VEGF111, a new VEGF-A isoform induced by genotoxic agents; resistance to proteolytic
; Colige, Alain ; DELGAUDINE, Marie et al
Conference (2010)Detailed reference viewed: 5 (4 ULg)
Alternative splicing: a promising target for pharmaceutical inhibition of pathological angiogenesis?
Munaut, Carine ; Colige, Alain ; Lambert, Charles
in Current Pharmaceutical Design (2010), 16(35), 3864-76
In eukaryotes, genes consist in coding sequences (exons) interspersed with non-coding ones (introns). The regulation of alternative inclusion/exclusion of exons, or part of exons, during the maturation of ... [more ▼]
In eukaryotes, genes consist in coding sequences (exons) interspersed with non-coding ones (introns). The regulation of alternative inclusion/exclusion of exons, or part of exons, during the maturation of the pre-mRNA into mRNA (alternative splicing) allows a dramatic increase of the protein versus the gene repertoire. In a number of cases, alternative splicing decision generates proteins with distinct, sometimes opposite, functions from a given gene. Angiogenesis is the process of vascularisation in physiological conditions and a series of pathologies, including cancer where it favours tumour progression and dissemination of metastasis. In this issue, we discuss some key examples showing how alternative splicing may induce a switch from anti-angiogenic to pro-angiogenic functions and reciprocally. For some of these splicing events, the molecular mechanisms that trigger alternative splicing toward one or the other direction start to be elucidated. The emergence of strategies enabling to regulate alternative splicing opens new routes for anti-angiogenic therapies. [less ▲]Detailed reference viewed: 7 (1 ULg)
Histological and transcriptional study of angiogenesis and lymphangiogenesis in uninvolved skin, acute pinpoint lesions and established psoriasis plaques: an approach of vascular development chronology in psoriasis
Henno, Audrey ; Blacher, Silvia ; Lambert, Charles et al
in Journal of Dermatological Science (2010), 57(3), 162-169
Background Dysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies ... [more ▼]
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 [less ▲]Detailed reference viewed: 49 (4 ULg)
Effects of mechanical stretch on human osteoblasts: role of the rho family GTPases in the mechanotransduction.
Neutelings, Thibaut ; ; Nusgens, Betty et al
Poster (2009)Detailed reference viewed: 4 (1 ULg)
Cold shock (25°C) and re-warming (37°C) affect cell phenotype: impac on space biology experiments.
Neutelings, Thibaut ; ; Nusgens, Betty et al
Conference (2009)Detailed reference viewed: 4 (0 ULg)
Role of the Rho family GTPases in the mechano-transduction and -reaction in human osteoblasts.
Lambert, Charles ; Neutelings, Thibaut ; et al
Poster (2009)Detailed reference viewed: 4 (0 ULg)
Altered expression of angiogenesis and lymphangiogenesis markers in the uninvolved skin of plaque-type psoriasis
Henno, Audrey ; Blacher, Silvia ; Lambert, Charles et al
in British Journal of Dermatology (2009), 160(3), 581-90
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 113 (52 ULg)
L’application de forces cycliques d’étirement diminue la production de médiateurs de l’inflammation par les chondrocytes arthrosiques
Mathy, Marianne ; ; Sanchez, Christelle et al
in Revue du Rhumatisme (2008), 75(10-11), 999Detailed reference viewed: 32 (5 ULg)
Human bone marrow adipocytes block granulopoiesis through neuropilin-1-induced granulocyte colony-stimulating factor inhibition.
