State-of-the-Art Lecture

Hypertension-Induced End-Organ Damage

A New Transgenic Approach to an Old Problem
Friedrich C. Luft, Eero Mervaala, Dominik N. Müller, Volkmar Gross, Folke Schmidt, Joon Keun Park,
Christian Schmitz, Andrea Lippoldt, Volker Breu, Ralph Dechend, Duska Dragun, Wolfgang Schneider,
Detlev Ganten, Hermann Haller

Abstract—Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson
and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human
angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ
cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the
hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on
the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the
vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-b and deposition of
extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and
kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted
pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure,
pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but
protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected
from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or
indirectly via endothelin 1 and that NFkB is upregulated in this model. We speculate that the transcription factors NFkB
and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique
model and our pharmacological probes will enable us to test these hypotheses. (Hypertension. 1999;33[part
II]:212-218.)
Key Words: angiotensin II n rats, transgenic n renin n nuclear factor-kB n monocyte chemoattractant protein-1
n muscle, smooth, vascular n endothelium

H ypertension injures blood vessels and thereby causes

end-organ damage. The mechanisms are complicated
and, although studied for decades in experimental animal
models,1 are only currently being elucidated. From the efforts
of many investigators, we are now in the position of con-
structing a chain of events from the endothelium to the
underlying matrix, to the vascular smooth muscle cells, and
beyond to the adventitia, and surrounding tissues. The endo-
thelial layer acts as a signal transduction interface for hemo-
dynamic forces in the regulation of vascular tone and chronic
structural remodeling of arteries.2 Effects of mechanical
forces on signal transduction and gene expression in endo-
thelial cells have been demonstrated.3 Mechanical stress
initiates numerous pathways including ion channels, integrin
interaction between cells and matrix, activation of various
tyrosine kinases, autocrine production, and release of growth
factors.4 Increased flow through small arteries has been
shown to increase connective tissue production and promote
medial hypertrophy, probably through proliferation of both
endothelial and vascular smooth muscle cells.5 Increased
pressure is also capable of inducing early response genes in
the arterial wall.6 Microvascular endothelium in hypertensive
animals has been shown to exhibit increased oxyradical
production attributable to xanthine oxidase.7 Oxyradical pro-
duction by endothelial cells can result in leukocyte-endothe-
lial adhesion responses that involve transcription-independent
and -dependent surface expression of different endothelial
cell adhesion molecules.8 Infiltration of the permeabilized
endothelium by leukocytes sets the stage for an inflammatory
cascade, involving cytokines, chemokines, growth factors,
and matrix metalloproteinases. Altered integrin signaling, the
production of tenacin, epidermal growth factor signaling,
tyrosine phosphorylation, and activation of downstream path-
ways culminate in vascular smooth muscle cell proliferation.9
Evidence is accumulating that matrix molecules provide an
environment which decreases the rate of programmed cell
death.10

Received September 17, 1998; first decision October 12, 1998; revision accepted October 23, 1998.
From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin,
Germany; and Department of Clinical Pharmacology, Benjamin Franklin University Hospital, Free University of Berlin.
Correspondence to Friedrich C. Luft, Franz Volhard Clinic, Wiltberg Str 50, 13122 Berlin, Germany. E-mail luft@fvk-berlin.de
© 1999 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org

