Figure 1.
Real-time images showing endothelial cell tube formation and pericyte recruitment using a serum-free defined system in 3D fibrin matrices.
Hematopoietic stem cell cytokines and FGF-2 stimulate these morphogenic responses which can be primed by EC pre-treatment with VEGF and FGF-2. Endothelial cells (ECs) were labeled with membrane targeted GFP while pericytes (PCs) were labeled with mCherry. The two cell types were randomly mixed into fibrin matrices in the presence of SCF, IL-3, SDF-1α, Flt-3L and FGF-2. Prior to harvesting the cells for suspension into fibrin gels, ECs were primed with VEGF and FGF-2 for 20 hr. After fibrin polymerization, cultures were imaged every 10 minutes and select fluorescent images are shown at the indicated times. Two different series (A,B) are illustrated. Bar equals 100 µm.
Figure 2.
Cross-sections of EC-pericyte co-cultures reveal marked EC tubulogenesis using a serum-free defined system in 3D fibrin matrices.
(A) EC-pericyte co-cultures were established and after 5 days, cultures were fixed, embedded into plastic, cross-sectioned, stained with toluidine blue, and photographed. Arrowheads indicate EC lumens in cross-section. (B) EC-pericyte co-cultures were established for 5 days, fixed and processed for transmission electron microscopy. EC lumen formation is observed and pericytes are demonstrated on the EC abluminal surface (arrows).
Figure 3.
Hematopoietic stem cell cytokines stimulate EC-pericyte tube co-assembly in 3D fibrin matrices.
Membrane-targeted GFP labeled ECs were mixed with pericytes in 3D fibrin matrices with various growth factor combinations. The upper panels (A-D) show light microscopic images while the lower panels show fluorescent images of the corresponding light image fields from cultures at 72 hr. (A) FGF-2 only; (B) VEGF+FGF-2; (C) Hematopoietic factors including SCF, IL-3, SDF-1α were added along with FGF-2; (D) Same factors added as C during the assay, but ECs were primed first with VEGF and FGF-2 overnight. Arrows indicate EC-lined tubes. Bar equals 25 µm. (E,F) EC-pericyte co-cultures were established with unprimed (E) or VEGF/FGF-2 primed (F) ECs in 3D fibrin matrices with the indicated growth factors. Cultures were fixed after 72 or 120 hr and tube area was quantitated using Metamorph software. Asterisks indicate significance of p< .01 relative to the FGF only condition (E) (n=20, values are averaged + SD) or relative to the unprimed+hematopoietic factors condition (F) (n=30, values are averaged + SD).
Figure 4.
Pericytes and Flt-3L enhance the ability of SCF, IL-3, SDF-1α and FGF-2 to stimulate EC tubulogenesis in 3D fibrin matrices.
ECs were seeded with or without pericytes and hematopoietic stem cell cytokines (SCF, IL-3, SDF-1α, FGF-2) were added into the fibrin matrices in the presence or absence of Flt-3L. After 72 hr, cultures were fixed, photographed (B), and quantitated for EC tube formation using Metamorph Software (A). Asterisks indicate significance at p< .01 with respect to the EC only cultures, while the triangles indicate significance at p< .01 with respect to cultures without Flt-3L addition (n=20, values are averaged + SD). Bar equals 25 µm.
Figure 5.
VEGF and FGF-2 prime ECs upstream of hematopoietic stem cell cytokines which act downstream to control EC tubulogenesis and EC-pericyte tube co-assembly in 3D fibrin matrices.
ECs were primed with hematopoietic cytokines (factors) or VEGF/FGF-2 overnight and then were seeded into fibrin matrices with hematopoietic factors including FGF-2 or with VEGF/FGF-2 alone so that all four possible combinations were examined. Pericytes were co-seeded with the hematopoietic factor or VEGF/FGF-2 primed ECs. Cultures were fixed after 72 or 120 hr and EC tube area was measured using Metamorph software. Asterisks indicate significance at p< .01 with respect to the Factor primed/ VEGF+FGF-2 in matrix conditions, while the triangles indicate significance at p< .05 with respect to the Factor primed/Hematopoietic factors in matrix conditions (n=15, values are averaged + SD).
