Background/Purpose: Systemic sclerosis (SSc, also known as scleroderma) is a systemic disease characterized by fibrosis, autoimmunity, and vasculopathy. Neutrophil extracellular traps (NETs) are web‐like chromatin structures released by activated neutrophils. They contribute to the vascular pathology in multiple diseases, but their role in SSc remains poorly understood. We recently demonstrated elevated plasma NET levels in SSc patients with vascular complications compared to those without vascular complications. Here, we sought to determine the direct effects of NET‐rich SSc plasma on microvascular endothelial cells (MVECs).

Methods: Human dermal MVECs were cultured in the presence of 2% pooled plasma from SSc patients with vascular complications exhibiting high circulating NET remnants (n=5), 2% pooled plasma from matched SSc patients without vascular complication exhibiting low circulating NET remnants (n=5), 2% pooled plasma from matched healthy controls (n=5), or purified NETs (0.1-0.5 µg/ml). Proliferation was quantified over 24 hours and calculated as the relative confluence change. Migration was evaluated via a scratch‐wound assay over 24 hours and quantified by wound confluence (%).

Results: We first stimulated MVECs with different concentrations of purified NETs (0.1 µg/ml versus 0.5 µg/ml) for 24 hours. Compared with vehicle control, NETs (0.5 µg/ml) increased MVEC proliferation (1.4-fold change, P=0.0001) and migration (mean 52% vs. 23% wound confluence, P=0.0001) at 24 hours in a concentration-dependent manner (Figure 1). Next, we stimulated MVECs with 2% NET‐rich SSc plasma compared with 2% NET-poor SSc plasma or 2% healthy control plasma. We found that NET-rich SSc plasma promoted MVECs proliferation (P=0.0003) and migration (P=0.0001) over 24 hours. In contrast, the effects of NET‐poor SSc plasma on proliferation (P=0.986) and migration (P=0.729) were comparable to healthy control plasma (Figure 2). Notably, NET-targeting agents, such as anti-histone 4 and a neutrophil elastase inhibitor, significantly reduced MVEC proliferation and migration induced by NET-rich SSc plasma.

Conclusion: Our study demonstrates that SSc plasma containing high levels of NETs robustly stimulates MVEC proliferation and migration in vitro, and that NET-targeting agents can reverse these effects significantly. These data preliminarily identify NETs as mediators of abnormal angiogenesis contributing to the aberrant and dysfunctional characteristics of the SSc microvasculature. Targeting NET formation may represent a novel therapeutic avenue to restore vascular homeostasis and ameliorate microvascular complications in SSc.

Figure 1: NETs increased MVECs proliferation and migration. MVECs were stimulated with NETs (0.1 or 0.5 µg/ml) or basic medium as a control for 24 hours. IncuCyte software was used to quantify (A) cell proliferation as the relative confluence change compared to time 0 hour and (B) cell migration by wound confluence (%). Data were represented as means ± sem. Two-way ANOVA test was applied; (**p < 0.01, ****p < 0.0001).

Figure 2: NET-rich SSc plasma increased MVECs proliferation and migration. MVECs were stimulated with 2% NET-rich SSc plasma, 2% NET-poor SSc plasma, 2% healthy control plasma for 24 hours. IncuCyte software was used to quantify (A) cell proliferation as the relative confluence change compared to time 0 hour and (B) cell migration by wound confluence (%). Data were represented as means ± sem with n=3 independent experiments. Two-way ANOVA test was applied; (**p < 0.01, ****p < 0.0001).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-trap-rich-systemic-sclerosis-plasma-promotes-microvascular-endothelial-cell-proliferation-and-migration-implications-for-aberrant-angiogenesis/