Expression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma

doi:10.1016/j.trsl.2006.12.007

Translational Research

Volume 149, Issue 5, May 2007, Pages 265-273

https://doi.org/10.1016/j.trsl.2006.12.007 Get rights and content

Angiogenesis is a crucial component of bone remodeling under both normal and pathophysiological conditions. Among the various mediators that regulate the angiogenic process is the angiopoietin (Ang) family of growth factors. Ang-1 stabilizes new blood vessels by recruiting surrounding mesenchymal cells and promoting their differentiation into vascular smooth muscle cells, whereas Ang-2 is a natural antagonist of Ang-1 and can inhibit angiogenesis. The expression of Ang-1 and Ang-2 in human osteoblasts (hOBs) isolated from rheumatoid arthritis (RA) and osteoarthritis (OA) patients and from healthy individuals has been examined. After incubation in the presence or absence of tumor necrosis factor-alpha (TNF-α) and/or interferon-gamma (IFN-γ), the culture supernatants were assayed for Ang using an enzyme-linked immunosorbent assay. In addition, expression of Ang protein and mRNA was examined using immunohistochemical techniques and quantitative real-time polymerase chain reaction, respectively. It was found that hOBs expressed Ang-1 but not Ang-2 protein, and cultured hOBs from RA and OA patients and from healthy individuals all spontaneously secreted significant amounts of Ang-1 in the absence of any stimulation. Although stimulation with TNF-α or IFN-γ had little or no effect on Ang-1 secretion, costimulation with IFN-γ plus TNF-α dose- and time-dependently diminished secretion of Ang-1 from hOBs. This inhibitory effect was mediated in part by nuclear factor-kappa B via upregulated expression of inducible nitric oxide synthase and enhanced synthesis of nitric oxide. Taken together, these findings suggest that OBs are an important cellular source of Ang-1 and may modulate bone remodeling through regulation of angiogenesis.

Section snippets

Reagent preparation

Completed medium consisted of Dulbecco’s modified eagle’s medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 2-mM L-glutamine, 100-U/mL penicillin, 100-μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY). Monoclonal and biotinylated polyclonal antibodies (Abs) against human Ang-1 and Ang-2 and TNF-α and interferon-gamma (IFN-γ) were from Genzyme/Techne (Cambridge, Mass). The NO scavenger L-nitro arginine methyl ester

Secretion of OB-derived angiopoietins and the effects of TNF-α and IFN-γ on levels of hOB-derived Ang-1

It was hypothesized that OBs are an important cellular source of the angiogenic factors needed during bone remodeling. To test this idea, hOBs were first isolated from RA (RA hOB) and OA patients (OA hOB) and healthy controls, after which secreted and cell-associated angiopoietins (Ang-1 and Ang-2) were assayed by ELISA. Significant amounts of Ang-1 were spontaneously secreted into the culture supernatants by RA, OA, and healthy hOBs (mean: 6206.7 pg/mL, 6349.9 pg/mL, and 5990.4 pg/mL,

Discussion

It has been shown here that OBs from patients with RA or OA as well as healthy hOBs constitutively express and secrete Ang-1. Furthermore, both the expression and the secretion of Ang-1 were negatively regulated by the combination of TNF-α and IFN-γ. This downregulation of OB-derived Ang-1 was mediated via an NF-κB-dependent pathway leading to induction of NO synthesis. Notably, it was previously reported that Ang-1 is upregulated in endothelial cells and RA synovial fibroblasts by TNF-α via an

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Original articleExpression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma

Section snippets

Reagent preparation

Secretion of OB-derived angiopoietins and the effects of TNF-α and IFN-γ on levels of hOB-derived Ang-1

Discussion

Microvasc Res

Biochem Biophys Res Commun

Bone

Cell

Bone

Trends Immunol

Bone

J Biol Chem

Bone

J Biol Chem

Bone

The osteoblast: a sophisticated fibroblast under central surveillance

Science

Osteoblasts and osteoclasts in bone remodeling and inflammation

Curr Drug Targets Inflamm Allergy

Immunobiology and regulatory mechanisms of the expression of angiogenic and angiostatic factors in arthritis inflammation

Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth, and repair

Curr Top Dev Biol

Angiogenesis in health and disease: insights into basic mechanisms and therapeutic opportunities

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The effects of angiopoietin-1 and -2 on tumor growth and angiogenesis in human colon cancer

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Original article
Expression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma