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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
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
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
models,1 are only currently being elucidated. From the efforts the arterial wall.6 Microvascular endothelium in hypertensive
of many investigators, we are now in the position of con- animals has been shown to exhibit increased oxyradical
structing a chain of events from the endothelium to the production attributable to xanthine oxidase.7 Oxyradical pro-
underlying matrix, to the vascular smooth muscle cells, and duction by endothelial cells can result in leukocyte-endothe-
beyond to the adventitia, and surrounding tissues. The endo- lial adhesion responses that involve transcription-independent
and -dependent surface expression of different endothelial
thelial layer acts as a signal transduction interface for hemo-
cell adhesion molecules.8 Infiltration of the permeabilized
dynamic forces in the regulation of vascular tone and chronic
endothelium by leukocytes sets the stage for an inflammatory
structural remodeling of arteries.2 Effects of mechanical cascade, involving cytokines, chemokines, growth factors,
forces on signal transduction and gene expression in endo- and matrix metalloproteinases. Altered integrin signaling, the
thelial cells have been demonstrated.3 Mechanical stress production of tenacin, epidermal growth factor signaling,
initiates numerous pathways including ion channels, integrin tyrosine phosphorylation, and activation of downstream path-
interaction between cells and matrix, activation of various ways culminate in vascular smooth muscle cell proliferation.9
tyrosine kinases, autocrine production, and release of growth Evidence is accumulating that matrix molecules provide an
factors.4 Increased flow through small arteries has been environment which decreases the rate of programmed cell
shown to increase connective tissue production and promote 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
© 1999 American Heart Association, Inc.
Hypertension is available at

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Luft et al January 1999 Part II 213

Mechanical forces alone are capable of initiating complex the shift in pressure-natriuresis and increase in blood pres-
events resulting in vascular remodeling and subsequent end- sure. The ACE inhibitor effects can be explained by kinin-
organ damage. However, hypertension is not merely a pro- related mechanisms. AT1 blockers are resistant to degrada-
cess of mechanical events. All forms of hypertension involve tion and reuptake following filtration and thus may affect AT
mediators, which elicit their own responses, independent of receptors at the tubular lumen. Our findings differ somewhat
arterial pressure. The first practicable model introduced by from a recent report by Ots et al,21 who studied combination
Goldblatt11 fascinated Wilson and Byrom,1 who appreciated therapy with enalapril and losartan on the rate of progression
much of which we regard as angiotensin (Ang) II-mediated of renal injury in a 5/6 nephrectomy renal mass ablation rat
damage today. Our group is interested in hypertension- model. They found similar degrees of blood pressure reduc-
induced and Ang II-mediated injury in the kidney and the tion with enalapril and losartan. However, combination ther-
heart. We have concentrated on a unique, double transgenic apy offered no clear-cut advantages that could not be attrib-
model in rats (dTGR) harboring the human renin and human uted to improved blood pressure reduction.
angiotensinogen genes.12 This model was developed by the
combined efforts of Ganten et al13 and collaborators from the Vascular and End-Organ Damage
laboratory of Murakami.14 This model permits studying local Wilson and Byrom1 performed a crucial experiment that
vascular effects of blood pressure and Ang II, while permit- showed that constriction of one renal artery produced severe
ting use of human renin inhibitors that otherwise would not hypertension in rats but that no vascular lesions in the clipped
function in a rat model. All animal studies reported here were kidney occurred. These findings indicated that increased
conducted according to American Physiological Association pressure preceded and elicited vascular damage. In their
guidelines and were duly approved. Similar models have been model, Ang II was responsible for pressure elevations and
