Cloning and characterization of a secreted form of angiotensin-converting enzyme 2.
Angiotensin-converting enzyme 2 (ACE2) is a newly discovered, membrane-bound aminopeptidase responsible for the production of vasodilatory peptides such as angiotensin 1-7 (Ang 1-7). Thus, ACE2 is important in counteracting the adverse, vasoconstrictor effects of angiotensin II (Ang II). The objective of the present study was to clone and characterize a constitutively secreted form of ACE2 as a prelude to an investigation into its therapeutic potential in hypertension. A truncated form of ACE2 was cloned into a lentiviral vector behind the human elongation factor 1 alpha promoter (lenti-shACE2). Transfection experiments demonstrated that secreted human ACE2 (shACE2) was secreted constitutively into the medium. The kinetic properties of shACE2 were comparable to the human recombinant enzyme (rACE2). Transduction of human coronary artery endothelial cells and rat cardiomyocytes with lenti-shACE2 showed a significant secretion of the enzyme into the medium compared to its native, membrane-bound homolog (human ACE2 [hACE2]). In addition, systemic administration of lenti-shACE2 into neonatal rats resulted in a eightfold increase in ACE2 activity in the serum above control values. These observations establish that lenti-shACE2 can be used to transduce cardiovascularly relevant cells for the secretion of functional ACE2 enzyme both in vitro and in vivo. Collectively, these results set the stage for the use of these vectors to investigate the consequences of ACE2 over-expression in the pathogenesis of hypertension.
Huentelman
Matthew J
MJ
Department of Physiology and Functional Genomics, College of Medicine, UF McKnight Brain Institute, University of Florida, 1600 SW Archer Road, P.O. Box 100274, Gainesville, FL 32610-0255, USA.
Zubcevic
Jasenka
J
Katovich
Michael J
MJ
Raizada
Mohan K
MK
D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D002268:Carboxypeptidases / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D007703:Peptidyl-Dipeptidase A; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection
D012333:RNA, Messenger; D002268:Carboxypeptidases; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; C000705329:Ace2 protein, rat; D000085962:Angiotensin-Converting Enzyme 2