Expression of Cell Cycle Proteins in Blood Vessels of
Angiotensin II–Infused Rats
Role of AT1 Receptors
Quy N. Diep, Mohammed El Mabrouk, Rhian M. Touyz, Ernesto L. Schiffrin
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract—Angiotensin II is an important modulator of cell growth through AT1 receptors, as demonstrated both in vivo
and in vitro. We investigated the role of proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase
4 (cdk4), and cyclin-dependent kinase inhibitors p21 and p27 in blood vessels of angiotensin II–infused rats and the
effect therein of the AT1-receptor antagonist losartan. Male Sprague-Dawley rats were infused for 7 days with
angiotensin II (120 ng/kg per minute SC) and/or treated with losartan (10 mg/kg per day orally). DNA synthesis in
mesenteric arteries was evaluated by radiolabeled 3H-thymidine incorporation. The expression of cyclin D1, cdk4, p21,
and p27, which play critical roles during the G1-phase of the cell cycle process, was examined by Western blot analysis.
Tail-cuff systolic blood pressure (mm Hg) was elevated (P⬍0.01, n⫽9) in angiotensin II–infused rats (161.3⫾8.2)
versus control rats (110.1⫾5.3) and normalized by losartan (104.4⫾3.2). Radiolabeled 3H-thymidine incorporation
(cpm/100 ␮g DNA) showed that angiotensin II infusion significantly increased DNA synthesis (152⫾5% versus
102⫾6% of control rats, P⬍0.05). Expression of cyclin D1 and cdk4 was significantly increased in the angiotensin II
group to 213.7⫾8% and 263.6⫾37% of control animals, respectively, whereas expression of p21 and p27 was
significantly decreased in the angiotensin II group to 23.2⫾10.4% and 10.3⫾5.3% of control animals, respectively.
These effects induced by angiotensin II were normalized in the presence of losartan. Thus, when AT1 receptors are
stimulated in vivo, DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell
cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT1 receptor stimulation.
(Hypertension. 2001;37[part 2]:604-608.)
Key Words: vasculature 䡲 muscle, smooth 䡲 hyperplasia 䡲 remodeling
D
proper sequence controls cell cycle progression.18 Progression through the G1 phase requires growth factor–induced
signals and must converge, in late G1, on the cell cycle
machinery to ensure the commitment of cells to enter the S
phase. The G1 phase is regulated, at least in part, by the action
of cyclin-dependent kinases (cdks) and their regulatory cyclin
subunits.19,20 Cyclin C, cyclins D1, D2, and D3, and cyclin E
play important roles in the G1 phase. Cyclin A is a key
molecule in the S and G2/M phases; cyclin B is essential in
the G2/M phase. A regulatory subunit of the G1 phase, cyclin
D1, forms a complex with the catalytic partners cdk4 or cdk6
to form an active holoenzyme that phosphorylates pRB.21–23
Cyclin D1 is required for progression of the G1 phase and is
therefore a critical target for proliferative signals in G1.21,22
Cyclin D1 expression is induced by several different growth
factors including colony stimulating factor-1, epidermal
growth factor, and Ang II.24 –27 It has been shown in cultured
cell lines that the cyclin D– cdk4/cdk6 complex regulates G1
progression, the cyclin E/cyclin A– cdk2 complex is essential
for G1/S transition, and cyclin A/cyclin B– cdc2 (cdk1)
uring development of hypertension, resistance arteries
undergo structural changes (remodeling) as an adaptation to increased wall stress.1 Vascular smooth muscle cell
(VSMC) proliferation is one of the important processes for
vascular remodeling.2 In blood vessels, angiotensin (Ang) II,
the most important peptide mediating the effects of the
renin-angiotensin system, contributes to development of
structure remodeling through its growth factor properties on
VSMCs.3,4 Ang II binds to its specific heterotrimeric Gprotein– coupled receptors, AT1 receptors,5,6 and exerts its
biological effects by modulating intracellular signaling pathways, including activation of phospholipase C, generation of
inositol trisphosphate, diacylglycerol, Ca2⫹, protein kinase C,
tyrosine kinases, Ras, Raf, and mitogen-activated protein
kinases,7–13 which in turn increase various immediate-early
genes, such as c-fos, c-jun, and c-myc14 –16 and DNA
synthesis.
Activation of VSMCs with Ang II has been shown to result
in entry of cells into the cell cycle.17 A network of biochemical pathways that ensure that each cell cycle event occurs in
Received October 24, 2000; first decision November 20, 2000; revision accepted December 8, 2000.
