Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases.
; ; Lambert, Charles et al
in Protoplasma (2006), 229(2-4), 215-20
Small GTP-binding proteins of the Rho family (RhoA, Cdc42, Rac1) regulate the organisation and the turnover of the cell's cytoskeleton and adhesion structures. A significant function of these cellular ... [more ▼]
Small GTP-binding proteins of the Rho family (RhoA, Cdc42, Rac1) regulate the organisation and the turnover of the cell's cytoskeleton and adhesion structures. A significant function of these cellular structures is to translate and counterbalance forces applied to, or generated by, cells in order to maintain homeostasis and control cell movement. We therefore hypothesised that Rho-GTPases are directly involved in cellular gravity perception and may participate in the alterations induced in microgravity. To define an adequate cellular model allowing to investigate this issue, we have established stable cell lines constitutively expressing active forms of either RhoA, Cdc42, or Rac1. The three cell lines differ by morphology and by their ability to form filopodia, lamellipodia, and bundles of actin stress fibers. Overexpression of the active form of either RhoA, Cdc42, or Rac1 is compatible with cell viability and does not affect cell population doubling time. Thus, our series of mutant cells appear well suited to gain further knowledge on the molecular mechanisms of cellular gravity perception. [less ▲]Detailed reference viewed: 43 (3 ULg)
Effects of constitutively active GTPases on fibroblast behavior.
; Lambert, Charles ; et al
in Cellular and Molecular Life Sciences : CMLS (2006), 63(1), 82-91
The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces ... [more ▼]
The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing. [less ▲]Detailed reference viewed: 23 (1 ULg)
Increased Mrna Expression of Decorin in the Prolapsing Posterior Leaflet of the Mitral Valve
; Limet, Raymond ; et al
in Interactive Cardiovascular and Thoracic Surgery (2003), 2(3), 389-94
To improve our understanding of myxomatous degeneration of the valvar tissue as seen in mitral valve prolapse, we have compared the biosynthetic phenotype of the connective tissue cells in myxomatous ... [more ▼]
To improve our understanding of myxomatous degeneration of the valvar tissue as seen in mitral valve prolapse, we have compared the biosynthetic phenotype of the connective tissue cells in myxomatous segments (n=4) resected during surgery with that of homologous segments of normal valves (n=4) harvested in age-matched organ donors. The steady-state level of mRNA for selected extracellular matrix macromolecules and metalloproteinases was assessed by quantitative (internal standard controlled) reverse transcriptase-polymerase chain reaction (RT-PCR). Among the investigated gene products, the decorin mRNA expression was significantly increased in degenerative valve compared with normal tissue (211+/-48 vs. 100+/-70, p<0.02). The level of fibrillin 2 also tended to be increased (194+/-88 vs. 100+/-81, p=0.08). These results suggest that myxomatous valvar tissue is characterized by an overexpression of mRNA for decorin. Owing to the role of this small leucine-rich proteoglycan in the regulation of fibril assembly and stability, this alteration may account for or is a result of a defective organization of the collagen and elastic fibers in this disease and contribute to the intrinsic distensibility and fragility of the myxomatous tissue. [less ▲]Detailed reference viewed: 27 (7 ULg)
Positron emission tomography (PET) evaluation of abdominal aortic aneurysm (AAA)
SAKALIHASAN, Natzi ; Van Damme, Hendrik ; et al
in European Journal of Vascular and Endovascular Surgery (2002), 23(5), 431-436
Background: aneurysmal disease is associated with all inflammatory Cell infiltrate and enzymatic degradation of the vessel wall. Aim of the study: to detect increased metabolic activity in abdominal ... [more ▼]
Background: aneurysmal disease is associated with all inflammatory Cell infiltrate and enzymatic degradation of the vessel wall. Aim of the study: to detect increased metabolic activity in abdominal aortic aneurysms (AAA) by means of positron emission tomography (PET-imaging). Study design: twenty-six patients with AAA underwent PET-imaging Results: in tell patients, PET-imaging revealed increased, fluoro-deoxy-glucose (18-FDG) uptake at the level of the aneurysm. Patients with positive PET-imaging had one or more of the following elements in their clinical history: history Of recent non-aortic surgery (n = 4) a painful inflammatory aortic aneurysm (n = 2). moderate low back pain (n = 2), rapid (>5 mm in 6 months) expansion (n = 4), discovery by PET-scan of a previously undiagnosed lung cancer (n = 3) or parotid tumour (n = 1). Five patients with a positive PET scan required urgent surgery within two to 30 days. Among the 16 patients with negative PET-imaging of their aneurysm, only one had recent non-aortic surgery, none of them required urgent surgery, only two had a rapidly expanding AAA, and in only one patient, PET-imaging revealed an unknown lung cancer. Conclusion: these data suggest a possible association between increased 18-FDG uptake and AAA expansion and rupture. [less ▲]Detailed reference viewed: 66 (5 ULg)
Stimulation of collagen biosynthesis by topically applied vitamin C.
