Role of CX3CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

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.单核细胞/巨噬细胞（Monocyte/Macrophages）与血管生成启动、组织/器官灌注及动脉粥样硬化的病理生理过程密切相关。我们此前的研究证实，趋化因子受体CX3CR1（chemokine receptor CX3CR1）是损伤后血管壁内单核细胞/巨噬细胞源性平滑肌细胞分化的关键调控因子。本研究假设CX3CR1-CX3CL1相互作用可参与体内血管新生，并探究了CX3CR1的遗传学与药理学靶向调控对微血管完整性的形成、成熟及维持所产生的功能性影响。在三维基质胶（3D Matrigel）实验中，CX3CR1功能缺陷的细胞丧失了基质隧道形成与微管组装能力。此类形态发生与细胞动力学生物学过程由CX3CL1-CX3CR1相互作用介导，并可被Rho拮抗剂（Rho antagonist）完全阻断。为在体内评估CX3CR1系统的生物学作用，我们将基质胶栓植入功能完整的CX3CR1+/gfp小鼠与功能缺陷型CX3CR1gfp/gfp小鼠体内。在CX3CR1gfp/gfp小鼠的植入基质胶中可观察到渗漏性微血管（microvessels, MV），而CX3CR1+/gfp小鼠则未出现此类现象。在实验性斑块血管新生模型中，与功能完整的CX3CR1+/gfp小鼠相比，CX3CR1gfp/gfp小鼠的微血管呈现未成熟表型，且缺乏CX3CR1阳性平滑肌样细胞、细胞外胶原与基底膜（basement membrane, BM）层粘连蛋白。该现象伴随CX3CR1gfp/gfp小鼠内膜（intima）内血小板（platelets）外渗增加，而功能完整的CX3CR1小鼠则无此变化。对野生型小鼠使用CX3CR1受体拮抗剂进行药理学靶向干预后，其斑块微血管的基底膜覆盖不良且呈现渗漏表型。本研究数据揭示了功能性CX3CR1在三维基质胶与体内实验性斑块血管新生中此前未被认知的生物学作用，该作用可协同巩固微血管，助力形成更稳定的非渗漏表型。