Mechanistic Role of the Calcium-Dependent Protease Calpain in the Endothelial Dysfunction Induced by MPO (Myeloperoxidase)

Abstract

MPO (myeloperoxidase) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the µ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 minutes. MPO denitrosylated µ-calpain C-terminus domain, and time dependently activated µ-calpain, but not the m-calpain isoform. MPO also reduced Thr¹⁷² AMPK (AMP-activated protein kinase) and Ser¹¹⁷⁷ eNOS (endothelial nitric oxide synthase) phosphorylation via upregulation of PP2A (protein phosphatase 2) expression. At the functional level, MPO increased endothelial VCAM-1 (vascular cell adhesion molecule 1) abundance and the adhesion of leukocytes to the mouse aorta. In MPO-treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr¹⁷² AMPK and Ser¹¹⁷⁷ eNOS phosphorylation. Compared with wild-type mice, µ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders.

Keywords: calpain; cell adhesion molecules; endothelial cells; inflammation; peroxidase.

Figure 1.. MPO activates μ-calpain in endothelial cells.

Upper panel: Serum starved MMVEC were incubated with 10 nmol/L MPO and calpain activity was measured at 60, 120, 180, and 240 min using the calpain specific fluorogenic substrate Succ-LLVY-AMC. Lower panels: MMVEC were then incubated with either vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L - 30 min pretreatment). The activity of the μ-calpain isoform was assessed by immunoblot analysis using a primary antibody that recognizes cleavage of the N-terminus domain of μ-calpain large subunit. Loss of N-terminus domain indicates calpain activation. An antibody against μ-calpain large subunit C-terminus domain was used to quantify total μ-calpain expression, β-Actin detection was used as a loading control. Representative immunoblot demonstrate cleavage of MMVEC μ-calpain N-terminus domain following incubation with 10 nmol/L MPO for 180 min. Bar graph summarizes densitometry quantification of μ-calpain N-terminus domain. Total μ-calpain expression level was not changed by MPO, as demonstrated C-terminus domain immunoblot. Data are mean±SEM of 4 (upper graph) and 6 to 8 (lower graph) independent experiments.

Figure 2.. MPO causes degradation of calpastatin via calpain activation.

Serum starved MMVEC were incubated with vehicle (8 μl PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment. The expression of the endogenous calpain inhibitor calpastatin was measured by immunoblot analysis and quantified by densitometry. Beta actin detection was used as a loading control. Data are mean±SEM of 6 to 7 independent experiments.

Figure 3.. MPO induces denitrosylation of μ-calpain.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min. Nitrosylation levels of μ-calpain in MMVEC was assessed by biotin switch assay. μ-Calpain was detected in the streptavidin-purified mixture using standard immunoblot techniques. Data are mean±SEM of 5 independent experiments.

Figure 4.. MPO/calpain signaling downregulates eNOS/AMPK phosphorylation.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment. Phosphorylation levels of Ser¹¹⁷⁷ eNOS and Thr¹⁷² AMPK, in MMVEC were assessed by immunoblot analysis. Data are mean±SEM of 6 to 8 independent experiments.

Figure 5.. Role of LKB1 and PP2A in the effect of MPO/calpain signaling on AMPK phosphorylation.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 30-180 min (LKB1 detection). Serum starved MMVEC were incubated with vehicle (X microL PBS) or 10 nmol/L MPO for 180 min (PP2A and pAMPK detection). with or without ZLLal (100 μmol/L, 30 min) pretreatment. Expression levels of LKB1 and PP2A in MMVEC were assessed by immunoblot analysis. Data are mean±SEM of 3 or 4 independent experiments for upper or lower panel, respectively.

Figure 6.. MPO increases endothelial adhesiveness to leukocytes.

Upper panels: Serum starved MMVEC were incubated with vehicle (8 μM PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment Expression levels of VCAM-1 in MMVEC were assessed by immunoblot analysis. Lower panel: Circulating leukocytes and thoracic aortas were isolated from C57BLK and μ-calpain^−/− donor mice. Leukocytes were fluorescently labeled as reported in the method section. Isolated 2 mm length aortic segments were first exposed to 10 nmol/L MPO for 180 min. Aortic segments where then washed and co-incubated endothelial surface up with leukocytes for an additional 60 min. In parallel experiments, inhibition of calpain activity by ZLLal also prevented adhesion of leukocytes to the wild-type aorta exposed to MPO. Data are mean±SEM of 6 aortic segments from 3 mice per group. A total of 54 aortic segments were studied.