Nusgens, Betty ; ; et al
in European Journal of Dermatology (2002), 12(4), -Detailed reference viewed: 65 (0 ULg)
Biochemical Study of Collagen in Adult Groin Hernias
Pans, Alain ; Albert, Adelin ; et al
in Journal of Surgical Research (2001), 95(2), 107-13
BACKGROUND: Previous works have suggested that a defect in collagen fiber structure may play a role in inguinal hernia formation. These studies focused mainly on the rectus sheath or the skin, while only ... [more ▼]
BACKGROUND: Previous works have suggested that a defect in collagen fiber structure may play a role in inguinal hernia formation. These studies focused mainly on the rectus sheath or the skin, while only few reports dealt with the transversalis fascia. According to these findings and to our previous biomechanical and histological studies suggesting that a connective tissue pathology could play a role in the genesis of groin hernias, we performed a biochemical investigation of the collagen in the transversalis fascia and rectus sheath. MATERIALS AND METHODS: The samples were collected from 40 adult patients with uni- or bilateral hernias and from 20 control subjects without hernia (autopsies and organ donors). A constant area of tissue was taken by using a calibrator. The wet and dry weights per 100 mm(2) were determined and the total collagen concentration as well as its sequential extractibility in NaCl, acetic acid, and pepsin was measured. The ratios of alpha(1)/alpha(2) chains (I) and of type I/III collagen were assessed by polyacrylamide gel electrophoresis. RESULTS: Samples collected in the control and patient sheaths showed an increased wet weight per 100 mm(2) in the patients. The wet and dry weights per unit area were increased in the patient fascias. The collagen concentration was increased in the indirect hernias. The fascias from the direct hernias (DH) presented a significantly increased collagen extractibility after pepsin digestion (5.6%), when compared to the control fascias (2.6%). The extractibility was 3.4% in the nonherniated (NH) sides. The qualitative study (ratios alpha(1)/alpha(2) (I) and I/III collagen) showed no difference between the fascia groups. CONCLUSIONS: The significant increase of collagen extractibility with pepsin in the DH fascias and at a lesser degree in the NH fascias suggests that molecular alterations of collagen could be involved in the genesis of groin hernias. This connective tissue pathology would express preferentially its effects in the inguinal region, since we have observed no major difference between the rectus sheaths of controls and those of patients. [less ▲]Detailed reference viewed: 22 (0 ULg)
In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton.
Deroanne, Christophe ; ; Nusgens, Betty
in Cardiovascular Research (2001), 49(3), 647-58
OBJECTIVE: This investigation aimed at determining the importance of the rigidity of the adhesive support and the participation of the cytoskeleton in tubulogenesis of endothelial cells in vitro. METHODS ... [more ▼]
OBJECTIVE: This investigation aimed at determining the importance of the rigidity of the adhesive support and the participation of the cytoskeleton in tubulogenesis of endothelial cells in vitro. METHODS: The morphotype, biosynthetic phenotype and cytoskeleton organization of human umbilical vein endothelial cells (HUVEC) were analyzed on supports of variable mechanical resistance. RESULTS: Western blot analysis revealed a strong reduction of the expression of actin and focal-adhesion plaque (FAP) proteins in HUVEC organized in tube-like structures (TLS) on soft matrigel or on matrigel co-polymerized with heat-denatured collagen as compared to HUVEC remaining in a monolayer pattern on rigid matrigel-coat or on matrigel co-polymerized with type I collagen. Human skin fibroblasts morphotype was not altered in these culture conditions and the pattern of FAP proteins and actin was not modulated. By using polyacrylamide gels polymerized with various concentrations of bis-acrylamide to modulate the mechanical resistance of the support and cross-linked to a constant amount of gelatin to provide an equal density of attachment sites, it was shown that the less rigid the support, the more endothelial cells switched to a tube-like pattern. Collagen type I-induced tubulogenesis was accompanied by a profound and reversible remodeling of the actin-FAP complex suggesting a weakening of the bridging between extracellular matrix (ECM) and the cytoskeleton. Human skin fibroblasts and smooth muscle cells, used as control cells, adhered strongly to the collagen, did not form TLS and their network of actin stress fibers was not remodeled. The inhibition of collagen type I-induced tubulogenesis by agents altering the actin cytoskeleton-FAP complex including calpain type I inhibitor, orthovanadate, KT5720 and jasplakinolide, further supports the determinant role of mechanical coupling between the cells and the matrix in tubulogenesis. CONCLUSIONS: A reduced tension between the endothelial cells and the extracellular matrix, originating in the support or within the cells is sufficient to trigger an intracellular signaling cascade leading to tubulogenesis, an event mimicking one of the last steps of angiogenesis. [less ▲]Detailed reference viewed: 19 (3 ULg)
Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts.
