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α-Lipoic acid reduces expression of vascular cell adhesion molecule-1 and endothelial adhesion of human monocytes after stimulation with advanced glycation end products

Authors:
  • Profil Neuss, Germany

Abstract and Figures

Advanced glycation end products (AGEs) have been identified as relevant mediators of late diabetic complications such as atherosclerotic disease. The endothelial migration of monocytes is one of the first steps in atherogenesis and monocyte-endothelial interaction itself is linked to the express ion of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1). Recently, stimulation of VCAM-1 by AG Es has been demonstrated. Since endothelial stimulation by AGEs is followed by generation of oxygen free radicals with subsequent activation of nuclear transcription factor kappa B, we investigated the influence of alpha-lipoic acid on the expression of VCAM-1 and monocyte adherence to endothelial cells in vitro by means of cell-associated chemiluminescence assays and quantitative reverse transcriptase polymerase chain reaction using a constructed recombinant RNA standard. We found that alpha-lipoic acid was able to decrease the number of VCAM-1 transcripts from 41.0+/-11.2 to 9.5+/-4.7 RNA copies per cell in AGE-stimulated cell cultures. Furthermore, expression of VCAM-1 was suppressed in a time- and dose-dependent manner by alpha-lipoic acid as shown by chemiluminescence endothelial cell assay. Pretreatment of endothelial cells with 0.5 mM or 5 mM alpha-lipoic acid reduced AGE-induced endothelial binding of monocytes from 22.5+/-2.9% to 18.3+/-1.9% and 13.8+/-1.8% respectively. Thus, we suggest that extracellularly administered alpha-lipoic acid reduces AGE-albumin-induced endothelial expression of VCAM-1and monocyte binding to endothelium in vitro. These in vitro results may contribute to the understanding of a potential antioxidative treatment of atherosclerosis.
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75Clinical Science (1999) 96, 75–82 (Printed in Great Britain)
α-Lipoic acid reduces expression of vascular cell
adhesion molecule-1 and endothelial adhesion
of human monocytes after stimulation with
advanced glycation end products
Thomas KUNT, Thomas FORST, Axel WILHELM, Hans TRITSCHLER†,
Andreas PFUETZNER, Oliver HARZER, Martin ENGELBACH, Albrecht ZSCHAEBITZ*,
Eckart STOFFT*and Juergen BEYER
Department of Endocrinology, Langenbeckstr. 1, University of Mainz, 55131 Mainz, Germany, *Institute of Anatomy, University
of Mainz, 55131 Mainz, Germany, and †ASTA Medica, Frankfurt, Germany
ABSTRACT
Advanced glycation end products (AGEs) have been identified as relevant mediators of late
diabetic complications such as atherosclerotic disease. The endothelial migration of monocytes
is one of the first steps in atherogenesis and monocyte–endothelial interaction itself is linked to
the expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1). Recently,
stimulation of VCAM-1 by AGEs has been demonstrated. Since endothelial stimulation by AGEs
is followed by generation of oxygen free radicals with subsequent activation of nuclear
transcription factor κB, we investigated the influence of α-lipoic acid on the expression of
VCAM-1 and monocyte adherence to endothelial cells in vitro by means of cell-associated
chemiluminescence assays and quantitative reverse transcriptase polymerase chain reaction
using a constructed recombinant RNA standard. We found that α-lipoic acid was able to decrease
the number of VCAM-1 transcripts from 41.0p11.2 to 9.5p4.7 RNA copies per cell in AGE-
stimulated cell cultures. Furthermore, expression of VCAM-1 was suppressed in a time- and
dose-dependent manner by α-lipoic acid as shown by chemiluminescence endothelial cell assay.
Pretreatment of endothelial cells with 0.5 mM or 5 mM α-lipoic acid reduced AGE-induced
endothelial binding of monocytes from 22.5p2.9 %to 18.3p1.9 %and 13.8p1.8 %respectively.
Thus, we suggest that extracellularly administered α-lipoic acid reduces AGE-albumin-induced
endothelial expression of VCAM-1 and monocyte binding to endothelium in vitro. These in vitro
results may contribute to the understanding of a potential antioxidative treatment of
atherosclerosis.
INTRODUCTION
Advanced glycation end products (AGEs) are believed to
play an important role in micro- and macrovascular
Key words: advanced glycation end products, α-lipoic acid, vascular cell adhesion molecule-1.
Abbreviations: AGE, advanced glycation end product; DMEM, Dulbecco’s modified Eagle’s medium ; HUVEC, human umbilical
vein endothelial cell; mAb, monoclonal antibody; NF-κB, nuclear transcription factor κB; RLU, relative light units ; RT–PCR,
reverse transcriptase–polymerase chain reaction; VCAM-1, vascular cell adhesion molecule-1.
