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Blood, Vol. 114, Issue 2, 237-247, July 9, 2009
Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP–dependent proteolysis in 3-dimensional collagen matrices
Blood Stratman et al.
114: 237
Supplemental materials for: Stratman et al
Files in this Data Supplement:
- Figure S1. MMP activity is required for EC lumen formation and vascular guidance tunnel formation in 3D collagen matrices (JPG, 128 KB)
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(A) ECs were seeded in FITC-labeled collagen matrices in the absence or presence of the MMP inhibitor, GM6001. Real time imaging was performed using both DIC and FITC settings and individual fields are shown over time, demonstrating the development of EC lumens and vascular guidance tunnels (Control panels). In contrast, this does not occur in the presence of the MMP inhibitor (GM6001 panels). Bar equals 50 µm. (B) EC cultures were established and allowed to form lumens and vascular guidance tunnels. Cultures were fixed and immunostained at varying intervals over a 24 hr time course. Quantification of EC lumen versus tunnel formation is shown demonstrating the concurrent nature of the two processes.
- Figure S2. Inhibitors of MT1-MMP markedly inhibit EC lumen and tube morphogenesis in 3D collagen matrices (JPG, 365 KB)
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(A) EC cultures were established in collagen matrices containing FITC-labeled collagen type I and media contained either no additive or GM6001 at 5 µM. After 48 hr of culture, fluorescent confocal images of cultures were imaged and then quantitated for lumenal areas by tracing the black areas representing the proteolyzed vascular guidance tunnels. (n=10 representative fields). Bar equals 100 µm. (B) MT1-MMP is required for mouse EC lumen formation in 3D collagen matrices. Mouse ECs (bEnd 3) were transfected with siRNAs to either MT1-MMP or Luciferase as a treatment control. ECs were then randomly seeded into a 3D collagen matrix for 48 hrs. Cultures were fixed, stained, and photographed. Bar equals 100 µm. (D) Average lumenal areas +SD per high powered field were measured after either 24 or 48 hrs from 3 independent cultures. MT1-MMP individual and Smartpool siRNAs were used to block MT1-MMP expression for this assay. p<.01.
- Figure S3. siRNA suppression of MT1-MMP markedly blocks EC lumenogenesis in 3D collagen matrices (JPG, 143 KB)
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(A) ECs were treated with the indicated siRNAs to MT1-MMP (Smartpool-sp and an individual-C1), MT2-MMP, and MT3-MMP as well as the control luciferase and EC lumen assays were performed for 48 hr. After fixation and staining, cultures were photographed at the indicated magnifications. Bar equals 100 µm. (B) Quantification of EC lumen formation was performed by image analysis of photographed fields using Metamorph software. The average lumen area +SD per high powered field is presented from 3 independent cultures. p< .01.
- Figure S4. Time-lapse analysis reveals a requirement for MT1-MMP in EC lumen and tube formation in 3D collagen matrices (JPG, 340 KB)
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ECs were treated with the indicated siRNAs and were suspended in 3D collagen matrices. Time-lapse movies were performed over a 48 hr period and images were obtained every 10 minutes. Representative images at the indicated time points show the progression of EC lumen formation following these siRNA treatments revealing that MT1-MMP, but not MT3-MMP or MMP-1 is necessary for EC lumen and tube formation in 3D collagen matrices. Bar equals 50 µm.
- Figure S5. ECs can migrate within vascular guidance tunnels in an MMP-independent manner (JPG, 322 KB)
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EC cultures were established for 48 hr and then were imaged for 48 hr in the presence of 5 µM GM6001 to block MMPs. A time-lapse series is shown at the indicated time points. White arrows indicate the borders of the EC-lined tube structure while black arrowheads indicate the borders of vascular guidance tunnels in which ECs migrate into and out of during tube remodeling events. Bar equals 25 µm.
