Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

Abstract

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs up-regulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.

Figure 1. Depiction of network visualization and tracing

A) Low magnification fluorescent images of day 7 capillary networks were segmented based on a constant threshold of a background corrected image. Minimum and maximum thresholds were set to delineate capillaries from noise or beads/nodes, respectively, before applying the Angiogenesis Tube Formation Application Module in Metamorph imaging software. B) A sample tracing generated by automated processing. The filament tracing function was used to visualize networks (blue) using Imaris.

Figure 2. MSCs and fibroblasts stimulate ECs to form mature capillary networks

MSCs (A–D) or fibroblasts (A’–D’) were interspersed throughout fibrin ECMs in the presence of microcarrier beads coated with mCherry-transduced ECs. A, A’) Beads were monitored over a 7 day period and imaged at days 1, 3, and 7. Red channel fluorescence (i.e., ECs) is shown. Scale = 200 µm. B, B’) Cultures were fixed and IF stained at day 7 for F-actin (green) and collagen IV (red). Basement membrane deposition (white arrows) was observed in both conditions. Scale = 50 µm. C, C’) Mesenchymal cells expressing GFP were interspersed with mCherry-transduced EC. Physical association of both cell types was observed in both conditions (white arrows). Scale = 25 µm. D, D’) Cultures containing mCherry-transduced ECs and either MSCs (D) or fibroblasts (D’) were fixed and IF stained for pericyte markers αSMA (aqua) and NG2 (white). DAPI-stained nuclei are visible in the blue channel. Scale = 50µm.

Figure 3. Interstitial cell-mediated capillary morphogenesis is accompanied by periendothelial proteolysis

mCherry-transduced ECs were cultured within fibrin ECMs containing 1% DQ Collagen either A) alone, B) with MSCs, or C) with fibroblasts. Red (mCherry-ECs), green (proteolyzed ECM), and merged channels are shown. Autofluorescence from the microcarrier bead is evident in A). Scales = 50 µm.

Figure 4. Onset of capillary morphogenesis coincides with upregulated expression of fibrinolytic enzyme transcripts by EC

A) Brightfield images (10x) of EC-coated microcarrier beads at days 1, 3, and 7 cultured either alone (top row), with overlaid MSCs (middle row), or with overlaid fibroblasts (bottom row). Lumen formation is first evident in both co-cultures by day 3 (white arrows). Scale = 100 µm. B) Fibrinolytic enzyme and GAPDH (internal control) transcript expression for mesenchymal cells and ECs cultured separately (“alone”) or together (“co-cultured”) probed via RT-PCR (day 3). This experiment was repeated twice and a representative sample is shown.

Figure 5. Broad spectrum inhibition reveals that MSC-mediated capillary morphogenesis is MMP dependent

A) mCherry-transduced ECs were coated onto microcarrier beads and cultured within fibrin matrices interspersed with unlabelled MSCs. Shown are fluorescent images at day 7 of capillary network formation in vehicle (DMSO) treated cultures, those treated with 10 µM, 20 µM, or 40 µM of the broad spectrum MMP inhibitor GM6001, with 2.2 µM of the serine protease inhibitor aprotinin, or with a combination of GM6001 (10 µM) and aprotinin (2.2 µM) (“both”). Scale = 200 µm. B) Total network lengths were quantified from a minimum of 10 images over 2–3 independent experiments per condition as described in Materials and Methods. Applied concentrations of BB2516 were 3.3 and 10 µM. * P<0.05 when comparing the indicated conditions to vehicle control. One-way ANOVA followed by post-test analysis using Dunn’s method was used to compare treatment groups.

Figure 6. Broad spectrum inhibition reveals that fibroblast-mediated capillary morphogenesis is both MMP and serine protease dependent

A) mCherry-transduced ECs were coated onto microcarrier beads and cultured within fibrin matrices interspersed with unlabelled fibroblasts. Shown are fluorescent images at day 7 of capillary network formation in vehicle (DMSO) treated cultures, those treated with 10 µM, 20 µM, or 40 µM of the broad spectrum MMP inhibitor GM6001, with 2.2 µM of the serine protease inhibitor aprotinin, or with a combination of GM6001 (10 µM) and aprotinin (2.2 µM) (“both”). Scale = 200 µm. B) Total network lengths were quantified from a minimum of 10 images over 2–3 independent experiments per condition as described in Materials and Methods. Applied concentrations of BB2516 were 3.3 and 10 µM. * P<0.05 when comparing the indicated conditions to vehicle control. One-way ANOVA followed by post-test analysis using Dunn’s method was used to compare treatment groups.

Figure 7. Endogenous inhibitors confirm that mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

A) mCherry-transduced ECs were coated onto microcarrier beads and cultured within fibrin matrices interspersed with unlabelled MSCs (top row) or fibroblasts (bottom row). Shown are fluorescent images at day 7 of capillary network formation in vehicle (PBS) treated cultures, or those treated with 5 µg/mL TIMP1 or TIMP2, or with 25 µg/mL of a function blocking antibody directed towards uPAR (α-uPAR). Scale = 200 µm. B, C) Total network lengths were quantified from a minimum of 5 images over 2–3 independent experiments per condition as described in Materials and Methods for EC-MSC (B) and EC-fibroblast (C) cultures. * P < 0.001 when compared to vehicle control. One-way ANOVA followed by post-test analysis using the Holm-Sidak method was used to compare treatment groups.