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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 27, 2001 as doi:10.1096/fj.00-0843fje. |
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B and protects from angiotensin II-induced organ damage1
,2,2
,||
* Franz Volhard Clinic, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany;
Max Delbrück Center for Molecular Medicine, Berlin, Germany;
Medizinische Hochschule Hannover, Hannover, Germany;
Hoffmann-La Roche, Basel, Switzerland;
|| Free University of Berlin, Berlin, Germany; and
Heinrich Heine University, Düsseldorf, Germany
3Correspondence: Franz Volhard Clinic, Wiltberg Strasse 50, 13125 Berlin, Germany. E-mail: luft{at}fvk-berlin.de
SPECIFIC AIMS
Aspirin inhibits IKKß in vitro; however, the in vivo relevance of the
phenomenon is unclear. We have developed a model of severe angiotensin
(Ang) II-induced vascular injury and now tested the hypothesis that
chronic aspirin treatment of transgenic rats overexpressing the human
renin and angiotensinogen genes (dTGR) protects from Ang II-induced end
organ damage by inhibiting NF-
B activation in vivo.
PRINCIPAL FINDINGS
High-dose aspirin reduces mortality and renal damage independent of
blood pressure
Renal glomerulus of untreated 7-wk-old dTGR shows mesangial
expansion and podocyte damage. Renal dTGR arterioles exhibit severe
changes in wall structure, including proliferation of smooth muscle
cells and deposition of dense vacuoles. In contrast, nontransgenic
Sprague-Dawley (SD) rats showed no vascular or glomerular damage.
Untreated dTGR show signs of glomerular sclerosis and vascular damage
with mortality > 50% at wk 7. High-dose aspirin reduced
mortality to < 10% whereas low-dose aspirin produced no effect
that differed from no treatment. Neither dose lowered blood pressure.
DTGR had a 24 h albumin excretion 150-fold greater than
SD rats. High-dose aspirin reduced albuminuria by
86% (P<0.0001) whereas low-dose aspirin did not. High-dose
aspirin also reduced renal fibrosis whereas low-dose aspirin had no
effect. Plasma thromboxane (TXB)2, a marker of cyclooxygenase activity,
increased significantly in dTGR compared with all other groups
(P<0.05). Low-dose and high-dose aspirin both reduced TXB2
to SD levels.
High-dose aspirin ameliorates cardiac damage
High-dose aspirin reduced cardiac fibronectin and collagen I
production whereas low-dose aspirin did not. Left ventricular
hypertrophy and diminished left ventricular cavity dimensions were
observed in untreated and low-dose-treated dTGR by M-mode
echocardiography. Cavity diameters of untreated dTGR and low-dose
aspirin were significantly lower vs. high-dose aspirin and
nontransgenic SD (P<0.05). Cardiac hypertrophy
index confirmed the data on matrix formation and echocardiography.
High-dose aspirin inhibits renal and cardiac NF-B and AP-1
Conflicting data about the specificity of NF-B inhibition
by aspirin was reported from various in vitro studies. We used
electrophoretic mobility shift assays (EMSA) to detect NF-B
(Fig. 1A
, C
) and AP-1
(Fig.1B
, D
) DNA binding activity in kidney and heart. Nuclear
extracts of untreated dTGR show increased NF-B DNA binding activity
in kidney and heart compared with SD
controls. High-dose aspirin reduced NF-B binding whereas
low-dose aspirin had no effect. High-dose aspirin also inhibited AP-1
DNA binding activity in kidney and heart. The effects of low-dose
aspirin could not be distinguished from untreated dTGR.
Semiquantification for NF-B and AP-1 is shown in Fig. 1E
, F
. The densitometric signal is expressed as a percentage
of untreated dTGR.
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Aspirin inhibits IB kinases (IKK) activity
To test whether the increased NF-B activity in dTGR kidneys was
caused by chronically activated IKK, we established an in vitro kinase
assay with extracts prepared from kidney tissue. After
immunoprecipitation against IKK
, the IKK activity in precipitates of
dTGR kidneys was clearly increased compared with activity from
SD controls. Specific phosphorylation of serines 32 and 36
of IB
from IKK complexes from high-dose and low-dose aspirin
groups was indistinguishable one from the other. The kinase activity in
both groups were reproducibly intermediate compared with untreated dTGR
and SD rats. IKK immunoprecipitation and subsequent
IKKß Western blotting consistently revealed more IKKß from dTGR
than SD kidneys. Based on these data, we assume that
increased IKK activity in dTGR was also determined by infiltrated
leukocytes.
Inflammatory responses in heart and kidney
Untreated dTGR showed leukocyte infiltration and dramatic
inflammatory injury. Monocytes/macrophages (Mo/Ma) infiltrated
predominantly around the damaged vessels whereas T helper
(CD4+) cells showed a perivascular location and
interstitial and cytotoxic (CD8+) T cells showed
interstitial, periglomerular, and glomerular locations.
Semiquantification revealed that high-dose and low-dose aspirin reduced
CD4+ and CD8+ cell
infiltration in the heart and kidney. However, the extent of the
reduction was significantly different between the aspirin groups.
Low-dose aspirin treatment led to more pronounced
CD4+ cell reduction, only a slight effect on
CD8+ cells, and no reduction of Mo/Ma. In
contrast, high-dose aspirin reduced all three infiltrated cell types.