developed in double transgenic mice by Shimokama et al15 vascular damage. Kincaid-Smith et al22 confirmed these
and by Merrill et al.16 The mouse model exhibits character- findings and observed that the constricted and dilated areas
istics also found in our rat model and is equally suitable. The along the vessels was accompanied by coagulation abnormal-
models provide an opportunity to study a cascade of events, ities and vascular lesions resembling those of the hemolytic-
in part briefly mentioned above, which results in vascular and uremic syndrome. More recently, Ruggenenti and Remuzzi23
subsequently end-organ damage. have drawn attention to endothelial cell swelling, detachment,
proliferation, fibrin deposits, fibrinoid necrosis, and the
Hypertensive Mechanisms in dTGR myointimal rearrangement resulting in irreversible vascular
The relationship between sodium chloride intake– excretion destruction. An example from a 6 week-old, salt-
and systemic blood pressure (pressure-natriuresis) is shifted supplemented dTGR exhibiting vascular changes indistin-
rightward in all forms of hypertension.17 Roman and Cowley guishable from those of the hemolytic uremic syndrome is
have developed a method which allows the determination of shown in Figure 1. A section from the heart of the same
pressure-natriuresis mechanisms intrinsic to the kidney, animal shows focal areas of myocardial necrosis.
thereby separating these mechanisms from extrarenal regula- The interplay between hypertension and Ang II appears
tors, which can also shift pressure-natriuresis.18 We studied responsible for these dramatic vascular changes. A direct
6-week-old dTGR and found that pressure-natriuresis was effect of Ang II on the endothelium has been appreciated for
shifted rightward and that only intrinsic renal mechanisms decades. Asscher and Anson24 demonstrated the existence of
accounted for the shift. The high expression of both trans- a vascular permeability factor which was responsible for the
genes within the kidney suggested Ang II acting locally may development of arterial necroses resembling those found in
be responsible.19 We tested for Ang II-related effects by malignant hypertension. Robertson and Khairallah25 subse-
blocking the action of Ang II at the Ang II (AT1) receptor and quently showed that Ang II increased the permeability of
by inhibiting the generation of Ang II through the actions of rabbit aortic endothelium to Evans blue, an effect that could
angiotensin converting enzyme (ACE).20 We found that both be blocked by competitive synthetic peptides. Increased
AT1 blockade and ACE inhibition lowered blood pressure vascular wall permeability undoubtedly is important to the
and shifted pressure-natriuresis leftward, but both did so only vasculopathy, as Wilson and Byrom1 conjectured. The effects
incompletely. When both agents were given together, blood of Ang II on endothelium are more complex than these
pressure could be normalized and pressure-natriuresis re- investigators could imagine. Bech Laursen et al26 showed
stored to normal levels. More importantly, we also observed recently that Ang II-induced hypertension involved vascular
that the action of ACE inhibition involved restoring renal zO22 production, whereas norepinephrine-induced hyperten-
blood flow and glomerular filtration rate to normal, while sion did not. Treatment with superoxide dismutase amelio-
AT1 receptor blockade diminished tubular sodium reabsorp- rated the Ang II-induced hypertension but not the norepi-
tion. Thus, two Ang II-related mechanisms appeared opera- nephrine-induced hypertension. The authors suggested that
tive: hemodynamic effects and tubular sodium reabsorption. the Ang II effect on zO22 production occurs via degradation of
Both human and rat renin and angiotensinogen genes were endothelium-derived NO. Reactive oxygen species were also
downregulated in dTGR and increased by AT1 blockade and implicated in Ang II-induced cardiomyocyte hypertrophy by
ACE inhibition, whereas no changes in the expression of rat Nakamura et al.27 These investigators found that they could
ACE and AT1 receptor genes were observed. We believe inhibit such hypertrophy by administering antioxidants.
these observations are novel because they point to two In addition to directly elevating blood pressure, zO22 pro-
distinct Ang II-related intrarenal mechanisms accounting for duction in the vessel wall, heart, kidney, and elsewhere may
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214 Hypertension and Damage Control

