Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors.

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.

MCP-1 promotes mural cell recruitment during angiogenesis in the aortic ring model.

Rings of rat or mouse aorta embedded in collagen gels produce angiogenic outgrowths in response to the injury of the dissection procedure. Aortic outgrowths are composed of branching endothelial tubes and surrounding mural cells. Mural cells emerge following endothelial sprouting and gradually increase during the maturation of the neovessels. Treatment of aortic cultures with angiopoietin-1 (Ang-1), an angiogenic factor implicated in vascular maturation and remodeling, stimulates the mural cell recruitment process. Ang-1 induces expression of many cytokines and chemokines including monocyte chemotactic protein-1 (MCP-1). Inhibition of p38 MAP kinase, a signaling molecule required for mural cell recruitment, blocks Ang1-induced MCP-1 expression. Recombinant MCP-1 dose-dependently increases mural cell number while an anti-MCP-1 blocking antibody reduces it. In addition, antibody mediated neutralization of MCP-1 abrogates the stimulatory effect of Ang-1 on mural cell recruitment. Aortic rings from genetically modified mice deficient in MCP-1 or its receptor CCR2 have fewer mural cells than controls. MCP-1 deficiency also impairs the mural cell recruitment activity of Ang-1. Our studies indicate that spontaneous and Ang1-induced mural cell recruitment in the aortic ring of model of angiogenesis are in part mediated by MCP-1. These results implicate MCP-1 as one of the mediators of mural cell recruitment in the aortic ring model, and suggest that chemokine pathways may contribute to the assembly of the vessel wall during the angiogenesis response to injury.

The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages.

The purpose of this study was to define early events during the angiogenic response of the aortic wall to injury. Rat aortic rings produced neovessels in collagen culture but lost this capacity over time. These quiescent rings responded to vascular endothelial growth factor but not to a mixture of macrophage-stimulatory cytokines and chemokines that was angiogenically active on fresh rings. Analysis of cytokine receptor expression revealed selective loss in quiescent rings of the proangiogenic chemokine receptor CXCR2, which was expressed predominantly in aortic macrophages. Pharmacologic inhibition of CXCR2 impaired angiogenesis from fresh rings but had no effect on vascular endothelial growth factor-induced angiogenesis from quiescent explants. Angiogenesis was also impaired in cultures of aortic rings from CXCR2-deficient mice. Reduced CXCR2 expression in quiescent rat aortic rings correlated with marked macrophage depletion. Pharmacologic ablation of macrophages from aortic explants blocked formation of neovessels in vitro and reduced aortic ring-induced angiogenesis in vivo. The angiogenic response of macrophage-depleted rings was completely restored by adding exogenous macrophages. Moreover, angiogenesis from fresh rings was promoted by macrophage CSF (CSF-1) and inhibited with anti-CSF-1 Ab. Thus, aortic angiogenic sprouting following injury is strongly influenced by conditions that modulate resident macrophage numbers and function.

The aortic ring model of angiogenesis.

Angiogenesis is regulated by a complex cascade of cellular and molecular events. The entire process can be reproduced in vitro by culturing rat or mouse aortic explants in three-dimensional biomatrices under chemically defined conditions. Angiogenesis in this system is driven by endogenous growth factors released by the aorta and its outgrowth. Sprouting endothelial cells closely interact with pericytes, macrophages, and fibroblasts in an orderly sequence of morphogenetic events that recapitulates all stages of angiogenesis. This model can be used to study the basic mechanisms of the angiogenic process and to test the efficacy of proangiogenic or antiangiogenic compounds. Aortic cultures can be evaluated with a range of morphologic and molecular techniques for the study of gene expression. In this chapter we describe basic protocols currently used in our laboratory to prepare, quantify, and analyze this assay.

Aortic rings stimulate inflammatory angiogenesis in a subcutaneous implant in vivo model.