Correspondence: Dr T. Kunt.
complications of diabetes mellitus [1]. They have been
characterized as irreversible products of a slow and
complex reaction of the aldehyde or keto group of sugars
with the terminal amino group of proteins [2]. Several
#1999 The Biochemical Society and the Medical Research Society
76 T. Kunt and others
studies have demonstrated that elevated levels of AGEs
are found in the serum of patients with diabetes [3], and
in patients with renal failure or receiving haemodialysis
treatment [4,5]. The cellular effect of AGEs is mediated
by specific receptors, and one of these, the receptor for
AGE (RAGE), has been identified on endothelial cells,
monocytes\macrophages, mesangial cells, neurons and
smooth muscle cells [6–11]. Binding of AGEs to this
receptor induces the generation of oxygen free radicals
[12,13] which activate nuclear transcription factor κB
(NF-κB). Activated NF-κB induces regulation of many
genes after its translocation to the nucleus. For example,
expression of vascular cell adhesion molecule-1 (VCAM-
1) is regulated by NF-κB and therefore, intracellular
oxidative stress induced by AGEs seems to be very
important for the understanding of the pathogenesis of
vascular disease in diabetes mellitus [6,13].
There are a lot of important intracellular antioxidant
defence mechanisms like the glutathione redox system,
the vitamin C\vitamin E cycle and the α-lipoic acid\
dihydrolipoic acid redox pair. Impairment of these
defence mechanisms in diabetes mellitus has been de-
scribed by several authors [14,15]. Furthermore, the
ability of α-lipoic acid, a cofactor in the α-ketoglutarate
dehydrogenase complex, to reduce lipid peroxidation, to
quench radicals, to regenerate vitamins C and E (in its
reduced form, dihydrolipoic acid), to increase intra-
cellular levels of glutathione and to prevent glycation of
serum albumin has been well documented [16–22].
The aim of this study was to characterize the influence
of antioxidative treatment with α-lipoic acid on the
AGE-mediated expression of VCAM-1 in cultured en-
dothelial cells and, as the functional task of the study, on
the endothelial adhesion of human monocytes in vitro
after stimulation with AGEs.
MATERIAL AND METHODS
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) were
isolated according to the method of Jaffe
!and co-workers
[23] by perfusion of the human umbilical veins with
0.1% collagenase for 20 min. The harvested cells were
washed with medium 199 and plated into 25-ml flasks
coated with gelatin. Cells were cultured in medium 199
containing 20% (v\v) fetal calf serum (Greiner, Fric-
kenhausen, Germany), 5 mg\ml endothelial growth
supplement (Sigma Aldrich, Deisenhofen, Germany),
100 units\ml penicillin and 100 µg\ml streptomycin (Life
Technologies, Eggenstein, Germany). Purity of endo-
thelial cells was checked by indirect immunofluorescence
microscopy using a monoclonal antibody (mAb) against
von Willebrand factor. Only early passages were used for
subsequent studies.
Preparation of AGE-BSA
The method of preparation of AGE-BSA has been
published many times (see [6,13]). Briefly, BSA (Sigma
Aldrich) was incubated in PBS with 0.5 M glucose at
37 mC for 6 weeks with 2 µg\ml PMSF, 2.5 mM EDTA
and antibiotics as described above. The sample was
dialysed against PBS and AGE-BSA was identified by
fluorescence spectrometry. The identity of AGE-BSA
has been checked by spectrofluorometry. The presence
of endotoxin was excluded by a Limulus polyphemus
assay (Sigma Aldrich). In addition, experiments were
performed with commercial glycated albumin (Sigma
Aldrich) in equal concentrations. There were no sig-
nificant differences between commercial and newly syn-
thesized glycated albumin concerning the expression of
endothelial adhesion molecules. The experiments report-
ed below were performed with the synthesized glycated
albumin.
Chemiluminescence endothelial cell assay
The second passages of HUVECs were cultured in
microtitre plates coated with gelatin. After the different
treatment procedures, cells were fixed with methanol\
ethanol (2:1, v\v) for 20 min. Incubation with VCAM-1
mAbs (mouse anti-human, Immunotech, Hamburg,
Germany) for 2 h at a concentration of 400 ng\ml and
washing steps with PBS were performed. After a second
incubation with a chemiluminescent-labelled secondary
antibody (flashlight-GxMIgG; Biotrend, Ko
$ln, Ger-
many) at a concentration of 25 ng\ml, the photonic
emission (relative light units) was measured in a lumino-
meter (EG & Bertold, Bad Wildbad, Germany) and
analysed after subtraction of the photonic emission of
non-specific binding.
