Diabetes Mellitus: A Fundamental and Clinical

Text
3rd Edition

© 2004 Lippincott Williams & Wilkins

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101
Oxidative Stress, Inflammation, and
Diabetic Complications
Milagros G. Huerta
Jerry L. Nadler
Increasing evidence suggests that oxidative stress (OS) and
inflammation play major roles in the complications of diabetes
mellitus (DM). In this chapter, an overview of free radical,
inflammation, and nitric oxide (NO) pathways is presented.
Subsequently, the potential mechanisms underlying DM-induced
alterations of the activity of these pathways are reviewed in the
context of the relevance of these changes to the development of
vascular complications of diabetes. Finally, the practical
application of this information and future considerations for
prevention of DM complications are discussed.

Overview of Free Radicals

Free radicals are highly reactive molecules with unpaired
electrons in the outer orbital. Free radicals perform beneficial
tasks, such as aiding in the destruction of microorganisms and
cancer cells. Excessive production of free radicals or inadequate
antioxidant defense mechanisms, however, can lead to damage
of cellular structures and enzymes (1 ). Damage to entire tissues
can result from free radical–mediated oxidative alteration of
fatty acids, also known as lipid peroxidation (2 ). There are well
characterized reactions that lead to the formation of the
superoxide anion, hydrogen peroxide and the highly toxic
hydroxyl radical (1 ). The cytotoxic potential of the superoxide
anion is derived mainly from its ability to be converted to the
hydroxyl radical directly or through interaction with hydrogen
peroxide. The superoxide anion can also interact with NO to
form peroxynitrite, which can degrade to form the hydroxyl
radical (3 ). Peroxy radicals can remove hydrogen from lipids,
such as polyunsaturated fatty acids, resulting in the formation
of lipid hydroperoxides and further propagation of the radical
pathways by regeneration of alkyl radicals (4 ). Enzyme systems
such as nicotinamide adenine dinucleotide phosphate (NADPH)
oxidases are important sources of superoxide in cells.
Hydroperoxides have direct toxic effects on endothelial cells and
can also degrade to form the hydroxyl radical (1 ).
Hydroperoxides may also react with transition metals to form
stable aldehydes, such as malonyldialdehyde (MDA), which
damages membranes by facilitating the formation of protein
cross-links and other end products (5 ). On the other hand, Rao
and Berk (6 ) have shown that active oxygen species can
stimulate vascular smooth muscle cell (VSMC) growth and
protooncogene expression, and suggest that arterial injury,
active oxygen species production, and VSMC proliferation are
strongly related. In support of this hypothesis is genetic
evidence for a common pathway mediating OS, inflammatory
gene induction, and aortic fatty streak formation in mice (7 ).
Increasing evidence suggests that certain enzymatic pathways
of arachidonic or linoleic acid metabolism can participate in the
formation of free radicals and lipid peroxides in the vascular
and renal systems. It has been suggested that certain
lipoxygenase (LO) enzymes that react with arachidonic or
linoleic acids play an important role in atherosclerosis by
inducing the oxidation of low-density lipoprotein (LDL) (8 ). A
15-LO was found to be colocalized with oxidized LDL in
macrophage-rich areas of human atherosclerotic lesions (9 ).
Furthermore, there is evidence that a leukocyte type of 12-LO is
expressed in human vascular and mononuclear cells (10 ). The
leukocyte-type 12-LO can be induced by VSMC growth factors
such as angiotensin II (10 ,11 ) and platelet-derived growth
factor (12 ), as well as by inflammatory cytokines (13 ). In
addition, 12-LO is an important mediator of the growth,
steroidogenic, and vasopressor effects of angiotensin II
(14 ,15 ,16 ,17 ) as well as the chemotactic effects of platelet-
derived growth factor (12 ). LO products such as
hydroperoxyeicosatetraenoic acids (HPETEs) and more stable
hydroxyeicosatetraenoic acids (HETEs) can also directly induce
VSMC migration (18 ). Also, 12-HPETE and 12-HETE are potent
direct inhibitors of renin secretion in isolated kidney cortical
slices (19 ). These LO products also activate many of the
pathways linked to increased vascular and renal disease,
including protein kinase C (PKC), oncogene activation, VSMC
hypertrophy, and increased matrix production (14 ,20 ,21 ). New
evidence has also shown that 12-HETE can induce activation of
key growth- and stress-related mitogen-activated protein
kinases (MAPKs) in VSMC cardiac cells and fibroblasts
(22 ,23 ,24 ,25 ). Furthermore, certain LO products have potent
angiogenic properties at subnanomolar concentrations (26 ), and
12-HETE can increase expression of the angiogenic vascular
endothelial growth factor (27 ). Of relevance to DM are data
showing that elevated glucose can increase the activity and
expression of 12-LO in VSMCs (11 ) and that the hypertrophic
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effects of 12-HETE in VSMCs are enhanced under hyperglycemic

conditions (14 ). Table 101.1 summarizes several potential
actions of these LO products that are relevant to vascular
complications of DM. The role of 12/15 LO in atherosclerosis
was clearly shown by demonstrating that targeted gene
disruption of 12/15 LO in mice markedly reduces the rate of
atherosclerosis (28 ). The role of 12-LO in vascular disease was
recently reviewed (29 ).

Table 101.1. Potential roles of the 12- and 15-lipoxygenase

pathway in cardiovascular disorders

1. Inhibition of renal renin release (particularly 12-lipoxygenase

pathway)
2. Inhibition of prostacyclin synthesis
3. Direct vasoconstriction of certain vascular beds
4. Mediation of angiotensin II action in blood vessels and adrenal
glomerulosa (particularly 12-lipoxygenase pathway)
5. Growth-promoting effects on smooth muscle cells and cardiac
cells
6. May be involved in oxidative modification of low-density
lipoprotein
7. Increased adhesion of monocytes to endothelial cells
8. Activation of protein kinase C and key growth- and stress-
related mitogen-activated protein kinases and transcription
factors

9. Regulation of macrophage cytokine production including

interleukin 12

Morrow and co-workers (30 ) have reported that a series of free

radical–catalyzed peroxidation products of arachidonic acid,
called isoprostanes, can be formed in vivo in models of OS.
These prostanoids are predominantly formed in a
cyclooxygenase-independent manner and remain associated with
membrane phospholipids until they are released by
phospholipases. One isoprostane, 8-epi-prostaglandin (PG) F , 2a

is potentially relevant to diabetic vascular disease based on its

potent vascular and renal vasoconstrictive properties and its
growth-promoting actions for vascular smooth muscle (31 ,32 ).
Evidence shows that 8-epi-PGF 2a levels are increased in VSMCs
cultured in elevated (25 mM) glucose (33 ) and in patients with
DM (34 ).
Antioxidant defense mechanisms are critically important for the
ultimate outcome of OS and free radical action on cells and
tissues. Nonenzymatic antioxidants that affect lipid peroxidation
(LPO) include vitamin E, which inhibits the initiation step;
vitamin C, which, along with vitamin E, inhibits hydroperoxide
formation; thiol-containing compounds, such as glutathione,
cysteine, methionine, ubiquinone, and urate, which degrade
hydroperoxides into nonradical metabolites; chelators, such as
penicillamine, which bind transition metals necessary for some
reactions involved in LPO; and vitamins A and E, which
scavenge free radicals to produce a less reactive species.
Glutathione peroxidase is an enzymatic antioxidant that
degrades hydroperoxides to less reactive products.
Nonenzymatic antioxidants involved in inorganic free radical
reactions include metal chelators that inhibit the Fenton and
Haber-Weiss–type reactions; scavengers of free radicals, such
as vitamin A, vitamin E, and urate (4 ,5 ); and inactivators of
inorganic reactions, such as glutathione. Enzymatic antioxidants
that promote inactivation of inorganically derived free radicals
include superoxide dismutase (SOD), catalase, glutathione
peroxidase, and glutathione reductase, which replenishes the
intracellular supply of glutathione (35 ).

Overview of Nitric Oxide

Nitric oxide has emerged as one of the most important
molecules released from the endothelium and a variety of other
tissues. Several excellent reviews have detailed aspects of NO
synthesis and function (36 ,37 ,38 ,39 ,40 ). NO is a free radical
that can act in a paracrine or autocrine manner to produce
diverse cellular responses, both beneficial and detrimental.
NO is generated from L-arginine by a family of NO synthases
(NOS). Many cells and tissues contain specific isoforms of NOS,
which oxidize the guanidino nitrogen of arginine to form
citrulline and NO. The human NOS have been isolated and
cloned, and can generally be divided into three major
categories: endothelial NOS (eNOS or type III NOS), inducible
NOS (iNOS or type II NOS), and neuronal NOS (ncNOS or type I
NOS). The ncNOS and eNOS are constitutive,
calcium/calmodulin–dependent enzymes that synthesize small
basal quantities of NO (35 ,38 ). Evidence suggests, however,
that eNOS activity can be increased by low concentrations of
oxidized LDL (41 ), physiologic levels of insulin (42 ,43 ,44 ), sex
hormones (45 ), and exercise (46 ). Additionally,
proinflammatory cytokines such as tumor necrosis factor-α
(TNFα) downregulate eNOS expression by shortening its
messenger RNA (mRNA) half-life (47 ). Furthermore, nerve
stimulation can directly increase the release of NO from isolated
rat skeletal muscle (48 ). In contrast to the constitutive forms,
the activity and expression of iNOS is low or absent in resting
cells but can be induced rapidly by the action of certain
cytokines and lipopolysaccharide (38 ). The activity of iNOS
appears to be largely independent of intracellular calcium
concentrations (38 ). iNOS can be expressed in many cells,
including pancreatic β-cells, macrophages, fibroblasts, vascular
endothelial cells and VSMCs, mesangial cells, and cardiac
myocytes (38 ). iNOS can produce large bursts of NO, which can
be cytotoxic or can inhibit pathogens.
Most of the vascular actions of NO are mediated via the
activation of the soluble form of guanylate cyclase, which in
turn leads to an increase in cyclic guanosine monophosphate
(cGMP) (40 ). However, NO may also exert its effects by a
mechanism that does not involve cGMP, such as through the
promotion of adenosine diphosphate ribosylation (40 ,49 ).
Nitric oxide produces many desirable effects that act to
maintain the normal vascular tone and reduce the rate of
atherosclerosis (Table 101.2 ). NO was originally identified as a
potent endothelial-derived relaxing factor for vascular smooth
muscle. Reduced NO bioavailability leads to endothelial
dysfunction, a key early event in the development of
atherosclerosis (50 ). Evidence also indicates that NO can
antagonize the actions of the pressor peptides, such as
angiotensin II (51 ). NO also inhibits platelet aggregation and
adhesion through a cGMP mechanism (52 ). On activation,
platelets release NO,
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resulting in a negative feedback loop to inhibit further

