Intravenous and gastric cerium dioxide nanoparticle exposure disrupts microvascular smooth muscle signaling

Abstract

Cerium dioxide nanoparticles (CeO2 NP) hold great therapeutic potential, but the in vivo effects of non-pulmonary exposure routes are unclear. The first aim was to determine whether microvascular function is impaired after intravenous and gastric CeO2 NP exposure. The second aim was to investigate the mechanism(s) of action underlying microvascular dysfunction following CeO2 NP exposure. Rats were exposed to CeO2 NP (primary diameter: 4 ± 1 nm, surface area: 81.36 m(2)/g) by intratracheal instillation, intravenous injection, or gastric gavage. Mesenteric arterioles were harvested 24 h post-exposure and vascular function was assessed using an isolated arteriole preparation. Endothelium-dependent and independent function and vascular smooth muscle (VSM) signaling (soluble guanylyl cyclase [sGC] and cyclic guanosine monophosphate [cGMP]) were assessed. Reactive oxygen species (ROS) generation and nitric oxide (NO) production were analyzed. Compared with controls, endothelium-dependent and independent dilation were impaired following intravenous injection (by 61% and 45%) and gastric gavage (by 63% and 49%). However, intravenous injection resulted in greater microvascular impairment (16% and 35%) compared with gastric gavage at an identical dose (100 µg). Furthermore, sGC activation and cGMP responsiveness were impaired following pulmonary, intravenous, and gastric CeO2 NP treatment. Finally, nanoparticle exposure resulted in route-dependent, increased ROS generation and decreased NO production. These results indicate that CeO2 NP exposure route differentially impairs microvascular function, which may be mechanistically linked to decreased NO production and subsequent VSM signaling. Fully understanding the mechanisms behind CeO2 NP in vivo effects is a critical step in the continued therapeutic development of this nanoparticle.

Keywords: cerium dioxide nanoparticles; microvascular function; nitric oxide; mesentery.

FIG. 1.

ACh-induced vasodilation was impaired in arterioles following intravenous injection (A, n = 8–15) and gastric gavage (B, n = 9–13) of CeO₂ NP. *p ≤ 0.05 versus control, ^†p ≤ 0.05 versus low dose CeO₂ NP (50 µg for intravenous injection and 100 µg for gastric gavage), ^‡p ≤ 0.05 versus middle dose CeO₂ NP (100 µg for intravenous injection and 300 µg for gastric gavage). The brackets indicate differences in the overall slope of the determination.

FIG. 2.

NO-induced vasodilation (via SPR) was impaired in mesenteric arterioles following intravenous injection (A, n = 8–12) and gastric gavage (B, n = 8–10) of CeO₂ NP. *p ≤ 0.05 versus control, ^†p ≤ 0.05 versus low dose CeO₂ NP (50 µg for intravenous injection and 100 µg for gastric gavage), ^‡p ≤ 0.05 versus middle dose CeO₂ NP (100 µg for intravenous injection and 300 µg for gastric gavage), ^p ≤ 0.05 versus high dose CeO₂ NP (900 µg for intravenous injection and 600 µg for gastric gavage). The brackets indicate differences in the overall slope of the determination.

FIG. 3.

Vasodilation in response increasing intraluminal pressure following intravenous injection (A, n = 8–13) and gastric gavage (B, n = 9–10) of CeO₂ NP was not significantly different. *p ≤ 0.05 versus control.

FIG. 4.

The left panels are vasodilation in response to increases in intraluminal flow following intravenous injection (A, n = 5–10) and gastric gavage (B, n = 6–8) of CeO₂ NP. The right panels are vasodilation in response to increasing shear stress following intravenous injection (C, n = 6–10) and gastric gavage (D, n = 6–8) of CeO₂ NP. *p ≤ 0.05 versus control.

FIG. 5.

Vasoconstriction stimulated by PE following intravenous injection (A, n = 7–12) and gastric gavage (B, n = 9–11) of CeO₂ NP was not significantly different. *p ≤ 0.05 versus control.

FIG. 6.

Calculation of the EC₅₀ for ACh (A) and SPR (B) for intratracheal instillation, intravenous injection, and gastric gavage. The scatter plot (C) shows the dilation response to ACh (10⁻⁴M) for all exposure routes. The following linear equations (y = mx + b) and r² values were obtained for each exposure route: intratracheal instillation: y = −0.07 x + 41.26, r²= 0.2513, intravenous injection: y = −0.03 x + 49.39, r²= 0.1994, and gastric gavage: y = −0.05 x + 54.71, r²= 0.2465.

FIG. 7.

Vasodilation in response to ACh following incubation with either L-NMMA (10⁻⁴M), INDO (10⁻⁵M), or both inhibitors. There was a significant impairment in dilation of control arterioles following incubation with L-NMMA and INDO (A, n = 14–34). There was a partial restoration in function following incubation with L-NMMA after intratracheal instillation of 65 µg CeO₂ NP (B, n = 5–14). Intravenous injection of 100 µg CeO₂ NP caused an attenuated response to ACh after incubation with INDO that was significantly different from the CeO₂ NP exposure alone (C, n = 5–9). Gastric gavage of 400 µg CeO₂ NP caused a significant impairment in arteriolar dilation following L-NMMA incubation compared with the CeO₂ NP exposure alone (D, n = 8–15). *p ≤ 0.05 versus L-NMMA (10⁻⁴M), ^†p ≤ 0.05 versus INDO (10⁻⁵M), ^‡p ≤ 0.05 versus both inhibitors, ^p ≤ 0.05 versus CeO₂ NP exposure. The brackets indicate differences in the overall slope of the determination.

FIG. 8.

CeO₂ NP reacted with NO in an acellular environment (A). There was a significant decrease in the amount of NO detected following intravenous injection (B, n = 14–21). NO was released with SNAP (1.6 × 10⁻⁴M) for the acellular assessments and with A23187 (10⁻⁵M) for the cellular assessments. *p ≤ 0.05 versus control, ^†p ≤ 0.05 versus intratracheal instillation.

FIG. 9.

CeO₂ NP ability to scavenge and/or generate free radicals was assessed in an acellular environment (A, n = 3), with control AM (B, n = 4–7), and with AM from CeO₂ exposed animals via different exposure routes (C, n = 7–9). Images of representative superoxide and hydroxyl free radical spectra are inset in panel A. *p ≤ 0.05 versus control, ^†p ≤ 0.05 versus Cr⁶⁺ alone, ^‡p ≤ 0.05 versus control AM + CeO₂ NP, ^p ≤ 0.05 versus gastric gavage AM + CeO₂ NP. NoP, no detectable ESR peaks.

FIG. 10.

VSM function was impaired following CeO₂ NP exposure and was not exposure route-dependent. Soluble GC activation was assessed with YC-1 (A, n = 8–11). Cyclic GMP responsiveness was assessed with 8-bromo-cGMP (B, n = 10). *p ≤ 0.05 versus control, ^†p ≤ 0.05 versus intratracheal instillation. The brackets indicate differences in the overall slope of the determination.