Upregulated Matrix Metalloproteinase 2/9 Activity in Murine Asthma Detected with Activatable Cell-Penetrating Peptides

Rationale: Asthma, an airway inflammatory disease characterized by intermittent airway obstruction, affects more than 300 million people worldwide. Matrix metalloproteinase 2 and 9 (MMP2/9) mRNA and protein levels correlate with asthma pathogenesis; however, the role of MMP2/9 in asthma is not well understood, particularly because previous studies have not evaluated protease activity. We report the application of activatable cell-penetrating peptides (ACPPs), injectable probes that highlight in vivo protease activity, to assess protease activity in murine asthma.

Methods: Asthma mice were sensitized (ovalbumin [OVA] + alum in saline [PBS]) and then challenged (OVA in PBS). Age-matched control mice were sensitized but not challenged. The day after the final OVA challenge, ACPPs (cleavable and uncleavable, n = 3-6 mice each) were administered intravenously 6 hr before mice were sacrificed. Lungs were imaged for Cy5 fluorescence (Maestro, CRi). Lung sections (10 μm, 8 images/mouse) were imaged for Cy5 fluorescence on a confocal microscope (5Live, Zeiss) and stained with hematoxylin and eosin.

Results: MMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8x10^-4). The same pattern was observed for ratiometric ACPPs. MMP2/9 activity localized around inflamed airways with 1.6-fold higher ACPP uptake surrounding airways compared to the normal lung parenchyma (p = 0.03). MMP2/9 activity detected by ACPPs co-localized with gelatinase activity measured with in situ DQ gelatin.

Conclusions: MMP-activated ACPPs allow for real-time detection of protease activity in a murine asthma model, improving our understanding of protease activation in asthma progression and elucidating novel therapy targets.