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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 14, 2001 as doi:10.1096/fj.01-0447fje. |
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Molecular Signaling Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia
3Correspondence: Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail: jun-ping.liu{at}baker.edu.au
SPECIFIC AIMS
In this study we addressed the hypothesis that telomerase activation and telomere maintenance mediate increased vascular smooth muscle cell proliferation subserving vascular wall remodeling in genetic hypertension. Telomerase activity and telomere length have been determined in vivo in the aorta of genetically hypertensive rats at different ages, and molecular targeting to telomerase and the tumor suppressor protein p53 in the reversal of vascular smooth muscle cell hyperproliferation has been studied in cultured cells.
PRINCIPAL FINDINGS
1. Telomerase is activated selectively in the aortic tissues of genetically hypertensive rats before the onset of hypertension
Analysis of telomerase activity in the aorta of spontaneously hypertensive rats (SHR) showed that telomerase activity was twice that in normotensive Wistar-Kyoto rat (WKY), but not in other tissues examined, and telomere was significantly longer in SHR aorta than WKY. The increased activity in the aorta was found in animals 3–6 wk after birth, well before the onset of hypertension. Immunoblotting also showed that expression of the catalytic subunit of telomerase (telomerase reverse transcriptase or TERT) was markedly increased in the aorta of immature SHR.
2. Telomerase activation underlies increased proliferation rate of VSMC from SHR
Subcellular fractionation of VSMC from aortic media of SHR and WKY rats aged 12 wk showed that telomerase activity was markedly elevated in the nucleus of SHR VSMC compared with WKY (Fig. 1
A). Consistent with the in vivo findings, the expression of TERT was significantly increased in the VSMC of SHR over that of WKY (Fig. 1B
). Continuous observation of cultured VSMC showed that VSMC from both WKY and SHR proliferated exponentially but at different growth rates over a period of 
6 months, with SHR at a significantly higher rate than WKY. The similar levels of 
actin up to 120 passages suggest no significant contamination by other cell types or dedifferentiation of the cultured VSMC (Fig. 1C
). Consistent with the increased telomerase activity, telomeres were also increased in the SHR VSMC and stabilized without progressive shortening up to 100 population doublings in both strains (Fig. 1D
). These data suggest that the maintenance of telomeres is involved in the increased proliferation of SHR VSMC. Immunofluorescent staining of cultured VSMC from SHR showed that at a relatively early stage of DNA damage in apoptotic cells, TERT was reduced in the nucleus; at a later stage of apoptotic cells, TERT was completely lost in the nucleus (Fig. 1E
). Thus, activation of telomerase and maintenance of telomeres may play an essential role in the hyperproliferative status of VSMC in genetically hypertensive animals, inhibition of which may inhibit proliferation and induce cell aging and/or apoptosis.
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3. Down-regulation of telomerase reverses the increased VSMC proliferation
Transient transfection of VSMC with wild-type TERT expression vectors increased TERT and telomerase activity, and transfection with TERT antisense plasmids reduced both (Fig. 2
A, B). Despite no significant change in telomere length after the transfection, significant cell death was observed in the cells with lowered TERT (Fig. 2C
) and a decrease of cell numbers was recorded in the VSMC cultures on days 3, 4, and 5 after forced expression of TERT antisense RNA (Fig. 2D
). Sporadic apoptosis was observed in the cell cultures at 24 h post-transfection with TERT antisense RNA, and the chromosome instability was also reflected by increased apoptosis in response to DNA damaging agents such as UV irradiation and oxidative insult with Fe2+ or the protein kinase inhibitor staurosporine. The apoptosis induced by TERT down-regulation is specific in that cotransfection with TERT wild-type abolished apoptosis. Thus, down-regulation of the elevated telomerase causes a reduction in cell proliferation in association with increased genomic instability and susceptibility to apoptosis in the hyperproliferative VSMC of SHR. The findings that telomerase inhibition reverses the increased VSMC proliferation seen in SHR together with the findings that telomerase activation occurs before significant vascular wall remodeling and the onset of hypertension in vivo suggest that telomerase activation is causal in SHR VSMC proliferation.
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4. SHR VSMC apoptosis induced by telomerase down-regulation is regulated by the tumor suppressor protein p53
Transfection of SHR VSMC with wild-type p53 or p53 antisense RNA resulted in increased or decreased cellular levels of p53, respectively. Lowering p53 triggered apoptosis and enhanced the apoptosis induced by lowering TERT, and increasing wild-type p53 unambiguously prevented the apoptosis induced by TERT antisense expression. Thus, in SHR VSMC cultures, reducing either TERT or p53 triggers apoptosis, possibly by compromising the genomic integrity of SHR VSMC, and overexpressing p53 effectively prevents the apoptosis initiated by lowering TERT. These findings suggest that malfunctioning of the p53 checkpoint is involved in telomerase down-regulation-induced apoptosis and that reintroduction of the wild-type p53 may rescue the apoptotic cells by allowing DNA repair or/and balancing telomerase-induced chromosomal instability.
CONCLUSIONS
We demonstrate that increased telomerase activity and telomere length play a causal role in the abnormally increased proliferation of VSMC in genetically hypertensive rats. Since telomerase activation occurs before the onset of hypertension and telomerase inhibition reverses increased proliferation, increased telomerase activity appears to be the driving force for the imbalance between cell proliferation and apoptosis that ultimately results in the vascular wall remodeling seen in genetic hypertension (Fig. 3
). In addition, we demonstrate an action of telomerase over and above telomere lengthening to regulate cell proliferation and apoptosis in the genetically hypertensive rat. Telomerase may thereby play multiple roles not only in up-regulating cell proliferative life span (or the timing of cell senescence) by stabilizing or/and elongating critically shortened telomeres, but also in supporting cell proliferative rate (or the rate of cell renewal) by a mechanism involving telomere lengthening-independent activity.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0447fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0447fje 
2 These authors contributed equally to this work.
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