Adhesion assays
Blood was drawn from 10 healthy donors and anti-
coagulated with 5000 i.u. of heparin. First, each blood
sample was diluted with an equal volume of PBS.
Mononuclear cells were obtained by density gradient
centrifugation using Ficoll (Pharmacia). The interface
was collected and washed twice with Dulbecco’s
modified Eagle’s medium (DMEM). These cells were
then incubated (in DMEM containing 20% fetal calf
serum) with magnetizable polystyrene beads
(20i10'beads\ml for 30 min; Dynal, Hamburg,
Germany) coated with a primary mAb specific for the
CD14 membrane antigen. The cell suspension was placed
in a magnetic separator rack (Dynal) and washed twice in
DMEM\20% fetal calf serum. Cell number was cali-
brated using a PC-based cell counter system (Casy 1,
Scha
$rfe Systems, Freiburg, Germany). The purity of
monocytes achieved by this method was 98% as
documented by microscopical counting after Giemsa
staining. The second passages of HUVECs were grown
to confluence in microtitre plates coated with gelatin.
#1999 The Biochemical Society and the Medical Research Society
77Vascular cell adhesion molecule-1 and antioxidative treatment
After co-incubation with the monocytes (100000 per
cm#, 45 min), cells were fixed with methanol\ethanol
(2:1) for 20 min and blocked with 1% blocking reagent
(Boehringer Mannheim, Germany) for 30 min. Incu-
bation with mAbs (mouse anti-human) against pan-
leucocyte membrane antigen CD45 (Immunotech) for
2 h at a concentration of 400 ng\ml and washing steps
with PBS were performed. After a second incubation
with a chemiluminescent-labelled secondary antibody
(flashlight-GxMIgG; Biotrend) at a concentration of
25 ng\ml, the photonic emission (relative light units) was
measured in a luminometer (EG & Bertold, Bad Wildbad,
Germany). Percentage of bound monocytes was analysed
according to the photonic emission of a standard curve
of chemiluminescent-labelled monocytes. Scanning
electron microscopy was performed according to stan-
dard procedures.
Quantitative reverse transcriptase–
polymerase chain reaction (RT–PCR)
Endothelial cells were harvested by digestion with 0.05 %
trypsin–EDTA and the cell number was determined in a
cell counter (Casy 1, Scha
$rfe Systems). RNA was isolated
using silica-gel-based membranes (Qiagen, Santa Clarita,
CA, U.S.A.) and its concentration was measured photo-
metrically (Pharmacia Biotech, Freiburg, Germany).
The internal standard was constructed by synthesizing
(Pharmacia, Freiburg, Germany) two oligonucleotides
(‘triple primers’) of approximately 60 bases containing
sequences for the T7 promoter, the target gene (VCAM-
1), the spacer gene (glutathione transferase-GSTM4 gene)
and a poly(dT) tail. The recombinant RNA forward
primer contained the T7 promoter sequence, the VCAM-
1 forward primer and the GSTM-4 forward primer (5h-
TAATACGACTCACTATAGGCGGGGAGCTAC-
AGCCTCTTTTAATGCCTTGAAGGCCAGGA-3h).
The reverse primer included the poly(dT) tail instead of
the T7 promoter (5h-TTTTTTTTTTTTTTTCTGTGT-
CTCCTGTCTCCGCTGGAAGTGAAGAGGCCC-
AATA-3h). Polymerase chain reaction (PCR) was per-
formed in a final volume of 50 µl: 40 cycles, 5 µlof
10iPCR buffer, 0.2 mM of each deoxyribonucleoside
triphosphate, 20 pmol of each recombinant RNA
forward and reverse primer, 2.5 mmol of MgCl#, 2 units
of Taq DNA polymerase (all by Boehringer Mannheim)
and 1 µg of endothelial cDNA obtained by reverse
transcription of isolated endothelial RNA (First Strand
cDNA Synthesis Kit, Boehringer Mannheim). The PCR
product was re-amplified under the same conditions. The
pooled PCR products were purified (gel purification kit,
Qiagen, Santa Clarita, CA, U.S.A.) and transcribed into
RNA by T7 polymerase (Boehringer Mannheim) in a
final volume of 30 µl: 0.2 pmol of PCR product, 3 µlof
10iT7 polymerase buffer, 0.5 mM of each NTP,
1 unit\µl RNase inhibitor and 1 unit\µl T7 polymerase.