212
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Mechanical forces alone are capable of initiating complex
events resulting in vascular remodeling and subsequent end-
organ damage. However, hypertension is not merely a pro-
cess of mechanical events. All forms of hypertension involve
mediators, which elicit their own responses, independent of
arterial pressure. The first practicable model introduced by
Goldblatt11 fascinated Wilson and Byrom,1 who appreciated
much of which we regard as angiotensin (Ang) II-mediated
damage today. Our group is interested in hypertension-
induced and Ang II-mediated injury in the kidney and the
heart. We have concentrated on a unique, double transgenic
model in rats (dTGR) harboring the human renin and human
angiotensinogen genes.12 This model was developed by the
combined efforts of Ganten et al13 and collaborators from the
laboratory of Murakami.14 This model permits studying local
vascular effects of blood pressure and Ang II, while permit-
ting use of human renin inhibitors that otherwise would not
function in a rat model. All animal studies reported here were
conducted according to American Physiological Association
guidelines and were duly approved. Similar models have been
developed in double transgenic mice by Shimokama et al15
and by Merrill et al.16 The mouse model exhibits character-
istics also found in our rat model and is equally suitable. The
models provide an opportunity to study a cascade of events,
in part briefly mentioned above, which results in vascular and
subsequently end-organ damage.
Hypertensive Mechanisms in dTGR
The relationship between sodium chloride intake– excretion
and systemic blood pressure (pressure-natriuresis) is shifted
rightward in all forms of hypertension.17 Roman and Cowley
have developed a method which allows the determination of
pressure-natriuresis mechanisms intrinsic to the kidney,
thereby separating these mechanisms from extrarenal regula-
tors, which can also shift pressure-natriuresis.18 We studied
6-week-old dTGR and found that pressure-natriuresis was
shifted rightward and that only intrinsic renal mechanisms
accounted for the shift. The high expression of both trans-
genes within the kidney suggested Ang II acting locally may
be responsible.19 We tested for Ang II-related effects by
blocking the action of Ang II at the Ang II (AT1) receptor and
by inhibiting the generation of Ang II through the actions of
angiotensin converting enzyme (ACE).20 We found that both
AT1 blockade and ACE inhibition lowered blood pressure
and shifted pressure-natriuresis leftward, but both did so only
incompletely. When both agents were given together, blood
pressure could be normalized and pressure-natriuresis re-
stored to normal levels. More importantly, we also observed
that the action of ACE inhibition involved restoring renal
blood flow and glomerular filtration rate to normal, while
AT1 receptor blockade diminished tubular sodium reabsorp-
tion. Thus, two Ang II-related mechanisms appeared opera-
tive: hemodynamic effects and tubular sodium reabsorption.
Both human and rat renin and angiotensinogen genes were
downregulated in dTGR and increased by AT1 blockade and
ACE inhibition, whereas no changes in the expression of rat
ACE and AT1 receptor genes were observed. We believe
these observations are novel because they point to two
distinct Ang II-related intrarenal mechanisms accounting for
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Luft et al January 1999 Part II 213
the shift in pressure-natriuresis and increase in blood pres-
sure. The ACE inhibitor effects can be explained by kinin-
related mechanisms. AT1 blockers are resistant to degrada-
tion and reuptake following filtration and thus may affect AT