Figure 6.
Neutralizing antibodies directed to the hematopoietic cytokines, SCF, IL-3 and SDF-1α markedly block EC tube formation, while antibodies to PDGF-BB and HB-EGF interfere with pericyte assembly on EC-lined tubes in 3D fibrin matrices under serum-free defined conditions.
GFP-ECs were primed with hematopoietic cytokines (factors) or VEGF/FGF-2 overnight and then were seeded into fibrin matrices with hematopoietic factors and FGF-2 in the presence of Cherry-pericytes and in the presence of the indicated neutralizing antibodies or chemical inhibitors. (A) The antibodies were added at 50 µg/ml (IL-6, SCF, IL-3, SDF-1α) or 100 µg/ml (VEGF) versus controls and after 72 hr, cultures were fixed, photographed and quantitated for EC tube formation (n=10, values are averaged + SD, asterisks indicate significance compared to control, p<.01). (B) Time course of pericyte recruitment to EC tubes using real-time movies. At the indicated times, the percentage of pericytes that were associated with EC tubes were quantitated (n=8, values are averaged + SD, asterisks indicate significance compared to control, p<.01). (C,D) EC-pericyte co-cultures were established in the absence or presence of the indicated blocking antibodies (each added at 50 µg/ml) or chemical inhibitors (each added at 1 µM). After 72 hr, cultures were fixed and photographed (C,D) or were quantitated for pericyte recruitment (E) (n=6, values are averaged + SD, asterisks indicate significance compared to control, p<.01). Bar equals 100 µm.
Figure 7.
Hematopoietic stem cell cytokines stimulate EC-pericyte tube co-assembly and vascular basement membrane matrix deposition under serum-free defined conditions in 3D fibrin matrices.
ECs were primed with VEGF/FGF-2 overnight and then were seeded with GFP-pericytes into 3D fibrin matrices containing SCF, IL-3, SDF-1α, Flt-3L, and FGF-2. After 120 hr, cultures were fixed and stained for extracellular antigens using various antibodies directed to: (A) Fibrin; (B) CD31; (C) Collagen type IV; (D) Laminin; (E) Fibronectin; (F) Perlecan; (G) Nidogen-1; (H) Nidogen-2; (K) Collagen type I; and (L) Collagen type III. In some cultures, the fibrin gel was supplemented with bovine fibronectin (I, J) and a monoclonal antibody that is specific for human fibronectin was used to assess fibronectin deposition within the vascular basement membrane (I). In this case, CD31 antibody staining was shown for comparison (J). In all other cases, the fibrin gels were supplemented with human fibronectin (A-H, K,L). Arrows indicate the borders of vascular guidance tunnels which are generated during EC tubulogenesis and both ECs and pericytes reside within these tunnel spaces (A). Arrowheads indicate vascular basement membrane deposition. Bar equals 25 µm.
Figure 8.
EC-pericyte tube co-assembly in 3D fibrin matrices under defined serum-free conditions leads to vascular basement membrane matrix formation.
ECs that were primed with VEGF/FGF-2 and GFP-pericytes were seeded together into 3D fibrin matrices in the presence of hematopoietic stem cell cytokines and after 120 hr, cultures were fixed for fluorescent immunomicroscopy (A,B), and for transmission electron microscopy (C,D). Cultures were stained with antibodies to collagen type IV (A) and laminin (B) demonstrating vascular basement membrane formation. Arrowheads indicate basement membrane matrix deposition. Bar equals 100 µm. (C,D) Transmission electron micrographs showing pericytes along an EC tube structure with a defined lumen space and vascular basement membrane deposition observed between the two cell types (arrowheads). Arrows indicate pericytes. Bars equal 2 µm (C) or 1 µm (D).
Figure 9.
Hematopoietic stem cell cytokines, FGF-2 and pericytes stimulate EC sprouting and tube morphogenesis in 3D fibrin matrices under defined serum-free conditions.
