Abstract
Monocyte/Macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX3CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX3CR1- CX3CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX3CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX3CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX3CL1-CX3CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX3CR1 system in vivo, Matrigel plugs were implanted in competent CX3CR1+/gfp and functionally deficient CX3CR1gfp/gfp mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX3CR1gfp/gfp but not in CX3CR1+/gfp mice. In experimental plaque neovascularization immature MV phenotype was observed in CX3CR1gfp/gfp mice, lacking CX3CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX3CR1+/gfp mice. This was associated with increased extravasation of platelets into the intima of CX3CR1gfp/gfp but not functionally competent CX3CR1 mice. Pharmacologic targeting using CX3CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX3CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.
Highlights
The monocyte–macrophage system (MPS) exhibits numerous functions with respect to vascular remodeling which include classical phagocytosis, regulation of antigen presentation and atherosclerotic plaque formation [1,2,3,4]
Myeloid phenotype of CX3CR1 expressing cells was established by FACS and tissue immunofluorescence (Figure S1 A, B.) indicating that the CX3CR1 transgenic models selected would be suitable for assessing structure function relationships between monocyte/macrophages and evolving neovascularization networks
Matrigels implanted in CX3CR1gfp/+ mice had significantly increased CX3CR1 positive cells integrated into MV wall (4.0960.34 cells/Matrigel MV/HPF) compared to Matrigels implanted in CX3CR1gfp/gfp mice (1.3360.05 cells/Matrigel MV/HPF) (Figure 1 B, p,0.05) and predominantly a perivascular MV association was observed for CX3CR1 positive cells in Matrigel of CX3CR1gfp/gfp mice (Figure 1B)
Summary
The monocyte–macrophage system (MPS) exhibits numerous functions with respect to vascular remodeling which include classical phagocytosis, regulation of antigen presentation and atherosclerotic plaque formation [1,2,3,4]. In the current study we selected the chemokine receptor CX3CR1 as a transgenic marker allowing baseline and loss of function status to be examined in mice that had green fluorescence protein knocked in at one or both CX3CR1 alleles [17,23] This facilitated simultaneous in vivo tracking of a major monocyte/macrophage subset into evolving neovascularization networks in solid matrix and in medium sized vessel wall microvasculature. These transgenic models facilitated interrogation of loss of CX3CR1 function effects on CX3CR1 cell recruitment to the perivascular space, mural cell integration into evolving microvasculature, microvessel maturation and extracellular matrix production and microvessel haemorrhage and permeability. In vitro studies evaluated the signaling pathway downstream of CX3CR1 activation and its effects on cytoskeletal re-organization and solid matrix tunneling of CX3CR1 positive cells
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