activation. NO can inhibit leukocyte adhesion to activated
endothelium (53 ), thus blocking a critical step in the
atherosclerotic process. Furthermore, NO can inhibit VSMC
growth and migration (47 ,54 ) and reduce the oxidation of LDL
by macrophages (3 ). Studies also indicate that NO can reduce
expression of endothelin and platelet-derived growth factor in
normal or hypoxic endothelium (55 ). Cooke et al. (56 ) have
shown that supplementation of L-arginine, the precursor for NO,
can reduce the rate of atherosclerosis in the
hypercholesterolemic rabbit model.

Table 101.2. Beneficial vascular action of nitric oxide

 1. Potent endothelium-dependent smooth muscle vasodilator
2. Inhibition of platelet aggregation and adhesion
3. Inhibition of leukocyte adhesion to activated endothelium
4. Inhibition of vascular smooth muscle cell migration and
proliferation
5. Reduction of macrophage-dependent oxidation of low-density
lipoprotein

6. Inhibition of expression endothelin and platelet-derived growth

factor by the vascular endothelium

A wide variety of studies, therefore, have demonstrated the

beneficial actions of NO in the prevention of cardiovascular
disease. In specific circumstances, however, NO, when
generated in large quantities for long periods, can be cytostatic
or cytotoxic for organisms or cells.
The oxidative state profoundly affects NO function (39 ).
Superoxide-generating systems can inhibit constitutive NOS
activity (57 ). The superoxide anion can also react with NO to
yield peroxynitrite, which decomposes to the toxic hydroxyl
radical (3 ), which in turn can lead to substantial vessel injury
(58 ). Peroxynitrite is also a mediator of lipoprotein oxidation
(59 ). One study has shown that, under certain circumstances,
derivatives of NO can lead to biologically active oxidized LDL,
which could accelerate atherosclerosis (60 ). Peroxynitrite is also
able to induce apoptosis in various cell types (61 ). Therefore,
under states of OS, as in DM, it is possible that a lack of NO
formation or NO conversion to toxic radicals could contribute to
the development and progression of cardiovascular disease.
Clearly, hypertension and atherosclerosis, in general, have been
characterized as states showing reduced ecNOS activity (49 ).
Mechanisms by Which Elevated Glucose Could
Lead to Increased Oxidative Stress,
Inflammation, and Diabetic Complications
The weight of experimental and human evidence supports a
clear role for increased OS in many of the proposed biochemical
pathways linked to microvascular and macrovascular
complications of DM (62 ). Recently a unifying hypothesis has
been proposed suggesting that overproduction of superoxide
may be involved in many of the pathways proposed for vascular
diabetic complications (63 ). For this to be true, the diabetic
milieu must encourage an enhanced oxidative state. Table
101.3 describes potential mechanisms by which hyperglycemia
could increase the formation of free radicals and lipid peroxides.
Glucose autoxidation, as described in cell-free systems, is a
means by which glucose itself initiates free radical production
and alters the ratio of reduced nicotinamide-adenine
dinucleotide (NADH) to NAD (64 ). Glucose, in its enediol form,
+

may be autooxidized in a transition metal–dependent reaction to

an enediol radical anion, which is then converted to
ketoaldehyde, which can yield the superoxide anion. Superoxide
anion then undergoes conversion to hydrogen peroxide and,
ultimately, to the hydroxyl radical (65 ,66 ). The hydroxyl radical
produced specifically by glucose autoxidation has been shown to
damage proteins (67 ). Evidence shows that culture of VSMC
under high glucose (HG; 25 mM) conditions significantly
increased the production of superoxide and, furthermore, HG
had an additive effect to that of the inflammatory cytokine TNF-
α on superoxide production (68 ) (Fig. 101.1 ).

Table 101.3. Potential mechanisms by which hyperglycemia can

lead to free radicals and lipid peroxidation
 1. Direct autoxidation of glucose
2. Induction and activation of various lipoxygenase enzymes
3. Activation of glycation pathways and receptor for advanced
glycation and products (RAGE)
4. Stimulation of protein kinase C activity
5. Promotion of the interaction of nitric oxide with superoxide
anions to produce peroxynitrite and hydroxyl radicals
6. Reduction of the activity of the antioxidant defense
mechanisms
7. Activation of the sorbitol pathway

8. Activation of NADPH oxidases

Glucose can also increase free radical production by intracellular

activation of the sorbitol pathway, which alters the
NADH/NAD ratio (69 ). Glucose is reduced to sorbitol by aldose
+

reductase, and this reaction uses NADPH as the hydrogen donor.

Then sorbitol is oxidized to fructose using NAD as the hydrogen
acceptor and leads to an increase in the NADH/ NAD ratio. +

Increased flux through the polyol pathway is associated with

decreased myoinositol uptake, decreased Na/K ATPase activity,
and increased production of vasodilatory prostraglandins. It has
been proposed that alterations in the NADH/ NAD ratio lead to
+

changes in vascular permeability and flow (69 ). It may also lead

to increases in diacylglycerol, which in turn activates the PKC
pathway.
In addition, glucose catalyzes LPO reactions (70 ). In particular,
studies have underscored the role of hyperglycemia in the
oxidative modification of LDL by a superoxide-dependent
pathway (71 ). High glucose can also upregulate cyclooxygenase
2 through PKC and OS in human aortic endothelial cell (72 ).
Elevated glucose has also been shown to increase the activity
and expression of the LO enzymes. Endothelial cells cultured in
HG have been found to produce more 15-HETE than cells
maintained in normal glucose concentrations (73 ). We have
P.1488

found that elevated glucose concentrations increase the rate of

porcine VSMC growth (74 ), and this accelerated growth was
partially attenuated by LO inhibitors. Elevated glucose markedly
increased basal 12-LO ribonucleic acid expression (11 ) using a
specific reverse transcriptase polymerase chain reaction
technique (Fig. 101.2 ). Furthermore, HG conditions markedly
enhanced the effects of angiotensin II to increase 12-LO activity
and expression (11 ) (Fig. 101.2 ) and to stimulate fibronectin
concentration in VSMCs (14 ). Moreover, angiotensin II and 12-
HETE increased fibronectin production to a greater extent in HG
(14 ).

Figure 101.1. Effect of elevated glucose concentrations on basal and

tumor necrosis factor-α (TNF-α)–induced superoxide generation by
porcine vascular smooth muscle cells (VSMCs). VSMCs growing in a
normal glucose (NG) medium were placed in medium containing 12.5
mM glucose or high glucose (HG; 25 mM) and 10% fetal calf serum (FCS)
for 1 week. Confluent cells were made quiescent for 24 hours in
Dulbecco’s modified Eagle medium (NG, 12.5 or 25 mM glucose) + 0.2%
bovine serum albumin (BSA) + 0.4% FCS. Washed cells were then placed
in fresh medium containing 0.2% BSA only. Cells were then incubated
with or without 5 ng/mL TNF-α for 4 hours and then processed for
superoxide measurement by the lucigenin chemiluminescence assay as
described (64). Results shown are the mean ± SEM (n = 6). a, p < 0.01
vs. 5.5 mM basal; b, p < 0.001 vs. 5.5 mM basal; c, p < 0.02 vs. 5.5
mM basal; d, p < 0.03 vs. 12.5 mM basal and p < 0.04 vs. 5.5 mM TNF-α;
e, p < 0.01 vs. 5.5 mM TNF-α (using analysis of variance and paired
Student’s t tests).

Figure 101.2. Regulation of porcine leukocyte-type 12-lipoxygenase (12-

LO) messenger RNA (mRNA)by angiotensin II (100 nM) treatment for 24
hours in porcine vascular smooth muscle cells cultured in normal glucose
(NG) or high glucose (HG) concentrations. mRNA samples (0.5 mg each)
were amplified for 25 cycles with porcine leukocyte 12-LO primers.
Hybridization was performed with 12-LO oligonucleotide (A) and
glyceraldehyde-3-phosphate dehydrogenase probes (B). (Reproduced
from
Natarajan R, Gu JL, Rossi J, et al. Elevated glucose and angiotensin II
increase 12-lipoxygenase activity and expression in porcine aortic smooth
muscle cells. Proc Natl Acad Sci USA 1993;90:4947
, with permission.)

