Introduction: Neutrophil extracellular traps (NETs) are originally described as proinflammatory, antimicrobial structures consisting of extracellular chromatin decorated with granular and cytoplasmic proteins. The formation of NETs requires peptidylarginine deiminase (PAD) 4. Recent studies revealed that NETs can play an important role in promoting inflammation in vascular diseases. Inflammation is emerging as a key component of the pathophysiology of intracranial aneurysm. In both human and mouse studies, activated neutrophils were shown to be associated with formation and rupture of intracranial aneurysms. Therefore, we hypothesized that the formation of NETs by PAD4 promotes the development of aneurysm rupture. We tested this hypothesis utilizing both pharmacological and genetic approaches in mice.
Methods: We used 8- to 10-week-old male C57BL/6J mice, global PAD4 knock out (KO) and granulocyte-specific PAD4 KO mice. Aneurysm was induced by a combination of elastase injection and hypertension as we previously describes. We used deoxycorticosterone acetate (DOCA)-salt hypertension. A single dose of elastase was injected into the cerebrospinal fluid using a stereotaxic approach.
Results: Cl-amidine, an inhibitor for PAD4, significantly decreased the rupture rates (92 vs 36%, P < 0.01). At the same time, Cl-amidine decreased the number of neutrophils and the formation of NETs analyzed by Ly-6G staining and citrullinated histone H3 staining, respectively. In the inhibitor treated mice, the messenger ribonucleic acid (RNA) level of ICAM-1, MCP-1 and TNFα in the cerebral arteries were lower than that of vehicle control treated mice. Furthermore, the rupture rates were decreased in the global PAD4 KO mice and the granulocyte-specific PAD4 KO mice than in the corresponding wild type mice groups. Finally, inhibition of the formation of NETs by DNase treatment also reduced the rupture rates.
Conclusions: Our data indicate that the formation of NETs by granulocyte PAD4 promotes the development of aneurysm rupture. NETs may be represented a potential therapeutic target for the prevention of aneurysm rupture.