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First published online 22 July 2003
doi: 10.1242/jcs.00629

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Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins

¹ Department of Pathology, University of Washington, Seattle, WA 98195, USA
² IFOM, Institute FIRC for Molecular Oncology, Milan 20139, Italy
³ Department of Engineered Biomaterials, University of Washington, Seattle, WA 98195, USA
⁴ Veterans Administration Puget Sound Health Care System, Seattle, WA 98108, USA

View larger version (30K): [in a new window]   Fig. 1. Effect of Ang-1 and Ang-2 on angiogenesis (A) and mural cell recruitment (B) in the rat aorta model. (A) Angiogenesis was measured by counting branch points under light microscopy in the living cultures. Notice that Ang-1 and Ang-2 promoted branching morphogenesis of aorta-derived microvessels in a dose-dependent fashion. Bars represent the SEM (n=4). *, P<0.05; **, P<0.01. (B) Mural cell recruitment was evaluated by counting the mural cells of the ten longest microvessels of each culture at day 7, under phase-contrast microscopy. Notice that Ang-1 (0.67 µg ml-1), Ang-2 (0.67 µg ml-1) and the Ang-1/Ang-2 combination stimulated mural cell recruitment. Soluble Tie2 blocked stimulation of mural cell recruitment by Ang-1 and Ang-2 used alone or in combination. Bars represent the SEM (n=40). *, P<0.01; **, P<0.001.

View larger version (123K): [in a new window]   Fig. 2. Photomicrographs of living cultures (A) and immunoperoxidase preparations of MPC stained for the smooth muscle cell markers calponin (B) and -SMA (C) or the endothelial cell marker FVIII-RA (D). Notice that MPCs are positive for calponin and -SMA, and negative for FVIII-RA. Scale bar, 100 µm.

View larger version (17K): [in a new window]   Fig. 3. Effect of Ang-1 and Ang-2 on chemotaxis of MPC in a Boyden chamber assay in which PDGF-BB was used as positive control. (A) Ang-1 (0.67 µg ml-1) and Ang-2 (0.67 µg ml-1) used alone or in combination stimulated the migration of MPCs. The stimulatory effect obtained with the Ang-1/Ang-2 combination was higher than but not significantly different from that obtained with the single molecules. Bars represent the SEM (n=6). *, P<0.0001; **, P<0.02; ***, P<0.03. (B) Ang-1-mediated stimulation of MPC migration (0.67 µg ml-1) was inhibited by soluble Tie2 (13.4 µg ml-1). Cells were responsive to the PDGF-BB positive control and showed no random migration (chemokinesis) when Ang-1 (0.67 µg ml-1) was loaded in both the upper and lower compartments of the Boyden chamber. Bars represent the SEM (n=6). *, P<0.001; **, P<0.01; ***, P<0.05. Values are expressed as optical density (O.D.) in both A and B.

View larger version (28K): [in a new window]   Fig. 4. Zymographic analysis of medium conditioned by MPCs (300,000 cells per culture well) without (control) or with Ang-1 (0.5 µg ml-1), Ang-2 (0.5 µg ml-1) or the Ang-1/Ang-2 combination (A). Bar graph shows quantitative evaluation of gelatinase bands by densitometry (B). Optical density values were normalized for the total protein content of each sample. Notice that Ang-1 and Ang-2 used alone or in combination stimulated MMP-2 production by MPCs.

View larger version (55K): [in a new window]   Fig. 5. Tie2 expression in MPCs. (Top) Western-blot analysis (WB) of MPC protein extract immunoreacted for Tie2 demonstrated a 140 kDa band that migrated with the Tie2 band of the endothelial cell control (EC). RT-PCR confirmed expression of Tie2 in MPCs at the mRNA level. Endothelial cells (EC) were used as positive control. No PCR bands were detected in the absence of reverse transcriptase (data not shown). Specificity of the PCR products was confirmed by sequence analysis. (Bottom) Analysis of MPC protein extracts immunoprecipitated (IP) with an anti-Tie2 antibody and reacted with an anti-phosphotyrosine antibody (-pY) showed phosphorylation of the MPC Tie2 receptor upon stimulation with both Ang-1 and Ang-2. Stripping and reprobing of the membrane with an anti-Tie2 antibody (-Tie2) showed that equal amounts of Tie2 were present in each lane.

View larger version (93K): [in a new window]   Fig. 6. Photomicrographs of planar cultures of isolated MPCs (A-E) and endothelial cells (F), and of histologic sections of native rat aorta (G,H) immunostained for Tie2 by immunoperoxidase (A,B,F-H) or immunofluorescence (C-E). Planar cultures: (A) shows a group of confluent MPCs whereas (B) shows a single MPC at higher magnification. Immunoperoxidase staining of cultured cells demonstrated Tie2 in both MPCs (A,B) and control endothelial cells (F). The positive staining reaction for Tie2 was predominantly localized at the cell periphery (arrows). Confocal images of MPCs double stained for Tie2 (C, green fluorescence) and -SMA (D, red fluorescence) showed coexpression of Tie2 and -SMA in the same cells (E, green and red fluorescence overlay). Cultured cells reacted with nonimmune IgG were negative (data not shown). Scale bars, 50 µM (A,C-E), 30 µM (B,F). Histological sections of native rat aorta: the intimal and subintimal layers of the aorta contain Tie2+ nonendothelial mesenchymal cells (G,H, arrows). Arrowheads highlight the endothelial lining of the aortic intima, which serves as a positive internal control. Scale bar, 100 µm.

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