The recombinant RNA produced was treated with
DNase to remove the DNA template and purified as
described above. The following competitive reverse
transcription was carried out with five reactions for each
sample, containing an aliquot of the prepared RNA
sample and a dilution series of the recombinant RNA
internal standard. Then, 5 µl of each transcription prod-
uct was used in a PCR in a final volume of 50 µl: 35
cycles, 5 µlof10iPCR buffer, 0.2 mM of each deoxy-
ribonucleoside triphosphate, 20 pmol of each VCAM-1
forward (5h-CGGGGAGCTACAGCCTCTTT-3h) and
reverse primer (5h-CTGTGTCTCCTGTCTCCGCT-
3h), 1.25 mmol of MgCl#and 2.5 units of Taq DNA
polymerase.
After agarose gel (1.5% plus ethidium bromide)
electrophoresis, quantification by PC-based densito-
metry was performed. The calculated amount of specific
mRNA was related to the cell number in order to
determine the number of RNA copies per cell.
Scanning electron microscopy
Fixation, dehydration, critical-point drying, and sput-
tering (polaron equipment) of the electron microscopy
samples were performed according to standard methods
with subsequent analysis by a stereoscan MK 250
(Cambridge) electron microscope.
Statistical analysis
Values are expressed as meanspS.D. The statistical
significance was assessed by non-parametric analysis
(Mann–Whitney U-test).
RESULTS
Since AGEs have been reported to induce oxygen free
radicals after binding to their receptor [12,13], we
investigated whether α-lipoic acid was able to influence
the NF-κB-related expression of VCAM-1 on human
endothelial cells after stimulation with AGE-BSA.
HUVECs were pretreated with α-lipoic acid in various
concentrations (0.05–10 mM) and with different pre-
incubation periods (0–24 h). Endothelial expression of
VCAM-1 antigen was assessed by a chemiluminescence
assay [photonic emission of bound mAbs was measured
over a period of 5 s and defined as relative light units
(RLU)]. Untreated HUVECs revealed 66.7p14.5 RLU
(Figure 1) whereas the photonic emission of AGE-
BSA-stimulated cells (1 µM; 5 h) was raised to
1014.0p179.0 RLU (P0.0001). Pretreatment of
HUVECs with 10 mM α-lipoic acid suppressed VCAM-
1 expression to baseline levels independent of the
duration of antioxidative treatment. A preincubation
period of 24 h was required to suppress VCAM-1 in
endothelial cells if α-lipoic acid was used at a concen-
#1999 The Biochemical Society and the Medical Research Society
78 T. Kunt and others
Figure 1 Chemiluminescence analysis of VCAM-1 antigen presentation
HUVECs were stimulated with 1 µM AGE-BSA for 5 h (A). Antioxidative treatment with 10 mM, 5 mM, 0.5 mM or 0.05 mM α-lipoic acid started 0–24 h before AGE
stimulation. Untreated HUVECs (C) served as a control. The time- and dose-dependent influence of α-lipoic acid is apparent. Statistical analysis was assessed by non-
parametric analysis (Mann–Whitney
U
-test).
Figure 2 Quantitative RT–PCR for VCAM-1 by competition of the target sequence (T) with a recombinant RNA standard (S)
Each lane represents the competition of an aliquot of the RNA sample (lower row: 205 bp) with a dilution of the recombinant RNA standard (upper row : 165 bp). The
dilution factor of the standard (895i108copies) is shown above each lane. An increasing number of VCAM-1 transcripts shifts the intensity of target sequence (T)
products (lower row) to the right because of better quantitative competition with the following higher concentration of standard (S).
tration of 5 mM (66.4p23.0 RLU ; P0.0001). Never-
theless, even if 5 mM α-lipoic acid was administered
simultaneously with AGE-BSA, the VCAM-1 signal
decreased to 233.1p27.8 RLU (P0.0001). The sim-
ultaneous treatment of HUVECs with 0.5 mM α-lipoic
did not decrease the expression of VCAM-1 antigen
significantly (912.5p106.9 RLU), but longer pre-
incubation periods were able to definitely attenuate the
VCAM-1 response of HUVECs to AGE-BSA. The
lowest concentration of α-lipoic acid (0.05 mM) used in
this study failed to affect VCAM-1 antigen presentation.
It should be emphasized that the influence of α-lipoic
acid on VCAM-1 increased constantly in a time-de-
pendent manner over 24 h without revealing a maximum
value.
