Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair: conversion of cell columns into fibrovascular bundles

Abstract

The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently become a topic of intense scrutiny. Here, we characterized the behavior of MC/Mph in cellular infiltrates, with emphasis on their spatial organization and localization in newly formed microvessels. To this end, we studied MC/Mph migration and assembly in basic fibroblast growth factor-supplemented Matrigel plugs placed in transgenic Tie2-beta-galactosidase mice for up to 4 weeks. In these plugs, along with Nile Red-positive adipocytes, we found MC/Mph distributed in cell cords, also containing various mature and progenitor tissue cells; and functional Tie2-positive or -negative microvessels embedded in bundles of fibrillar collagen surrounded by F4/80-positive MC/Mph. At earlier stages of infiltration, we found tubular destruction of the matrix (tunnels) and MC/Mph-lined capillary-like structures occasionally containing erythrocytes, indicating their propensity for endothelial trans-differentiation. We also analyzed in vitro the MCP-1-induced chemotactic migration of fluorescently labeled peritoneal MC/Mph incorporated in Matrigel-containing fluorescent protease substrates. Many of these MC/Mph produced MMP-12- and TIMP-1-dependent tunnels coupled with acquisition of a lumen. In conclusion, long-term implantation of Matrigel plugs qualifies as a novel experimental model of tissue regeneration, in which neovascularization intimately couples with fibrosis and organogenesis and in which cells of MC/Mph phenotype play a key structural role.

Figure 1

MC/Mph migration leads to tunnel formation in Matrigel. A: Matrigel retrieved after 1 week from a subcutaneously implanted, Nuclepore filter-limited chamber. Note the cells accumulated underneath the filter (arrowheads) and the tunnels left behind the front of migrating cells (arrow). B: Most of the cells present in the tunnels are F4/80⁺ MC/Mph (brown, arrows). C: Mph-lined, capillary-like structures (arrow) detected at 1 week in Matrigel plugs in Tie2-βGal mice (no Tie2-βGal-positive cells present). Note the branching (arrowheads) and the F4/80⁺cells in the lumen. Inset: Enlargement of the F4/80⁺ cell marked with dashed lines, showing a lumen-like vacuole and a low-density tunnel in Matrigel (arrowhead). D: Negative control for the F4/80 immunostaining (omission of the primary antibody). The cell columns line a collagen bundle cut longitudinally, visible in the background of this phase contrast micrograph (arrow, compare with Figure 3A). In this field, a Tie2-βGal-positive cell (light blue) is wrapped by another cell (arrowhead, compare with Figure 4, D and E). Counterstaining with hematoxylin. Original magnifications, ×120.

Figure 2

Cellular organization and vascularization of subcutaneous Matrigel plugs in Tie2-βGal mice. Matrigel plugs containing bFGF (A, C, D) or VEGF-165 and SDF-1α (B) retrieved after 4 weeks from Tie2-βGal mice, sectioned, and stained for LacZ (light blue) and counterstained with H&E (A, B, D) or for the Mph marker F4/80 (brown) (C). A: Massive adipogenesis throughout the whole plug. B: Adipocytes and blood vessels are missing in the plug, and the overall infiltration is reduced. The lower portion in A and B is the attached tissue. C: Adipocyte cluster containing microvessels positive (arrow) and negative (arrowhead, containing erythrocytes) for Tie2-βGal. D: Section running obliquely through microvessels that are Tie2-βGal-positive (arrow) or -negative (arrowhead; containing pale erythrocytes). Note the oriented distribution of rows of adipocytes (asterisks), flanked by F4/80⁺ Mph. Hematoxylin counterstaining. Original magnifications: ×40 (A, B); ×120 (C, D).

Figure 3

Lipid accumulation and formation of MC/Mph-lined fibrovascular bundles in bFGF-supplemented Matrigel plugs (4 weeks old). A: Nile Red-stained lipid droplets (arrows) in a Matrigel cryosection. B and C: Structures containing collagen in the Matrigel plug (Masson Trichrome staining) B: Cell column containing fibrillar collagen (arrow, light blue) and an adipocyte (asterisk). Note the bundle branching (arrowhead) and other adipocytes that align with the main bundle (asterisks). C: Cells present inside the collagen bundles (arrows); the upper structure displays a lumen. D–F: Anti-F4/80⁺ immunostaining of the Matrigel plug. D: F4/80⁺ MC/Mph distributed in a capillary-like pattern (arrows). Note the Tie2-βGal⁺ cell aggregate without a lumen (arrowhead, light blue). E: Tie2-βGal⁺ cell column inside a collagen bundle lined by F4/80⁺ Mph (arrow). Other Mph-wrapped collagen bundles are nearby (arrowheads). F: Erythrocytes present in a Tie2-βGal⁻ microvessel, within a collagen bundle lined by F4/80⁺ cells (arrow). G: Erythrocytes inside a collagen bundle (arrow) (H&E staining). H and I: Anti-smooth muscle α-actin immunostaining. H: α-Actin-expressing cell within a cell-lined collagen bundle. I: α-Actin-positive cells surrounding a Tie2-βGal⁺ cell aggregate (light blue) without a lumen (arrow). Note the presence of a nearby actin-positive single cell (arrowhead). B–I, Hematoxylin counterstaining. Original magnifications: ×80 (A); ×120 (B, D); ×200 (C, E–I).

