Background/Purpose:

Scleroderma (SSc) is a complex disease characterized by inflammation, vasculopathy, and excessive deposition 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. 8-isoprostane is an oxidized lipid created by excessive oxidative stress, and has been shown to be elevated in SSc. The thromboxane A2 receptor (TXAR) and ROCK pathway, which can be activated by 8-isoprostane (8-IP), inhibits VEGF-induced endothelial cell (EC) differentiation and migration. However, its role in SSc has not been examined. In this study we determined whether the TXAR pathway was activated by 8-IP in SSc ECs. Its effect on VEGF-induced angiogenesis was also determined.

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. TXAR expression was determined by qPCR and Western blotting. Knockdown studies were performed using TXAR siRNAs.

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). Increased oxidative stress was detected in SSc ECs as increased 8-IP in SSc EC conditioned media and excessive superoxide in SSc ECs were observed. In healthy ECs, 8-IP inhibited VEGF-induced EC migration, and the inactivation of TXAR or ROCK pathways restored VEGF-induced angiogenesis inhibited by 8-IP. In SSc ECs, VEGF did not induce EC migration, however, addition of the TXAR or ROCK inhibitors restored the pro-angiogenic effect of VEGF. This was further confirmed by TXAR siRNA experiments which showed that TXAR-knockdowned SSc ECs migrated towards VEGF while the SHAM-transfected ECs did not. We then measured ROCK activity in healthy and SSc ECs before and after VEGF or 8-IP stimulation. Basal ROCK activity was significantly higher in SSc ECs compared to healthy ECs. Moreover, 8-IP-induced ROCK activation was significantly higher in SSc ECs while VEGF induced significantly higher ROCK activation in healthy ECs. The expression of key players in this pathway was also examined. The protein expression of TXAR, RhoA, ROCK1/2 were all elevated in SSc ECs compared to healthy ECs.

Conclusion:

We show that 8-IP inhibits VEGF-induced migration in healthy ECs through the TXAR/ROCK pathway. ECs not only produce high levels of 8-IP, but also show elevated expression of TXAR and RhoA/ROCK levels. This could explain the increased activation of the TXAR pathway in terms of ROCK activity in SSc ECs compared to healthy ECs. This hyper-activation leads to inhibition of VEGF-induced EC migration, as using the TXAR or ROCK inhibitor, as well as specific knockdown of TXAR, results in restoration of VEGF activity. These results suggest that the TXAR pathway plays a crucial role in angiogenesis and that 8-IP is not just a by-product as a result of oxidative stress, but instead plays a significant role in impaired angiogenesis that characterizes SSc.

---

« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/activation-of-the-thromboxane-a2-receptor-by-8-isoprostane-inhibits-the-pro-angiogenic-effect-of-vascular-endothelial-growth-factor-in-scleroderma/