(188) THE ROLE OF CELL-FREE MITOCHONDRIAL DNA IN NLRP3 INFLAMMASOME ACTIVATION IN STRESS-INDUCED VASCULAR AND ERECTILE DYSFUNCTION
Authors: Rodrigues Dos Passos R , Priviero F , Dos Anjos Moraes R , Arishe O , Vieira Dos Santos C , Wilczynski S , Crockett A , Frambes N , Cavalli E , Stegmann K , Churillo A , Wood S , Spinale F , Ryan M , Hollis F , Webb RC 
Published: December 2024

Abstract

Introduction
Stress causes vascular and erectile dysfunction, with inflammation being a major factor. Cell-free mitochondrial DNA (mtDNA) activates the NLRP3 inflammasome, promoting pro-inflammatory cytokine release via gasdermin-D pore formation, causing inflammation. However, the mechanisms by which chronic stress-induced inflammation affects the vasculature and leads to vasculogenic erectile dysfunction remain unclear.

Objective
This study aimed to test the hypothesis that chronic stress promotes mtDNA release, activating the NLRP3 inflammasome pathway and causing vascular and erectile dysfunction in male mice.

Methods
Male C57Bl/6 mice were submitted to chronic unpredictable stress (CUS) via daily exposure to different stressors for 28 days, while control animals were submitted to handling (non-CUS). Anxiety-like behavior, body weight, and systolic blood pressure (SBP) were assessed. Mesenteric resistance arteries (MRA), pudendal arteries (PA), and corpus cavernosum (CC) were removed and mounted in myographs to evaluate reactivity. Concentration-response curves to acetylcholine (ACh; 1 nM–30 uM) and phenylephrine (PE; 1 nM–30 uM) were performed. Non-linear regression provided maximal response (Emax) and potency (pEC50) values for ACh and PE. Isolation of mtDNA from the bladder was performed using a purified mtDNA isolation kit following the manufacturer’s instructions. In vitro studies we performed by incubating MRA endothelial cells with 4 μg/mL of mtDNA, isolated from the liver of control animals, for 24 hours. This was done in the presence or absence of 10 μM MCC950, an NLRP3 inhibitor. NLRP3 and gasdermin-D expression were assessed by Western blot and flow cytometry. Group comparisons used Student’s t-test, with data presented as mean ± S.E.M. Significance was set at p &lt; 0.05.

Results
Stress increased SBP by 17 ± 4.2% (p = 0.003), decreased body weight gain by 53% (p = 0.04), and induced anxiety-like behavior (p = 0.01) in mice exposed to CUS when compared to non-CUS. In the vasculature, CUS led to endothelial dysfunction observed by reduced potency of ACh in the MRA (pEC50; CUS: 6.17 ± 0.09 vs non-CUS: 6.81 ± 0.11; p = 0.003) and PA (pEC50; CUS: 6.31 ± 0.07 vs non-CUS: 6.65 ± 0.09; p = 0.008). Moreover, CUS led to a significant decrease in the maximal relaxation induced by ACh in the CC (CUS: 75 ± 4% vs. non-CUS: 88 ± 4%) as well as an impaired contraction to PE (CUS: 1.07 ± 0.15% vs. non-CUS: 1.72 ± 0.17%). To assess mtDNA content, we isolated mtDNA from bladder tissue (due to limited sample availability) and found that CUS mice had significantly higher mtDNA levels in their bladders compared to controls. In endothelial cells, 24-hour incubation with mtDNA increased NLRP3 expression (78% vs 84%) compared to the vehicle, but this was reversed by co-incubation with MCC950 (70%). Similarly, gasdermin-D expression increased after 24 hours of mtDNA incubation (11% vs 16%) and was also reversed by MCC950 co-incubation (10%). Finally, in the MRA, we observed increased expression of NLRP3 (CUS: 1.5 ± 0.6 vs non-CUS: 0.7 ± 0.2) and gasdermin-D (CUS: 0.42 ± 0.1 vs non-CUS: 0.16 ± 0.07) and, in the CC, gasdermin-D expression (CUS: 0.41 ± 0.07 vs non-CUS: 0.18 ± 0.08) was found to be increased in mice exposed to CUS.

Conclusions
These data suggest that CUS led to increased mtDNA release and NLRP3 inflammasome pathway activation, promoting vascular and erectile dysfunction in male mice.

Disclosure
No.