Hemodynamic regulation of metalloendopeptidases EC3.4; 24.15 and EC3.4; 24.16: expression and function in the vascular endothelium
Cotter, Eoin J. (2005) Hemodynamic regulation of metalloendopeptidases EC3.4; 24.15 and EC3.4; 24.16: expression and function in the vascular endothelium. PhD thesis, Dublin City University.
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Hemodynamic forces, namely shear stress and cyclic strain, have been well characterised as modulators of vascular endothelial function, and have been assigned an important role in the maintainence of vascular tone, haemostasis, and regulation of vascular growth and health. They exert their influence in part by effecting changes in the production and release of vasoactive compounds by the endothelium, and by effecting changes in the levels and activity of various enzymes. Thimet oligopeptidase (EC188.8.131.52, EP24.15) and neurolysin (EC184.108.40.206, EP24.15) are closely related zinc metalloendopeptidases that have been shown to be expressed and active in the vascular endothelium. Their substrates include the vasoactive peptides bradykinin and angiotensin I, which have been identified as important regulators of both blood pressure and angiogenic processes. Other related peptidases, namely endothelin converting enzyme (ECE) and angiotensin converting enzyme (ACE), have been shown to be regulated by hemodynamic forces in the vascular endothelium. As such EP24.15 and EP24.16 are likely candidates for regulation by hemodynamic forces.
In this regard we have investigated the effect of cyclic strain on the expression and activty of EP24.15 and EP24.16 in cultured bovine aortic endothelial cells (BAECs). We have shown that exposure to cyclic strain significantly increases the mRNA expression as well as both the cellular and secreted activity of both enzymes. We have demonstrated that up-regulation of both enzymes is dependent on Gi-protein mediated signalling, although with varying Gia/G(3y subunit specificity for either enzyme. Using immunocytochemistry, we have also demonstrated a strain-dependent increase in EP24.15 protein expression within the nucleus and cytoplasm in parallel with an increase in membrane associated EP 24.15
The effects of strain on the ability of BAECs in culture to cleave both Ang I, and BK in an EC24.15/EC24.16 specific manner was also studied. We observed that exposure to cyclic strain induces a significant increase in the EP24.15 specific hydrolysis of both exogenously added BK and Ang I.
The potential of the observed effects of cyclic strain on EP24.15 to effect changes in endothelial cell function were also examined. Use of the dual EP24.15/EP24.16 inhibitor, cFP-AAF-pAB, and the EP24.15 specific antisense, FLIP, was seen to significantly attenuate cyclic strain-induced endothelial cell tubule formation and migration. We also found that the effects of FLIP transfection on cyclic strain-induced endothelial cell tubule formation could be largely reversed by addition of exogenous Ang-(l-7). Taken together these results suggest that strain-induced endothelial cell angiogenesis and migration putatively involves EP24.15 cleavage of vasoactive peptide substrates.
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