From the Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada.
Correspondence to Ernesto L. Schiffrin, MD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7. E-mail
[email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
604
Diep et al
promotes entry into mitosis. Activity of cdks is regulated not
only by binding of cyclins but also by phosphorylation of
threonine and tyrosine residues and by binding of cdk
inhibitors, such as p21, p27, p57, and the INK4 family.28 –30
Although the molecular mechanisms of cell cycle regulation
have been extensively studied, it is not fully understood how
Ang II starts the cell cycle regulatory machinery.
How Ang II induces cellular proliferation and DNA synthesis in VSMCs and a role for cyclin D1 and cdk4 in Ang II
signaling in vivo, to our knowledge, has not been examined.
We used Ang II–infused rats as a model to examine the effect
of Ang II on proliferation of smooth muscle cells from small
vessels in vivo and to investigate the role of cell cycle
proteins in the Ang II–induced proliferative response. Blockade of AT1 receptors was used to determine the role of AT1
receptors in Ang–II induced proliferation.
AT1 Receptors and Cell Cycle
605
Body Weight and Blood Pressure of Rats Treated or Not
Treated With Ang II With or Without Losartan
Parameter
Body wt, g
SBP, mm Hg
Control
324⫾3.5
110.1⫾5.3
Ang II
Ang II⫹Los
Los
343.2⫾4.9
337.2⫾8.2
322⫾9.4
161.3⫾8.2*
104.4⫾3.2†
106.1⫾5.3†
n⫽6.
*P⬍0.01 vs control rats.
†P⬍0.01 vs Ang II group.
Results
Body Weight and SBP
Body weight was unchanged in Ang II–infused rats treated
with or without losartan compared with normotensive rats
(Table). The increase in SBP induced by Ang II infusion
(P⬍0.01 versus control) was completely prevented by treatment with losartan (Table). Treatment of normotensive rats
with losartan alone had no effect on SBP and body weight.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Methods
Animal Experiments
The study was approved by the Animal Care Committee of the
Clinical Research Institute of Montreal and was performed according
to the guidelines of the Canadian Council for Animal Care. As
previously described,31 male Sprague-Dawley rats 7 weeks of age
(weight, 200 g; n⫽9) were infused subcutaneously with Alzet
osmotic minipumps (Alza Corp) with Ile5–Ang II (Peninsula) at a
dose of 120 ng/kg per minute. Losartan (AT1 receptor antagonist)
was given in the drinking water at a dose of 10 mg/kg per day. After
7 days of treatment, systolic blood pressure (SBP) was measured by
the tail-cuff method. Rats were killed by decapitation. The entire
mesenteric bed was dissected, cleaned of fat and adventitia, and
immediately frozen in dry ice and kept at ⫺70°C until it was studied.
The fraction of smooth muscle cells present in the samples exceeds
85%.
Evaluation of DNA Synthesis
DNA synthesis in mesenteric arteries was evaluated by radiolabeled
3
H-thymidine incorporation. Rats were given an intraperitoneal
injection of [methyl-3H]thymidine (0.5 mCi/kg, ICN Biomedicals
Inc) 24 hours before being killed. DNA was extracted by phenol and
chloroform as previously described.31 DNA concentration was determined by spectrophotometry. Equal amounts of DNA (100 ␮g)
were counted by scintillation counter. DNA specific activity (cpm/
100 ␮g DNA) reflects the incorporation of 3H-thymidine into smooth
muscle DNA over the last 24 hours in vivo.
DNA Synthesis
Figure 1 shows a significant increase in DNA synthesis as
demonstrated by increased 3H-thymidine incorporation in the
Ang II–infused group (152.0⫾5.0%) in comparison to control
rats (102⫾6%, P⬍0.05). In Ang–II infused rats that received
losartan, DNA synthesis was similar to that of control rats
(108.5⫾5.9%). Losartan alone decreased DNA synthesis
slightly (to 80.7⫾3.3%).
Expression of Cell Cycle Proteins
Expression of cyclin D1 and cdk4 was increased 2- to 3-fold
in Ang II–infused rats compared with normotensive rats
(Figures 2 and 3). Expression of cyclin D1 was similar to that
of control rats in Ang–II infused rats treated with losartan
(Figure 2). However, the expression of cdk4 was slightly
reduced but not back to normal levels. Losartan on its own
had no effect on expression of cyclin D1 or cdk4. As shown
in Figures 4 and 5, expression of p21 and p27 was reduced to
23.2⫾10.4% and 10.3⫾5.3% of that in control rats. Losartan-treated Ang II–infused rats exhibited normal levels of p27
(78.3⫾15.6%). However, the expression of p21 in Ang
II–infused rats treated with losartan did not return to normal.
