Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation

Abstract

We show that endothelial cell (EC)-generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, alpha(5)beta(1), alpha(3)beta(1), alpha(6)beta(1), and alpha(1)beta(1), which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Figure 1

Recruitment of pericytes to the ablumenal surface of EC-lined tubes within vascular guidance tunnels using a new model of EC-pericyte tube coassembly in 3D collagen matrices. EC-pericyte cocultures were allowed to assemble randomly within a 3D collagen matrix for 5 days, fixed, and processed for immunofluorescence or cross-sectioning (HUVECs-bovine pericytes). (A) ECs were stained with the endothelial specific marker, CD31, whereas pericytes were GFP labeled. Bar equals 25 μm. (B) Plastic sections show association of pericytes with endothelial tubes on the abluminal face. Bar equals 25 μm. (C-D) EC-pericyte cocultures were allowed to form, and a thin plastic section of the monolayer was examined along an EC tube surface. ECs form a continuous layer with pericytes recruited to the basal surface. Black arrows indicate the border of the vascular guidance tunnel and the entrance of pericytes within these borders. Bar equals 50 μm. (E) Randomly placed ECs and GFP pericytes were allowed to form within the 3D collagen matrices for 5 days. These cultures were then immunostained with an anti-collagen type I antibody. (F) Images of the GFP pericytes were also obtained and overlaid with the corresponding collagen type I stain. Images show the clear presence of pericytes within vascular guidance tunnels. Bar equals 50 μm.

Figure 2

Extracellular deposition of basement membrane components is observed only when EC are cocultured with pericytes during EC-pericyte tube coassembly. ECs were cultured alone or with pericytes and allowed to randomly assemble in a 3D collagen for 5 days, fixed, and processed for immunofluorescence (HUVECs-bovine pericytes). Staining was done without detergent to assure extracellular staining only. (A) Basement membrane matrices were stained for the following: collagen IV, laminin, nidogens 1 and 2, perlecan, and fibronectin (red staining), and GFP pericytes (green staining). EC-only cultures show very little extracellular deposition of the indicated molecules. EC-pericyte cocultures show a dramatic increase in extracellular deposition of the basement membrane components along with fibronectin. (B) α-Tubulin staining was conducted as a methods control. (C) Pericytes are localized within vascular guidance tunnels; EC-GFP pericyte cocultures in 3D collagen gels were allowed to coassemble, and then stained with an anti-collagen type I antibody. Bar equals 25 μm. (D) Separate gels from the same cultures were then immunostained with an anti-laminin antibody that shows localized laminin staining between the 2 cell types. Bar equals 25 μm. (E-F) Electron microscopy was performed, and representative images are shown revealing the deposition of basement membrane matrix material (arrowheads) between ECs and pericytes, corresponding to the position of anti-laminin staining shown in (D); this deposition and organization is not seen when ECs are cultured alone (F). Bar equals 2 μm.

Figure 3

Pericyte recruitment to microvascular tubes during vasculogenesis in quail CAMs correlates with basement membrane matrix assembly. (A) Quail CAMs were isolated at 5 and 7 days of development and fixed for detergent-free immunostaining. Images reveal basement membrane deposition (arrowheads) around developing microvessels at the 7-day time point. Bars equal 10 μm. (B-C) Double staining of quail CAMs at 7 days for QH1, an EC-specific marker in red, versus SMA, recognizing pericytes at the microvascular level in green. (B) Reveals the coassociation of the 2 cells at this time point. (C) Staining of SMA (in green) versus the basement membrane component protein, fibronectin (in red), reveals the location of the pericytes in relation to the basement membrane. (D-E) Electron microscopy of quail CAMs reveals a continuous basement membrane (arrows) along the ablumenal surface of developing microvessels and between the perivascular cells (closed arrowheads) and the endothelium (open arrowheads). Bar equals 2 μm.