One of the active areas of research is examination of the signal

transduction mechanisms of hyperglycemia-induced vascular cell
dysfunction and diabetic complications. It is well established
that HG increases the activity of PKC. Other studies have shown
that culture of VSMC under HG conditions significantly increases
the activity of the MAPKs, extracellular signal-regulated kinase
(ERK1/2), C-jun amino-terminal kinase, and p38 MAPK (75 ,76 ).
Furthermore, HG and angiotensin II had additive effects on
ERK1/2 and p38 MAPK activation (75 ). Because these MAPKs are
key transducers of signals to the nucleus and effectors of gene
transcription (77 ), their activation represents an important
mechanism by which hyperglycemia can alter cellular behavior.
Recent studies have shown a clear relationship of MAPK and 12-
LO pathways in matrix protein expression in response to glucose
and Ang 2 (78 ).
It has also been shown that HG culture of VSMC can lead to
increased expression of the oxidant-sensitive transcription
factors, activator protein-1 (AP-1) (75 ) and nuclear factor κB
(NFκB) (68 ). Figure 101.3 shows that basal activity of NFκB is
increased nearly twofold in cells cultured in HG. Furthermore,
HG culture of VSMC increased the stimulatory effects of
angiotensin II on AP-1 activation (75 ) and also increased the
stimulatory effects of TNF-α on NF-κB activation (68 ) (Fig.
101.3 ). Increased PKC activation was shown to be a potential
mechanism (68 ) for HG-induced NFκB activation. NF-κB
regulates the transcription of a large number of genes, including
vascular endothelial growth factor (VEGF), proinflammatory
cytokines (TNFα and IL-1β), adhesion molecules [vascular cell
adhesion molecule-1 (VCAM-1)], and advanced glycosylation
end product (AGE) receptor (68 ,78 ). VEGF has been identified
as a mediator in the development of proliferative diabetic
retinopathy, nephropathy, and neuropathy (79 ,80 ).
Proinflammatory cytokines and adhesion molecules play an
important role in the development of atherosclerosis (81 ). A
central role of the NFκB pathway in the association between
increased OS and the development of diabetic complications has
been proposed. In bovine endothelial cells, hyperglycemia
causes an initial increase in intracellular reactive oxygen
species (ROS) and activation of NFκB, with a subsequent
increase in PKC activity, AGE, and sorbitol levels. Disruption of
mitochondrial production of ROS by either overexpression of
manganese SOD, the mitochondrial form of SOD, or
overexpression of uncoupling protein-1 production, leads to
suppression of the hyperglycemia-induced effects on NFκB, PKC,
AGE, and sorbitol (63 ). Furthermore, in streptozotocin-induced
diabetic mice, overexpression of SOD attenuated early diabetic
glomerular changes (82 ). Glucose challenge in humans can lead
to clear increases in ROS in leukocytes (83 ), and postprandial
hyperglycemia thus may be a factor in complications (84 ).
These findings indicate that OS may be the initial change
induced by hyperglycemia and that it leads to activation of
stress-activated signaling pathways, mainly NFκB, that regulate
gene expression, resulting in cellular damage.
Culture of endothelial cells under HG conditions leads to
increased adhesion and transmigration of monocytes (83 ,84 ),
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and increased LO products have been shown to be contributing

factors (85 ). Figure 101.4 shows that chronic culture of
endothelial cells in HG (25 mM) led to increased adhesion to
monocytes relative to acute HG, normal glucose, or chronic
mannitol. The effects of minimally oxidized LDL and
lipopolysaccharide are shown for comparison. Glucose and
diabetes also lead to endothelial dysfunction by increasing
superoxide via NADPH oxidase through a PKC-dependent
mechanism (86 ).
Figure 101.3. Hyperglycemia-induced modulation of nuclear factor κB
(NF-kB) DNA binding activity in vascular smooth muscle cells
(VSMCs). A: Serum-starved VSMCs growing for two passages in normal-
glucose (NG) or high-glucose (HG) media were treated for 3 hours alone
or with 5 ng/mL of tumor necrosis factor (TNF)-α. Nuclear proteins were
prepared and subjected to electromobility shift assays (EMSAs) to
determine activation of NF-MB using an NF-MB consensus vascular cell
adhesion molecule (VCAM) oligonucleotide. For the competition studies
shown in the last two lanes, nuclear extracts (5 mg each, from TNF-α–
treated HG cells) were pretreated with either 40× excess cold wild-type
(wt) VCAM promoter sequence oligonucleotide or 40× excess cold mutant
(m) VCAM oligonucleotide. These samples were then subjected to DNA
binding reactions with the labeled VCAM oligonucleotide and EMSA.
Results demonstrate specificity of the binding. For the supershift
experiments with p65 and p50 antibodies (fifth and sixth lanes), nuclear
extracts from TNF-α–treated cells in HG were incubated with the
respective antibodies for 1 hour at 4 °C and then EMSAs run as usual.
Specific complexes, X and Y, are indicated. Results indicate that the
binding complex is composed of p65 and p50 subunits. B: Bar graph
showing the mean ± SEM of results from the phosphorimager quantitation
of the EMSA results obtained from six experiments. *p < 0.005 vs. NG
basal; **p < 0.01 vs. NG TNF-α, by analysis of variance using Prism
software (GraphPad, San Diego, CA, U.S.A.).

Antioxidant defense mechanisms may also be reduced under

high glucose conditions as well as in DM. Hyperglycemia can
lower the activity of several enzymes, including glutathione
reductase and SOD, presumably by glycation (86 ). Endothelial
cell growth was inhibited by HG (87 ), but these effects were
reversed by glutathione, SOD, and catalase (88 ), suggesting
that increased OS coupled with impaired degradation of
superoxide and hydrogen peroxide are important mechanisms
for the glucose-induced decline in endothelial cell growth (89 ).
Similar results have been observed in VSMCs (90 ).
Recent evidence indicates that these effects of hyperglycemia
on OS are seen not only in conditions of chronic hyperglycemia
but also in acute hyperglycemia such as that observed
postprandially or during an oral glucose challenge test (91 ). In
diabetic subjects, LDL oxidation increases during the
postprandial phase and is directly related to the degree of
hyperglycemia (92 ). Further studies are needed to determine
the relevance of postprandial hyperglycemia on increased OS
and the development of diabetic complications.

Figure 101.4. Effect of high-glucose (HG) culture of human aortic

endothelial cells (HAECs) on monocyte binding to HAECs. HAECs were
cultured in HG (25 mM) for 14 days (chronic, CH-HG) or for 4 days
(acute, AC-HG) or for the same period in normal glucose (NG; 5.5 mM) or
high mannitol (CH-HM; osmolality control). Monocyte binding (with human
monocytes) experiments were then performed as described (72,74).
Minimally oxidized low-density lipoprotein (MM-LDL) and
lipopolysaccharide (LPS) were used as positive controls.

Association of Free Radicals and Advanced

Glycosylation End Products
Nonenzymatic glycosylation of proteins, or the Maillard reaction,
begins with the interaction of glucose with protein to form early
glycosylation products, known as Schiff bases and Amadori
products. Amadori products may be degraded oxidatively to
form carboxymethyl-lysine (93 ), or they may form glucose-
derived protein cross-links known as AGEs (94 ). Protein and
glucose mixtures in cell-free systems generate nanomolar
quantities of H O (65 ), whereas Schiff bases and Amadori
2 2

products are sources of the superoxide radical (95 ). In addition,

superoxide anion production by glycated polylysine, a glycated
protein, is suppressed by SOD (96 ). Glycosylated proteins drive
other free radical reactions, as evidenced by the catalysis of
LPO by glycated collagen and glucose-treated LDL (97 ). Vitamin
E was found to inhibit completely, and SOD only partially, LPO
catalyzed by glycated polylysine. However, catalase was found
to have no effect, which demonstrates the nonuniformity of
antioxidant effects on LPO induced by this process.
Carboxymethyl-lysine and pentosidine, which are sugar-derived
autoxidation products known as glycoxidation products, may
initiate and propagate free radical reactions (98 ). Interaction of
AGEs with their endothelial surface receptors (RAGEs) generates
intracellular OS, resulting in activation of NFκB, which induces
the expression of endothelin-1 and tissue factor, leading to
endothelial dysfunction (99 ). Another means by which AGE
formation plays a role in DM-related OS is through glycation and
resultant inactivation of antioxidant enzymes, such as copper-
zinc (Cu-Zn) SOD (100 ). It has been suggested that an increase
in the steady-state levels of reactive carbonyl compounds
formed from oxidative and nonoxidative reactions results in
increased
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“carbonyl stress,” which leads to increased glycoxidation and

lipoxidation of tissue protein in DM (101 ).
Maillard intermediates are capable of promoting free radical
production. However, free radical reactions may also promote
AGE formation. Glucose autoxidation, for example, enhances the
covalent attachment of glucose to protein (102 ). MDA, an end
product of LPO, facilitates protein cross-linking, a destructive
and final step in AGE formation. Conversely, the antioxidant
vitamin E prevents protein glycosylation (103 ,104 ). Therefore,
blockade of free radical formation could provide a mechanism
for preventing AGE formation or blocking AGE action. In
vivo relevance of AGEs and oxidant stress in diabetic renal
disease was demonstrated by the observation of colocalization
of AGE structures such as carboxymethyl-lysine and pentosidine
with markers of lipid peroxidation and oxidant stress in diabetic
glomerular lesions (104 ).
In endothelial cells, AGE content increases 13.8-fold after only
1 week of hyperglycemia (105 ). Basic fibroblast growth factor is
the major protein modified by AGEs in endothelial cells (69 ).
Incubation of human umbilical vein endothelial cells (HUVECs)
with AGE or HG has been shown to lead to apoptosis (61 ). The
increase in caspase 3 activity, an early marker for induction of
apoptosis, caused by HG is prevented by incubation with
antioxidants such as α-tocopherol or lipoic acid (LA).
AGE formation also alters the functional properties of several
important matrix molecules altering the structure and function
of intact vessels (69 ). In diabetic animal models, AGE
accumulation is associated with decreased vasodilatory response
to NO (106 ).
Additional evidence on the role of AGEs in the development of
diabetic complications was obtained by using aminoguanidine,
an inhibitor of AGE formation, in animal models of diabetes.
Treatment with aminoguanidine resulted in a significant
inhibition of the development of retinal acellular capillaries,
retinal microaneurysms, increased urinary albumin excretion
and mesangial fraction volume, decreased motor and sensory
nerve conduction velocity and action-potential amplitude, and
diminished arterial elasticity (107 ).

