Cardiovascular disease and the associated endothelial dysfunction are characterized by leukocyte activation, decrease endothelial nitric oxide synthase (eNOS) activity, and increased endothelial cell adhesion molecules expression. This leads to the release of myeloperoxidase (MPO) by activated neutrophils and monocytes. MPO is a peroxidase enzyme that plays an important role in innate immune host defense, however it has been shown to play a pathogenic role in cardiovascular disease, mainly by causing endothelial dysfunction. The molecular mechanisms through which MPO induces endothelial damage are not fully understood. Calpains are a family of calcium-dependent proteases. Two calpain isoforms, µ- and m-calpain, are expressed in the vascular wall, including endothelial cells. Activation of calpains has been recently implicated in inflammatory disorders of the vasculature. The goal of this study was to test the hypothesis of a role for calpains in the molecular mechanism(s) through which MPO causes endothelial dysfunction and vascular inflammation. To explore if MPO activates calpain and to identify the calpain isoform(s) involved, we first studied the effects of MPO treatment on calpain activity in mouse lung microvascular endothelial cells (MMVEC). MMVECs were stimulated with 10 nmol/L MPO for 60, 120, 180, and 240 min. Using a fluorescent calpain activity assay, we found that MPO time dependently activates calpain in endothelial cells, with peak activity reached within 180 min. Using immunoblot analysis techniques we demonstrated that the calpain isoform targeted by MPO is µ-calpain. Interestingly, using a biotin switch assay,10 nmol/L MPO appears to activate the µ-calpain isoform via denitrosylation of its C-terminus domain. Using MMVECs, we studied the effects of the MPO/µ-calpain signaling on endothelial dysfunction. MMVECs were stimulated with 10 nmol/L MPO for 180 min. Expression levels of Protein Phosphatase 2 (PP2A), total 5' AMP-activated protein kinase (AMPK), Thr172 phospho-AMPK, total endothelial nitric oxide synthase (eNOS),Ser1177 phospho-eNOS, total protein kinase B (AKT), Ser473 phospho AKT, Adiponectin receptor 1 (AdipoR1), and Adiponectin receptor 2 (AdipoR2), were measured by immunoblot analysis. Interestingly, MPO impaired Thr172AMPK, Ser1177eNOS, but not Ser 473 AKT phosphorylation in a calpain dependent manner. On the other hand, MPO significantly increased the expression levels of PP2A. Inhibition of PP2A with okadiak acid decreased the phosphorylation levels of AMPK, and eNOS, indicating that PP2A is a downstream target of the MPO/calpain system. MPO treatment significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Pharmacological inhibition of calpain activity attenuated expression of VCAM-1. MPO also decreased protein levels of AdipoR1, and AdipoR2 in a calpain dependent manner. The treatment of MMVEC with adiponectin in the presence of MPO was not able to restore AdipoR2 expression levels. Using genetically modified mice, we found evidence of reduced leukocyte adhesion to the aortic endothelium in response to MPO in µ-calpain deficient mice, compared to wild-type mice . These effects appears to be attributed to the endothelial calpain, since incubating wild type aortas with calpain deficient leukocytes did not reduce leukocyte adhesion to the endothelium. The data in this study first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO, with MPO activating the µ-calpain isoform via denitrosylation. Our data also report that increased calpain activity downregulats the expression of a number of signaling molecules important for endothelial cell function. This study may provide the MPO/calpain/PP2A signaling pathway as a novel pharmacological targets for the treatment of inflammation-driven vascular disorders. / Biomedical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/2836 |
| Date | January 2018 |
| Creators | Etwebi, Zienab |
| Contributors | Scalia, Rosario, Autieri, Michael V., Eguchi, Satoru, Soprano, Dianne R., Goldfinger, Lawrence |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 104 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/2818, Theses and Dissertations |
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