To further confirm the critical role of ZO-1 degradation in EC barrier disruption in vitro and pulmonary inflammation in vivo, we next examined the beneficial effects of over-expressing ZO-1 protein in endothelial cells in vitro and in vivo. ZO-1 over-expression ( Figure 6A ) significantly (but not completely) attenuated PM-induced EC barrier disruption ( Figure 6B ). These facts demonstrate that endothelial ZO-1 loss contributes to PM-mediated EC barrier disruption. We next over-expressed ZO-1 in vivo by a liposome delivery system labeled with ACE antibody, which successfully over-expressed ZO-1 in Human lung microvascular ECs were plated on glass cover slips and loaded with 5 μM Fura-2 AM in 1 ml of basic medium. ECs were rinsed twice and treated with PM (100 μg/ml). Fura-2 fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm (0-60 min). Fura-2 has excitation wavelengths of 380 nm in its free form and 340 nm when it is bound to calcium; therefore, relative calcium concentrations are represented by the ratio of 340/ 380. (B) ECs plated on glass cover slips were treated with TRPM2 antibody or control IgG (5 μg/ml, 4 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). *p < 0.05 compared to control IgG group at the same time point. (C) ECs were transfected with TRPM2 siRNA or control siRNA for 48 hrs. ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). EC cell lysates were analyzed by Western blot with antibodies to TRPM2 and β-actin to confirm silencing. *p < 0.05 compared to control siRNA group at the same time point. (D-E) ECs plated on glass cover slips (95% confluent) were treated with NAC (5 mM, 1 hr pretreatment) or 3-AB (1 mM, 1 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). *p < 0.05 compared to PM-only group at the same time point. murine lung tissues ( Figure 6C ). ZO-1 over-expression significantly attenuated BAL protein leakage ( Figure 6D) , BAL white blood cell infiltration ( Figure 6E) , and the release of proinflammatory cytokine IL-6 into BAL ( Figure 6F ), indicating the crucial role of ZO-1 loss in mediating PM-induced pulmonary inflammation and lung vascular hyperpermeability.