Figure 4

Up-regulation of fibronectin, nidogen-1, laminin isoforms, and perlecan occurs selectively in EC-pericyte cocultures to regulate EC basement membrane assembly. ECs alone, pericytes alone, or both ECs and pericytes were seeded randomly in 3D collagen matrices and allowed to assemble into tube networks. These networks were then either isolated for RNA or lysed for Western blot analysis. (A) mRNA expression of basement membrane components (using species-specific primer sets to HUVECs versus bovine pericytes) was examined at 1, 3, and 5 days of culture. ECs and pericytes alone in culture were examined as well as EC-pericyte cocultures. Relative expression patterns are shown, with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a control. (B) Western blot analysis of collagen IV, laminin isoforms, nidogen 1/2, and fibronectin are shown demonstrating protein levels in EC-only (HUVECs), EC-pericyte coculture (HUVECs, bovine pericytes), and pericyte-only cultures (bovine pericytes). CM refers to conditioned medium; L refers to lysates. Actin blots are included as a loading control.

Figure 5

Selective induction and functional requirement of integrins with affinity for basement membrane matrices during EC-pericyte tube coassembly. (A) PCR analysis of selected α integrin chains was performed to examine expression in both the bovine pericytes and ECs (HUVECs) when cultured alone or in coculture. G3PDH is shown as a loading control. (B) Five-day images from time lapse movies are shown, highlighting the differences in vessel width between control EC-only cultures and EC-pericyte cocultures. Arrows show the differences in width. (C) Average vessel widths were measured from images obtained from time lapse movies for quantitation. (D) Integrin-blocking antibodies were added to the culture media of EC alone versus EC-pericyte cocultures either from the beginning of the experiment or starting at day 3 or 5 at 20 μg/mL. Analysis of total vessel area is shown, demonstrating a requirement for α₂β₁ using EC-only cultures. Analysis of average vessel width is reported showing increases in EC tube width with blockade of α₅β₁, α₃β₁, and α₆β₁ only when pericytes are present, and more significantly increased tube widths when antibodies to α₁β₁, α₃β₁, and α₆β₁ are mixed (bottom panels). Statistical significance, P < .01. Species specificity of the antibodies can be found in supplemental Table 2.

Figure 6

Fibronectin matrix assembly is required for vascular basement membrane formation during EC-pericyte tube coassembly. EC-pericyte cocultures were allowed to assemble for 5 days in a 3D collagen matrix either in the presence or absence of 50 μg/mL 70-kDa fragment of fibronectin (using HUVECs-bovine pericytes). (A) Immunofluorescent staining of fibronectin, collagen IV, and laminin demonstrates that disruption of fibronectin assembly leads to disrupted collagen IV assembly (first column in red, with the second column showing overlays denoting the position of GFP-pericytes). (B) Quantification of average vessel width reveals that blockade of fibronectin assembly leads to increased vessel width of EC tubes in the cocultures, but not EC-only cultures (P < .01). (C) Intensity mapping of representative fibronectin and collagen IV stains is shown to demonstrate the reduced levels of assembly/deposition of these proteins.

Figure 7

Pericyte TIMP-3 regulates basement membrane formation during EC-pericyte coassembly events within vascular guidance tunnels during tube remodeling and maturation events. (A) Immunostaining of basement membrane components reveals that in cocultures in which pericytes were treated with a siRNA to TIMP-3, there is marked disruption of collagen IV (first column in red, with the second column showing overlays denoting the position of GFP-pericytes). (B) Furthermore, suppression of pericyte TIMP-3 leads to increased vessel width (P < .01). (C) RT-PCR analysis of pericyte TIMP-3 suggests regulation over time in EC-pericyte cocultures. G3PDH is shown as a loading control. (D) Western blot analysis of TIMP-3 is shown to demonstrate siRNA suppression versus controls. (E) This schematic diagram illustrates the function of vascular guidance tunnels that affect EC tube remodeling, recruitment of pericytes, and dynamic EC-pericyte interactions that are necessary for deposition of basement membrane matrix as well as EC and pericyte integrin expression changes that control vascular tube maturation events. Vascular guidance tunnels form as a consequence of EC lumen and tube formation that occurs through a signaling cascade involving Cdc42, MT1-MMP, and α₂β₁ integrin and the indicated downstream kinase effectors.^, Asterisks indicate basement membrane components and integrins that are up-regulated specifically by EC-pericyte interactions during tube coassembly in 3D collagen matrices. Cross indicates the down-regulation of α₂β₁, which is observed in EC-pericyte cocultures and not EC-only cultures.