Belaid-Choucair, Zakia ; ; Poncin, Géraldine et al
in Stem Cells (2008), 26(6), 1556-64
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral ... [more ▼]
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article. [less ▲]Detailed reference viewed: 86 (28 ULg)
Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents
Mineur, Pierre ; Colige, Alain ; Deroanne, Christophe et al
in Journal of Cell Biology (2007), 179(6), 1261-1273
Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1-4 and 8 in many cultured cells. Although not detected ... [more ▼]
Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1-4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix-binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases. [less ▲]Detailed reference viewed: 66 (20 ULg)
Signaling through Rho GTPases in microgravity (Rho signaling) on ISS (Soyuz TMA-1) Belgian Soyuz Mission "Odissea"
Nusgens, Betty ; Lambert, Charles ;
in Microgravity Science and Technology (2007), XIX(5-6),Detailed reference viewed: 14 (5 ULg)
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: 42 (2 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: 21 (1 ULg)
Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated protein kinase signaling
; Lambert, Charles ; Colige, Alain et al
in Journal of Biological Chemistry (2005), 280(12), 10938-10944
The extracellular pH (pHe) of tumor tissues is often acidic, which can induce the expression of several proteins. We previously showed that production of matrix metalloproteinase-9 (MMP-9) was induced by ... [more ▼]
The extracellular pH (pHe) of tumor tissues is often acidic, which can induce the expression of several proteins. We previously showed that production of matrix metalloproteinase-9 (MMP-9) was induced by culturing cells at acidic pHe (5.4-6.5). Here we have investigated the signal transduction pathway by which acidic pHe induces MMP-9 expression. We found that acidic pHe (5.9) activated phospholipase D (PLD), and inhibition of PLD activity by 1-butanol and Myr-ARF6 suppressed the acidic pHe-induced MMP-9 expression. Exogenous PLD, but not phosphatidylinositol-specific PLC or PLA(2), mimicked MMP-9 induction by acidic pHe. Western blot analysis revealed that acidic pHe increased the steady-state levels of phosphorylated extracellular signal-regulated kinases 1/2 and p38 and that the PLD inhibitors suppressed these increases. Using 5'-deletion mutant constructs of the MMP-9 promoter, we found that the acidic pHe-responsive region was located at nucleotide -670 to -531, a region containing the NF kappa B binding site. A mutation into the NF kappa B binding site reduced, but not completely, the acidic pHe- induced MMP-9 promoter activity, pand NF kappa B activity was induced by acidic pHe. Pharmacological inhibitors specific for mitogen-activated protein kinase kinase 1/2 (PD098059) and p38 (SB203580) attenuated the acidic pHe- induced NF kappa B activity and MMP-9 expression. These data suggest that PLD, mitogen-activated protein kinases (extracellular signal-regulated kinases 1/2 and p38), and NF kappa B mediate the acidic pHe signaling to induce MMP-9 expression. A transcription factor(s) other than NF kappa B may also be involved in the MMP-9 expression. [less ▲]Detailed reference viewed: 38 (2 ULg)
Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.
Deroanne, Christophe ; ; Ho, Thi Thanh Giang et al
in Journal of Cell Science (2005), 118(Pt 6), 1173-83
The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small ... [more ▼]
The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity. [less ▲]Detailed reference viewed: 45 (4 ULg)
Role of the RhoGTPases in the cellular receptivity and reactivity to mechanical signals including microgravity
Nusgens, Betty ; ; et al
in Journal of Gravitational Physiology : A Journal of the International Society for Gravitational Physiology (2005), 12(1), 269-270
The small G proteins of the Rho Family (RhoGTPases) are key operators in the signaling arising from extracellular matrix through integrin receptors and from membrane receptors for soluble ligands. FLight ... [more ▼]
The small G proteins of the Rho Family (RhoGTPases) are key operators in the signaling arising from extracellular matrix through integrin receptors and from membrane receptors for soluble ligands. FLight data show that microgravity affects cell architecture and gene expression leading us to assume that the signaling pathways(s) involving the RhoGTPAses might disturbed in a weightlessness environment. TO test this hypothesis in microgravityu, we created genetically engineered human fibroblasts that will be used in Biolab on the ISS. [less ▲]Detailed reference viewed: 10 (3 ULg)
Microgravity-induced matrix remodelling is linked to reduction in tension of human dermal fibroblasts
; Lambert, Charles ; et al
in Journal of Gravitational Physiology : A Journal of the International Society for Gravitational Physiology (2005), 12(1),Detailed reference viewed: 6 (0 ULg)
RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts.
Mineur, Pierre ; ; Lambert, Charles et al
in Biochimica et Biophysica Acta (2005), 1746(1), 28-37
A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that ... [more ▼]
A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways. [less ▲]Detailed reference viewed: 17 (2 ULg)
Cell survival and preservation of siRNA-mediated protein knock-down upon serum-free cryopreservation (-80 degrees C).
Lambert, Charles ; Deroanne, Christophe ; et al
in Gravitational and Space Biology Bulletin (2005), 18(2), 103-4Detailed reference viewed: 8 (1 ULg)