The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time- and context-dependent inhibitory and stimulatory properties.

BACKGROUND: Tissues respond to injury by releasing acute phase reaction (APR) proteins which regulate inflammation and angiogenesis. Among the genes upregulated in wounded tissues are tumor necrosis factor-alpha (TNFα) and the acute phase reactant orosomucoid-1 (ORM1). ORM1 has been shown to modulate the response of immune cells to TNFα, but its role on injury- and TNFα-induced angiogenesis has not been investigated. This study was designed to characterize the role of ORM1 in the angiogenic response to injury and TNFα. METHODS AND RESULTS: Angiogenesis was studied with in vitro, ex vivo, and in vivo angiogenesis assays. Injured rat aortic rings cultured in collagen gels produced an angiogenic response driven by macrophage-derived TNFα. Microarray analysis and qRT-PCR showed that TNFα and ORM1 were upregulated prior to angiogenic sprouting. Exogenous ORM1 delayed the angiogenic response to injury and inhibited the proangiogenic effect of TNFα in cultures of aortic rings or isolated endothelial cells, but stimulated aortic angiogenesis over time while promoting VEGF production and activity. ORM1 inhibited injury- and TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in aortic rings, but not of NFκB. This effect was injury/TNFα-specific since ORM1 did not inhibit VEGF-induced signaling, and cell-specific since ORM1 inhibited TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in macrophages and endothelial cells, but not mural cells. Experiments with specific inhibitors demonstrated that the MEK/ERK pathway was required for angiogenesis. ORM1 inhibited angiogenesis in a subcutaneous in vivo assay of aortic ring-induced angiogenesis, but stimulated developmental angiogenesis in the chorioallantoic membrane (CAM) assay. CONCLUSION: ORM1 regulates injury-induced angiogenesis in a time- and context-dependent manner by sequentially dampening the initial TNFα-induced angiogenic response and promoting the downstream stimulation of the angiogenic process by VEGF. The context-dependent nature of ORM1 angioregulatory function is further demonstrated in the CAM assay where ORM1 stimulates developmental angiogenesis without exerting any inhibitory activity.

Paracrine regulation of angiogenesis by different cell types in the aorta ring model.

The development of blood vessels during angiogenesis is the result of paracrine interactions between tube-forming endothelial cells and angiogenic factor-producing nonendothelial cells. This process can be reproduced and studied under chemically defined culture conditions by culturing vascular explants in three-dimensional gels of extracellular matrix. Rings of rat or mouse aorta cultured in collagen, fibrin or basement membrane gels produce angiogenic outgrowths composed of a mixed population of endothelial cells and nonendothelial cells. Aortic angiogenesis is regulated by endogenous angiogenic factors, inflammatory cytokines, chemokines, extracellular matrix molecules, and proteolytic enzymes produced by cells of the vessel wall in response to the injury of the dissection procedure. In this paper, we review how macrophages, mural cells and fibroblasts regulate different stages of the angiogenic process, from the formation of immature endothelial sprouts to the reabsorption of the neovessels. We also describe how aortic cultures can be used to study interactions between angiogenic outgrowths and nonvascular cell types such as bone marrow macrophages, platelets or cancer cells. Morphologic, genetic and functional studies of this model have provided invaluable information on how vessels form, mature, interact with nonvascular cell types, and are eventually reabsorbed. Further analysis of the paracrine cross-talk between aortic endothelial and nonendothelial cells is likely to provide new insights into the angiogenic process and its key mechanisms.

Macrophage-derived tumor necrosis factor-alpha is an early component of the molecular cascade leading to angiogenesis in response to aortic injury.

OBJECTIVE: The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the cascade of gene activation that regulates aortic angiogenesis in response to injury. METHODS AND RESULTS: Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNFα gene disruption and recombinant TNFα or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNFα-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNFα and TNFα-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNFα. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNFα gene disruption and promoted by exogenous TNFα. Antibody-mediated inhibition of TNF receptor 1 significantly inhibited angiogenesis. The proangiogenic effect of TNFα was suppressed by blocking VEGF or by ablating aortic macrophages. Exogenous TNFα, however, maintained a limited proangiogenic capacity in the absence of macrophages and macrophage-mediated VEGF production. CONCLUSIONS: Overexpression of TNFα is required for optimal VEGF production and angiogenesis in response to injury. This TNFα/VEGF-mediated angiogenic pathway requires macrophages. The residual capacity of TNFα to stimulate angiogenesis in macrophage-depleted aortic cultures implies the existence of a VEGF-independent alternate pathway of TNFα-induced angiogenesis.