Liver-Derived, Circulating Xanthine Oxidoreductase Drives Vascular Impairment Associated with Inhalation of Ultrafine Particulates

Abstract

Inhalation of ultrafine particles (UFP) mediates systemic vascular impairment which is, in part, driven by elevated rates of oxidant generation. One significant source of oxidant production in the vascular compartment is the purine catabolizing enzyme, xanthine oxidoreductase (XOR). However, mechanisms linking XOR and/or endothelial glycosaminoglycan (GAG)-sequestered XOR to vessel dysfunction allied to UFP inhalation remain underexplored. Based on known interactions between UFP and the liver, we hypothesized that exposure could lead to hepatic release of XOR to the circulation which subsequently contributes to vascular impairment. Utilizing our murine hepatocyte-specific XOR knockout (XOR^Hep-/-) model (loss of function) in conjunction with reintroducing exogenous XOR (restoration of function) we demonstrate a specific role for liver-derived XOR in the pathogenesis of UFP-induced vascular impairment. Exposure of mice as well as in vitro exposure of hepatocytes to our model UFP, nano titanium dioxide (nTiO₂) results in the upregulation and active release of XOR. Drinking water supplemented with the XOR inhibitor febuxostat or nitrite ( $\left({\text{NaNO}}_2^{-}\right)$ ) partially prevented nTiO₂-induced impairment of vascular reactivity. Interestingly, nitrite appears to cause a down-regulation of hepatic XOR. XOR^Hep-/- mice were partially protected against both impairment of endothelial dependent dilation and augmented angiotensin II constriction. To further demonstrate the role of circulating XOR in nTiO₂-induced impairment of vessel reactivity, XOR^Hep-/- mice had circulating XOR restored by i.v. injection prior to exposure, which eliminated the protection of the hepatic knockout. It is important to note that acute restoration of intraluminal XOR in isolated vessels did not alter endothelial-dependent dilation or angiotensin II constriction. As such, we interrogated potential downstream mediators of XOR effects on endothelial function and found a decrease in the repressive trimethylation of lysine 9 on histone 3. Together these findings demonstrate that circulating XOR is a key contributor to endothelial dysfunction caused by UFP exposure. However, the impairment is not acute in nature and might involve epigenetic-mediated alterations in gene expression.

Keywords: glycosaminoglycans; nano-TiO2; nitrate; nitric oxide; nitrite; oxidants; xanthine oxidase.

Fig. 1.. UFPs mediate impairment of endothelial-dependent dilation.

A&B) Pressurized 2^nd order arterioles from sham and nTiO₂ exposed mice (n=5–6) and rats (n=6–10). Below active resting tone of the pressurized arterioles is shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 with, ], indicating group X concentration interaction.

Fig. 2.. UFPs induced XOR expression and subsequent export from hepatocytes.

A&B) Activity of XO in plasma and liver from sham and nTiO₂ exposed mice (n=36–42). C&D) Activity of XO in plasma and liver from sham and nTiO₂ exposed rats (n=6). E&F) Gene and protein expression of XOR in cultured AML12 hepatocytes exposed to 0.5 μg/ml of nTiO₂ for either 6 h (PCR, n=4 in triplicate) or 24 h (WB, n=5–6). G) XO release from AML12 hepatocytes measured in the culture media after 24 h (n=3). H) H₂O₂ production rate measured by CBA following 24 h of 0.5μg/ml of nTiO₂ (n=8). * p<0.05, *** p<0.001, **** p<0.0001.

Fig. 3.. XOR inhibition with febuxostat partially prevents (in vivo) or restores (ex vivo) UFP-associated vascular impairment.

A) Maximal dilation to 1μM acetylcholine in pressurized 2^nd order arterioles with or without febuxostat administered via drink water (starting 5 days before exposer) in sham and nTiO₂ single exposed mice (n=5–13). B&C) Liver and plasma activity of XO in single exposed mice with or without febuxostat administered via drink water (control as in figure 2 shown again for comparison) (febuxostat treated mice n=11–12). D) In-vivo intravital assessment of endothelial-dependent dilation of 3^rd order mesenteric arterioles. Selected arterioles were stimulated with iontophoresis delivery of acetylcholine then Febuxostat delivered via syringe pump and endothelial-dependent dilation reassessed (Sham+febuxostat had no significant effect and is not depicted on the graph) (n=3). E) Ex-vivo administration of febuxostat to pressurized 2^nd order rat arterioles (n=6–10). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, † p<0.005 compared to nTiO₂. nA, nano Amps; Feb, febuxostat.

Fig. 4.. Nitrite supplementation prevents UFP-induced XO upregulation and impairment of endothelial-dependent dilation.

