Pulmonary delivery of the broad-spectrum matrix metalloproteinase inhibitor marimastat diminishes multiwalled carbon nanotube-induced circulating bioactivity without reducing pulmonary inflammation

Figures

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  Fig. 1. Experimental Design. Male C57BL/6 mice aged 6–8 weeks were randomized into 6 groups of 5 mice each. Animals were weighed and dosed with 10 mg/kg body weight Marimastat via oropharyngeal aspiration 1 h prior to dosing with dispersion media (DM; 0 µg), 10 µg, or 40 µg of MWCNT via oropharyngeal aspiration. Animals were euthanized 24-h post MWCNT exposure and tissues collected to assess pulmonary inflammation, serum peptide profile and serum bioactivity. (Figure generated in BioRender)

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  Fig. 2. Electron microscopy images of the Mitsui-7 MWCNT used for this study, at increasing magnification

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  Fig. 3. MWCNT-mediated lung inflammation. Bronchoalveolar lavage fluid (BALF) total protein and infiltrating inflammatory cell count. MWCNT exposure-induced pulmonary inflammation was not altered by MMP blockade. a Total lavage fluid protein concentration assessed via Bradford protein assay. MWCNT-exposure increased BALF total protein in a dose-dependent manner when compared with controls. b and c BALF cell differentials; Polymorphonuclear neutrophils (PMN) cells were significantly increased by MWCNT exposure at both low and high doses; Macrophages in BALF were decreased in MWCNT treated cells. d Total BALF cells were unchanged by MWCNT exposure. p values based on 2-way ANOVA presented for each figure. N = 4–5 per group. Data presented are means ± SEM

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  Fig. 4. MWCNT-mediated pulmonary inflammatory activation. Bronchoalveolar lavage fluid (BALF) inflammatory cytokine profile assessed via Meso Scale Discovery multiplex cytokine assay. MWCNT-exposure increased BALF cytokine protein expression in a dose-dependent manner which was not significantly altered by MMP blockade. a IL-1β cytokine protein expression. b IL-2 cytokine protein expression. c IL-4 cytokine protein expression. d IL-5 cytokine protein expression. e IL-6 cytokine protein expression. f IL-10 cytokine protein expression. g IL12p70 cytokine protein expression. h IFN-γ cytokine protein expression. i TNF-α cytokine protein expression. j KC-Gro/CXCL1/GROα chemokine protein expression. p values based on 2-way ANOVA presented for each figure. N = 4–5 per group. Data presented are means ± SEM

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  Fig. 5. Lung cytokine gene expression profile. Cytokine expression in response to MWCNT was largely unaltered by lung MMP blockade. a Il1b gene expression. b Il6 gene expression. c Ccl2 gene expression. d Tgfb gene expression. e Tnfa gene expression. f Icam1 gene expression. g Vcam1 gene expression. Marimistat only inhibited Tnfa expression (e). p values based on 2-way ANOVA presented for each figure. N = 4–5 per group. Data presented are means ± SEM

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  Fig. 6. Serum peptidomics heatmap. MWCNT-induced peptide serum profile changes assessed via Mass Spectrometry. MWCNT exposure increased total serum peptide composition in a differential dose-dependent manner. Left panel: Serum peptide changes resulting from MWCNT exposure. Blue box: Serum peptide composition induced exclusively by high dose (40 µg) MWCNT exposure; Red box: Serum peptide composition induced by exclusively low dose (10 µg) MWCNT exposure; Purple box: Serum peptide composition induced by both low and high dose MWCNT exposure. Right panel: Serum peptide profile generated by MWCNT and Marimastat exposure. MWCNT-induced serum peptide changes were abrogated by broad spectrum MMP inhibitor, Marimastat. These findings indicate an MMP-mediated mechanism of action for MWCNT in generating lung-derived serum bioactivity. N = 5 per group

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  Fig. 7. MWCNT in vitro serum bioactivity evaluation. Contribution of CD36 to MWCNT-mediated serum cumulative inflammatory potential in mouse cerebrovascular endothelial cells (mCEC). Cells in monolayer were treated with a CD36 blocking antibody or IgG control antibody, followed by 5% v/v serum from C57BL/6 mice exposed to MWCNT in conjunction with MMP blockade. qPCR performed on cell isolates to access cytokine gene expression changes. Shown are gene expression for a Ccl2, b Il6, c Tgfb, d Icam1, and e Vcam1. p values based on 3-way ANOVA presented for each figure. N = 4–5 per group. Data presented are means ± SEM

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  Fig. 8. Evaluation of the role of MWCNT-exposure generated serum bioactivity on brain endothelial barrier integrity. Mouse cerebrovascular endothelial cells (MCEC) grown in a confluent monolayer were treated with serum (5% v/v) from C57BL/6 mice exposed to MWCNT in conjunction with Marimastat (Mar) or Vehicle (Veh). Barrier integrity changes were accessed via Electric Cell-substrate Impedance Sensing (ECIS). a Normalized electric resistance changes across endothelial monolayer were recorded over the course of 24 h. after serum treatment. b Averages of normalized resistance from different groups were compared after 24 h of serum treatment. Serum from MWCNT-treated mice decreased barrier integrity compared to controls. Marimastat partially recovered barrier integrity in MWCNT exposed group although not back to DM/Veh control serum levels. Data presented are means ± SEM. *p < 0.05 compared to DM/Mar. **p < 0.01 compared to DM/Veh. ***p < 0.0001 compared to DM/Veh and DM/Mar. p values based on 2-way ANOVA with Tukey’s multiple comparison test presented for each figure. n = 4 per group

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  Fig. 9. Graphical Summary. Consequences of MWCNT oropharyngeal exposure in C57BL/6 mice. MWCNT induced pulmonary inflammatory activation as evidenced by increased neutrophilia and dose-dependent cytokine BALF profile and whole lung gene expression. MMP blockade did not play a role in the observed pulmonary findings. However, MMP activity was shown in induce serum peptide profile changes in a dose-dependent differential manner. Administration of broad-spectrum MMP inhibitor Marimastat abrogated these serum changes and return peptide profiles to near unexposed levels. The systemic consequences of these serum changes were evaluated in vitro. Serum from MWCNT exposed mice decreased endothelial barrier integrity as assessed via ECIS. Marimastat treatment, presumably through blockade of MMP activity in the lung, resulted in serum with considerably less potential for endothelial cell activation. These findings provide a mechanism for MWCNT action in the lung and starts to elucidate the systemic consequences of inhalation exposures to MWCNT. Additionally, serum findings provide a base for the exploration of MWCNT exposure and pathological systemic consequences, as well as the identification of…