Fig 1.
Vascular structures on de-cellularized matrix express high fibronectin.
Confocal images of vascular structures illustrate the localization of ECM proteins following vascular morphogenesis of ECs cultured atop de-cellularized co-culture ECM. Images of vascular lumens, indicative of the 3D nature of the structures, were rendered from confocal z stacks. The dashed line indicates the corresponding position of the lumen. Scale bars for cross sectional lumens are 10μM.
Fig 2.
Vascular structures on de-cellularized matrix express active MMPs.
(A) qRT-PCR illustrated up-regulated expression of MMPs in CLS (ECs on ECM) as compared to ECs cultured on tissue culture plastic. Significance was assessed based on the following p values: *p≤0.05; **p≤0.01; ***p≤0.001. (Bi) Western blot did not detect differences in the expression of MT1-MMP between control ECs and CLS. (Bii) Enzyme zymography revealed the expression of the active form of MMP2 and the pro-form of MMP9 in CLS. (Biii) Active forms of MMP1 were observed in control ECs and CLS although CLS contained additional bands of active forms.
Fig 3.
Fibronectin inhibition during vascular morphogenesis reduces CLS formation.
(A) Representative immunofluorescence images of vascular structures treated with 500μM pUR4B or control, III-11C peptides at the time of EC seeding on ECM. Corresponding high magnification images are shown as insets. Arrows indicate the presence of CLS in high magnification merged inset images. Images for fibronectin were acquired using the same exposure settings. (B) Quantification of CLS showed a significantly higher percentage of CLS in structures treated with control III-11C versus pUR4B. (C) Confocal images of reconstructed z stacks illustrate the presence of lumens indicative of the 3D nature of the CLS in both control III-11C and pUR4B treated vascular structures. The dashed line indicates the corresponding position of the lumen. Scale bars for cross sectional lumens are 10μM. *p≤0.05; **p≤0.01; ***p≤0.001.
Fig 4.
Fibronectin inhibition reduces the deposition of matrix proteins from co-cultures.
(A) Representative immunofluorescence images of de-cellularized matrix from pUR4B and control III-11C treated co-cultures reveal the absence of collagen I and tenascin-C in co-culture ECM lacking a polymerized fibronectin matrix. (B) Ultrastructural high magnification images of ECM deposited by pUR4B and control III-11C treated co-cultures illustrate a marked reduction in the matrix of co-culture ECM lacking a polymerized fibronectin matrix.
Fig 5.
Matrix proteins are highly co-localized with a polymerized fibronectin matrix.
(A) Representative immunofluorescence images of matrix proteins co-localized with the polymerized fibronectin matrix show co-localization of collagens I and IV, laminin and tenascin-C with fibronectin. (B) Quantification of matrix proteins co-localized with the polymerized fibronectin matrix. Fn: fibronectin; Ten-C: tenascin-C; Col I, IV; collagens I or IV. *p≤0.05; **p≤0.01; ***p≤0.001.
Fig 6.
Vascular kinetics is altered following culture of GFP+ ECs atop pUR4B or III-11C ECM.
(A) GFP+ ECs were seeded on Chambers, III-11C-ECM and pUR4B-ECM and were assessed for differences in vascular kinetics between hours 7 and 12 post-seeding. Graphs depict (B) total distance traveled, (C) net displacement, (D) average velocity, and (E) mean square displacement of the GFP+ ECs. *p≤0.05; **p≤0.01; ***p≤0.001. The error bars represent the SEM.
Fig 7.
Inhibition of vascular integrin adhesion to a polymerized fibronectin matrix disrupts CLS formation.
(A) Representative immunofluorescence images of vascular structures treated with blocking antibodies to the fibronectin integrins α5β1;αvβ3, α5β1, and αvβ3 and control IgG. Top panel; low magnification. Bottom panel; high magnification. Vascular structures are indicated with white arrows. (B) Quantification of CLS revealed near absent vascular morphogenesis in ECs treated with anti-α5β1;αvβ3 antibodies while ECs treated with control IgG antibody possessed the greatest percentage of CLS. (C) Vascular lumens were present in structures treated with anti-αvβ3 and IgG control antibody only. The dashed line indicates the corresponding position of the lumen. Scale bars for cross sectional lumens are 10μM. *p≤0.05; **p≤0.01; ***p≤0.001.