A) Maximal dilation to 1μM acetylcholine in pressurized 2^nd order arterioles with or without nitrite supplementation via drink water (starting 5 days before exposer) in sham and nTiO₂ single exposed mice (n=5–13). B&C) Liver and plasma activity of XO in single exposed mice with or without nitrite supplementation via drink water (control as in figure 2 shown again for comparison) (nitrite treated mice n=9–10). D&E) Expression of XOR measured by WB and PCR in liver lysates of single exposed mice with or without nitrite supplementation. F) Cultured hepatocytes treated with nitrite for 6 hours and Xdh gene expression measured by PCR (n=3 in triplicate). *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 5.. Liver-derived XOR contributes to UFP-associated vessel dysfunction.

A) Endothelial-dependent dilation in pressurized 2^nd order arterioles from sham and nTiO₂ exposed (3), XOR^fl/fl and XOR^Hep−/− mice (n=5–6). B) Endothelial-dependent dilation in pressurized 2^nd order arterioles of XOR^Hep−/− mice that had circulating XO reintroduced by iv injections immediately preceding each exposure (n=5). C) Endothelial-independent dilation in pressurized 2^nd order arterioles from sham and nTiO₂ exposed (3), XOR^fl/fl and XOR^Hep−/− mice. In A) * p<0.05 compared to nTiO₂ XOR^fl/fl, in B) * p<0.05 compared to nTiO₂ XOR^Hep−/−, ], indicates 2-way ANOVA group X concentration analysis.

Fig. 6.. Acute, luminal delivery of XO (ex vivo) does not alter endothelial-dependent dilation.

Endothelial-dependent dilation in pressurized 2^nd order arterioles pre and post lumen delivery of 5mU/ml of XO + 10μM hypoxanthine in A) Sham XOR^fl/fl, B) Sham XOR^Hep−/−, and C) nTiO₂ XOR^Hep−/− mice. ], indicates 2-way ANOVA group X concentration analysis

Fig. 7.. Hepatic-derived XO augments response to angiotensin II.

Angiotensin II response in pressurized 2^nd order arterioles from sham and nTiO₂ exposed (3), A) XOR^fl/fl mice and B) XOR^Hep−/− mice (n=5–6). C) Angiotensin II response in pressurized 2^nd order arterioles of XOR^Hep−/− mice that had circulating XO reintroduced by iv injections immediately preceding each exposure (n=5). Angiotensin II response in pressurized 2^nd order arterioles pre and post lumen delivery of 5mU/ml of XO + 10μM hypoxanthine in D) Sham XOR^fl/fl, E) Sham XOR^Hep−/−, and F) nTiO₂ XOR^Hep−/− mice. For (A) *p<0.05 compared to Sham, for (B) *p<0.05 compared to nTiO₂ XOR^fl/fl, and for (C) * p<0.05 compared to nTiO₂ XOR^Hep−/−. ] indicates 2-way ANOVA group X concentration analysis

Fig. 8.. Endothelial bound XO induces Xdh and Nox1 expression and alters histone methylation.

Human aortic endothelial cells between passage 3–7 underwent XO binding for 1h then washed to remove unbound enzyme then cultured for 24h. A) After 24h H₂O₂ production was measured by CBA +/− heparin wash and +/− febuxostat treatment. B&C) After 24 h treatment gene expression for Xdh and Nox1 were analyzed in HAEC. D) EC-XOR treatment effect on eNOS expression in HAEC. Finally, western blot analysis for trimethylation of lysine 9 in HAEC 24 h after EC-XO treatment. *p<0.05, ** p<0.01, **** p<0.0001

Fig. 9.. H3k9me³ CUT&Tag in human aortic endothelial cells.

A) Combined peak tracks for vehicle, 5mU/ml GOx, and 5mU/ml of XO depicted in the Integrative Genome Visualizer (IGV). B-C) Relative genomic sight and peak width of H3K9me³ peaks. D-E) Total number of called peaks in the combined data set with differential peak identification venn diagram with overlapping area represents shared peaks while nonoverlapping areas represent peaks unique to that treatment. F-I) Representative peaks showing loss of H3K9me³ on ICAM, IL-1β, IL-6, and Nox1, IGV. n=4 per with >40 million aligned reads (>98% alignment)

Fig. 9.. H3k9me³ CUT&Tag in human aortic endothelial cells.

A) Combined peak tracks for vehicle, 5mU/ml GOx, and 5mU/ml of XO depicted in the Integrative Genome Visualizer (IGV). B-C) Relative genomic sight and peak width of H3K9me³ peaks. D-E) Total number of called peaks in the combined data set with differential peak identification venn diagram with overlapping area represents shared peaks while nonoverlapping areas represent peaks unique to that treatment. F-I) Representative peaks showing loss of H3K9me³ on ICAM, IL-1β, IL-6, and Nox1, IGV. n=4 per with >40 million aligned reads (>98% alignment)