Evidence for an Enhanced Oxidative State in

Diabetes Mellitus
A number of studies indicate that DM is associated with a state
of enhanced OS, resulting from the combination of increased
ROS generation and decreased antioxidant capacity, particularly
in the poorly controlled state (86 ). Many of these studies
support an association between glycemic control and free
radical load, which is consistent with the pathways outlined
in Table 101.3 . In uncontrolled diabetes, the level of SOD, the
enzyme responsible for inactivating the superoxide radical,
along with the levels of the antioxidants vitamin E and α-LA, are
decreased (108 ,109 ,110 ,111 ). Superoxide anion production, as
determined by the ferricytochrome C method, is greater in the
serum of patients with type 1 DM compared with nondiabetic
subjects, and it correlates with glycemic control (112 ). In
patients with poorly controlled type 1 DM, increased LDL
oxidation associated with reduced antioxidant defenses has
been described (113 ). Plasma thiobarbituric acid levels, a
measure of MDA that is an indirect index of LPO, are
significantly higher in patients with poorly controlled type 2 DM
than in patients with well-controlled disease or in control
subjects (114 ). No significant difference in plasma thiobarbituric
acid levels was found between well-controlled diabetic patients
and control subjects.
Conjugated dienes, early products of the reaction of hydroxyl
radicals with polyunsaturated fatty acyl chains, are higher in
patients with type 1 diabetes compared with controls (115 ). Jain
et al. (116 ) have demonstrated increased LPO in erythrocyte
membranes of patients with type 1 DM, and other studies found
that erythrocytes from patients with type 2 DM show an 8- to
10-fold increase in lipid MDA and 13-fold higher levels of
phospholipid MDA adduct (Fig. 101.5 ) compared with healthy
controls (117 ). Furthermore, as seen in Fig. 101.5 , glucose
further increases MDA and phospholipid MDA adduct. In the
same study, it was found that LO inhibitors, but not
cyclooxygenase inhibitors, could reduce LPO induced by
glucose. In vivo relevance of the LO pathway to diabetic
complications was shown in a study that demonstrated
increased urinary excretion of 12-HETE in diabetic patients with
incipient and early renal disease (118 ). In addition, increased
oxidant stress and vascular 12-LO expression was noted in a
porcine model of DM-induced accelerated atherosclerosis (119 ).
We have developed a porcine model of accelerated
atherosclerosis due to diabetes and high-fat feeding. This model
shows clear evidence of increases in 12-LO expression and OS
(120 ).
Poor glycemic control increases NFκB activity in peripheral blood
monocytes from patients with type 1 DM (121 ). In diabetic
patients with nephropathy, a correlation exists between the
severity of albuminuria and mononuclear NFκB binding activity
(122 ). Furthermore, treatment with LA, an antioxidant, results
in significant suppresion of NFκB and plasma markers of lipid
oxidation.
Recent evidence suggests that hyperketonemia may be an
additional risk factor leading to the development of OS in
patients with type 1 diabetes (123 ). In vitro, acetoacetate has
been shown to cause lipid peroxidation in cultured human
endothelial cells at concentrations frequently found in diabetic
patients (123 ).
Diabetes mellitus is also associated with a decrease in
antioxidant defenses. Lowered total antioxidant capacity has
been demonstrated in patients with type 1 diabetes compared
with healthy controls (115 ,124 ). Vucic et al. reported that
polymorphonuclear cells of patients with both type 1 and type 2
diabetes exhibit a twofold decrease in SOD activity (125 ).
Yoshida et al. reported reduced total glutathione levels in type 2
diabetes, which were restored by treatment with an
antihyperglycemic agent (126 ).
Additional evidence exists that enhanced OS is present in target
organs during the development of diabetic complications. In the
streptozotocin-diabetic rat, there is evidence of enhanced OS in
the renal cortex at a very early stage of diabetes (127 ). In a
clinical study, diabetic nephropathy correlated with mononuclear
NFκB activation (122 ). It has been proposed that susceptibility
of the kidney to OS is an important factor in the development of
diabetic nephropathy (128 ). In experimental diabetic
neuropathy, free radical activity in the sciatic nerve is
P.1491

increased (129 ). Decreased mRNA levels of glutathione

reductase and SOD were found in preapoptotic pericytes from
human diabetic retinas compared with those from nondiabetic
subjects (130 ). Altomare et al. reported decreased glutathione
peroxidase activity and ascorbic acid levels in the lens of
diabetic patients, especially those with retinal damage (131 ).
Gurler et al. found that patients with type 2 DM and diabetic
retinopathy exhibited higher levels of MDA, a marker of lipid
peroxidation, than those without diabetic retinopathy (132 ).
They also described a significant correlation between markers of
lipid peroxidation and duration of DM. Valabhji et al. found that
antioxidant status was reduced in patients with type 1 DM and
correlated with coronary calcification, a correlate of prevalent
coronary heart disease (124 ).

Figure 101.5. Effect of glucose on malonyldialdehyde (MDA) (A) and

phospholipid-MDA (PL-MDA) (B) adduct formation in erythrocytes of
diabetes mellitus (dotted lines) and normal healthy control subjects (bold
lines). Erythrocytes (45% hematocrit) in phosphate-buffered saline (PBS)
were incubated with glucose (0–35 mM) for 24 hours at 37 °C. At the end
of incubation, erythrocytes were washed three times in PBS, and from the
aliquots, the formation of MDA and PL-MDA adduct was determined. Each
value represents mean ± SD (n = 25 for type 2 diabetes mellitus; n = 10
for normal healthy control subjects). TBA, thiobarbituric acid. (Reproduced
from
Rajeswari P, Natarajan R, Nadler JL, et al. Glucose induces lipid
perioxidation and inactivation of membrane-associated ion-transport
enzymes in human erythrocytes in vivo and in vitro. J Cell
Physiol 1991;40:100
, with permission.)

Nitric Oxide: Effects of Diabetes Mellitus

Evidence suggests that DM can produce major changes in NO
production or action. There are several likely mechanisms that
explain how DM can alter NO pathways (Table 101.4 ). In the
diabetic rat and rabbit aorta, HG conditions result in an
impaired relaxation response to acetylcholine, implying a
reduced release or action of NO (133 ,134 ). This impaired
dilation response is reversed by SOD, again suggesting an
important role of free radicals in NO pathway dysfunction in DM
(134 ). Blockade of PKC has been found to reverse the HG-
induced impairment of endothelium-dependent relaxation (135 ).
Furthermore, reduced NO-mediated increases in cGMP in
glomeruli from diabetic rats are mediated, in part, by PKC
activation (136 ), suggesting that glucose-induced increases in
PKC could be a factor in reduced NO action in DM. Data also
show that PKC inhibition can reduce superoxide formation and
restore normal activity of NOS III (137 ).
It has been suggested that inhibition of Na /K -adenosine
+ +

triphosphatase (ATPase) activity by elevated glucose could be a

factor contributing to both microvascular and macrovascular
disease (138 ). It has been shown that the glucose-induced
reduction of Na /K -ATPase activity can be completely reversed
+ +

by L-arginine or sodium nitroprusside (Fig. 101.6 ), implying

that glucose effects are secondary to inhibition of NO formation
(138 ). High glucose was demonstrated to reduce NOS activity in
endothelial cells (139 ). Studies in porcine aortic endothelial
cells exposed to HG conditions, however, actually demonstrated
a net increase in NO formation owing to an enhanced free
calcium concentration (140 ). Furthermore, spontaneous NO
release was greater in diabetic rat aorta than in controls,
although NO activity was reduced (141 ). This increase in
P.1492

NO release could represent a compensatory mechanism for the

reduced bioavailability of NO. In untreated streptozotocin-
induced diabetic rats, increased OS was associated with
decreased expression of eNOS and nNOS in the renal cortex and
eNOS in the left ventricle (142 ). Insulin therapy resulted in
upregulation of NOS isoforms and reduction in lipid and glucose
oxidation, whereas insulin therapy plus antioxidant
supplementation resulted in normalization of all these
parameters. Additional carefully controlled studies in
appropriate models evaluating NO expression, enzyme activity,
and NO release are required to further clarify the effects of DM
on NO production. LO enzymes including 12-LO, can act as a
catalytic sink for NO, inactivating its activity (143 ).

Table 101.4. Mechanisms by which diabetes can alter nitric oxide

(NO) pathways

1. Reduction of NO production
2. Reduction of NO action by interaction with advanced
glycosylation end products
3. Reaction of NO with superoxide anions to produce peroxynitrite,
which can promote oxidation of low-density lipoprotein and lead
to lipid peroxidation
4. Increased renal production of or sensitivity to NO in early
diabetic nephropathy

5. Quenching of NO by lipoxygenases and NADPH oxidases

Figure 101.6. Reversal of hyperglycemia-induced inhibition of ouabain-
sensitive Rb-uptake by L-arginine and sodium nitroprusside (SNP) in
86

endothelium-intact aorta. L-arginine (0.3 mM) and SNP (10 mM) were
added to the incubation media during the final 30 and 10 minutes,
respectively, of the 3-hour incubation; hyperglycemia failed to decrease
ouabain-sensitive Rb-uptake. The asterisk denotes values that are
86

significantly different from those in aorta incubated in 5.5 or 44

mM glucose (p < 0.05). Cont, control. (Reproduced from
Gupta S, Sussman L, McArthur CS, et al. Endothelium-dependent
inhibition of Na , K ATPase activity in rabbit aorta by hyperglycemia:
+ +

possible role of endothelium-derived nitric oxide. J Clin Invest 1992;90:

727
, with permission.)