receptors at the tubular lumen. Our findings differ somewhat
from a recent report by Ots et al,21 who studied combination
therapy with enalapril and losartan on the rate of progression
of renal injury in a 5/6 nephrectomy renal mass ablation rat
model. They found similar degrees of blood pressure reduc-
tion with enalapril and losartan. However, combination ther-
apy offered no clear-cut advantages that could not be attrib-
uted to improved blood pressure reduction.
Vascular and End-Organ Damage
Wilson and Byrom1 performed a crucial experiment that
showed that constriction of one renal artery produced severe
hypertension in rats but that no vascular lesions in the clipped
kidney occurred. These findings indicated that increased
pressure preceded and elicited vascular damage. In their
model, Ang II was responsible for pressure elevations and
vascular damage. Kincaid-Smith et al22 confirmed these
findings and observed that the constricted and dilated areas
along the vessels was accompanied by coagulation abnormal-
ities and vascular lesions resembling those of the hemolytic-
uremic syndrome. More recently, Ruggenenti and Remuzzi23
have drawn attention to endothelial cell swelling, detachment,
proliferation, fibrin deposits, fibrinoid necrosis, and the
myointimal rearrangement resulting in irreversible vascular
destruction. An example from a 6 week-old, salt-
supplemented dTGR exhibiting vascular changes indistin-
guishable from those of the hemolytic uremic syndrome is
shown in Figure 1. A section from the heart of the same
animal shows focal areas of myocardial necrosis.
The interplay between hypertension and Ang II appears
responsible for these dramatic vascular changes. A direct
effect of Ang II on the endothelium has been appreciated for
decades. Asscher and Anson24 demonstrated the existence of
a vascular permeability factor which was responsible for the
development of arterial necroses resembling those found in
malignant hypertension. Robertson and Khairallah25 subse-
quently showed that Ang II increased the permeability of
rabbit aortic endothelium to Evans blue, an effect that could
be blocked by competitive synthetic peptides. Increased
vascular wall permeability undoubtedly is important to the
vasculopathy, as Wilson and Byrom1 conjectured. The effects
of Ang II on endothelium are more complex than these
investigators could imagine. Bech Laursen et al26 showed
recently that Ang II-induced hypertension involved vascular
zO22 production, whereas norepinephrine-induced hyperten-
sion did not. Treatment with superoxide dismutase amelio-
rated the Ang II-induced hypertension but not the norepi-
nephrine-induced hypertension. The authors suggested that
the Ang II effect on zO22 production occurs via degradation of
endothelium-derived NO. Reactive oxygen species were also
implicated in Ang II-induced cardiomyocyte hypertrophy by
Nakamura et al.27 These investigators found that they could
inhibit such hypertrophy by administering antioxidants.
In addition to directly elevating blood pressure, zO22 pro-
duction in the vessel wall, heart, kidney, and elsewhere may
214 Hypertension and Damage Control
Figure 1. Top, Section of kidney from a salt-supplemented
dTGR at 6 weeks showing fibrinoid thrombi in small arteries and
arterioles, fibrinoid wall necroses, and a fibrinoid thrombus with
necrosis within the glomerulus upper right. The picture is con-
sistent with hemolytic uremic syndrome (hematoxylin and eosin).
Bottom, Section of myocardium from the same animal with
hemorrhages and patchy areas of necrosis, as well as an inter-
stitial fibroblastic reaction.
have been responsible for a host of other consequences. The
role of oxidative stress and the mediation of arterial inflam-