Rat or mouse aortic rings produce angiogenic outgrowths in vitro through endogenous production of growth factors and inflammatory cytokines. To further investigate this process in vivo, collagen-Gelfoam constructs containing aortic rings were implanted subcutaneously in syngeneic animals. Aortic rings stimulated a prominent angiogenic response characterized by peri- and intra-aortic accumulation of florid granulation tissue. Conversely, implants without rings elicited a non-specific inflammatory reaction without significant angiogenesis. The angiogenic response to the rings peaked at day 14 and was followed by regression of neovessels, which were mostly reabsorbed by day 28. Gene expression studies showed upregulated expression of angiogenic growth factors and cytokines in implants with rings. Tracking experiments with LacZ expressing ROSA26 transgenic mice demonstrated that both the aorta and the host contributed to the angiogenic response. These studies show that the angiogenic properties of the rodent aorta can be studied in the live animal under conditions that can be monitored and quantified. This in vivo assay can be used to study the molecular mechanisms by which the arterial wall and its proangiogenic cytokines regulate formation of granulation tissue during wound healing.

Regulation of postangiogenic neovessel survival by beta1 and beta3 integrins in collagen and fibrin matrices.

We used the aortic ring model of angiogenesis to investigate the role of beta(1) and beta(3) integrins in postangiogenic vascular survival in collagen and fibrin matrices. Confocal microscopy studies showed that both beta(1) and beta(3) integrins were expressed in endothelial cells and pericytes of sprouting neovessels. Antibody blocking experiments demonstrated that beta(1) integrins but not beta(3) integrins were required for angiogenic sprouting in collagen. Conversely, in fibrin, blockade of both integrins was needed to inhibit angiogenesis whereas treatment with either antibody alone was ineffective. Antibody-mediated blockade of beta(1) but not beta(3) integrins accelerated vascular regression in collagen. In contrast, both anti-beta(1) and -beta(3) integrin antibodies were required to promote neovessel breakdown in fibrin. These results demonstrate that angiogenic sprouting and postangiogenic neovessel survival in collagen are critically dependent on beta(1) integrins. They also indicate that these processes involve a redundant repertoire of beta(1) and beta(3) integrins when angiogenesis occurs in fibrin. Thus, pharmacologic targeting of integrin receptors aimed at blocking neovessel formation and survival must be tailored to the specific extracellular matrix environment in which angiogenesis takes place.

Angiopoietin-1 and vascular endothelial growth factor induce expression of inflammatory cytokines before angiogenesis.

The purpose of this study was to identify novel transcriptional events occurring in the aortic wall before angiogenesis. We used a defined tissue culture system that takes advantage of the capacity of rat aortic rings to generate neovessels ex vivo in response to angiogenic factor stimulation. Total RNA isolated from aortic rings 18 h posttreatment with angiopoietin (Ang)-1 or vascular endothelial growth factor (VEGF) was used to probe oligonucleotide microarrays. Many genes were up- or downregulated by either Ang-1 or VEGF, with a subset being affected by treatment with both growth factors. Grouping of genes by biological function revealed that Ang-1 and VEGF both upregulated a host of immune-related genes including many inflammatory cytokines. A mixture of the Ang-1- and VEGF-induced cytokines stimulated the spontaneous angiogenic response of aortic rings and was synergistic with a low dose of recombinant VEGF. This effect was associated with enhanced recruitment of adventitial macrophages and dendritic cells in the angiogenic outgrowths. Thus Ang-1 and VEGF activate the innate immune system of the vessel wall, stimulating the production of proangiogenic inflammatory cytokines before the emergence of neovessels. This hitherto unreported feature of the angiogenic response might represent an important early component of the cellular and molecular cascade responsible for the angiogenic response of the aortic wall.

A new ex vivo model to study venous angiogenesis and arterio-venous anastomosis formation.