There have been several important human studies indicating

that DM may alter NO action. In a study of 15 patients with type
1 DM (144 ), it was found that forearm vasodilatory responses to
methacholine were reduced in this population compared with
control subjects (Fig. 101.7 ). In another study of patients with
type 1 DM, it was found that blockade of NO with an NO
inhibitor or exogenous NO administration with nitroprusside
produced less of a forearm flow response in diabetic patients
than in control subjects (145 ). In this study, no difference in
stimulated NO action was demonstrated between the diabetic
patients and control subjects, suggesting that abnormalities in
NO may not directly occur in DM unless another factor, such as
enhanced AGEs or free radicals, is also present.
It is likely that NO action is reduced in type 2 DM. This could be
mediated by several factors, including hyperlipidemia, insulin
resistance, hypertension, and altered ions, such as calcium and
magnesium, which in turn could alter NO production and action.

Figure 101.7. Plot of forearm blood flow response to intraarterial infusion

of methacholine chloride in normal and diabetic subjects. Cholinergic
vasodilation was less in the diabetic group than in the normal group. The
difference between groups was significant at the 3- and 10-mg/min doses.
(Reproduced from
Johnstone MT, Craeger SJ, Scales KM, et al. Impaired endothelium
dependent vasodilation in patients with insulin-dependent diabetes
mellitus. Circulation 1993;88: 2510
, with permission.)

Diabetes mellitus can also have a profound influence on NO

action and metabolism through effects of free radicals and AGEs
on NO. In an earlier section, evidence was reviewed showing
that NO can react with superoxide anions to produce
peroxynitrite, which can lead to membrane damage and LPO.
AGEs also exert substantial effects on NO. AGEs have been
shown to quench NO in vitro and in rat models (106 ), most
likely because of an enhanced free radical load induced by
glucose. AGEs also have been shown to block the
antiproliferative effect of NO in rat aortic smooth muscle and
murine glomerular mesangial cells (146 ). In some studies,
aminoguanidine has been found to be an inhibitor of NO, thus
confounding the relationship between NO and AGEs. Bucala et
al. (106 ) demonstrated that when aminoguanidine was
administered to rats that were diabetic for less than 1 month,
the vasodilatory impairment otherwise observed in these
diabetic animals was ameliorated, suggesting that
aminoguanidine, presumably by blocking AGE formation,
increased NO. Other studies have found that aminoguanidine
exerts its beneficial effects by inhibiting the formation of NO. In
support of this, Tilton et al. (147 ) have demonstrated that
aminoguanidine inhibits NOS. Methylguanidine, which is
equipotent to aminoguanidine as an inhibitor of NOS, but which
has limited ability to prevent AGE formation, was found to
reduce regional vascular albumin hyperpermeation induced by
DM to levels comparable with those of aminoguanidine. These
data suggest that, in some instances, the mechanism by which
aminoguanidine normalizes DM-induced vascular dysfunction is
related to its ability to inhibit NO production instead of its
action to prevent AGE formation.

Therapeutic Implications of Antioxidants for the

Prevention of Diabetic Complications
Most of the studies in the literature have focused on the role of
OS as it relates to the effects of hyperglycemia on diabetic
complications. It has been established, however, that insulin
resistance
P.1493

and hyperinsulinemia are important factors linked to

hypertension and atherosclerotic cardiovascular disease
(reviewed in other chapters). Several lines of evidence now
support the concept that NO activation may be an important
mechanism in insulin-induced vasodilatory effects. Studies using
inhibitors of NOS have demonstrated a reduction in insulin’s
vasodilatory actions (44 ,148 ), although the source of NO in
response to insulin was not fully evaluated in these studies. It
is now clear, however, that skeletal muscle can synthesize NO
(48 ), suggesting that blood vessels and skeletal muscle may be
potential sources of NO release in response to insulin. One area
for future investigation will be to determine whether altered NO
release or action could be involved in altered vascular function
and hypertension in insulin-resistant states. The results of these
types of studies could provide a rationale for modulation of the
NO system to reduce diabetic complications associated with
hyperinsulinemia.
One obvious question that arises from the information available
is whether supplementation with antioxidants, such as vitamins
C and E, and LA is warranted to prevent diabetic complications.
Studies in nondiabetic subjects support the potential benefit of
vitamin E to reduce vascular disease (149 ,150 ). However, the
results of clinical trials evaluating the effect of vitamin E
supplementation in diabetics have varied. Vitamin E
supplementation has been shown to decrease or have no effect
on glycemic control, to reduce or have no effect on
triglycerides, to lower levels of lipid peroxides and thromboxane
B , and to reduce the ex vivo oxidative susceptibility of LDL
2

(151 ). Bursell et al. demonstrated that vitamin E treatment

(1,800 IU/day) was effective in normalizing abnormalities in
retinal hemodynamics and improving renal hyperfiltration in
patients with type 1 DM, particularly in those with the poorest
glycemic control and the most impaired retinal and renal
hemodynamics (152 ). A potential mechanism for this effect was
proposed by Kunisaki et al. (153 ), who demonstrated that
vitamin E and another antioxidant, probucol, normalize the
changes in diacylglycerol and PKC activation in diabetic rats.
Other studies reported that vitamin E supplementation in
diabetic patients did not exhibit a protective effect on vascular
endothelial function (154 ), and did not decrease the risk to
develop cardiovascular and renal disease (155 ). Due to these
controversial results, caution must be used in recommending
antioxidant supplements to people with DM. One basis for this
caution is that, under certain circumstances, vitamin E or C can
actually act as a prooxidant (156 ,157 ,158 ). Vitamin C shares
several cellular transport mechanisms with glucose (159 ), and it
can increase the rate of absorption of iron, which is a
prooxidant. In fact, a study in patients with type 2 diabetes
showed that increased intake of antioxidant nutrients had no
beneficial effect and, in patients taking insulin, had a potential
deleterious effect, on severity of diabetic retinopathy (160 ). The
American Diabetes Association has published a consensus
statement on this issue that states that supplementation with
antioxidant vitamins cannot be recommended for all diabetic
patients at this time (161 ).
LA is an antioxidant that combines free radical scavenging and
metal chelating properties with an ability to regenerate the
levels of other enzymatic and nonenzymatic antioxidants
(127 ). In vitro, LA has been shown to suppress TNF- and AGE-
induced activation of NFκB in cultured human aortic endothelial
cells (99 ,162 ). In vivo, 3-day oral treatment with 600 mg of LA
reduced NFκB activation in peripheral blood mononuclear cells
and was paralleled by a decrease in OS in plasma of diabetic
patients with nephropathy (122 ). In streptozotocin-diabetic
rats, LA counteracts OS in the lens, retina, renal cortex, and
peripheral nerve, and prevents manifestations of diabetic
nephropathy, neuropathy, and retinopathy (127 ). Intravenous
or oral treatment with LA in patients with type 2 diabetes has
been shown to reduce OS (163 ). In recent clinical trials, oral
and intravenous treatment with LA has proven to be beneficial
in the treatment of diabetic peripheral and cardiac autonomic
neuropathy, and it is currently used in Europe for the treatment
of this condition (164 ,165 ). There is a multicenter trial of oral
treatment with LA currently being conducted in North America
and Europe, aimed at slowing the progression of diabetic
polyneuropathy. A pilot open-labeled nonrandomized clinical
study demonstrated a potential beneficial effect of LA
supplementation in preventing the progression of endothelial
cell damage and diabetic nephropathy in patients with type 1
and type 2 diabetes (166 ).
Activation of NFκB can also be blocked by several other
antioxidants, including N-acetyl-cysteine, the glutathione
precursor L-2 oxothiazolidine-4-carboxylic acid, and resveratrol
(62 ).
Concentrations of coenzyme Q , a critical intermediate of the
10

mitochondrial electron transport chain, have been negatively

correlated with poor glycemic control and diabetic complications
(167 ). It has been shown to inhibit superoxide generation by
endothelial cells (168 ). Additionally, coenzyme
Q 10 supplementation for 12 weeks in dyslipidemic patients with
type 2 diabetes was shown to improve endothelial function
(167 ).
Additional studies in diabetic patients evaluating the effect of
these and other novel antioxidants, such as compounds that
mimic SOD or catalase activity (169 ), on OS, NO action, and
NF-κB activation will be needed to fully address their role in the
prevention of diabetic complications.
Insulin may also play a role in the development of diabetic
macrovascular complications. It has been proposed that low
physiologic concentrations of insulin induce the expression of
eNOS by activation of phosphatidylinositol-3 kinase in
endothelial cells and microvessels, resulting in vasodilation
(43 ). In contrast, high levels of insulin, such as those observed
in insulin-resistant subjects, may have proatherogenic actions,
including induction of c-myc, MAPK, and cell growth (69 ). The
possibility that hyperinsulinemic states are associated with
selective insulin resistance in the vasculature leading to
increased cardiovascular risk warrants further evaluation.
The role of nutrition should be considered as a factor related to
increased OS in DM. Evidence in diabetic animals shows that
oxidized lipids in the diet make a major contribution to the
levels of oxidized lipids in lipoproteins, and that DM increases
the rate of oxidized lipid absorption (170 ). Furthermore,
magnesium deficiency, which is a common problem in patients
with type 2 DM (171 ,172 ), has been associated with increased
free radical damage, insulin resistance, and increased
vasomotor tone (173 ,174 ). One study in diabetic patients from
St. Louis (175 ), and our data in 50 nonselected patients with
type 2 DM in Duarte (unpublished observations, 1995), indicate
that more than 50% of diabetic patients consume less than the
recommended dietary allowance of magnesium. Short-term
magnesium
P.1494

supplementation has been shown to improve endothelial

function (176 ). A recent study reported that 6 months of oral
magnesium supplementation in patients with coronary artery
disease resulted in a significant improvement in exercise
tolerance, exercise-induced chest pain, and quality of life (177 ).
Thus, studies are warranted that would address the effect on
diabetic complications of modified dietary intake of factors that
reduce oxidant stress.