Western Blot Analysis of Cyclin D1, cdk4, p21,
and p27
Protein was extracted from frozen tissue as previously described.31
Protein concentration was determined by the BioRad protein assay
(Bio-Rad Laboratories Inc). Equal amounts of protein were separated
by electrophoresis on a 15% polyacrylamide gel at 100 V for 1 hour
and transferred onto a polyvinylidene difluoride membrane in a
cooling system at 100 V for 1 hour. Membranes were incubated with
specific antibody to cyclin D1, cdk4, p21, and p27 (Santa Cruz
Biotechnology Inc) at a dilution 1:500, 1:1500, 1:500, and 1:1000,
respectively, for 1 hour at room temperature. Signals were revealed
with chemiluminescence and visualized by autoradiography.
Statistical Analysis
Results are presented as mean⫾SEM. Data were analyzed by 1-way
ANOVA followed by a Newman-Keuls test. A value of P⬍0.05 was
considered statistically significant.
Figure 1. Bar graph shows 3H-thymidine incorporation into DNA
from mesenteric arteries from each group expressed as percent
of control (Ctrl). Los indicates losartan. Error bars indicate SEM
(n⫽4). *P⬍0.05 vs control.
606
Hypertension
February 2001 Part II
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Figure 2. Top, Representative Western blot of cyclin D1. Ctrl
indicates control; Los, losartan. Bottom, Bar graph shows
mean⫾SEM of results from 3 rats. *P⬍0.05 vs control.
Figure 4. Top, Representative Western blot of p21. Ctrl indicates control; Los, losartan. Bottom, Bar graph shows
mean⫾SEM of results from 3 rats. *P⬍0.05 vs control.
Losartan alone did not affect the expression of p21 but
reduced the expression of p27.
suggesting that Ang II–induced proliferation was mostly
mediated by AT1 receptors. Our results also showed that the
increase in cyclin D1 and cdk4 induced by Ang II was
reversed to normal levels in the presence of losartan, which
suggests that Ang II through AT1 receptors stimulates DNA
synthesis by regulating cyclin D1 and cdk4. However, Ang
II–induced downregulation of p21 was not inhibited by
losartan. Thus, regulation of p21 may occur by other mechanisms, independent of AT1 receptors.
In addition to their contractile function, VSMCs can
increase their mass through cellular proliferation, cellular
hypertrophy, and production of extracellular matrix proteins.
Changes in growth rates occur normally during development
of the vascular system and after vascular injury but also under
pathological conditions such as hypertension.32 In animal
models of hypertension, the increase in vascular mass has
been reported to be associated primarily with SMC hypertrophy in large arteries and with hyperplasia or proliferation in
small resistance vessels. The growth response of VSMCs is
clearly dependent on the nature of the growth stimulus. There
Discussion
To evaluate the hypothesis that AT1-receptor–induced smooth
muscle cell growth in vivo is associated with increased cell
proliferation (DNA synthesis) and changes in cell cycle,
particularly the G1 phase, we examined Ang II–infused rats
treated without or with the AT1 receptor antagonist losartan.
Our results show that AT1 stimulation is associated with
enhanced proliferation of smooth muscle cells in resistance
arteries of rats, as shown by increased DNA synthesis.
Furthermore, we also show that AT1-receptor activation
induces proliferation in blood vessels by stimulating cyclin
D1 and cyclin-dependent kinases (cdk4) in G1 phase of cell
cycle. These findings extend our understanding of the role of
Ang II and its receptors, particularly AT1 receptors, as
important contributors and regulators of cell growth contributing to vascular remodeling in hypertension.
Ang II–induced increase in 3H-thymidine uptake was
completely inhibited by the AT1 receptor antagonist losartan,
Figure 3. Top, Representative Western blot of cdk4. Ctrl indicates control; Los, losartan. Bottom, Bar graph shows
mean⫾SEM of results from 3 rats. *P⬍0.05 vs control.
Figure 5. Top, Representative Western blot of p27. Ctrl indicates control; Los, losartan. Bottom, Bar graph shows
mean⫾SEM of results from 3 rats. *P⬍0.05 vs control.