Quantitative RT–PCR analysis using an internal
recombinant RNA standard was performed in order to
quantify the single cell-associated number of VCAM-1
#1999 The Biochemical Society and the Medical Research Society
79Vascular cell adhesion molecule-1 and antioxidative treatment
Table 1 Densitometric analysis of the recombinant RNA
standard and the VCAM-1 target sequence of three different
samples: untreated HUVECs (control), HUVECs stimulated
with 1 µM AGE-BSA and HUVECs treated with 0.5 mM α-lipoic
acid for 6 h before AGE-BSA stimulation
AGE-BSA (1 µm)j
Control AGE-BSA (1 µM) α-lipoic acid (0.5 mM)
Standard Target Standard Target Standard Target
1167 0 1176 0 911 0
1190 0 616 585 676 0
559 174 42 1112 311 336
0 543 0 1212 0 738
0 500 0 1214 0 810
Table 2 Influence of α-lipoic acid on VCAM-1 transcripts
after stimulation with AGE-BSA
Quantitative RT–PCR analysis of VCAM-1 transcripts was performed in 28 samples
(control:
n
l12; AGE-BSA:
n
l8; AGE-BSAjα-lipoic acid:
n
l8) or 140
competitive PCR products respectively. HUVECs were treated with 1 µM AGE-BSA
for 45 min with or without preincubation of 0.5 mM α-lipoic acid for 6 h.
Unstimulated HUVECs served as a control.
VCAM-1 RNA
P
value
copies/cell (cf. AGE-BSA)
Control 4.3p3.1 0.0001
AGE-BSA (1 µM) 41.0p11.2 –
AGE-BSA (1 µM)j9.5p4.7 0.0001
α-lipoic acid (0.5 mM)
Figure 3 Scanning electron microscopy of a HUVEC mono-
layer after co-incubation with separated monocytes under
control conditions
transcripts. Representative samples are shown after
agarose gel electrophoresis (Figure 2) or densitometric
analysis (Table 1). The analysis of 28 samples, i.e. 140
competitive RT–PCR reactions (Table 2), detected
4.3p3.1 copies\cell in untreated HUVECs. The VCAM-
Figure 4 Monocyte binding to HUVECs increases significantly
after stimulation of endothelial cells with AGE-BSA (see
Figure 5) as demonstrated here by a representative scanning
electron microscopy image
Figure 5 Chemiluminescence analysis showing that α-lipoic
acid decreases endothelial binding of monocytes after stimu-
lation with AGE-BSA
The amount of bound monocytes was calculated with reference to the photonic
emission of a monocyte standard used in each assay. Values are meanspS.D. of
24 samples in each column.
1 transcription was increased to 41.0p11.2 copies\cell
after stimulation with AGE-BSA for 45 min. According
to the results of the chemiluminescence assays of VCAM-
1 antigen, the amount of detectable VCAM-1 transcripts
was reduced after pretreatment of HUVECs with 0.5 mM
α-lipoic acid for 6 h (9.5p4.7; P0.0001).
It was a special interest of this study to characterize the
functional impact of antioxidative down-regulation of
VCAM-1 on monocyte adhesion to endothelium in vitro.
Human monocytes were purified using mAbs against
the CD14 membrane antigen and co-incubated with
confluent endothelial monolayers (Figures 3 and 4).
Chemiluminescence analysis revealed that monocyte
binding to AGE-BSA-stimulated HUVECs increased to
#1999 The Biochemical Society and the Medical Research Society
80 T. Kunt and others
22.5p2.9% versus 11.2p1.4% in controls (Figure 5). In
order to examine the influence of antioxidative treatment
on the adhesion of monocytes, HUVECs were incubated
with 0.5 mM or 5 mM α-lipoic acid 6 h before stimulation
with AGE-BSA. The percentage of bound monocytes
decreased to 18.3p1.9% (Pl0.01) and 13.8p1.8%
(P0.0001) respectively.
DISCUSSION
Atherosclerotic diseases like coronary heart disease are
the major cause of morbidity and mortality in diabetes
mellitus [24,25]. Atherosclerosis itself is characterized by
focal thickening of the intima of arteries and it is well
known that more than 50% of the cells in the lipid core
of atherosclerotic plaques are derived from monocytes
[26]. Furthermore, the migration of monocytes in the
arterial wall is an early step in the pathogenesis of
atherosclerosis [27]. This recruitment of monocytes is
related to the expression of leucocyte-specific integrins
and endothelial adhesion molecules like E-selectin, in-
tercellular adhesion molecule-1 (ICAM-1) or VCAM-1
[28–31]. Although there is widespread agreement that
development of atherosclerosis is accelerated in both
Type 1 and Type 2 diabetes mellitus, the mechanisms
leading to increased monocyte binding to endothelium in
diabetes are only partially known. A number of potential
mechanisms have been described that may explain the
increase in monocyte accumulation in diabetic vessels.
