Neutrophils: Novel Contributors to Estrogen-Dependent Intracranial Aneurysm Rupture Via Neutrophil Extracellular Traps

Abstract

Background Intracranial aneurysms (IAs) are more prevalent in women than men, and aneurysmal subarachnoid hemorrhage disproportionately affects postmenopausal women. These sex differences suggest estrogen protects against IA progression that can lead to rupture, but the underlying mechanisms are not fully understood. Although studies have demonstrated estrogen regulates inflammatory processes that contribute to IA pathogenesis, the role of neutrophils remains to be characterized. Using a murine model, we tested our hypothesis that neutrophils contribute to IA pathophysiology in an estrogen-dependent manner. Methods and Results We compared neutrophil infiltration in C57BL/6 female mice that develop IAs to those with a normal circle of Willis. Next, we investigated the estrogen-dependent role of neutrophils in IA formation, rupture, and symptom-free survival using a neutrophil depletion antibody. Finally, we studied the role of neutrophil extracellular trap formation (NETosis) as an underlying mechanism of aneurysm progression. Mice that developed aneurysms had increased neutrophil infiltration compared with those with a normal circle of Willis. In estrogen-deficient female mice, both neutrophil depletion and NETosis inhibition decreased aneurysm rupture. In estrogen-deficient female mice treated with estrogen rescue and estrogen-intact female mice, neither neutrophil depletion nor NETosis inhibition affected IA formation, rupture, or symptom-free survival. Conclusions Neutrophils contribute to aneurysm rupture in an estrogen-dependent manner. NETosis appears to be an underlying mechanism for neutrophil-mediated IA rupture in estrogen deficiency. Targeting NETosis may lead to the development of novel therapeutics to protect against IA rupture in the setting of estrogen deficiency.

Keywords: NETosis; aneurysm formation; aneurysm rupture; estrogen deficiency; neutrophils.

Figure 1. Neutrophil infiltration in murine intracranial aneurysms (IAs).

A, Representative images of immunohistochemistry for neutrophils in normal intracranial arteries (top) compared with aneurysms (bottom) in female ovariectomy (OVE) mice and female sham OVE mice. Red=neutrophil elastase, blue=4′,6‐diamidino‐2‐phenylindole nuclear counterstain, ×40. Scale bar=50 μm. B, There are significantly more neutrophils in aneurysms compared with normal arteries in estrogen‐intact female mice (40.42.0±9.7 vs 1.92±1.28 relative fluorescence units [RFU]; n=5 each; P=0.0079) and estrogen‐deficient female mice (48.65±7.98 vs 2.30±3.04 RFU; n=5; P=0.0079).

Figure 2. Effect of neutrophil depletion on intracranial aneurysm formation, rupture, and symptom‐free survival.

A, In estrogen‐deficient female mice, neutrophil depletion (antipolymorphonuclear) significantly decreased aneurysm rupture compared with rabbit serum control (RS control) (50% [6/12] vs 92% [12/13]; P=0.03). Neutrophil depletion had no significant effect on aneurysm formation (75% [12/16] vs 87% [13/15]; P=0.65) or symptom‐free survival (69% vs 47%; P=0.19). B, Estradiol rescue in neutrophil‐depleted, estrogen‐deficient female mice resulted in no difference compared with the control group in intracranial aneurysm formation (58% [7/12] vs 67% [8/12]; P=1.00), rupture (57% [4/7] vs 63% [5/8]; P=1.00), or symptom‐free survival (80% vs 67%; P=0.81). C, In estrogen‐intact female mice, neutrophil depletion did not affect aneurysm formation (52% [11/21] vs 63% [12/19]; P=0.54), rupture (45% [5/11] vs 55% [6/11]; P=1.00), or symptom‐free survival (71% vs 74%; P=0.93). E2 indicates estradiol; and PMN, polymorphonuclear.

Figure 3. Neutrophil extracellular trap formation (NETosis) in murine IAs.

Immunohistochemistry was used to identify NETosis in female sham OVE mice and female OVE mice. Co‐staining of histone H3 and neutrophil elastase, a marker for NETosis, was seen in murine intracranial aneurysm tissue (bottom row, white arrows) but not in normal intracranial vasculature (top row). Red=histone H3, green=neutrophil elastase, blue=4′,6‐diamidino‐2‐phenylindole nuclear counterstain. ×40. Scale bar=50 μm. Neu indicates neutrophil; and OVE, ovariectomy.

Figure 4. NETosis inhibition in estrogen‐deficient female mice and estrogen‐intact female mice.

A, In estrogen‐deficient female mice, NETosis inhibition (Cl‐amidine+DNase I) significantly reduced aneurysm rupture compared with dimethyl sulfoxide control (17% [1/6] vs 73% [8/11]; P=0.0498) but did not affect aneurysm formation (40% [6/15] vs 73% [11/15]; P=0.14) or symptom‐free survival (93% vs 67%; P=0.07). B, NETosis inhibition in estrogen‐intact female mice did not affect aneurysm formation (47% [7/15] vs 60% [9/15]; P=0.72), rupture (43% [3/7] vs 56% [5/9]; P=1.00), or symptom‐free survival (80% vs 73%; P=0.69). DMSO indicates dimethyl sulfoxide; DNase, deoxyribonuclease; and NETosis, neutrophil extracellular trap formation.