Figure 4

Cellular phenotypes at the distal margin of a Matrigel plug 4 weeks after implantation. A: F4/80⁺ cell with vacuole (arrow) and one displaying a fibroblastic morphology (arrowhead), in a field containing immature adipocytes (asterisks). B: F4/80⁺ MC/Mph displaying vacuoles (arrows) or lumen (arrowhead), and immature adipocytes (asterisks). C: F4/80⁺ Mph presenting a large vacuole (arrow) comparable to the size of a Tie2-βGal⁺ capillary lumen (arrowhead, light blue). D: Intimate apposition between an F4/80⁺ Mph (arrow) and a Tie2-βGal⁺ cell without lumen (arrowhead, light blue). Inset: Tie2-βGal⁺ cell within an F4/80⁺ lumen. E: F4/80⁺ Mph (arrow) wrapping cells that are Tie2-βGal-positive (arrowhead, light blue). F: Erythrocytes present in a lumen lined by F4/80⁺ MC/Mph (arrow and inset). Also, note the presence of an erythrocyte in an F4/80⁻ structure (arrowhead). Hematoxylin counterstaining. Original magnifications: ×120 (A, B, F); ×200 (C, D, E).

Figure 5

Connectivity of the fibrovascular bundles developing in Matrigel plugs with the main blood circulation. A: Erythrocyte-filled microvessel (arrow) found at the core of a fibrovascular bundle. B: Erythrocytes (arrow) and dense India ink (carbon particles, arrowheads) aggregates present in a fibrovascular bundle at the interface of Matrigel plug (top right) with the tissue (left side of the image). C: India ink-filled microvessel in a fibrovascular bundle found deeper in the Matrigel plug. D: Uptake of carbon particles in a phagocytic cell (arrowhead), close to an India ink-filled space inside a fibrovascular bundle (arrow). Original magnifications: ×200 (A, B, D); ×120 (C).

Figure 6

Coupling of matrix degradation with lumen formation in MC/Mph. A–C: High-resolution histology of peritoneal Mph embedded in Matrigel and exposed to a MCP-1 gradient for 24 hours. A: Ruffled zone and secretion granules at one pole of the cell (arrow). B: Large intracellular vacuole (arrow); arrowhead, the nucleus. C: Tunnel (arrow) half-covered by an Mph cytoplasmic fold that contains an array of dense intracellular granules (arrowhead). D–F: Virtual sectioning through a three-dimensional confocal reconstitution of a pair of CTG-labeled Mph, embedded in Matrigel containing DQ Red BSA, for 24 hours. D: Proteolysis was observed closest to the source of MCP-1 (arrow). E: Distribution of CTG fluorescence in Matrigel-embedded Mph: the right-hand cell displays a vacuole or a lumen (arrow). F: Overlay of the red (DQ BSA) and green (CTG) fluorescence channels. Note the presence of degradation product in the empty space (arrowhead). G–J: DQ Red BSA proteolysis and Mph morphology after 5 days in Matrigel. G: Lateral view of a three-dimensional reconstitution of optical sections (see also Movie 1, Supplemental data, at http://ajp.amjpathol.org). The dashed lines indicate where the planes in H and I were taken. H: Virtual section through the image in G. I: Frontal section of the matrix modification in G, showing proteolytic degradation (red) of a size similar to a single cell diameter (the tunnel). J: Mph displaying a lumen and containing large amounts of intracellular proteolysis product, both on the inner face of the lumen (arrow) and within plasmalemmal vesicles (arrowhead). Scale bars: 10 μm (D–F); 10 μm (G–I); 5 μm (J). Original magnifications: ×200 (A–C).

Figure 7

Factors controlling the chemotactic migration of peritoneal Mph and Matrigel proteolysis. Mph chemotaxis through a Matrigel-coated filter in response to MCP-1 (100 ng/ml), VEGF (10 ng/ml), or MCP-1 plus folimycin (50 nmol/L). *P < 0.05; NS, not significant.

Figure 8

Diagram illustrating the proposed stages of conversion of MC/Mph-based cell columns into microvessels containing tissular units. Color code: brown, F4/80⁺ Mph and F4/80⁺ fibrocytes; blue, Tie2-βGal⁺ EPC/EC; light green, Tie2-βGal⁻ EPC/EC; yellow, (pre)adipocyte; orange, smooth muscle/pericyte precursors; pink, fibroblasts; red, erythrocytes; black lines, collagen. The numbers indicate figures in which these cells were described. Note: Because Tie2-βGal-positive and -negative microvessels were never found in the same fibrovascular bundle, separate EPC precursors of arteriolar (Tie2-βGal⁺) and venular (Tie2-βGal⁻) endothelium could be hypothesized.