For example, increased levels of diacylglycerol due to
enhanced glycolysis in hyperglycaemia activate protein
kinase C which influences vascular functions and haemo-
dynamic changes in diabetes [32,33]. Furthermore,
glycosylation of low-density lipoproteins has been
shown to result in increased oxidation, and oxidized low-
density lipoprotein stimulates endothelial cells to bind
monocytes [34]. In recent years, many groups have
focused on the influence of AGEs and oxidative stress on
the development of diabetic macrovascular disease
[2,35–39]. AGEs result from non-enzymic glycation of
proteins or lipids, initially forming reversible early
glycation products (Schiff bases and Amadori products).
These early glycation products can undergo further
complex molecular rearrangements and become irre-
versible AGEs [2]. AGEs are found in the plasma and
accumulate in the extracellular matrix of the vessel wall in
diabetes [2,3]. The early discovery of a macrophage
receptor system for the internalization and degradation
of AGEs began the speculation about receptor-mediated
interactions [40]. Receptors for AGEs (RAGEs) have
been characterized on several cell types and it is known
that their binding to RAGE induces oxidative stress with
subsequent radical-dependent activation of NF-κB [13].
Activated NF-κB is translocated to the nucleus and
initiates changes in vascular homoeostasis and endothelial
dysfunction due to its ability to promote synthesis of
defence and signalling proteins [41]. Although this study
has only been performed with AGE-BSA, convincing
data have been published which demonstrate that other
free-radical generator systems can also stimulate the
expression of VCAM-1 [42,43]. Under physiological
conditions, free radicals are rapidly eliminated by anti-
oxidative defence mechanisms, e.g. the glutathione redox
system, the vitamin C\vitamin E cycle and the α-lipoic
acid\dihydrolipoic acid redox pair [14,15]. In this con-
text, studies demonstrating the impairment of anti-
oxidative systems [37,38] in diabetes mellitus are of
considerable interest.
Since AGEs have been shown to induce NF-κB
activation and expression of VCAM-1 [6], the potential
influence of α-lipoic acid on transcription and expression
of VCAM-1 has been investigated in this study using
HUVECs. Even if HUVECs never undergo athero-
sclerotic processes they are a common source of research
on atherosclerosis because of their comparable behaviour
concerning the expression of adhesion molecules. We
were able to demonstrate a time- and dose-dependent
effect of α-lipoic acid on the expression of VCAM-1
antigen in cultured endothelial cells by means of a cell-
associated chemiluminescence assay. The highest con-
centration of α-lipoic acid (10 mM) suppressed VCAM-
1 presentation even when administered simultaneously
with AGE-BSA. Furthermore, the results of the experi-
ments performed with 5 mM and 0.5 mM α-lipoic acid
revealed that the increase of the suppression of VCAM-
1 response to AGE-BSA continued over time and was
not saturable. This does not correlate with the findings of
Bierhaus et al. [13] who demonstrated that the
suppressing effect of α-lipoic acid on NF-κB activation
was reduced after a preincubation period of 8 h. They
discussed the loss of α-lipoic acid inhibitory capacity on
the basis of metabolic degradation. However, Bierhaus et
al. were working with bovine aortic endothelial cells
whereas we performed our studies with human umbilical
vein endothelial cells. Thus, the experimental setting is
not comparable. It is noteworthy that Bierhaus et al. were
able to show that α-lipoic acid inhibits translocation of
NF-κB after its inhibitory protein IκB has been
phosphorylated. They speculate that α-lipoic acid exerts
its inhibitory effect a step behind phosphorylation, e.g.
by acting on the phosphorylated NF-κB\IκB complex or
the proposed IκB-protease [13].
We constructed a recombinant internal RNA standard
according to the method described by vanden Heuvel et
al. [44] in order to measure the expression of VCAM-1-
mRNA in endothelial cells by means of quantitative
RT–PCR which has become a valuable technique for
detection and quantification of mRNA levels, especially
for products of immunological interest [45]. The number
of VCAM-1 transcripts increased from 4.3p3.1 to
41.0p11.2 copies per cell after stimulation with AGE-
#1999 The Biochemical Society and the Medical Research Society
81Vascular cell adhesion molecule-1 and antioxidative treatment
BSA. Preincubation of endothelial cells with 0.5 mM α-
lipoic acid 6 h before AGE-BSA treatment decreased the
mRNA level to 9.5p4.7 (P0.0001) copies per cell.
Thus, the inhibitory effect of α-lipoic acid could also be
demonstrated at the level of transcription.