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
Figure 1. Top, Section of kidney from a salt-supplemented control kidneys is shown in Figure 2. We are particularly
dTGR at 6 weeks showing fibrinoid thrombi in small arteries and
arterioles, fibrinoid wall necroses, and a fibrinoid thrombus with
interested in this interconnection because of the considerable
necrosis within the glomerulus upper right. The picture is con- MCP-1, ICAM-1, and VCAM-1 expression we were able to
sistent with hemolytic uremic syndrome (hematoxylin and eosin). detect in our model.
Bottom, Section of myocardium from the same animal with We present evidence that surface adhesion molecules and
hemorrhages and patchy areas of necrosis, as well as an inter-
stitial fibroblastic reaction. cell migration into the vessel wall are important to the
vasculopathy. With fluorescent antibody cell-sorting analysis,
have been responsible for a host of other consequences. The we observed that LFA-1 and VLA-4, the ligands to ICAM-1
role of oxidative stress and the mediation of arterial inflam- and VCAM-1 were expressed on circulating leukocytes and
matory responses in hypertension and atherosclerosis have that both ICAM-1 and VCAM-1 were expressed on the
endothelial cell surface, cardiac vessels, and elsewhere.35
been recently reviewed.28 Reactive oxygen species may act as
Komatsu et al36 found that chronic hypertension in spontane-
signal transduction messengers for several important tran-
ously hypertensive rats also resulted in increased ICAM-1
scription factors, including NFkB and activator protein (AP)-
expression on the endothelium and emphasized the role of
1.29 Binding sites of the redox-regulated transcription factors
ICAM-1 in end-organ damage. Simultaneously, MCP-1 was
NFkB and AP-1 are located in the promoter region of a large
increased and ED-1-positive cells appeared within the vascu-
variety of genes that are directly involved in the pathogenesis lar wall in significant numbers. We and others have described
of vascular disease. NFkB-regulated proteins include proin- surface adhesion molecule expression on the vascular wall in
flammatory cytokines such as tumor necrosis factor, certain high renin models of hypertension.37,38 The appearance is
interleukins, and granulocyte-macrophage colony–stimulat- reminiscent of histological findings observed in models of
ing factor; chemokines such as macrophage chemotactic reperfusion injury.39 Mononuclear cell recruitment via adhe-
protein (MCP)-1; lipoxygenases; receptors such as the IL-2 sion molecules, GM-CSF, and MCP-1, as in our study, is
and T-cell receptor; and adhesion molecules such as intercel- likely to be important to the vasculopathy on the basis of
lular adhesion molecule (ICAM)-1, vascular-cell adhesion cytokine release, leading to increased expression of extracel-
molecule (VCAM)-1, and E-selectin.30,31 Cyclic strain in- lular matrix and vascular smooth muscle cell proliferation.
duces an oxidative stress in endothelial cells.32 Ang II can The remarkable MCP-1 expression we observed, to the point
activate p38 mitogen-activated protein kinase, which is a that we could measure an increase in this chemokine in urine,
critical component of the redox-sensitive signaling path- is in accord with recent findings reported by Capers et al.40
way.33 Further evidence supporting these mechanisms is We observed increased extracellular matrix production in this
provided by a model of atherosclerosis, which included and in previous models.41 Kim and Iwao42 have recently
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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-
Figure 3. Upper, Blood pressure values (tail cuff) in control rats,
untreated dTGR, ACE inhibitor (cilazapril) treated, AT1 blocker erable amounts of the changes we observed may have been
(valsartan) treated, renin inhibitor treated, and bosentan treated primarily related to Ang II and less to blood pressure.48
rats. Lower, Urinary albumin excretion from these same groups. The human renin inhibitor we used had a shorter duration
Blood pressure was only marginally reduced by the renin inhibi-
tor and bosentan. All treatments markedly reduced albuminuria.
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
reviewed their findings on TGF-b1, fibronectin, and collagen effectively decreased end-organ damage in dTGR. The pro-
expression in heart and kidney in several rat models of tection appeared to be greater than we would have predicted
hypertension and the effects of AT1 receptor blockade. Their from the reduction in blood pressure alone. It is likely that the
findings are in accord with those observed in our dTGR renin inhibitor acted not only on circulating renin, but also
model. We previously observed increased expression for the renin incorporated into the vasculature,49 within the intersti-
gene encoding the GM-CSF receptor in the hearts of hyper- tium of the kidney,50 or even within certain cell types. De
tensive rats, concomitant with cardiac macrophage infiltra- Mello51 has shown that intracellular renin may influence
tion.43 It is likely that GM-CSF-related mechanisms also cell-to-cell communications, an effect which could be inhib-
played a role in macrophage proliferation in dTGR. Finally, ited with enalaprilat. These observations are particularly
we have not yet investigated whether or not the infiltrating interesting, since we have shown that Ang II operates
macrophages contain, or even produce, Ang II. Circulating intracellularly in terms of initiating signaling and that this
macrophages and macrophages infiltrating atherosclerotic signaling can be spread to adjacent cells, probably through
plaques, have been found to contain generous amounts of the second messenger IP-3 acting through tight junctions.52
Ang II.44 Whether they take Ang II up from circulating However, understanding the nature of an intracellular renin-
plasma or whether they actually make their own, cannot be angiotensin system leaves much to be elucidated.
answered for certain. The clinical significance of endothelin in cardiovascular
disease has recently been reviewed.53 Ang II stimulates the
dTGR as a Model for Interventions expression of endothelin by endothelial cells.54 Furthermore,
Figure 3 (upper panel) shows the effect on blood pressure Ang II increases tissue endothelin and induces vascular
exerted by the chronic daily gavage-administered ACE-inhib- hypertrophy.55 In vitro studies on vascular smooth muscle
itor cilazapril, the AT1 receptor blocker valsartan, the human cells suggest that endothelin has a stimulating effect on cell
renin inhibitor RO 65 to 7219, and the endothelin receptor proliferation. We were thus not surprised to find that dTGR
blocker bosentan in dTGR. The animals were treated for 3 had increased endothelin concentrations in kidney and heart

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

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Luft et al January 1999 Part II 217

Deutsche Forschungsgemeinschaft (Bonn, FRG), EM is the recipient 21. Ots M, Mackenzie HS, Troy JL, Rennke HG, Brenner BM. Effects of
of a Humboldt Fellowship. DM are CS supported by the Klinisch- combination therapy with enalapril and losartan on the rate of progression
Pharmacologischer Verbund, Berlin-Brandenburg, FRG. of renal injury in rats with 5/6 renal mass ablation. J Am Soc Nephrol.
1998;9:224 –230.
22. Kincaid-Smith P, Hobbs JB, Friedman A, Mathews DC. Structured and
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Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old
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
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1999 American Heart Association, Inc. All rights reserved.
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