matory responses in hypertension and atherosclerosis have
been recently reviewed.28 Reactive oxygen species may act as
signal transduction messengers for several important tran-
scription factors, including NFkB and activator protein (AP)-
1.29 Binding sites of the redox-regulated transcription factors
NFkB and AP-1 are located in the promoter region of a large
variety of genes that are directly involved in the pathogenesis
of vascular disease. NFkB-regulated proteins include proin-
flammatory cytokines such as tumor necrosis factor, certain
interleukins, and granulocyte-macrophage colony–stimulat-
ing factor; chemokines such as macrophage chemotactic
protein (MCP)-1; lipoxygenases; receptors such as the IL-2
and T-cell receptor; and adhesion molecules such as intercel-
lular adhesion molecule (ICAM)-1, vascular-cell adhesion
molecule (VCAM)-1, and E-selectin.30,31 Cyclic strain in-
duces an oxidative stress in endothelial cells.32 Ang II can
activate p38 mitogen-activated protein kinase, which is a
critical component of the redox-sensitive signaling path-
way.33 Further evidence supporting these mechanisms is
provided by a model of atherosclerosis, which included
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Figure 2. Gel mobility shift assay for NFkB expression is
shown. Left, Enhanced DNA binding activity of NFkB in dTGR
and positive control Hodgkin tumor cell line versus control rats.
Middle, Control CAAT enhancer binding protein as control tran-
scription factor. Controls and dTGR are not different. Right,
Western blot for IKB-a. Controls show increased IKB-a com-
pared with dTGR. The results are consistent with enhanced
IKB-a degradation in dTGR, permitting more NFkB translocation
to the nucleus. Methods similar to those described by Hernan-
dez et al.30
amelioration by ACE inhibition. 34 We have accrued evidence
of increased zO22 production and NFkB upregulation in the
dTGR model. A mobility shift assay, documenting increased
NFkB expression in kidney tissue from dTGR compared with
control kidneys is shown in Figure 2. We are particularly
interested in this interconnection because of the considerable
MCP-1, ICAM-1, and VCAM-1 expression we were able to
detect in our model.
We present evidence that surface adhesion molecules and
cell migration into the vessel wall are important to the
vasculopathy. With fluorescent antibody cell-sorting analysis,
we observed that LFA-1 and VLA-4, the ligands to ICAM-1
and VCAM-1 were expressed on circulating leukocytes and
that both ICAM-1 and VCAM-1 were expressed on the
endothelial cell surface, cardiac vessels, and elsewhere.35
Komatsu et al36 found that chronic hypertension in spontane-
ously hypertensive rats also resulted in increased ICAM-1
expression on the endothelium and emphasized the role of
ICAM-1 in end-organ damage. Simultaneously, MCP-1 was
increased and ED-1-positive cells appeared within the vascu-
lar wall in significant numbers. We and others have described
surface adhesion molecule expression on the vascular wall in
high renin models of hypertension.37,38 The appearance is
reminiscent of histological findings observed in models of
reperfusion injury.39 Mononuclear cell recruitment via adhe-
sion molecules, GM-CSF, and MCP-1, as in our study, is
likely to be important to the vasculopathy on the basis of
cytokine release, leading to increased expression of extracel-
lular matrix and vascular smooth muscle cell proliferation.
The remarkable MCP-1 expression we observed, to the point
that we could measure an increase in this chemokine in urine,
is in accord with recent findings reported by Capers et al.40
We observed increased extracellular matrix production in this
and in previous models.41 Kim and Iwao42 have recently

