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First published online 4 January 2005
doi: 10.1242/jcs.01613

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Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP

Arnaud Robinet^1,*, Abdel Fahem^1,*, Jean-Hubert Cauchard¹, Eric Huet¹, Loïc Vincent², Sandrine Lorimier³, Franck Antonicelli¹, Claudine Soria², Michel Crepin⁴, William Hornebeck¹ and Georges Bellon^1,

¹ Laboratoire de Biochimie et Biologie Moléculaire, CNRS UMR 6198, IFR 53 Biomolécules, Faculté de Médecine, Université de Reims Champagne-Ardenne, 51 rue Cognacq Jay, 51095 Reims CEDEX, France
² Groupe de Recherche MERCI, EA CNRS 2122, Faculté de Médecine et de Pharmacie, 22 Boulevard Gambetta, 78183 Rouen, France
³ Laboratoire Biomatériaux, INSERM EMI, IFR 53 Biomolécules, Faculté d'Odontologie, Université de Reims-Champagne-Ardenne, 1 rue du Maréchal Juin, 51095 Reims CEDEX, France
⁴ Laboratoire Hémostase, Endothélium et Angiogenèse, INSERM U553, Hôpital St Louis, 1 Avenue Claude Vellefaux, 75475 Paris CEDEX 10, France

View larger version (102K): [in a new window]   Fig. 1. Elastin-derived peptides enhanced in vivo and ex vivo angiogenesis. (A) Representative photomicrographs of CAM from chick embryos at day 7 (D7) and 8 (D8) of embryonic development treated daily from day 6 of embryonic development with PBS (control), 50 ng of -elastin (KE) or 200 ng VGVAPG peptide. (B) Representative photomicrographs of capillary tube formation in three-dimensional type I collagen gel by HMECs incubated in the absence or presence of 100 ng/ml KE. (C) Representative photomicrographs of capillary-like structures from HUVECs cultured on matrigel for 14 hours in the absence (control) or presence of KE (10 and 100 ng/ml). As a positive control, VEGF (20 ng/ml)-induced tubulogenesis is shown in comparison to KE (100 ng/ml). (D) Semi-quantitative evaluation of pseudotube formation was performed by determining the number of black pixels per field. Results, each based on ten randomly selected fields, are expressed as mean±s.d. of four experiments. ***P<0.001 compared to the control level.

View larger version (82K): [in a new window]   Fig. 2. Morphological changes induced by elastin-derived peptides. (A) Confocal microscopy analysis of actin filaments stained with tetra methyl rhodamine isothiocyanate-labeled phalloidin from HUVECs and HMECs cultured on matrigel for 24 hours in the absence (control) or presence of KE (50 µg/ml) or presence of VEGF (20 ng/ml). (B) Scanning electron micrographs of HUVECs cultured for 14 hours on matrigel in the absence (a and c) or presence (b and d) of 50 µg/ml KE. Samples were fixed, dehydrated and desiccated as described in the methods section. KE induces a dense honeycomb network (arrowheads in a and b) where several cell-cell interactions with close contacts are frequently observed (arrows in d), whereas it is sparse in the control with a number of rounded cells (arrow in c). (C) Quantitative evaluation of the cell form factor after 4 and 14 hours' incubation of HUVECs in the absence or presence of KE was performed by computerized image analysis. Cell form factor was calculated according to the formula 4S/P2 where S is the surface of cell and P its perimeter. Results represent the mean±s.d. obtained from 60 cells. **P<0.01; ***P<0.001 when compared to levels in the relevant controls.

View larger version (44K): [in a new window]   Fig. 3. Influence of elastin-derived peptides on cell proliferation, survival and migration. (A,B) Cell proliferation and survival: HMECs (104 cells) were seeded in 24-well culture plates and cultured in FCS-free ECGM MV for various periods (A) or for five days (B) in the absence of KE (control, C) and growth factor (none) or in the presence of KE and VEGF (20 ng/ml) (b) or bFGF (20 ng/ml) (c). Cell number was determined at the end of each period of incubation. (C) To examine cell migration, HMEC monolayers were disrupted with a 1mm cell scraper and incubated in the absence or presence of KE or VGVAPG peptide and in the absence (a, none) or presence of VEGF (20 ng/ml) (b) or bFGF (20 ng/ml) (c) for 24 hours. Cell migration was determined in triplicate experiments as percentage of migrating cells relative to control value in the absence of growth factors and expressed as mean±s.d. NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

View larger version (61K): [in a new window]   Fig. 4. Elastin-derived peptide-mediated angiogenesis is triggered by occupancy of S-Gal (EBP). (A) Representative photomicrographs of capillary-like structure from HUVECs cultured on matrigel for 14 hours in the absence or presence of elastin-derived peptides (100 ng/ml KE or 200 ng/ml VGVAPG peptide). Cells were also concomitantly incubated in the absence or presence of lactose (10-4 M) or V14 peptide (10 µg/ml). (B) Semi-quantitative evaluation of pseudotube formation was performed as in Fig. 1. Results, each based on ten randomly selected fields, are expressed as mean±s.d. of four experiments. Insert shows a western blot analysis of EBP from cell extracts of fibroblasts (FC), HUVECs and HMECs using polyclonal antibodies directed against the binding sequence of elastin (V14 peptide) on EBP. **P<0.01; ***P<0.001.