Diep et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
is evidence that Ang II induces both cellular hypertrophy and
cellular hyperplasia as a result of increased protein and DNA
synthesis, respectively. Still, much remains to be learned
about the molecular determinants of vascular SMC hypertrophic versus hyperplastic growth responses, particularly in
vivo. In cell culture, previous studies have shown that Ang II
induced cell growth by stimulation of cyclin D1.17,27 However, it has also been shown that Ang II induces cell cycle
entry but fails to downregulate the level of p27 protein,17,33
resulting in blocking of the progression through the cell cycle
toward DNA synthesis and mitosis. It has been speculated
that not only may commitment to hyperplasia versus hypertrophy be made during the G1 phase, but the response to
stimuli of cellular activation and programmed cell death may
also be affected by early cell-cycle entry. Our present study
shows that Ang II increases DNA synthesis by decreasing
expression of p21 and p27. In the presence of losartan, the
change in DNA synthesis, cyclin D1, cdk4, and p27 was
completely or partly reversed. However, the expression of
p21 remains the same in the Ang II group with or without
losartan, suggesting that p21 may play a role not only in DNA
synthesis but also protein synthesis. A previous study has also
shown that Ang II, through AT1 receptors, may simultaneously induce cell growth and apoptosis, although the latter
may be a reactive response to cell growth independent of
direct effects of AT1 receptors and involving different molecular mechanisms.31 We have also shown that Ang II
stimulated DNA synthesis by increasing expression of cyclin
D1 and cdk4. Cyclin D1– cdk4 complexes promote G1 phase
progression through phosphorylation and inactivation of the
retinoblastoma (Rb) gene product.30,34 However, the role of
Rb in Ang II–stimulated DNA synthesis in vivo remains to be
clarified. The extent to which normalization of cell-cycle
protein expression by AT1 antagonism with losartan results
from blood pressure reduction or blockade of Ang II effects
is unclear. Answering this question will require comparison
with results of blood pressure reduction with agents that do
not block Ang II action.
Conclusions
We have investigated molecular steps involved in the cell
cycle induced by Ang II in resistance arteries. Cell growth in
blood vessels, which may play an important role in vascular
remodeling in hypertension, may be regulated in vivo by Ang
receptors, specifically by AT1 receptors, starting cell cycle
progression. Activation of AT1 receptors in vivo in rats results
in SBP increase and blood vessel growth as well by stimulation of cyclin D1 and cdk4 in the cell cycle. Thus, the
present results extend our knowledge on the essential role of
AT1 receptors in blood pressure control and VSMC growth,
as shown by increases in blood pressure, cell proliferation,
and expression of cyclin D1 and cdk4.
Acknowledgments
This work was supported by a Group Grant from the Medical
Research Council of Canada (now the Canadian Institutes of Health
Research) to the Multidisciplinary Research Group on Hypertension.
Dr Q.N. Diep holds a postdoctoral fellowship from the Canadian
Institutes of Health Research. The authors are grateful to Suzanne
Diebold for excellent technical assistance.
AT1 Receptors and Cell Cycle
607
References
1. Heistad DD, Baumbach GL. Cerebral vascular changes during chronic
hypertension: good guys and bad guys. J Hypertens. 1992;10(suppl
7):S71–S75.
2. Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany MJ. Small
artery structure in hypertension: dual process of remodeling and growth.
Hypertension. 1993;21:391–397.
3. Egido J. Vasoactive hormones and renal sclerosis. Kidney Int. 1996;49:
578 –597.
4. Dubey RK, Jackson EK, Rupprecht HD, Sterzel RB. Factors controlling
growth and matrix production in vascular smooth muscle and glomerular
mesangial cells. Curr Opin Nephrol Hypertens. 1997;6:88 –105.
5. Neer EJ. Heterotrimeric G proteins: organizers of transmembrane signals.
Cell. 1995;80:249 –257.
6. Clapham DE. Calcium signaling. Cell. 1995;80:259 –268.
7. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H. Role of
calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev.
1989;10:495–518.
8. Chao TS, Foster DA, Rapp ER, Rosner MR. Differential Raf requirement
for activation of mitogen-activated protein kinase by growth factors,
phorbol esters, and calcium. J Biol Chem. 1994;269:7337–7341.
9. Chao TS, Byron KL, Lee KM, Villereal M, Rosner MR. Activation of
MAP kinases by calcium-dependent and calcium-independent pathways:
stimulation by thapsigargin and epidermal growth factor. J Biol Chem.
1992;267:19876 –19883.
10. Lin LL, Wartman M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is
phosphorylated and activated by MAP kinase. Cell. 1993;72:269 –278.