Figure 3. Upper, Blood pressure values (tail cuff) in control rats,
untreated dTGR, ACE inhibitor (cilazapril) treated, AT1 blocker
(valsartan) treated, renin inhibitor treated, and bosentan treated
rats. Lower, Urinary albumin excretion from these same groups.
Blood pressure was only marginally reduced by the renin inhibi-
tor and bosentan. All treatments markedly reduced albuminuria.
reviewed their findings on TGF-b1, fibronectin, and collagen
expression in heart and kidney in several rat models of
hypertension and the effects of AT1 receptor blockade. Their
findings are in accord with those observed in our dTGR
model. We previously observed increased expression for the
gene encoding the GM-CSF receptor in the hearts of hyper-
tensive rats, concomitant with cardiac macrophage infiltra-
tion.43 It is likely that GM-CSF-related mechanisms also
played a role in macrophage proliferation in dTGR. Finally,
we have not yet investigated whether or not the infiltrating
macrophages contain, or even produce, Ang II. Circulating
macrophages and macrophages infiltrating atherosclerotic
plaques, have been found to contain generous amounts of
Ang II.44 Whether they take Ang II up from circulating
plasma or whether they actually make their own, cannot be
answered for certain.
dTGR as a Model for Interventions
Figure 3 (upper panel) shows the effect on blood pressure
exerted by the chronic daily gavage-administered ACE-inhib-
itor cilazapril, the AT1 receptor blocker valsartan, the human
renin inhibitor RO 65 to 7219, and the endothelin receptor
blocker bosentan in dTGR. The animals were treated for 3
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Luft et al January 1999 Part II 215
weeks between weeks 4 and 7. The ACE inhibitor and the
AT1 receptor blocker effectively lowered blood pressure, the
human renin inhibitor lowered blood pressure significantly
less, and bosentan lowered blood pressure slightly but not
significantly. The ACE inhibitor, AT1 receptor blocker, and
the endothelin receptor blocker were effective in ameliorating
histological damage. Figure 3 (lower panel) shows the effect
of treatment on albuminuria. The albuminuria of dTGR was
1000-fold greater than control rats. All drug treatments
markedly reduced albuminuria. Importantly, complete regres-
sion of renal and cardiac injury was also observed with
human renin inhibition and endothelin receptor blockade,
although the blood pressure-lowering effects were modest.
These latter two agents therefore appeared to exert positive
effects on renal damage independent of blood pressure.
In untreated dTGR, we found severe left ventricular hy-
pertrophy with focal areas of necrosis, probably on the basis
of fibrinoid necrosis and vascular occlusion. Rarefaction of
capillaries and arteriolar growth have recently been described
in Ang II-dependent cardiac hypertrophy.45 The cardiac
changes we observed were as severe as those observed in the
kidneys and responded similarly to treatment. Interestingly,
although the human angiotensinogen gene was expressed in
considerable abundance in the heart, the human renin gene
mRNA was barely detectable, even with a quantitative
polymerase chain reaction determination. We believe that
Ang II is generated locally in the heart and that the renin
necessary for this purpose is taken up from the plasma and
perhaps processed. Evidence for such uptake has been pro-
vided by our group and others in earlier studies.46,47 Consid-
erable amounts of the changes we observed may have been
primarily related to Ang II and less to blood pressure.48
The human renin inhibitor we used had a shorter duration
of action compared with the ACE inhibitor and the AT1
receptor blocker, which in part accounts for its lesser effect
on blood pressure. Nevertheless, the human renin inhibitor
effectively decreased end-organ damage in dTGR. The pro-
tection appeared to be greater than we would have predicted
from the reduction in blood pressure alone. It is likely that the
renin inhibitor acted not only on circulating renin, but also
renin incorporated into the vasculature,49 within the intersti-
tium of the kidney,50 or even within certain cell types. De
Mello51 has shown that intracellular renin may influence
cell-to-cell communications, an effect which could be inhib-
ited with enalaprilat. These observations are particularly
interesting, since we have shown that Ang II operates
intracellularly in terms of initiating signaling and that this
signaling can be spread to adjacent cells, probably through
the second messenger IP-3 acting through tight junctions.52
However, understanding the nature of an intracellular renin-
angiotensin system leaves much to be elucidated.
The clinical significance of endothelin in cardiovascular
disease has recently been reviewed.53 Ang II stimulates the
expression of endothelin by endothelial cells.54 Furthermore,
Ang II increases tissue endothelin and induces vascular
hypertrophy.55 In vitro studies on vascular smooth muscle
cells suggest that endothelin has a stimulating effect on cell
proliferation. We were thus not surprised to find that dTGR
had increased endothelin concentrations in kidney and heart
216 Hypertension and Damage Control

