Angiopoietin 2 in inflammation, a primary role in the invasive process?
Authors: Trevor J Markham, Ursula Fearon, Oliver FitzGerald, Douglas Veale
Journal: Journal of the American Academy of Dermatology (2004)

Angiopoietin 2 (Ang2) is a member of a family of angiogenic factors that, in the presence of VEGF, may be critical for early tube formation and blood vessel maturation. We have previously shown high expression in early inflammatory arthritis correlates with increased synovial membrane (SM) vascularity. The aim of the present study is to examine the expression and the functional role of Ang2 in inflammatory cells. Methods: Ang2 mRNA expression was quantified using Real Time PCR in psoriasis (Ps) skin from psoriatic arthritis patients, early SM; synovial T cells, 1o synovial fibroblast (SF) cultures from RA and dermal microvascular endothelial cells cultures (ECs). Protein expression was demonstrated in skin and SM sections using immunohistology. SM explant cultures, SFs and ECs were stimulated with Ang2 (200ng/ml) alone and in combination with VEGF (20ng/ml). MMP-1, MMP-3 and TIMP-1 expression was measured by ELISA in culture supernatants. Cell surface expression of ICAM and PECAM was measured in SFs and ECs using Flow cytometry. EC tubule formation was examined using matrigels. Results: Ang2 mRNA was expressed as a ratio of Ang2 /B-Actin and was demonstrated in all Ps skin, SM, SFs, T cells, and ECs. Ang2 mRNA levels were higher in SM biopsies (Ang2/B-actin: 1.5) compared to Ps skin biopsies (1.7). In the SM, protein expression was localised to the lining layer and perivascular regions, with patchy expression in the synovial infiltrate, while in the Ps skin, Ang2 was localised to the epidermis and perivascular regions. Ang2 mRNA levels were significantly higher in the ECs with a ratio of (Ang2/B-actin; 1.1) compared to SFs (1.7) and T cells which had the lowest expression with a ratio of 2. This reflected Ang2 protein level and localisation in the skin and the SM. MMP-1 and TIMP-1 expression was increased 2-3-fold following stimulation with Ang2 in the presence of VEGF, in SM explant cultures, SFs and ECs, but MMP-3 expression was only minimally increased. Tubule formation increased following stimulation with Ang2 or VEGF, however the greatest increase followed stimulation with Ang2 in the presence of VEGF. There was no significant difference in ICAM and PECAM expression. Conclusion. These data demonstrate increased Ang2 expression in inflamed skin and SM, epsecially in ECs and SFs. Ang2 increased ECs tubule formation and matrix turnover in SM explant, EC and SF cultures suggesting it plays a dual role. In the EC it is critical for angiogenesis and advancement of the new blood vessel through the dermis and SM by matrix turnover, and in the SFs at the site of erosion, where the lining layer invades and destroys cartilage and bone. These results suggest Ang2 is an important factor for angiogenesis and joint erosion in inflammation. Angiopoietin 2 (Ang2) is a member of a family of angiogenic factors that, in the presence of VEGF, may be critical for early tube formation and blood vessel maturation. We have previously shown high expression in early inflammatory arthritis correlates with increased synovial membrane (SM) vascularity. The aim of the present study is to examine the expression and the functional role of Ang2 in inflammatory cells. Methods: Ang2 mRNA expression was quantified using Real Time PCR in psoriasis (Ps) skin from psoriatic arthritis patients, early SM; synovial T cells, 1o synovial fibroblast (SF) cultures from RA and dermal microvascular endothelial cells cultures (ECs). Protein expression was demonstrated in skin and SM sections using immunohistology. SM explant cultures, SFs and ECs were stimulated with Ang2 (200ng/ml) alone and in combination with VEGF (20ng/ml). MMP-1, MMP-3 and TIMP-1 expression was measured by ELISA in culture supernatants. Cell surface expression of ICAM and PECAM was measured in SFs and ECs using Flow cytometry. EC tubule formation was examined using matrigels. Results: Ang2 mRNA was expressed as a ratio of Ang2 /B-Actin and was demonstrated in all Ps skin, SM, SFs, T cells, and ECs. Ang2 mRNA levels were higher in SM biopsies (Ang2/B-actin: 1.5) compared to Ps skin biopsies (1.7). In the SM, protein expression was localised to the lining layer and perivascular regions, with patchy expression in the synovial infiltrate, while in the Ps skin, Ang2 was localised to the epidermis and perivascular regions. Ang2 mRNA levels were significantly higher in the ECs with a ratio of (Ang2/B-actin; 1.1) compared to SFs (1.7) and T cells which had the lowest expression with a ratio of 2. This reflected Ang2 protein level and localisation in the skin and the SM. MMP-1 and TIMP-1 expression was increased 2-3-fold following stimulation with Ang2 in the presence of VEGF, in SM explant cultures, SFs and ECs, but MMP-3 expression was only minimally increased. Tubule formation increased following stimulation with Ang2 or VEGF, however the greatest increase followed stimulation with Ang2 in the presence of VEGF. There was no significant difference in ICAM and PECAM expression. Conclusion. These data demonstrate increased Ang2 expression in inflamed skin and SM, epsecially in ECs and SFs. Ang2 increased ECs tubule formation and matrix turnover in SM explant, EC and SF cultures suggesting it plays a dual role. In the EC it is critical for angiogenesis and advancement of the new blood vessel through the dermis and SM by matrix turnover, and in the SFs at the site of erosion, where the lining layer invades and destroys cartilage and bone. These results suggest Ang2 is an important factor for angiogenesis and joint erosion in inflammation.