Background/Purpose:

Scleroderma (SSc) is a complex disease characterized by inflammation, vasculopathy, and excessive disposition of extracellular matrix. Various studies have demonstrated a paradoxical increase in angiogenic mediators, such as vascular endothelial growth factor (VEGF), in both the skin and serum of patients with SSc. Despite this, angiogenesis does not occur normally. The signaling of the thromboxane A2 receptor (TXAR), which can be activated by 8-isoprostane (8-IP), has been shown to inhibit angiogenesis as well as VEGF-induced endothelial cell (EC) differentiation and migration. However, its role in SSc has not been examined. In this study we examined whether 8-IP was elevated in patient plasma and conditioned medium (CM) from ECs, and examined its’ effect on VEGF-induced angiogenesis in SSc.

Methods:

Dermal ECs were isolated from punch biopsies from healthy subjects or patients with diffuse cutaneous SSc. Angiogenesis was assessed by chemotaxis and in vitroMatrigel tube formation assays. 

Results:

SSc patients had significantly higher 8-IP plasma levels (60.9±8.4 pg/ml) compared to healthy subjects (24.9±5.0 pg/ml, p<0.05). When divided into disease subtypes, patients with diffuse SSc or interstitial lung disease showed elevated 8-IP, while those with limited SSc or pulmonary hypertension did not. Increased oxidative stress was detected in SSc ECs as increased 8-IP in SSc EC CM was observed (15.1±2.8 vs. 27.8±3.6 pg/ml, p<0.05). To test whether the increased 8-IP levels in CM affected angiogenesis, we performed Matrigel tube formation assay using NL ECs. SSc EC CM decreased VEGF-induced tube formation in NL ECs while addition of vitamin E restored it. In addition, 8-IP inhibited VEGF-induced healthy EC migration, and the inhibition of TXAR or ROCK pathways restored VEGF-induced angiogenesis inhibited by 8-IP. We then measured ROCK activity in NL and SSc ECs before and after VEGF or 8-IP stimulation. Basal ROCK activity was significantly higher in SSc ECs compared to healthy ECs. 8-IP-induced ROCK activation was significantly higher in SSc ECs while VEGF induced significantly higher ROCK activation in healthy ECs. 

Conclusion:

We show that 8-IP inhibits VEGF-induced migration in healthy ECs through the TXAR/ROCK pathway. SSc ECs produce high levels of 8-IP and exhibit elevated ROCK activity compared to healthy ECs, suggesting that this pathway plays a crucial role in impaired angiogenesis in SSc. This is supported by using vitamin E, which decreases 8-IP, in the Matrigel in vitro assay, in which it restores the inhibitory effect of SSc EC CM on VEGF-induced angiogenesis in healthy ECs. This study provides a potential link between oxidative stress and impaired angiogenesis in SSc, and shows that 8-IP is not just a by-product as a result of oxidative stress, rather it plays a significant role in impaired angiogenesis that characterizes SSc.

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« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/elevated-8-isoprostane-in-scleroderma-implications-of-its-role-in-inhibiting-vascular-endothelial-growth-factor-induced-angiogenesis/