Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis
Syed H.E. Zaidi, … , Mansoor Husain, Marlene Rabinovitch
Syed H.E. Zaidi, … , Mansoor Husain, Marlene Rabinovitch
Published April 15, 1999
Citation Information: J Clin Invest. 1999;103(8):1211-1219. https://doi.org/10.1172/JCI5099.
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Article

Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis

Abstract

Serine elastases degrade elastin, stimulate vascular smooth muscle cell migration and proliferation, and are associated with myocardial damage. To evaluate the impact of elastase inhibition on cardiovascular development and disease, transgenic mice were created in which the mouse preproendothelin-1 promoter was used to target elafin overexpression to the cardiovascular system. To distinguish the transgene from endogenous elafin, constructs were made incorporating a FLAG sequence; the COOH-terminus FLAG-tagged elafin construct produced a stable, functionally active gene product and was used to create transgenic mice. Consistent with endothelin expression, abundant elafin mRNA was observed in transgenic F1 embryos (embryonic day 13.5) and in adult transgenic mice heart, trachea, aorta, kidney, lung, and skin, but not in liver, spleen, and intestine. Functional activity of the transgene was confirmed by heightened myocardial elastase inhibitory activity. No tissue abnormalities were detected by light microscopy or elastin content. However, injection of 10 plaque-forming units (PFU) of encephalomyocarditis virus resulted in death within 11 days in 10 out of 12 nontransgenic mice compared with one out of nine transgenic littermates. This reduced mortality was associated with better cardiac function and less myocardial inflammatory damage. Thus, elafin expression may confer a protective advantage in myocarditis and other inflammatory diseases.

Authors

Syed H.E. Zaidi, Chi-Chung Hui, Alexander Y.L. Cheah, Xiao-Mang You, Mansoor Husain, Marlene Rabinovitch

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Figure 1

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Human elafin expressed by the preproendothelin promoter is processed and...
Human elafin expressed by the preproendothelin promoter is processed and secreted by pulmonary artery endothelial cells. (a) Expression vectors contain 5.9-kb mouse preproendothelin-1 promoter, luciferase, or full-length human elafin cDNA, SV40 polyadenylation signals and intron, endothelin-1 (ET-1) intron, and the pBluescript SK (BlSK). Positions of signal peptide (SP), transglutaminase (TG) substrate domain, and elafin inhibitory domain are shown at the top. (b) Bovine (CPAE) and 100-day gestation fetal ovine (LPAE) pulmonary artery endothelial cells were transfected with pHL3 (L, luciferase) or pHZ5 (E, human elafin) constructs using calcium phosphate, and culture medium was analyzed by Western immunoblotting using anti–human elafin antisera. Migration of molecular weight markers is denoted along the left. (c) Addition of FLAG epitope to human elafin. Several constructs were generated and contained FLAG sequences at the NH2-terminus of signal peptide (pHZ9), COOH-terminus of elafin (pHZ10), and NH2-terminus of mature elafin (pHZ11). These constructs were then evaluated for FLAG-tagged elafin processing in CPAE cells, using the transfection conditions described for the pHZ5 construct. Anti-FLAG monoclonal antibody M2 was used for the Western immunoblot and demonstrated that the precursor and the processed elafin forms were detectable in culture medium of CPAE cells transfected with COOH-terminus and NH2-terminus FLAG-tagged mature elafin constructs. L, 5, 9, 10, and 11, along the top of the Western blot, represent cells transfected with pHL3 (luciferase), pHZ5, pHZ9, pHZ10, and pHZ11, respectively. Schematic representation of processing, shown on the left, indicates signal peptide cleavage, secretion of precursor, and processing to release mature elafin.