The last part of the study concerned the functional
impact of the above findings on monocyte adhesion to
endothelium in vitro. Increased binding of monocytes in
vitro is found in leucocytes isolated from patients with
Type 1 and Type 2 diabetes and has been associated
with the degree of hyperlipidaemia and hypergly-
caemia ([46,47]; T. Kunt, T. Forst, B. Fru
$h, O. Harzer,
A. Pfu
$etzner, M. Engelbach, H. Lo
$big and J. Beyer, un-
published work). Furthermore, the effect of AGEs on the
endothelial adhesion of monocytes or monocyte-derived
cell lines has also been demonstrated [6]. In our study
the percentage of bound monocytes increased from
11.2p1.4 % in control cultures to 22.5p2.9% in cultures
stimulated with AGE-BSA. We were able to show that α-
lipoic acid not only decreased the transcription and
expression of VCAM-1 but also attenuated the adhesion
of monocytes to 18.3p1.9% (0.5 mM α-lipoic acid) and
13.8p1.8% (5 mM α-lipoic acid).
The naturally occurring α-lipoic acid is used in the
therapy of diabetic polyneuropathy [19]. Dietary sup-
plied α-lipoic acid is readily absorbed and has been
proven to be relatively free of side effects [15]. It has been
introduced into polyneuropathy treatment because of its
ability to reduce hyperglycaemia-induced neurological
failure via improvement of ATP production and energy
supply [48]. Nevertheless, neuronal dysfunction in dia-
betes is also supposed to be mediated by endothelial
dysfunction of nutritional capillaries of the nerve fibres
[16]. As mentioned already, α-lipoic acid has been
demonstrated to be a potent radical scavenger (coenzyme
of the pyruvate and α-ketoglutarate dehydrogenase
complex) and may therefore contribute to the restoration
of oxygen free-radical-related cellular disorders such as
endothelial dysfunction due to expression of proteins
that are mediated by NF-κB activation, e.g. endothelin-1
or tissue factor [13].
Although these data cannot be extrapolated to in vivo
human pathology, our findings have characterized the
inhibitory effect of α-lipoic acid transcription on ex-
pression of VCAM-1 and on AGE-mediated monocyte
adhesion in vitro. Further studies are necessary in order
to validate this finding in vivo and the potential role of α-
lipoic acid in the treatment of diabetes-associated macro-
vascular disease.
ACKNOWLEDGMENTS
We wish to thank Dr. V. Krahn and Anke Bastelberger
(Institute of Anatomy, Mainz, Germany) for their pro-
fessional assistance in scanning electron microscopy; α-
lipoic acid (Thioctacid) was provided by ASTA Medica
(Frankfurt, Germany). Part of this work was presented
at the annual meeting of the German Society for Dia-
betes (abstract no. 76) in Lu
$beck, Germany, 1997, and
at the annual meeting of the American Diabetes
Association (abstract no. 440) in Boston, U.S.A., 1997.
Methodological aspects of this work were honoured
with the Albert-Knoll Award in 1997 (T.K.).
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... Aside from the antioxidant and pro-cognitive effects described in the brain, ALA may exert systemic effects; at the cellular level, ALA alters the nuclear factor kappa-beta related signal transduction cascade, which, in turn, regulates the expression of pro-inflammatory cytokines, metalloproteases, and cell adhesion molecules resulting in an anti-inflammatory effect [40,41]. Plus, ALA and its active metabolite DHLA can both exert a strong chelating effect on redox-active metal ions, such as zinc, copper, iron, and magnesium: this may be relevant as metal ion accumulation constitutes a molecular hallmark of aging processes [42,43]. ...
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... Moreover, ALA could markedly suppress AGEs-induced activation of NF-kB in cultured vascular endothelial cells and in retinal endothelial cells 37 . Exogenous administration of ALA diminished AGEs-induced endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte binding to endothelium 38 . Furthermore, ALA prevented the up-regulation of AGEs-induced inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in murine microglial cells 39 . ...
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The therapeutic potential of α-lipoic acid (thioctic acid) was evaluated with respect to its influence on cellular reducing equivalent homeostasis. The requirement of NADH and NADPH as cofactors in the cellular reduction of α-lipoic acid to dihydrolipoate has been reported in various cells and tissues. However, there is no direct evidence describing the influence of such reduction of α-lipoate on the levels of cellular reducing equivalents and homeostasis of the NAD(P)HNAD(P) ratio. Treatment of the human Wurzburg T-cell line with 0.5 mM α-lipoate for 24 hr resulted in a 30% decrease in cellular NADH levels. α-Lipoate treatment also decreased cellular NADPH, but this effect was relatively less and slower compared with that of NADH. A concentration-dependent increase in glucose uptake was observed in Wurzburg cells treated with α-lipoate. Parallel decreases (30%) in cellular NADHNAD+ and in lactate/pyruvate ratios were observed in α-lipoate-treated cells. Such a decrease in the NADHNAD+ ratio following treatment with α-lipoate may have direct implications in diabetes, ischemia-reperfusion injury, and other pathologies where reductive (high NADHNAD+ ratio) and oxidant (excess reactive oxygen species) imbalances are considered as major factors contributing to metabolic disorders. Under conditions of reductive stress, α-lipoate decreases high NADH levels in the cell by utilizing it as a co-factor for its own reduction process, whereas in oxidative stress both α-lipoate and its reduced form, dihydrolipoate, may protect by direct scavenging of free radicals and recycling other antioxidants from their oxidized forms.
