Oxygen-Loaded Nanodroplets Effectively Abrogate Hypoxia Dysregulating Effects on Secretion of MMP-9 and TIMP-1 by Human Monocytes

Abstract

Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation.

Figure 1

Hypoxia and OLN effects on human monocyte viability. Human adherent monocytes (10⁶ cells/2 mL Panserin 601 medium) were left untreated or treated with increasing doses (100–400 μL) of OLNs or with 200 μL OFNs or OSS for 24 h in normoxia (20% O₂, black columns/squared-lines) or hypoxia (1% O₂, white columns/squared-lines). After collection of cell supernatants and lysates, cytotoxicity percentage was measured through LDH assay (panels (a) and (c)), whereas cell viability percentage was measured through MTT assay (panels (b) and (d)). Results are shown as means + SEM from three independent experiments. Data were also evaluated for significance by ANOVA. No significant differences between normoxic and hypoxic control cells or between OLN-treated and untreated cells were observed (all panels).

Figure 2

OLN internalization by human monocytes. Human adherent monocytes (10⁶ cells/2 mL Panserin 601 medium) were left untreated (a) or treated with 200 μL rhodamine B-labeled dextran-shelled OLNs (b) for 24 h in normoxia (20% O₂). After DAPI staining, cells were checked by confocal microscopy. Results are shown as representative images from three independent experiments. Left panels: cell nuclei after DAPI staining (blue). Central panels: rhodamine B-labeled dextran-shelled OLNs (red). Right panels: merged images. Magnification: 63x.

Figure 3

Hypoxia and OLN effects on MMP-9 secretion by human monocytes. Human adherent monocytes (10⁶ cells/2 mL Panserin 601 medium) were left untreated or treated with 200 μL OLNs, OFNs, or OSS for 24 h in normoxia (20% O₂; panels (a), (c), and (d): black columns; panel (b): odd lanes) or hypoxia (1% O₂; panels (a), (c), and (d): white columns; panel (b): even lanes). After collection of cell supernatants, MMP-9 protein levels were quantified by ELISA (panel (a)), whereas MMP-9 latent/active forms were analyzed by gelatin zymography (panel (b)) and subsequent densitometry (panels (c)-(d)). For gelatin zymography, recombinant human MMP-9 (83 kDa) was employed as a standard marker (st). Results are shown as means + SEM (panels (a), (c)-(d)) or as a representative gel (panel (b)) from three independent experiments. ELISA and densitometric data were also evaluated for significance by ANOVA: ∗ versus normoxic control cells: P < 0.0001 (panel (a)), P < 0.02 (panel (c)), and P < 0.0001 (panel (d)); ° versus hypoxic control cells: P < 0.0001 (panel (b)), P < 0.005 (panel (c)), and P < 0.0001 (panel (d)).

Figure 4

Hypoxia and OLN effects on protein levels of gelatinase inhibitors (TIMP-1 and TIMP-2) secreted by human monocytes. Human adherent monocytes (10⁶ cells/2 mL Panserin 601 medium) were left untreated or treated with 200 μL OLNs, OFNs, or OSS for 24 h in normoxia (20% O₂; black columns, both panels) or hypoxia (1% O₂; white columns, both panels). After collection of cell supernatants, TIMP-1 (panel (a)) and TIMP-2 (panel (b)), protein levels were quantified by ELISA. Results are shown as means + SEM from three independent experiments. Data were also evaluated for significance by ANOVA: ∗ versus normoxic control cells: P < 0.02 (panel (a)) and P not significant (panel (b)); ° versus hypoxic control cells: P < 0.02 (panel (a)) and P not significant (panel (b)).

Figure 5

Hypoxia and OLN effects on MMP-9/TIMP-1 balances upon secretion by human monocytes. MMP-9/TIMP-1 stoichiometric ratio was calculated after results from ELISA investigation (see Figures 3 and 4). Results are shown as means + SEM from three independent experiments. Data were also evaluated for significance by ANOVA: ∗ versus normoxic control cells: P < 0.0001; ° versus hypoxic control cells: P < 0.0001.