Inflammation and Macrovascular Diabetic

Complications
Over the past decade, a significant amount of evidence has
been reported indicating that inflammation plays an important
role in the development of atherosclerosis. Several studies have
shown an association between inflammatory markers (evidence
of chronic subclinical inflammation) and increased incidence of
cardiovascular disease in diabetic patients. The “response to
injury” hypothesis (Fig. 101.8 ) proposes that extravascular or
intravascular proinflammatory conditions such as oxidized LDL,
advanced glycosilation end-products, or chronic infection lead to
an increased secretion of proinflammatory cytokines such as
interleukin-1 (IL-1), TNF, and IL-6 (178 ). These in turn will
influence all processes of atherogenesis from increased
monocyte adhesion to endothelial cells to increased risk of
atherosclerotic plaque rupture (81 ).
Figure 101.8. The role of inflammation in atherogenesis. Vascular and
intravascular proinflammatory conditions lead to release of
proinflammatory cytokines, which in turn influence all stages of
atherosclerosis from monocyte endothelial cell interactions to plaque
rupture. Reproduced from
Huerta MG, Nadler JL. Role of inflammatory pathways in the development
and cardiovascular complications of type 2 diabetes. Current Diabetes
Reports 2002;2:396–402
, with permission.)

Monocyte binding to endothelial cells is a crucial early event in

the development of atherosclerosis. It has been shown that
hyperglycemia increases monocyte adhesion to human aortic
endothelial cells in vitro (83 ). Patients with type 2 diabetes
exhibit increased monocyte binding to endothelial cells (179 ).
Inflammation and OS have been proposed as potential
mechanisms leading to such increased monocyte binding to
endothelial cells associated with hyperglycemia and diabetes.
IL-8 is a chemokine produced by endothelial cells in response to
inflammatory stimuli. It has been shown to be chemotactic to
neutrophils and an important mediator of monocyte–endothelial
cell interactions. Glucose regulates IL-8 production at the level
of transcription and this effect is mediated at least in part by
AP-1 and CHO-RE elements located within the IL-8 promoter
(180 ). Both oxidized LDL and TNF-α can induce IL-8 mRNA in
endothelial cells, and inhibition of ROS production reduces IL-8
production.
A correlation has been shown between plasma lipid peroxide
levels and monocyte binding in patients with type 2 diabetes
(181 ). Short-term administration of a second-generation
sulfonylurea with free-radical scavenging properties, to patients
with type 2 diabetes has been shown to decrease plasma lipid
peroxides and to decrease monocyte adhesion to cultured
bovine aortic endothelial and human aortic smooth muscle cells
by a mechanism unrelated to glycemic control, but rather
through its effect inhibiting LDL oxidation (181 ). Similarly, α-
tocopherol has been found to decrease LDL susceptibility to
oxidation and to decrease monocyte binding when administered
to patients with type 2 diabetes (179 ).
AGEs stimulate macrophage production of IL-1, TNF-α, and
granulocyte-macrophage colony-stimulating factor (182 ). In
vascular endothelial cells, AGEs induce generation of free
radicals leading to activation of the stress-sensitive NFκB
pathway (69 ).
Monocyte–endothelial cell interactions are regulated by
adhesion molecules, cell surface proteins present in both cells
(85 ,183 ). These include intercellular adhesion molecule-1
(ICAM), vascular cell adhesion molecule-1 (VCAM), E-selectin,
P-selectin, and their ligands: LFA-1, Mac-1, VLA-4, and PSGL-1.
Soluble cell adhesion molecules (sCAMs) are cleaved forms of
the adhesion molecules present in the circulation. Their function
remains unknown, but it has been suggested that sCAMs may
serve as molecular markers of atherosclerosis (184 ). Elevated
levels of sCAMs, mainly ICAM, have been found in patients with
type 2 diabetes and shown to be associated with increased risk
for death (185 ,186 ,187 ). Serum from patients with type 1
diabetes has recently been shown to induce the expression of
VCAM-1 in cultured endothelial cells, indicating that circulating
factors, possibly AGEs or cytokines, may contribute to increased
risk for atherosclerosis in these patients (188 ).
TNF-α and IL-6 stimulate hepatic synthesis of acute-phase
proteins. C reactive protein (CRP) is the principal downstream
mediator of the acute phase response. In healthy lean
individuals, CRP circulates at low concentrations in plasma (<3
mg/L). Slightly increased CRP concentrations, detected with
high sensitivity
P.1495

assays (hs-CRP), but still within the traditionally considered

normal range (1–10 mg/L), may reflect chronic low-grade
inflammation (189 ). Because CRP has a longer half-life than IL-
6 and because there are diurnal variations in IL-6 release, hs-
CRP has been proposed as the most potentially useful marker of
chronic subclinical inflammation in clinical practice (189 ). A
remarkably consistent series of prospective data support the
use of hs-CRP as a predictor of future coronary events
(189 ,190 ).
Experimental studies suggest a role for CRP as a marker or in
the initiation or progression of atherosclerosis. CRP has been
found in early atherosclerotic lesions in human aorta and
coronary arteries, and it has been shown to promote tissue
factor production by macrophages, to activate complement, to
induce increased expression of adhesion molecules in human
aortic endothelial cells, and to promote lipid accumulation in the
atherosclerotic plaque (191 ,192 ). A consistent series of
prospective data is available for both hs-CRP and fibrinogen in
regard to their ability to predict future coronary events (178 ).
Highly sensitive CRP levels were found to be a strong
independent predictor of risk for future myocardial infarction
and stroke among apparently healthy men and women
(193 ,194 ) and of overall mortality in patients with diabetes
(185 ). Increased serum levels of CRP and IL-6 have been found
in patients with diabetes (185 ,195 ). The Physician’s Health
Study and the Women’s Health Study showed that those in the
highest quartile of both hs-CRP and total cholesterol (TC)/high-
density lipoprotein cholesterol (HDL-C) are at the highest risk
for future coronary events, indicating an additive risk for
elevated hs-CRP over lipid abnormalities (196 ,197 ). Data also
suggest that hs-CRP can predict future development of type 2
DM, particularly in women (198 ). Ridker et al. recently reported
that CRP values greater than 3 mg/L also add prognostic
information regarding risk for cardiovascular events in
apparently healthy women with and without features of the
metabolic syndrome (199 ).

Role of Peroxisome Proliferator–Activated

Receptors
Peroxisome proliferator–activated receptors (PPARs) are lipid-
activated transcription factors that regulate the expression of
genes that control lipid and lipoprotein metabolism, glucose
homeostasis, and cellular differentiation (200 ).
The PPAR subfamily includes (201 ):

• PPAR-a: widely expressed but highest in the liver, kidney,

heart, and skeletal muscle, where it controls fatty acid
catabolism.
• PPAR-γ: highly expressed in adipose tissue, intestine,
mammary gland and a number of other tissues; is a central
mediator of adipogenesis, lipid metabolism, and glucose
regulation.
• PPAR-δ: ubiquitously expressed, controls brain lipid
metabolism, fatty acid-induced adipogenesis, and
preadipocyte proliferation.

Both PPAR-α and -γ are expressed in primary cultures of

endothelial and smooth muscle cells and in foam cells that are
resident in atherosclerotic lesions, where they exert
antiinflammatory activities (200 ,201 ). PPARs can repress gene
transcription by antagonizing NFκB, STAT, and AP-1
inflammatory signaling pathways.
PPAR expression is under the control of a wide variety of
factors. It has been recently demonstrated that specific
cytokines regulate the expression of PPAR-γ in different tissues:
TNF, IL-1α and β, and IL-6 decrease PPAR-γ in mature rat
adipocytes; IL-4 induces PPAR-γ expression in monocytes and
macrophages; and 9- and 13-HODE (inflammatory mediators
derived from oxidized LDL) increase PPAR-γ mRNA levels in
human macrophages (200 ).
PPAR-α and -γ activation limits the expression of
proinflammatory cytokines and as such may reduce
atherosclerosis (201 ). Several clinical trials show that fibrates
(PPAR-α agonists) reduce the progression of coronary
atherosclerosis and reduce acute coronary events, especially in
patients with low HDL-C, high triglyceride, and only moderately
increased LDL cholesterol, a lipid profile commonly seen in
patients with diabetes (202 ). Preclinical studies with
thiazolidinediones (TZDs; PPAR-γ agonists) support their
potential beneficial role in reducing cardiovascular risk by
acting at multiple levels of the inflammatory pathways that lead
to atherogenesis. The effects of TZDs include reduction in ROS
generation by leukocytes, downregulation of plasminogen
activator inhibitor-1 expression in human endothelial cells,
downregulation of CCR2 (MCP-1 receptor) in lesional and
circulating monocytes, and inhibition of arteriolar smooth
muscle cell proliferation (201 ,203 ).

