited. At the surface of a collagen gel, protein synthesis was reduced, while collagen synthesis and degradation were slightly stimulated. When embedded in a lattice, the overall biosynthetic activity of fibroblasts was largely depressed. The addition of EGF to cultures on plastic and on the various coated macromolecules resulted in a further repression of collagen synthesis while cell multiplication was slightly stimulated. On the contrary, the addition of EGF to fibroblasts in a collagen lattice resulted in a stimulation of both NCP and collagen synthesis as observed in vivo. These opposite effects of EGF in a two- or three-dimensional culture system are not related to modification in number or affinity of the EGF receptors at the cell surface. These results further support the similarity in the state of differentiation of fibroblasts in a three-dimensional lattice and in vivo.

Development of pH-responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery.

RNA interference-based therapies are dependent on intracellular delivery of siRNA. The release of siRNA from the endosomal compartment may be a rate limiting step in the transfection process. The purpose of this study was to produce pH-responsive nanocarriers made of trimethylchitosan (TMC). To this end, pH-sensitive methacrylic acid (MAA) copolymer was added to TMC-siRNA formulations. Four different TMCs associated or not with MAA were evaluated as siRNA carriers. Nanoparticles were characterized in terms of size, surface charge, morphology and interaction with siRNA. A swelling behaviour due to a decrease in pH was observed and was found to be dependent on MAA content in the complexes. In vitro experiments aimed at evaluating how the capacity of the nanocarriers to transfect siRNA in L929 cells was affected by MAA content. Confocal microscopy experiments showed that fluorescent MAA-containing particles exhibit a different distribution pattern inside the cells comparing to their counterpart without this pH-sensitive polymer. Transfection efficiency was investigated by RhoA mRNA expression inhibition. MAA-TMC-siRNA complexes were able to transfect L929 cells with greater efficiency than corresponding TMC-siRNA complexes. This study gives an insight into the opportunity of pH-sensitive nanocarriers for siRNA delivery. Such formulations may represent an attractive strategy to improve endosomal escape of siRNA.

Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro.

Chitosan and trimethylchitosan (TMC)-siRNA nanoparticles were produced by simple complexation technique or by ionic gelation using tripolyphosphate (TPP). The obtained complexes were characterized in terms of physicochemical properties such as size, zeta potential, complexation efficiency and stability. Furthermore, cytotoxicity, cell uptake and transfection efficiency of polyplexes were evaluated in vitro. Under pH condition of cell culture medium, a strong decrease in siRNA condensation efficiency was observed with chitosan nanoparticles. This characteristic resulted in low transfection efficiencies in HEK293 cell line. Formulation of chitosan polyplexes with TPP led to improvement of polyplexes stability but no significant increase in transfection efficiency was observed compared to simple chitosan complexes. By contrast, TMC complexes did not have pH dependency on siRNA complexation. TMC-siRNA nanoparticles were stable in physiological condition. Accordingly, cellular uptake was increased compared to chitosan polyplexes. However, improvement of transfection efficiency was low regarding to cellular uptake of these complexes. Chitosan and TMC complexes present some characteristics favourable for siRNA delivery, such as ability to integrate siRNA into small discrete particles or low toxicity of the complexes. This study also highlights the importance of complexes stability in physiological environment for siRNA transfection purposes.

Altered response of progeria fibroblasts to epidermal growth factor.

The Hutchinson-Gilford syndrome (progeria) is a rare disorder in childhood characterized by premature and accelerated aging. This study reports the effect of a potent growth factor, EGF, on the proliferative capacities and extracellular matrix macromolecules and collagenase expression of two strains of progeria skin-derived cells. At low population doubling levels (PDL less than 10), confluent cultures of progeria fibroblasts made quiescent by lowering the concentration of serum in the medium did not respond to EGF while the mitotic activity of normal PDL-matched fibroblasts was almost maximally restored upon addition of EGF. No obvious difference between normal and low PDL progeria fibroblasts was observed in the number and in the affinity of the receptors measured by [125I]EGF binding. The synthesis of collagen and non-collagen proteins was similar in normal and affected cells at low and high serum concentration and both types of cells responded to EGF by a specific inhibition of collagen synthesis. Besides a normal level of mRNA coding for type I and type III collagens, collagenase and laminin, progeria fibroblasts expressed a high level of elastin and type IV collagen mRNA. Like normal fibroblasts, progeria cells responded to EGF by a decrease in the level of mRNA for fibrillar collagens and elastin. In contrast, a complete lack of response to EGF was observed for collagenase mRNA whereas the expression of this enzyme was strikingly induced by EGF in normal PDL-matched cells. The abnormal expression of type IV collagen was not significantly modified by EGF. At PDL greater than 10, progeria cells exhibited features of senescence. A significant reduction of collagen synthesis was observed and no further inhibition by EGF was recorded.

Response to epidermal growth factor of skin fibroblasts from donors of varying age is modulated by the extracellular matrix.

The present study was undertaken to investigate the effect of epidermal growth factor (EGF) on the biosynthetic activity of skin fibroblasts from donors of varying age and the modulation of their response to this growth factor by culture in a three-dimensional extracellular matrix. When cultured in monolayer on plastic or at the surface of a collagen gel, EGF specifically inhibited collagen synthesis whatever the age of the donor (from 17 to 84 years, n = 11). This inhibition was paralleled by a significant decrease in the steady-state level of procollagen type I mRNAs. When embedded in a three-dimensional floating collagen lattice, EGF stimulated the non-collagen protein (NCP) synthesis in fibroblasts from younger donors (5 out of 6) while fibroblasts from the older ones were not affected. Collagen production by fibroblasts from younger donors was not inhibited as in monolayer (some being even stimulated) while that of the older donors was inhibited as observed in monolayer. The steady-state level of procollagen type I mRNA was not modified by EGF in the three-dimensional culture. No significant difference was observed in the affinity and the number of EGF receptors of the fibroblasts on plastic or embedded in a collagen lattice between young and aged donors. Our results suggest that the environment of the cells can modulate the reactivity to EGF and reveal differences related to in vivo aging.