Date of Award

Spring 2026

Degree Type

Thesis

Department

Cell Biology and Anatomy

Director of Thesis

Dr. R. Clinton Webb

Second Reader

Dr. Rinaldo R. dos Passos

Abstract

Hypertension is the leading global risk factor for mortality and is closely associated with vascular inflammation and dysfunction. Damaged mitochondria release cell-free mitochondrial DNA (cf-mtDNA), which activates toll-like receptor 9 (TLR9). The binding of TLR9 to mtDNA activates the NLRP3 inflammasome pathway, promoting the release of pro-inflammatory cytokines via gasdermin D (GSDMD), therefore worsening inflammation. However, the mechanisms linking mitochondrial injury to inflammation-induced vascular dysfunction remain unclear. We hypothesize that cf-mtDNA triggers TLR9-dependent NLRP3 inflammasome activation contributing to vascular and endothelial dysfunction. To assess that, adult male C57BL/6 were exposed to 28 consecutive days of chronic unpredictable stress (CUS) or handling (CON). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were assessed. Also, circulating mtDNA was measured in plasma via mtND1 expression, while hepatic tissue mtDNA levels were measured and quantified using a commercial kit. The vascular reactivity of mesenteric resistance arteries (MRA) was assessed in response to phenylephrine or acetylcholine and then collected to assess gene expression via RT-qPCR. We isolated mtDNA from C57BL/6 mouse livers using a commercial kit. Primary endothelial cells (ECs) from C57BL/6 mice were cultured for 24 hours in culture media containing mtDNA (1 µg/mL) in the presence or absence of the TLR9 antagonist ODN2088 (3 µM). Another set of endothelial cells were incubated with the pro-inflammatory cytokine TNF-α (10ng/mL) for 24h. Following incubation, cells were collected to perform protein expression via Western blot or gene expression via RT-qPCR. Statistical significance was assessed using one-way ANOVA or Student’s t-test with statistical significance set at p < 0.05. Both SBP and DBP, as well as circulating and hepatic mtDNA levels, were elevated in CUS mice compared to CON. In the vasculature, MRAs from CUS mice had increased contraction to phenylephrine and decreased relaxation to acetylcholine. CUS significantly increased the expression of Tlr9, Nlrp3, Caspase-1, and Gsdmd genes. In ECs, incubation with mtDNA promoted increased protein expression of NLRP3 (p< 0.05) and GSDMD (p< 0.05), which was prevented by the concomitant incubation with TLR9 antagonist. Additionally, incubation of ECs with TNF-α promoted the upregulation of the inflammasome gene Nlrp3 and Tlr9 as well as the vascular cell adhesion molecule 1 (Vcam1), a key marker for endothelial activation and dysfunction. These findings suggest that the mtDNA-TLR9 signaling activates the inflammasome pathway in vascular cells, contributing to endothelial and vascular dysfunction.

First Page

1

Last Page

25

Rights

© 2026, Mohin Amin

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