Role of the Renin-Angiotensin System

It has been proposed that the renin-angiotensin system (RAS)
may contribute to the inflammatory process underlying the
onset and progression of atherosclerosis. Angiotensin II,
angiotensin II type 1 (AT1) receptor, and angiotensin-
converting enzyme (ACE) are expressed at strategic sites of
human atherosclerotic coronary arteries (204 ). Angiotensin II
activates various nuclear transcription factors including AP-1,
the STAT family of transcription factors, and NFκB (205 ). NFκB
plays a pivotal role in the control of several genes, including
proinflammatory cytokines and adhesion molecules. Use of
fosinopril, an ACE inhibitor, decreased the level of soluble
adhesion molecule VCAM-1 in patients with type 2 DM and
microalbuminuria (205 ). There are two different types of
receptors for angiotensin II. AT1 is involved in cell proliferation
and in the production of cytokines and extracellular matrix
proteins in cultured cells. AT2 regulates blood pressure control
and renal natriuresis, and causes an inhibition of cell
proliferation and neointimal formation after vascular injury. It
appears that angiotensin II regulates several NFκB-related
genes, mainly via AT1, and, in certain conditions, through AT2.
Several clinical trials have shown that administration of ACE
inhibitors after myocardial infarction dramatically reduces the
cumulative incidence of heart failure, reoccurrence of
myocardial infarction, and mortality (206 ,207 ). The HOPE trial
clearly demonstrated that ACE inhibition with ramipril was
associated with long-term reductions in myocardial infarction,
stroke, cardiac arrest, heart failure, and mortality in patients
P.1496

who were at high risk for cardiovascular events but did not have
left ventricular dysfunction or heart failure (155 ). These
findings were consistent for both the overall cohort (9,541
subjects) and patients with type 2 DM with one additional
cardiovascular risk factor (3,654 subjects, 39% of total cohort),
with a 22% and 25% overall risk reduction in the incidence of
cardiovascular events, respectively (155 ). This beneficial effect
was only partially related to the blood pressure–lowering effect,
suggesting a direct vascular protective effect of ramipril. The
Losartan Intervention for Endpoint Reduction (LIFE) study, a
double-masked, randomized, parallel-group trial, showed that
losartan, an AT1 receptor blocker, was more effective than
atenolol in reducing cardiovascular morbidity and mortality in
diabetic patients with hypertension and left ventricular
hypertrophy (208 ). It is intriguing that studies also suggest that
ACE inhibition or AT1 receptor blockade can also reduce the
development of type 2 diabetes. The hypothesis proposed is
that there are common inflammatory cascades that lead to
development of progressive β-cell failure and macrovascular
disease. Therefore, targeting these pathways could have
therapeutic effects to prevent type 2 onset and cardiovascular
disease (Fig. 101.9 ).

Figure 101.9. Common inflammatory pathways have been proposed as

the underlying mechanism leading to both development of type 2 diabetes
and its complications.

Conclusion
Significant evidence from experimental, animal, and human
studies supports the role of OS and inflammation in the
development of diabetic microvascular and macrovascular
complications. A promising area of drug development is the
search for agents that can target inflammatory and stress-
sensitive pathways, including the LO pathway. It is likely that,
in the near future, new pharmacologic or nutritional approaches
for reducing diabetic complications will become available as we
further our knowledge of the mechanisms by which diabetes
mellitus may lead to OS and altered function or synthesis of NO.

Acknowledgments
This chapter is dedicated to the memory of Rachmiel Levine,
M.D., who was an inspiration to all of us. The authors thank
Terry Howell and Marit Kington for their help with this chapter
and Rama Natarajan, Ph.D., and Jiali Gu, Ph.D., for the many
years of collaboration in this area. Research was supported in
part by grants from the National Institutes of Health (DK 39721
and PO1 HL55798) and the Juvenile Diabetes Foundation.

References
1. Southorn PA. Free radicals in medicine: 1. chemical nature
and biologic reactions. Mayo Clin Proc 1988;63:381.
2. Halliwell B, Gutteridge JMC. Free radicals in biology and
medicine. New York: Oxford University Press, 1985:139.
3. Hogg N, Kalyanaraman B, Joseph J, et al. Inhibition of low-
density lipoprotein oxidation by nitric oxide: potential role in
atherogenesis. FEBS Lett 1993;334:170.
4. Halliwell B, Gutteridge JM. Role of free radicals and catalytic
metal ions in human diseases: an overview. Methods
Enzymol 1990;186:1.
5. Freeman BA, Crapo JD. Biology of disease: free radicals and
tissue injury. Lab Invest 1982;47:412.
6. Rao GN, Berk BC. Active oxygen species stimulate vascular
smooth muscle cell growth and proto-oncogene expression. Circ
Res 1992; 70:593.
7. Feng L, Andalibi A, Qiao JH, et al. Genetic evidence for a
common pathway mediating oxidative stress, inflammatory gene
induction, and aortic fatty streak formation in mice. J Clin
Invest1994;94:877.
8. Kuhn H, Belkner J, Suzuki H, et al. Oxidative modification of
human lipoproteins by lipoxygenases of different positional
specificities. J Lipid Res 1994;35:1749.
9. Yla-Hertuala S, Rosenfeld ME, Parthasarathy S, et al.
Colocalization of 15-lipoxygenase mRNA and protein with
epitopes of oxidized low density lipoprotein in macrophage-rich
areas of atherosclerotic lesions. Proc Natl Acad Sci
USA 1990;87:6959.
10. Kim J, Gu J, Natarajan R, et al. A leukocyte type of 12-
lipoxygenase is expressed in human vascular and mononuclear
cells. Arterioscler Thromb Vasc Biol 1995;15:942.
11. Natarajan R, Gu JL, Rossi J, et al. Elevated glucose and
angiotensin II increase 12-lipoxygenase activity and expression
in porcine aortic smooth muscle cells. Proc Natl Acad Sci
USA 1993;90:4947.
12. Natarajan R, Bai W, Rangarajan V, et al. Platelet-derived
growth factor BB mediated regulation of 12-lipoxygenase in
porcine VSMC. J Cell Physiol 1996;169:391.
13. Natarajan R, Rosdahl J, Gonzales N, et al. Regulation of 12-
lipoxygenase by cytokines in vascular smooth muscle
cells. Hypertension 1997;30:873.
14. Natarajan R, Gonzales N, Lanting L, et al. Role of the
lipoxygenase pathway in angiotensin II–induced vascular
smooth muscle cell hypertrophy. Hypertension 1994;23:I-142.
15. Nadler JL, Natarajan R, Stern N. Specific action of the
lipoxygenase pathway in mediating angiotensin II–induced
aldosterone synthesis in isolated adrenal glomerulosa cells. J
Clin Invest1987;80:1763.
16. Gu J, Natarajan R, Ben-Ezra J, et al. Evidence that a
leukocyte type of 12-lipoxygenase is expressed and regulated
by angiotensin II in human adrenal glomerulosa
cells. Endocrinology 1994;134:70.
17. Nozawa K, Tuck M, Golub M, et al. Inhibition of the
lipoxygenase pathway reduces blood pressure in renovascular
hypertensive rats. Am J Physiol 1990;259:H174.
18. Nakao J, Ooyama T, Ito H, et al. Comparative effect of
lipoxygenase products of arachidonic acid on rat aortic smooth
muscle cell migration. Atherosclerosis 1982;44:339.
19. Antonipillai I, Nadler JL, Robin EC, et al. The inhibitory role
of 12- and 15-lipoxygenase products on renin
release. Hypertension 1987;10:61.
20. Natarajan R, Lanting L, Xu L, et al. Role of specific isoforms
of protein kinase C in angiotensin II and lipoxygenase action in
rat adrenal glomerulosa cells. Mol Cell Endocrinol 1994;101:59.
P.1497

21. Haliday E, Ramesha C, Ringold G. TNF induces c-fos via a

novel pathway requiring conversion of arachidonic acid to a
lipoxygenase metabolite. EMBO J 1991;10:109.
22. Rao G, Bass AS, Glasgow WC, et al. Activation of MAP
kinases by arachidonic acid and its metabolites in vascular
smooth muscle cells. J Biol Chem 1994;269:32586.
23. Wen Y, Nadler JL, Gonzales N, et al. Mechanisms of ANG II–
induced mitogenic responses: role of 12-lipoxygenase and
biphasic MAP kinase. Am J Physiol 1996;271:C1212.
24. Wen Y, Scott S, Liu Y, et al. Evidence that angiotensin II
and lipoxygenase products activate c-jun NH -terminal
2

kinase. Circ Res 1997; 81:651.

