Bortezomib Increased Vascular Permeability by Decreasing Cell-Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells

Abstract

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and β-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Keywords: adhesion molecule; bortezomib; vascular permeability.

Figure 1

Effect of BTZ on the viability of the HPMEC monolayer. (a) HPMEC monolayer treated with 0–10 ng/mL BTZ for 72 h. Viability was determined in a PrestoBlue cell viability assay, and the fluorescent intensity was measured. Experiments were repeated three times, and data are expressed as the mean ± SD. Significant differences were analyzed using one-way analysis of variance, followed by Dunnett’s multiple comparisons test. **** p < 0.001, *** p < 0.005. RFU, relative fluorescence intensity. (b) Morphology of the HPMEC monolayer treated with 0–10 ng/mL for 72 h. Magnification: 20×; scale bar: 50 μm.

Figure 2

Effect of BTZ on the permeability of HPMEC monolayers. (a,b) Permeability of NaF (a) and EBA (b) through HPMEC monolayers treated with the indicated concentrations of BTZ for 72 h. P, permeability efficiency. The calculation method has been described previously [17]. Both experiments were conducted three times, and data are expressed as the mean ± SD. Significant differences were analyzed via one-way analysis of variance followed by Dunnett’s multiple comparisons test. **** p < 0.001, *** p < 0.005, ** p < 0.01.; ns = not significant.

Figure 3

Effect of BTZ on the expression of adhesion molecules in HPMECs (a–c) HPMECs were treated with BTZ at the indicated concentrations for 72 h, and the protein (a,b) and mRNA (c) expression levels of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin were analyzed. (a) Representative blot for Western blot analysis. β-Actin was detected as a reference. (b) Relative expression of the five proteins normalized to the expression level of β-actin. (c) mRNA expression level of the five adhesion molecule genes normalized to that of the β-actin gene. Both protein and mRNA expression experiments were conducted three times, and data are expressed as the mean ± SD. Significant differences were analyzed via one-way analysis of variance, followed by Dunnett’s multiple comparisons test. **** p < 0.001, *** p < 0.005, ** p < 0.01, and * p < 0.05; ns = not significant.