Lambert, Charles ; Colige, Alain ; et al
in European Journal of Cell Biology (2001), 80(7), 479-85
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 10 (0 ULg)
Apoptosis in v-myc-transfected MSU-1.1 fibroblasts is induced by cell-matrix contact and differs from that of normal dermal fibroblasts.
; ; et al
in In Vitro Cellular & Developmental Biology. Animal (2001), 37(9), 606-12
In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and ... [more ▼]
In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3-4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF. MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors alpha1beta1 and alpha2beta1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.l fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF. [less ▲]Detailed reference viewed: 4 (2 ULg)
Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.
Lambert, Charles ; Colige, Alain ; Munaut, Carine et al
in Matrix Biology (2001), 20(7), 397-408
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 26 (2 ULg)
Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.
Colige, Alain ; ; et al
in American Journal of Human Genetics (1999), 65(2), 308-17
Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia ... [more ▼]
Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene. [less ▲]Detailed reference viewed: 23 (1 ULg)
Mecanisme de croissance et de rupture des anevrysmes de l'aorte abdominale.
Limet, Raymond ; SAKALIHASAN, Natzi ;
in Bulletin et Mémoires de l'Académie Royale de Médecine de Belgique (1997), 152(7-9), 295-302302-3
The relationship between atherosclerosis and abdominal aortic aneurysm development is well known. Atherosclerosis cannot explain the whole mechanism. Genetic characters of mechanisms leading to abdominal ... [more ▼]
The relationship between atherosclerosis and abdominal aortic aneurysm development is well known. Atherosclerosis cannot explain the whole mechanism. Genetic characters of mechanisms leading to abdominal aortic development is obvious from this study and others. Our study evidences an increased metalloproteases activity in aortic wall proportionally to the size of the abdominal aortic aneurysm. A decrease of aortic wall elastin is evidenced proportionally to the AAA size. Extractable collagen is significantly increased in the aortic wall of patients operated on for aortic rupture. [less ▲]Detailed reference viewed: 13 (3 ULg)
cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components.
Colige, Alain ; ; et al
in Proceedings of the National Academy of Sciences of the United States of America (1997), 94(6), 2374-9
Procollagen N-proteinase (EC 188.8.131.52) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and ... [more ▼]
Procollagen N-proteinase (EC 184.108.40.206) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor beta or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis. [less ▲]Detailed reference viewed: 7 (0 ULg)
Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms.