View larger version (43K): [in a new window]   Fig. 5. KE increased proMMP-2 and proMT1-MMP expression and activation in HMECs. (A) HMECs were cultured as monolayers and incubated in the presence of various concentrations of KE for 24 hours. ProMMP-2 and MMP-2 in conditioned media (a) and in cell extracts (b) were assessed by gelatin zymography analysis. Arrows indicate the positions of proMMP-2 and MMP-2. (c) TIMPs in conditioned medium of HMECs were analyzed by reverse zymography analysis. Positions of TIMP-1 and TIMP-2 are shown. (d) Representative western blot of MT1-MMP from cell extracts of HMECs cultured in the absence or presence of KE. Rabbit polyclonal antibodies were used to reveal MT1-MMP (hinge region). Molecular masses of the bands are indicated, the 66-kDa species corresponds to proMT1-MMP, the 60 and 55-kDa species to MT1-MMP and the band at 44 kDa corresponds to a species processed further (Ellerbroek et al., 1999). (B) Semi-quantitative RT-PCR analysis of proMMP-2 and proMT1-MMP was performed in HMECs incubated for various periods in the absence (white column) or presence of 10 µg/ml KE (black column) and with various concentrations of KE for 24 hours. The time- and concentration-dependent histograms, each based on four experiments, display the mean±s.d. of proMMP-2 and proMT1-MMP values that have been normalized to the levels of GAPDH mRNA and expressed relative to control cell value (time 0 and not KE treated). NS, not significant; *P<0.05; **P<0.01; P<0.001.

View larger version (60K): [in a new window]   Fig. 6. Subcellular distribution of siRNA107 and time- and concentration-dependent inhibition of MT1-MMP expression in HMECs transfected with siRNA107. (A) HMECs were cultured to 60% subconfluency in ECGM MV and then transfected with Cy3-labeled siRNA107 for 72 hours. (a,d) Confocal microscopy analysis of scrambled siRNA107 (ssiRNA107) and siRNA107, respectively. (b,e) Phase-contrast microscopy analysis of scrambled siRNA107 and siRNA107, respectively. (c,f) Overlays of fluorescence and phase-contrast photomicrographs. Perinuclear distribution of siRNA107 and nucleus membrane is indicated by the arrows. (B) HMECs were cultured in ECGM MV to 60% subconfluency and then transfected with various concentrations of siRNA107 or scrambled siRNA107 (ssiRNA107) for several periods in the presence of oligofectamine as a transfecting reagent. MT1-MMP expression was determined by semi-quantitative RT-PCR and expressed relative to GAPDH expression. PCR products were resolved by 1% (w/v) agarose gel electrophoresis. Control cells (C) were incubated with oligofectamine alone. The X174/HaeIII markers (M) were used to evaluate the DNA fragment length. (C,D) Time- and concentration-dependent inhibition of MT1-MMP expression by siRNA. Semi-quantitative evaluation of MT1-MMP expression was performed by fluorometric scanning of the gel and computerized using BioProfile software (Vilbert-Lourmat, Marne la Vallée, France). Data represent the mean±s.d. of four experiments. NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

View larger version (76K): [in a new window]   Fig. 7. SiRNA107 decreases the amount of MT1-MMP, suppresses proMMP-2 activation and abolishes pseudotube formation by HMECs cultured on matrigel. HMECs were cultured in six-well culture plates and transfected at 60% confluency with 25 nM siRNA107 or scrambled siRNA107 (ssiRNA107) or oligofectamine alone (OL) for 72 hours. Then, the medium was replaced with fresh medium containing 2% (v/v) FCS and cells were incubated in the absence (-KE) or presence (+KE) of 10 µg/ml KE or 10-7 M phorbol myristate acetate (PMA) for 24 hours. Total MT1-MMP (A) and proMMP-2 activation (B) was analyzed by ELISA and zymography analysis, respectively. (C,D) HMECs were transfected with 25 nM siRNA107 or scrambled siRNA107 (ssiRNA107) for 72 hours and then cultured on matrigel for 24 hours in ECGM MV in the presence of 1 µg/ml KE. OF, HMECs incubated with oligofectamine alone. Non-transfected HMECs were cultured on matrigel in the absence (control) or presence of 1 µg/ml KE (+KE). (D) Tube formation was observed with a phase-contrast microscope equipped with a digital camera. Images were taken using a phase-contrast microscope with a x10 lens. (C) Semi-quantitative evaluation of pseudotubes was performed after white and black pixellization of images and was expressed as black pixels relative to total pixels from 20 selected fields. Data represent the mean±s.d. of four experiments. ***P<0.001.

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