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

Abstract

RationaleAsthma, 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.MethodsAsthma 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.ResultsMMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). 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.ConclusionsMMP-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. RationaleAsthma, 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. 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. MethodsAsthma 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. 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. ResultsMMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). 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. MMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). 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. ConclusionsMMP-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. 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.