11. Ghosh A, Greenberg ME. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science. 1995;268:239 –246.
12. Schorb W, Peeler TC, Madigan NN, Conrad KM, Baker KM. Angiotensin
II-induced protein tyrosine phosphorylation in neonatal rat cardiac fibroblasts. J Biol Chem. 1994;269:19626 –19632.
13. Marrero MB, Paxton WG, Duff JL, Berk BC, Bernstein KE. Angiotensin
II stimulates tyrosine phosphorylation of phospholipase C-gamma 1 in
vascular smooth muscle cells. J Biol Chem. 1994;269:10935–10939.
14. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nidal-Ginard
B. Angiotensin II induces c-fos mRNA in aortic smooth muscle: role of
Ca2⫹ mobilization and protein kinase C activation. J Biol Chem. 1989;
264:526 –530.
15. Naftilan AJ, Gilliland GK, Eldridge CS, Kraft AS. Induction of the
proto-oncogene c-jun by angiotensin II. Mol Cell Biol. 1990;10:
5536 –5540.
16. Viard I, Hall SH, Jaillard C, Berthelon MC, Saez JM. Regulation of c-fos,
c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells. Endocrinology. 1992;
130:1193–1200.
17. Servant MJ, Coulombe P, Turgeon B, Meloche S. Differential regulation
of p27kip1 expression by mitogenic and hypertrophic factors: involvement
of transcriptional and posttranscriptional mechanism. J Cell Biol. 2000;
148:543–556.
18. Murray A, Hunt T. The Cell Cycle: An Introduction. New York, NY: WH
Freeman & Co Press; 1993.
19. Draetta GF. Mammalian G1 cyclins. Curr Opin Cell Biol. 1994;6:
842– 846.
20. Grana X, Reddy EP. Cell cycle control in mammalian cells: role of
cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and
cyclin dependent kinase inhibitors (CKIs). Oncogene. 1995;11:211–219.
21. Hunter T, Pines J. Cyclins and cancer, II: cyclin D and CDK inhibitors
come of age. Cell. 1994;79:573–582.
22. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell.
1995;81:323–330.
23. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994;79:551–555.
24. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating
factor 1 regulates novel cyclins during the G1 phase of the cell cycle.
Cell. 1991;65:701–713.
25. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell
RG. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1
promoter through distinguishable regions. J Biol Chem. 1995;270:
23589 –23597.
26. Sewing A, Burger C, Brusselbach S, Schalk C, Lucibello FC, Muller R.
Human cyclin D1 encodes a labile nuclear protein whose synthesis is
directly induced by growth factors and suppressed by cyclic AMP. J Cell
Sci. 1993;104:545–554.
608
Hypertension
February 2001 Part II
27. Watanabe G, Lee RJ, Albanese C, Rainey WE, Batlle D, Pestell RG.
Angiotensin II activation of cyclin D1-dependent kinase Activity. J Biol
Chem. 1996;271:22570 –22577.
28. Nigg EA. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays. 1995;17:471– 480.
29. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent
kinases. Genes Dev. 1995;9:1149 –1163.
30. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding
of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 1993;7:
331–342.
31. Diep QN, Li JS, Schiffrin EL. In vivo study of AT1 and AT2 angiotensin
receptors in apoptosis in rat blood vessels. Hypertension. 1999;34:
617– 624.
32. Schwartz SM, Campell GR, Campell JH. Replication of smooth muscle
cells in vascular disease. Circ Res. 1986;58:427– 444.
33. Braun-Dullanaeus RC, Mann MJ, Ziegler A, von der Leyen HE, Dzau VJ.
A novel role for the cyclin-dependent kinase inhibitor p27kip1 in angiotensin II-stimulated vascular smooth muscle cell hypertrophy. J Clin
Invest. 1999;104:815– 823.
34. Lukas J, Bartkova J, Rohde M, Strass M, Bartek J. Cyclin D1 is dispensable for G1 control in retinoblasma gene-deficient cells independently of cdk4 activity. Mol Cell Biol. 1995;15:2600 –2611.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Expression of Cell Cycle Proteins in Blood Vessels of Angiotensin II−Infused Rats: Role of
AT1 Receptors
Quy N. Diep, Mohammed El Mabrouk, Rhian M. Touyz and Ernesto L. Schiffrin
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Hypertension. 2001;37:604-608
doi: 10.1161/01.HYP.37.2.604
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/37/2/604
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/