and that bosentan ameliorated the severity of vascular dam-

age, even though blood pressure was scarcely influenced by
this intervention. Moreau et al55 were able to show that Ang
II stimulates endothelin under in vivo conditions and that
endothelin thus represents a paracrine local system that
interacts with the renin-angiotensin system. The interaction
appears more prominent in the vascular wall than in the
plasma. Our findings would support that view. Furthermore,
the amelioration of vascular damage suggests that endothelin
receptor blockade may provide an additional therapeutic
avenue. Nephroprotection of an ETA-receptor blocker in
salt-loaded uninephrectomized stroke-prone spontaneously
hypertensive rats has been demonstrated by Orth et al.56 In
spontaneously hypertensive rats, bosentan ameliorated car-
diac hypertrophy and fibrosis and improved creatinine clear-
ance, independent of blood pressure-lowering effects.57

dTGR as a Model of Hypertension and Ang

II-Related Effects
We observed that leukocyte infiltration in the vascular wall
accompanies PAI-1, MCP-1, and VEGF expression. PAI-1 is
a major physiological inhibitor of the plasminogen activator
(PA)/plasmin system, a key regulator of fibrinolysis and
extracellular matrix turnover.58 Activation of the renin-angio-
tensin system can disturb the balance of the fibrinolytic
system by stimulating excess production of PAI-1 and
thereby increasing the risk of thrombotic events. We believe
that the resemblance of the kidneys in our untreated, salt-
supplemented dTGR to the hemolytic uremic syndrome,
reflects that effect. In our model, we were able to show that
ACE inhibition, AT1 receptor blockade, and renin inhibition
decreased PAI-1 expression. We observed similar effects on
VEGF expression. A stimulatory interaction between VEGF
and endothelin-1 on each gene expression has recently been
described.59 This interaction could have an important con-
comitant effect on proliferation of endothelial and smooth
muscle cells in the vascular wall.
Ang II is mitogenic for several renal cell types.60 For
instance, Ang II stimulates expression of the chemokine
RANTES in rat glomerular endothelial cells; the AT2 recep-
tor may be involved in this response.61 We are currently
investigating this issue in our model. The growth-promoting
action of Ang II is largely mediated by autocrine and
paracrine factors such as platelet-derived growth factor
(PDGF) and basic fibroblast growth factor (bFGF).62 ACE
inhibition abolishes medial smooth muscle PDGF-AB bio-
synthesis and attenuates cell proliferation in injured carotid
arteries.63 In the rat carotid artery and in certain mesenteric
microvessels, the mitogenic effects of Ang II are mediated by
bFGF.64 The importance of tyrosine phosphorylation in Ang
II signaling has been demonstrated by Schieffer et al65 as well
as by Schmitz et al66 Ang II-regulated tyrosine kinases are
required for proto-oncogene expression, protein synthesis,
and proliferation. Ang II stimulates expression of transform-
ing growth factor-b (TGF-b) in cultured renal cells. How-
ever, in vivo, TGF-b expression can also be up-regulated by
blood pressure increases, independent of Ang II.67 Ang II
upregulates VEGF expression in cardiac endothelial cells68
while potentiating VEGF-related effects in microcapillary
Figure 4. Hypothetical schema of vascular injury in the dTGR
model, representing a series of testable hypotheses.
endothelial cells.69 In vivo studies to investigate the role of
growth factors in this model are planned. Interactions be-
tween Ang II, nitric oxide, and endothelin remain to be
explored.70 The role of disturbed carbohydrate metabolism in
altering the sensitivity to Ang II in the kidney warrants
attention.71 Finally, apoptosis and its induction appear impor-
tant in protection from, and regression of, vascular disease.72
Pollman et al73 recently showed that down-regulation of
intimal bcl-xl expression with antisense oligodesoxynucleo-
tides induced acute regression of vascular lesions in a rabbit
model of balloon-induced vascular injury. Similarly, Yaoita
et al showed attenuation of ischemia/reperfusion injury in rats
by a caspase inhibitor.74 The role of apoptosis or its inhibition
in our model is yet to be explored. New therapeutic avenues
may be introduced by influencing apoptosis,75,76
In summary, we are in the process of investigating a high
human renin double transgenic rat model, characterized by
severe nephrosclerosis and cardiac injury. We have devel-
oped a hypothetical schema shown in Figure 4. We postulate
that forces acting on the vascular wall and Ang II stimulate
oxidative stress directly or indirectly via endothelin 1. We
speculate that the transcription factors NFkB and AP-1 are
involved with initiating chemokine and cytokine expression,
leading to the above cascade. Adhesion molecule expression,
attraction of leukocytes, release of cytokines and chemokines,
factors favoring coagulation, cell proliferation, and growth
factor-induced matrix production all are likely to promote
vascular injury. This rapidly moving area of research will
permit novel approaches to test new hypotheses and to
develop experimental therapies for hypertension-induced vas-
cular injury.
Acknowledgments
These studies were supported by a grant-in-aid from Hoffmann La
Roche (Basel, CH). FCL, VG, AL, DG, and HH are supported by the

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Deutsche Forschungsgemeinschaft (Bonn, FRG), EM is the recipient
of a Humboldt Fellowship. DM are CS supported by the Klinisch-
Pharmacologischer Verbund, Berlin-Brandenburg, FRG.
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 Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old
Problem
Friedrich C. Luft, Eero Mervaala, Dominik N. Müller, Volkmar Gross, Folke Schmidt, Joon
Keun Park, Christian Schmitz, Andrea Lippoldt, Volker Breu, Ralph Dechend, Duska Dragun,
Wolfgang Schneider, Detlev Ganten and Hermann Haller

Hypertension. 1999;33:212-218
doi: 10.1161/01.HYP.33.1.212
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