- Figure S6. Vascular guidance tunnels remain in the matrix as patent conduits for EC tube regrowth following regression events (JPG, 340 KB)
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EC cultures were established for 48 hr and then were imaged by time-lapse microscopy before and after thrombin-induced tube collapse and regrowth of tubes. Note the appearance of the vascular guidance tunnel (indicated by the white arrowheads) following tube collapse induced by. thrombin (5 µg/ml) treatment for 30 minutes, an effect which was reversed by the addition of the thrombin inhibitor, hirudin. Select panels from the time-lapse movie are shown revealing that ECs repopulate the vascular guidance tunnel over time. Black arrows indicate collapsed ECs, while the black arrowhead indicates ECs spreading out and migrating within the vascular guidance tunnel. Bar equals 25 µm.
- Video 1. Time-lapse microscopy of EC lumenogenesis in 3D collagen matrices (MOV, 4.18 MB)
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EC cultures were established in 3D collagen matrices in assays mimicking vasculogenesis and were imaged by time-lapse microscopy over a 48 hr period. Frames were collected every 10 minutes and are shown at 10 frames/sec.
- Video 2. EC lumen formation occurs normally in the presence of TIMP-1 due to its inability to block MT1-MMP (MOV, 4.83 MB)
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EC cultures were established in 3D collagen matrices in the presence of 5 µg/ml of recombinant TIMP-1 and were imaged by time-lapse microscopy over a 48 hr period. Frames were collected every 10 minutes and are shown at 24 frames/sec.
- Video 3. The MMP inhibitor, GM6001, markedly blocks EC lumen formation in 3D matrices due to its ability to inhibit MT1-MMP (MOV, 2.32 MB)
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EC cultures were established in 3D collagen matrices in the presence of 5 µM of GM6001 and were imaged by time-lapse microscopy over a 48 hr period. Frames were collected every 10 minutes and are shown at 24 frames/sec.
- Video 4. TIMP-3 markedly blocks EC lumen formation in 3D matrices due to its ability to inhibit MT1-MMP (MOV, 2.79 MB)
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EC cultures were established in 3D collagen matrices in the presence of 5 µg/ml of recombinant TIMP-3 and were imaged by time-lapse microscopy over a 48 hr period. Frames were collected every 10 minutes and are shown at 24 frames/sec.
- Video 5. EC motility in conjunction with EC lumen and vascular guidance tunnel formation in 3D collagen matrices (MOV, 4.8 MB)
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EC cultures were established with nuclear GFP-labeled ECs in 3D collagen matrices and were imaged by time-lapse fluorescence and light microscopy over a 24 hr period. Frames were collected every 10 minutes, were overlaid, and are shown at 8 frames/sec.
- Video 6. Blockade of EC motility as well as EC lumen and tunnel formation in the presence of GM6001 in 3D collagen matrices (MOV, 1.51 MB)
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EC cultures were established with nuclear GFP-labeled ECs in 3D collagen matrices and were imaged by time-lapse fluorescence and light microscopy over a 24 hr period in the presence of GM6001 at 5 µM. Frames were collected every 10 minutes, were overlaid, and are shown at 8 frames/sec.
- Video 7. EC motility on 2D collagen matrices (MOV, 2.4 MB)
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EC cultures were established using nuclear GFP-labeled ECs on 2D collagen coated plastic microwells (50 µg/ml) and were imaged by time-lapse fluorescence and light microscopy over a 24 hr period. Frames were collected every 10 minutes, were overlaid, and are shown at 5 frames/sec.
- Video 8. EC motility on 2D collagen matrices is unaffected in the presence of the MMP inhibitor, GM6001 (MOV, 3.23 MB)
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EC cultures were established using nuclear GFP-labeled ECs on 2D collagen coated plastic microwells (50 µg/ml) and were imaged by time-lapse fluorescence and light microscopy over a 24 hr period in the presence of GM6001 at 5 µM. Frames were collected every 10 minutes, were overlaid, and are shown at 5 frames/sec.
- Video 9. Control siRNA treated EC motility in conjunction with EC lumen and vascular guidance tunnel formation in 3D collagen matrices (MOV, 4.42 MB)
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EC cultures were established with nuclear GFP-labeled ECs in 3D collagen matrices (after treatment with the control luciferase siRNA) and were imaged by time-lapse fluorescence and light microscopy over a 24 hr period. Frames were collected every 10 minutes, were overlaid, and are shown at 8 frames/sec.
Additional videos can be found here.
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