The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

Background: Acute lung injury (ALI) is a severe respiratory disease with high rates of morbidity and mortality. Many mediators regarding endogenous or exogenous are involved in the pathophysiology of ALI. Here, we have uncovered the involvement of integrins and Matrix Metalloproteinases, as critical determinants of excessive inflammation and endothelial permeability, in the regulation of ALI.

Methods: Inflammatory cytokines were measured by quantitative Real-Time PCR for mRNA levels and ELISA for secretion levels. Endothelial permeability assay was detected by the passage of rhodamine B isothiocyanate-dextran. Mice lung permeability was assayed by Evans blue albumin (EBA). Western blot was used for protein level measurements. The intracellular Reactive Oxygen Species (ROS) were evaluated using a cell-permeable probe, DCFH-DA. Intratracheal injection of lipopolysaccharide (LPS) into mice was conducted to establish the lung injury model.

Results: Exogenous MMP-9 significantly aggravated the inflammatory response and permeability in mouse pulmonary microvascular endothelial cells (PMVECs) treated by LPS, whereas knockdown of MMP-9 exhibited the opposite phenotypes. Knockdown of Integrin β3 or β5 in LPS-treated PMVECs significantly downregulated MMP-9 expression and decreased inflammatory response and permeability in the presence or absence of exogenous MMP-9. Additionally, the interaction of MMP-9 and Integrin β5 was impaired by a ROS scavenger, which further decreased the pro-inflammatory cytokines production and endothelial leakage in PMVECs subjected to co-treatment (LPS with exogenous MMP-9). In vivo studies, exogenous MMP-9 treatment or knockdown β3 Integrin significantly decreased survival in ALI mice. Notably, knockdown of β5 Integrin alone had no remarkable effect on survival, but which combined with anti-MMP-9 treatment significantly improved the survival by ameliorating excessive lung inflammation and permeability in ALI mice.

Conclusion: These findings support the β3/5 integrin-MMP-9 axis as an endogenous signal that could play a pivotal role in regulating inflammatory response and alveolar-capillary permeability in ALI.