GFP-pericytes were seeded into 3D fibrin matrices which contained SCF, IL-3, SDF-1α, Flt-3L and FGF-2. ECs were primed with VEGF/FGF-2 and were seeded on the surface of the polymerized fibrin gels that contained growth factors and pericytes. ECs were allowed to sprout and form tubes for 120 hr prior to fixation and immunofluorescence staining with anti-CD31 antibodies (red). (A) Monolayer surface with visible EC-EC junctions as well as out of focus sprouts invading beneath the EC monolayer. (B,C) Cross sections of the cultures showing the monolayer surface (arrowheads) and invading EC sprouts and tubes (arrows). (D) Invading EC sprouts are observed (arrows) beneath the monolayer surface. (E) Overlay image of (D) showing EC sprouts (red) with associated and surrounding GFP-pericytes (green) and Hoechst dye-stained nuclei (blue). (F) Overlay image showing a cross-section of the EC monolayer (arrowheads) and invading EC sprouts (red) (arrows) with associated and surrounding GFP-pericytes (green) as well as Hoechst dye-stained nuclei (blue). Bar equals 100 µm (A,D,E,F) or 500 µm (B,C).
Figure 10.
Pericytes recruit to invading EC sprouts/tubes and induce vascular basement membrane assembly on these EC tubes in 3D fibrin matrices under serum-free defined conditions.
GFP-pericytes were seeded into 3D fibrin matrices which contained SCF, IL-3, SDF-1α, Flt-3L and FGF-2 while ECs were primed with VEGF/FGF-2 and then seeded on the surface of polymerized fibrin gels that contained growth factors and pericytes. ECs were allowed to sprout and form tubes for 120 hr prior to fixation and immunofluorescence staining with antibodies to CD31 (A,B); laminin (C,D); collagen type IV (E,F); or fibronectin (G,H). The basement membrane matrix antigens were stained in the absence of detergent to examine only extracellular deposition of these molecules. The right sided images (B,D,F,H) are overlay images for each stain showing the antigen stain in red, while GFP-pericytes are green and nuclei are blue (stained with Hoechst dye). Arrowheads indicate vascular basement membrane matrix deposition. Bar equals 100 µm.
Figure 11.
Time lapse images of EC sprouting and tubulogenesis in 3D fibrin matrices under serum-free defined conditions which are stimulated by hematopoietic stem cell cytokines, FGF-2 and pericytes.
mCherry-pericytes, hematopoietic cytokines and FGF-2 were incorporated into the fibrin matrix, and VEGF/FGF-2 primed ECs (membrane AcGFP-labeled) were seeded onto the surface of fibrin gels and real-time movies were made. Select time points are shown from a representative field showing marked EC sprouting and tube formation as well as EC-pericyte interactions. Interestingly, it appears that EC sprouts might be preferentially orienting toward mCherry-pericytes in these movies suggesting the presence of pericyte-derived directional cues. Bar equals 100 µm.
Figure 12.
Integrin α5β1 controls EC sprouting and tubulogenesis in 3D fibrin matrices under serum-free defined conditions.
(A) EC-pericyte co-cultures were established by suspending both cell types into fibrin matrices along with hematopoietic cytokines and FGF-2. The indicated anti-integrin blocking antibodies were added at 20 µg/ml and after 72 hr, EC tube area was determined using Metamorph software. Asterisk indicates significance at p< .01 compared to the control condition (n=18, values are averaged + SD). (B) EC sprouting assays were established by seeding ECs on the surface of fibrin gels which contained pericytes as well as hematopoietic cytokines and FGF-2. The indicated anti-integrin blocking antibodies were added at 20 µg/ml and after 72 hr, EC tube area in the invading sprouts was quantitated using Metamorph software. Asterisks indicate significance at p< .01 compared to the control condition (n=6, values are averaged + SD). Representative photographs of invading sprouts and EC tubulogenesis in the control condition (C); anti-α5 integrin subunit (D); and anti-β1 integrin subunit (E). Bar equals 100 µm.