Explants of rat inferior vena cava embedded in collagen gel and cultured under serum-free conditions produced microvascular outgrowths composed of endothelial cells and pericytes. Exogenous vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently stimulated angiogenesis and induced the formation of complex networks of highly branched microvessels. VEGF and the VEGF/bFGF combination also promoted pericyte recruitment. Medium conditioned by untreated vena cava cultures contained endogenous VEGF, and a blocking antibody against VEGF significantly reduced the spontaneous angiogenic response of the explants. Vena cava explants exhibited a greater capacity to form neovessels than aortic rings when tested in parallel cultures from the same animal. When compared with aorta-derived microvessels, neovessels of vena cava origin were longer and had fewer pericytes. Vena cava-aorta cocultures produced extensive anastomosing networks of microvessels, which were primarily contributed by the venous explants. Because of its florid angiogenesis and exquisite sensitivity to angiogenic factor stimulation, the vena cava model may provide novel insights into the regulation of the angiogenic process, which typically initiates from the venous side of the vascular bed. Combined with the aortic ring model, this new assay may also enhance our understanding of the mechanisms of anastomosis formation between the arterial and the venous circulations.

Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

Recent studies have implicated the Tie2 tyrosine-kinase receptor and its main ligands--angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2)--as crucial regulators of mural cell recruitment during angiogenesis. Angiopoietin-mediated activation of Tie2 promotes perivascular mural cell assembly, but the mechanisms regulating this process are poorly understood because differentiated mural cells do not have the Tie2 receptor, which is reportedly expressed only in endothelial cells. There is also no direct evidence that Tie2 activation results in production of mural cell chemoattractants by the endothelium. In the rat aorta model of angiogenesis, developing microvessels recruit mural cells from the intimal/subintimal layers of the aortic wall. Ang-1 and Ang-2 promote angiogenesis in this system, stimulating branching morphogenesis and mural cell assembly. Mural precursor cells (MPCs) isolated with a nonenzymatic method from the intimal aspect of the rat aorta were positive for smooth muscle cell markers (alpha-smooth muscle actin and calponin) and negative for endothelial markers (factor-VIII-related antigen and CD31). These cells responded chemotactically to Ang-1 and Ang-2, and secreted MMP-2 when treated with these factors. Western-blot analysis, immunocytochemistry and RT-PCR demonstrated that MPCs express the Tie2 receptor. Immunoprecipitation showed phosphorylation of MPC Tie2 on tyrosine residues upon stimulation with Ang-1 or Ang-2. Surface expression of Tie2 was further demonstrated by isolating Tie2+/alpha-smooth muscle actin+ MPCs from primary aortic outgrowths with anti-Tie2-IgG-coated magnetic beads. Immunostaining of the rat aorta confirmed expression of Tie2 not only in endothelial cells but also in nonendothelial mesenchymal cells located in the aortic intimal/subintimal layers, which are the source of MPCs. These data indicate that the aortic wall contains Tie2+ nonendothelial mesenchymal cells and suggest that Tie2-related recruitment of mural cells during angiogenesis may occur through angiopoietin-mediated direct stimulation of these cells.

The mouse aorta model: influence of genetic background and aging on bFGF- and VEGF-induced angiogenic sprouting.

Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unlike rat aorta explants, unstimulated mouse aortic rings were unable to spontaneously produce an angiogenic response under serum-free conditions. They, however, responded to bFGF and VEGF, generating networks of branching neovessels. Aortic rings from GFP-Tie2-transgenic mice generated GFP-labeled neovessels that could be easily identified by their distinctly green fluorescence. Aortic rings from 1- to 2-month-old mice produced microvessels faster, more uniformly and in greater number than aortic rings from 6- to 10-month-old mice, particularly in VEGF-treated cultures. Aortic rings from 129/SVJ mice were capable of a much stronger and sustained angiogenic response to bFGF than those of C57BL/6 or BALB/c mice, which were in turn more angiogenic than aortic rings from FVB mice. The same strains of mice responded differently to VEGF, as C57BL/6 mouse aortic rings produced more microvessels than those of BALB/c, FVB, and 129/SVJ mice, which were capable of only a limited response. The significant impact that aging and genetic background have on mouse aortic angiogenesis should be taken into account when the aortic-ring assay is used to evaluate function of genes that have been deleted or overexpressed in genetically modified mice.