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The therapeutic potential of α-lipoic acid (thioctic acid) was evaluated with respect to its influence on cellular reducing equivalent homeostasis. The requirement of NADH and NADPH as cofactors in the cellular reduction of α-lipoic acid to dihydrolipoate has been reported in various cells and tissues. However, there is no direct evidence describing the influence of such reduction of α-lipoate on the levels of cellular reducing equivalents and homeostasis of the ratio. Treatment of the human Wurzburg T-cell line with 0.5 mM α-lipoate for 24 hr resulted in a 30% decrease in cellular NADH levels. α-Lipoate treatment also decreased cellular NADPH, but this effect was relatively less and slower compared with that of NADH. A concentration-dependent increase in glucose uptake was observed in Wurzburg cells treated with α-lipoate. Parallel decreases (30%) in cellular and in lactate/pyruvate ratios were observed in α-lipoate-treated cells. Such a decrease in the ratio following treatment with α-lipoate may have direct implications in diabetes, ischemia-reperfusion injury, and other pathologies where reductive (high ratio) and oxidant (excess reactive oxygen species) imbalances are considered as major factors contributing to metabolic disorders. Under conditions of reductive stress, α-lipoate decreases high NADH levels in the cell by utilizing it as a co-factor for its own reduction process, whereas in oxidative stress both α-lipoate and its reduced form, dihydrolipoate, may protect by direct scavenging of free radicals and recycling other antioxidants from their oxidized forms.
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Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.
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The influence of alpha-lipoic acid (CAS 62-46-4) on the amount of intracellular glutathione (GSH) was investigated in vitro and in vivo. Using murine neuroblastoma as well as melanoma cell lines in vitro, a dose-dependent increase of GSH content was observed. Dependent on the source of tumor cells the increase was 30-70% compared to untreated controls. Normal lung tissue of mice also revealed about 50% increase in glutathione upon treatment with lipoic acid. This corresponds with protection from irradiation damage in these in vitro studies. Survival rate of irradiated murine neuroblastoma was increased at doses of 100 micrograms lipoic acid/d from 2% to about 10%. In agreement with the in vitro studies, in vivo experiments with whole body irradiation (5 and 8 Gy) in mice revealed that the number of surviving animals was doubled at a dose of 16 mg lipoic acid/kg. Improvement of cell viability and irradiation protection by the physiological compound lipoic acid runs parallel with an increase of intracellular GSH/GSSG ratio.
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Advanced glycosylation endproducts (AGEs), the glucose-derived adducts that form nonenzymatically and accumulate on tissue proteins, are implicated in many chronic complications associated with diabetes and aging. We have previously described a monocyte/macrophage surface receptor system thought to coordinate AGE protein removal and tissue remodeling, and purified a corresponding 90-kD AGE-binding protein from the murine RAW 264.7 cell line. To identify AGE-binding proteins in normal animals, the tissue distribution of 125I-AGE rat serum albumin taken up from the blood was determined in rats in vivo. These uptake studies demonstrated that the liver was a major site of AGE protein sequestration. Using a solid-phase assay system involving the immobilization of solubilized membrane proteins onto nitrocellulose to monitor binding activity, and several purification steps including affinity chromatography over an AGE bovine serum albumin matrix, two rat liver membrane proteins were isolated that specifically bound AGEs, one migrating at 60 kD (p60) and the other at 90 kD (p90) on SDS-PAGE. NH2-terminal sequence analysis revealed no significant homology between these two proteins nor to any molecules available in sequence databases. Flow cytometric analyses using avian antibodies to purified rat p60 and p90 demonstrated that both proteins are present on rat monocytes and macrophages. Competition studies revealed no crossreactivity between the two antisera; anti-p60 and anti-p90 antisera prevented AGE-protein binding to rat macrophages when added alone or in combination. These results indicate that rat liver contains at least two novel and distinct proteins that recognize AGE-modified macromolecules, although p90 may be related to the previously described 90-kD AGE receptor isolated from RAW 264.7 cells. The constitutive expression of AGE-binding proteins on rat monocytes and macrophages, and the sequestration of circulating AGE-modified proteins by the liver, provides further evidence in support of a role for these molecules in the normal removal of proteins marked as senescent by accumulated glucose-derived covalent addition products, or AGEs.