High-dose aspirin also reduced immunofluorescence staining of the
NF-B/AP-1-regulated vascular cell adhesion molecule (VCAM-1) toward
SD levels. Western blotting of kidney and heart extracts confirmed the
VCAM-1 expression. Semiquantification of kidney and heart sections
showed a reduction of very late antigen VLA-4+
cells by high-dose aspirin vs. low-dose aspirin and no treatment.
CONCLUSIONS
We present the first in vivo data on IKK/NF-B
inhibition by aspirin in Ang II-induced organ damage. The effects of
high-dose aspirin were not only limited to the IKK/NF-B pathway, but
also involved AP-1 inhibition. Because NF-B functions in concert
with AP-1, the finding that high-dose aspirin inhibits both
transcription factors may have therapeutic importance. Our transgenic
rat model showed severe inflammatory end organ damage with increased
NF-B and AP-1 DNA binding activity. We treated the rats with both
high-and low-dose aspirin to distinguish between direct effects on the
NF-B-inflammatory system and possible organ protection due to
cyclooxygenase inhibition. We found that only high-dose aspirin reduced
mortality, cardiac hypertrophy, fibrosis, and albuminuria, independent
of blood pressure. In contrast, both doses blocked plasma TXB2 levels.
Ang II has been investigated for decades for causing peripheral
vascular constriction, aldosterone release, and renal sodium
reabsorption. The fact that Ang II activates NF-B through both Ang
receptors (AT1 and AT2) is a more recent finding that has a bearing on
inflammation. We documented a chain of inflammatory events involving
NF-B activation in our dTGR model earlier and showed that the
cascade could be modified by treating the rats with a NF-B
inhibitor, pyrrolidine dithiocarbamate. NF-B plays an important role
in the pathogenesis of numerous cardiovascular diseases with an
activated renin-angiotensin system. Morishita et al. demonstrated that
specific NF-B inhibition by a decoy technique reduced the extent of
myocardial infarction after reperfusion. In renal allografts,
pretreatment of donor kidneys with NF-B decoys led to reduced
NF-B activity and expression of VCAM-1, leading to reduced cell
infiltration. Inhibition of NF-B in various models with renal damage
resulted in reduced inflammation leading to improved renal function. In
the present study, only high-dose aspirin treatment was sufficient to
reduce NF-B activity, decreased mortality, cardiac hypertrophy,
albuminuria, and inflammation.
Kopp et al. and Pierce et al. showed that sodium salicylate and aspirin
can both inhibit the activation of NF-B by preventing the
phosphorylation and degradation of IB. Pierce et al. also
reported that sodium salicylate inhibited NF-B-regulated adhesion
molecules and neutrophil transmigration in endothelial cells. Our data
agree with and extend the observation by Pierce et al. from the cell
culture to the in vivo situation. A recent cell culture study provided
a cellular target for the aspirin/NF-B intervention. The ATP binding
site of IKKß was identified as target for reversible, competitive
aspirin binding whereas IKK was not affected. However, the
half-maximal inhibitory concentration IC 50 for aspirin required to
inhibit endogenous IKKß was relatively high, ranging from 0.1–5 mM.
Therefore, the in vivo relevance of their observations is still
unclear. Although serum aspirin levels of 1–2 mM seem to be too low
for NF-B inhibition, local concentrations at the site of
inflammation may reach sufficient levels. Salicylates are organic acids
and could accumulate at the mildly acidic environments occurring at
sites of inflammation. At low pH values, salicylates are uncharged and
can enter the cell membrane with subsequent deprotonation in the cell.
Anionic salicylates are trapped in a more neutral environment and could
reach concentrations that exceed the IC 50 for NF-B inhibition in
vivo. Our results show that chronic high-dose aspirin treatment was
sufficient to inhibit NF-B. Despite the reports on the direct
aspirin/NF-B interaction, various investigators have questioned the
specificity of aspirin/NF-B interaction. Frantz et al. demonstrated
the effect of sodium salicylate on CRE and AP-1 promoter activity and
kinase activity. Since AP-1 is also activated by Ang II, we addressed
the possibility that aspirin may also inhibit AP-1 activity. Our data
clearly show that high-dose aspirin reduced AP-1 activity and
AP-1-regulated fibrosis in the kidney and heart and might explain the
marked reduction in cardiac hypertrophy. It was recently shown that
salicylates inhibited smooth muscle cell proliferation by cell cycle
arrest at the G1-S phase. In addition, Wang and Brecher showed that
salicylate inhibits c-src phosphorylation. Since c-src plays an
important role in Ang II signaling, high-dose aspirin might also
inhibit c-src phosphorylation in vivo.
In summary, our results are the first to demonstrate that IKK/NF-B,
as well as AP-1 inhibition, by aspirin leads to organ protection in
vivo. High-dose aspirin reduced mortality, cardiac hypertrophy,
fibrosis, and albuminuria independent of blood pressure. Aspirin or its
derivatives may have therapeutic utility in ameliorating Ang II-related
effects. Thus, more potent IKKß inhibitors may be an important
therapeutic option in cardio-vascular disease.
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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.00-0843fje ; to cite
this article, use FASEB J. (June 27, 2001)
10.1096/fj.00-0843fje 
2 These authors contributed equally to this
work.
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