SAKALIHASAN, Natzi ; Delvenne, Philippe ; Richelle, Betty et al
in Journal of Vascular Surgery (1996), 24(1), 127-33
PURPOSE: This consistent observation of a reduction of the elastin concentration in abdominal aortic aneurysms (AAAs) has led us to investigate in AAA specimens two metalloproteinases that display ... [more ▼]
PURPOSE: This consistent observation of a reduction of the elastin concentration in abdominal aortic aneurysms (AAAs) has led us to investigate in AAA specimens two metalloproteinases that display elastase activity, MMP2 (gelatinase A/72kDa) and MMP9 (gelatinase B/92 kDa). METHODS: Samples of full-thickness aortic wall, adherent thrombus, and serum were collected in 10 patients with AAAs. Samples of normal aortic wall and serum were taken from 6 age-matched control patients. Quantitative gelatin-zymography and gelatinolytic soluble assays after acetyl-phenyl mercuric acid activation were performed on serum and tissue extracts, and the results were expressed in units on a comparative wet-weight basis. Histologic analysis was performed in parallel to score the inflammatory infiltrate. RESULTS: The luminal and parietal parts of the thrombus contained, respectively, 20- and 10-fold more gelantinolytic activity than the serum. The predominate form was MMP9. Although the total gelatinolytic activity was in the same range both in AAAs and in normal walls, a significantly higher proportion of MMP9 was found in the aneurysmal aortic walls. Furthermore, a significant proportion of MMP9 was under its processed active form, which was never observed in normal samples. A significantly higher proportion of MMP2 was also present as processed active form in AAA wall. This latter parameter positively correlated with the inflammatory score. CONCLUSIONS: The presence of activated MMP9 and MMP2 might contribute to the degradation of the extracellular matrix proteins that occurs during the development of aneurysms. [less ▲]Detailed reference viewed: 18 (6 ULg)
Les champs électromagnétiques de faible intensité produisent une vague calcique dans les fibroblastes
; ; et al
in Bulletin et Mémoires de l'Académie Royale de Médecine de Belgique (1996), 151(3-4), 243-9250-2
Human fibroblasts display a Ca2+ wave after irradiation with an electromagnetic field (EMF) of low intensity (100 to 900 microT) as seen by LASER confocal microscopy and excitation of Fluo 3. The number ... [more ▼]
Human fibroblasts display a Ca2+ wave after irradiation with an electromagnetic field (EMF) of low intensity (100 to 900 microT) as seen by LASER confocal microscopy and excitation of Fluo 3. The number of excited cells is proportional to the intensity of EMF between 100 and 900 microT. Cellular activation by a dialysable serum factor is required to induce the Ca2+ wave. It also depends on extracellular Ca2+ and active tyrosine kinases and phospholipase C gamma. [less ▲]Detailed reference viewed: 19 (1 ULg)
Modulation of expression and assembly of vinculin during in vitro fibrillar collagen-induced angiogenesis and its reversal.
Deroanne, Christophe ; Colige, Alain ; Nusgens, Betty et al
in Experimental Cell Research (1996), 224(2), 215-23
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 20 (6 ULg)
Investigation of the Relationship between Osteoporosis and the Collagenase Gene by Means of Polymorphism of the 5'upstream Region of This Gene
; ; Reginster, Jean-Yves et al
in Calcified Tissue International (1995), 56
Osteoporosis is a slowly progressing disease resulting from an imbalance between bone accretion and degradation. As interstitial collagenase is a key enzyme in the degradation of bone matrix, we ... [more ▼]
Osteoporosis is a slowly progressing disease resulting from an imbalance between bone accretion and degradation. As interstitial collagenase is a key enzyme in the degradation of bone matrix, we investigated a possible relationship between the collagenase gene and osteoporosis. Analysis of an amplified genomic DNA fragment from -524 to +52 by denaturing gradient gel electrophoresis and sequencing allowed us to detect three dimorphic sites upstream of base -300, one of them leading to a BanI restriction site. None of the sites could be directly associated with osteoporosis. The allele frequencies of the three dimorphic sites were estimated. The interallelic ratios were high, thus providing new useful genetic markers for linkage analysis. When comparing these ratios in osteoporotic and nonosteoporotic subjects, no significant differences could be observed. [less ▲]Detailed reference viewed: 7 (2 ULg)
Characterization and partial amino acid sequencing of a 107-kDa procollagen I N-proteinase purified by affinity chromatography on immobilized type XIV collagen.
Colige, Alain ; ; et al
in Journal of Biological Chemistry (1995), 270(28), 16724-30
Procollagen I N-proteinase (EC 220.127.116.11), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic ... [more ▼]
Procollagen I N-proteinase (EC 18.104.22.168), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (plH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 degrees C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, Km and Vmax values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH2-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the alpha 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance. [less ▲]Detailed reference viewed: 17 (7 ULg)
Enhancement of Tumorigenicity of Human Breast Adenocarcinoma Cells in Nude Mice by Matrigel and Fibroblasts
Noël, Agnès ; Gillet, Marie-Claire ; et al
in British Journal of Cancer (1993), 68(5), 909-15
The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane ... [more ▼]
The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors. [less ▲]Detailed reference viewed: 19 (1 ULg)