25. Roy P, Roy SK, Mitra A, et al. Superoxide generation by
lipoxygenase in the presence of NADH and NADPH. Biochim
Biophys Acta 1994; 1214:171.
26. Laniado-Schwartzman M, Lavrovsky Y, Stoltz R, et al.
Activation of nuclear factor kB and oncogene expression by
12(R)-hydroxyeicosatrienoic acid, and angiogenic factor in
microvessel endothelial cells. J Biol Chem 1994;269:24321.
27. Natarajan R, Wei B, Lanting L, et al. Effects of high glucose
on vascular endothelial growth factor expression in vascular
smooth muscle cells. Am J Physiol 1997;273:H2224.
28. Zhao L, Cuff CA, Moss E, et al. Selective interleukin-12
synthesis defect in 12/15-lipoxygenase–deficient macrophages
associated with reduced atherosclerosis in a mouse model of
familial hypercholesterolemia. J Biol
Chem 2002;277(38):35350–35356.
29. Natarajan R, Nadler JL. Lipoxygenase and lipid signaling in
vascular cells in diabetes cellular and molecular mechanisms of
diabetes-accelerated atherosclerosis: frontiers in bioscience. In:
Bornfeldt KE, ed. Webjournal: Frontiers in
Bioscience, http://www.Bioscience.org .
30. Morrow JD, Awad JA, Hollis JB, et al. Non–cyclooxygenase-
derived prostanoids (F2-isoprostanes) are formed in situ on
phospholipids. Proc Natl Acad Sci USA 1992;89:10721.
31. Patrono C. Isoprostanes: potential markers of oxidant stress
in atherothrombotic disease. Arterioscler Thromb Vasc
Biol 1997;17: 2309.
32. Fukunaga M, Makita N, Roberts JL, et al. Evidence for the
existence of F2-isoprostane receptors on rat vascular smooth
muscle cells. Am J Physiol 1993;264:1619.
33. Natarajan R, Lanting L, Nadler J. Formation of a F2-
isoprostane, 8-epi-prostaglandin F2a in vascular smooth muscle
cells (VSMC) by elevated glucose and growth factors. Am J
Physiol1996;271:H159.
34. Davi G, Ciabattoni G, Consoli A, et al. In vivo formation of
8-iso-prostaglandin F2alpha and platelet activation in diabetes
mellitus: effects of improved metabolic control and vitamin E
supplementation. Circulation 1999;99:224.
35. Frank L, Massaro D. Oxygen toxicity. Am J
Med 1980;69:117.
36. Murad F. Nitric oxide signaling: would you believe that a
simple free radical could be a second messenger, autacoid,
paracrine substance, neurotransmitter, and hormone? Rec Prog
Horm Res1998;53:43.
37. Ignarro LJ. Physiology and pathophysiology of nitric
oxide. Kidney Int Suppl 1996;55:S2.
38. Xie O, Nathan C. The high output nitric oxide pathway: role
and regulation. J Leukoc Biol 1994;56:576.
39. Stamler JS. Redox signalling: nitrosylation and related
target interactions of nitric oxide. Cell 1994;78:931.
40. Schmidt HH, Lohmann S, Walter U. The nitric oxide and
cGMP signal transduction system. Biochim Biophys
Acta 1993;1178:153.
41. Hirata K, Miki N, Kuroda Y, et al. Low concentration of
oxidized low-density lipoprotein and lysophosphatidylcholine
upregulate constitutive nitric oxide synthase mRNA expression
in bovine aortic endothelial cells. Circ Res 1995;76:958–962.
42. Zeng G, Nystrom FH, Ravichandran LV, et al. Roles for
insulin receptor, P13-kinase, and Akt in insulin-signaling
pathways related to production of nitric oxide in human vascular
endothelial cells.Circulation 2000;101:1539–1545.
43. Kuboki K, Jiang ZY, Takahara N, et al. Regulation of
endothelial constitutive nitric oxide synthase gene expression in
endothelial cells and in vivo: a specific vascular action of
insulin. Circulation2000; 101:676–681.
44. Steinberg H, Brechtel G, Johnson A, et al. Insulin-mediated
skeletal muscle vasodilation is nitric oxide dependent: a novel
action of insulin to increase nitric oxide release. J Clin
Invest1994;94:1172.
45. Weiner C, Lizasoain I, Baylis S, et al. Induction of calcium-
dependent nitric oxide by sex hormones. Proc Natl Acad Sci
USA 1994;91: 5212.
46. Hambrecht R, Wolf A, Gielen S, et al. Effect of exercise on
coronary endothelial function in patients with coronary artery
disease. N Engl J Med 2000;342:454–460.
47. Yoshizumi M, Perrella MA, Burnett JC Jr, et al. Tumor
necrosis factor downregulates an endothelial nitric oxide
synthase mRNA by shortening its half-life. Circ
Res 1993;73:205–209.
48. Balon TW, Nadler JL. Nitric oxide release is present in
skeletal muscle. J Appl Physiol 1994;77:2519.
49. Dinerman JL, Lowenstein CJ, Snyder SH. Molecular
mechanisms of nitric oxide regulation: potential relevance to
cardiovascular disease. Circ Res 1993;73:217.
50. Williams IL, Wheatcroft SB, Shah AM, et al. Obesity,
atherosclerosis and the vascular endothelium: mechanisms of
reduced nitric oxide bioavailability in obese humans. Int J
Obesity 2002;26:754–764.
51. Ito S, Johnson CS, Carretero OA. Modulation of angiotensin
II–induced vasoconstriction by endothelium-derived relaxing
factor in the isolated microperfused rabbit afferent arteriole. J
Clin Invest1991;87:1656.
52. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N
Engl J Med 1993;329:2002.
53. Kubes P, Suzuki M, Granger DN. Nitric oxide: an
endogenous modulator of leukocyte adhesion. Proc Natl Acad
Sci USA 1991;88:4651.
54. Garg UC, Hassid A. Nitric oxide-generating vasodilators and
8-bromo-cyclic guanosine monophosphate inhibit mitogenesis
and proliferation of cultured rat vascular smooth muscle cells. J
Clin Invest1989;83:1774.
55. Kourembanas S, McQuillan LP, Leung GK, et al. Nitric oxide
regulates the expression of vasoconstrictors and growth factors
by vascular endothelium under both normoxia and hypoxia. J
Clin Invest1993;92:99.
56. Cooke JP, Singer AH, Tsao P, et al. Antiatherogenic effects
of L-arginine in the hypercholesterolemic rabbit. J Clin
Invest 1992;90:1168.
57. Rengasamy A, Johns RA. Inhibition of nitric oxide synthase
by a superoxide generating system. J Pharmacol Exp
Ther 1993;267:1024.
58. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl
radical production by peroxynitrite: implications for endothelial
injury from nitric oxide and superoxide. Proc Natl Acad Sci
USA 1990; 87:1620.
59. White CR, Brock TA, Chang L-Y, et al. Superoxide and
peroxynitrite in atherosclerosis. Proc Natl Acad Sci
USA 1994;91:1044.
60. Chang GJ, Woo P, Honda HM, et al. Oxidation of LDL to a
biologically active form by derivatives of nitric oxide and nitrite
in the absence of superoxide. Arterioscler
Thromb 1994;14:1808.
61. Rösen P, Du X, Sui GZ. Molecular mechanisms of endothelial
dysfunction in the diabetic heart. In: Angel A, Dhalla NS, Pierce
GN et al., eds. Diabetes and cardiovascular disease: etiology,
treatment and outcomes. New York: Kluwer Academic/Plenum
Publishers 2001:75–86.
62. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress
and stress-activated signaling pathways: a unifying hypothesis
of type 2 diabetes. Endocr Rev 2002;23:599–622.
63. Brownlee M. Biochemistry and molecular cell biology of
diabetic complications. Nature 2001;414:813–820.
64. Ido Y, Kilo C, Williamson JR. Cytosolic NADH/NAD , free
+

radicals, and vascular dysfunction in early diabetes

mellitus. Diabetologia 1997;40(suppl):115.
65. Jiang Z-Y, Woolard ACS, Wolff SP. Hydrogen peroxide
production during experimental protein glycation. FEBS
Lett 1990;268:69.
66. Thornalley P, Wolff S, Crabbe M, et al. The autoxidation of
glyceraldehyde and other simple monosaccharides under
physiological conditions catalysed by buffer ions. Biochim
Biophys Acta1984;797:276.
67. Hunt JV, Dean RT, Wolff SP. Hydroxyl radical production and
autoxidative glycosylation: glucose autoxidation as the cause of
protein damage in the experimental glycation model of diabetes
mellitus and aging. Biochem J 1988;256:205.
P.1498

68. Yerneni KKV, Bai W, Khan BV, et al. Hyperglycemia-induced

activation of nuclear transcription factor κB in vascular smooth
muscle cells. Diabetes 1999;48:855.
69. King GL, Brownlee M. The cellular and molecular
mechanisms of diabetic complications. Endocrinol Metab Clin
North Am 1996;25: 255–270.
70. Hicks M, Delbridge L, Yue DK, et al. Catalysis of lipid
peroxidation by glucose and glycosylated collagen. Biochem
Biophys Res Commun 1988;151:649.
71. Kawamura M, Heinecke JW, Chait A. Pathophysiological
concentrations of glucose promote oxidative modification of low
density lipoprotein by a superoxide-dependent pathway. J Clin
Invest 1994; 94:771.
72. Cosentino F, Eto M, De Paolis P, et al. High glucose causes
upregulation of cyclooxygenase-2 and alters prostanoid profile
in human endothelial cells. Circulation 2003;107:1017–1023.
73. Natarajan R, Gonzales N, Xu L, et al. Vascular smooth
muscle cells exhibit increased growth in response to elevated
glucose. Biochem Biophys Res Commun 1992;187:552.
74. Koya D, King GL. Protein kinase C activation and the
development of diabetic complications. Diabetes 1998;47:859.
75. Natarajan R, Scott S, Bai W, et al. Angiotensin II signaling
in vascular smooth muscle cells under high glucose
conditions. Hypertension 1999;33:378.
76. Igarashi M, Wakasaki H, Takahara N, et al. Glucose or
diabetes activates p38 mitogen-activated protein kinase via
different pathways. J Clin Invest 1999;103:185.
77. Force T, Bonventre JV. Growth factors and mitogen-
activated protein kinases. Hypertension 1998;31:152.
78. Morigi M, Angioletti S, Imberti B, et al. Leukocyte-
endothelial interaction is augmented by high glucose
concentrations and hyperglycemia in a NF-κB dependent
fashion. J Clin Invest 1998;101: 1905–1915.
79. Aiello LP, Wong JS. Role of vascular endothelial growth
factor in diabetic vascular complications. Kidney
Int 2000;58(suppl 77):113–119.
80. Chiarelli F, Santilli F, Mohn A. Role of growth factors in the
development of diabetic complications. Horm Res 2000;53:53–
67.
81. Ross R. Atherosclerosis: an inflammatory disease. N Engl J
Med 1999;340:115–126. [Review of the inflammatory
mechanisms that contribute to the development and progression
of atherosclerosis.]
82. Craven PA, Melham MF, Phillip SL. Overexpression of
Cu /Zn
2+ 2+
superoxide dismutase protects against early diabetic
glomerular injury in transgenic mice. Diabetes 2001;50:2114–
2125.
83. Kim JA, Berliner JA, Natarajan R, et al. Evidence that
glucose increases monocyte binding to human aortic endothelial
cells. Diabetes 1994;43:1103.
84. Rattan V, Yamin S, Sultana C, et al. Glucose-induced
transmigration of monocytes is linked to phosphorylation of
PECAM-1 in culture endothelial cells. Am J
Physiol 1996;271:E711.
85. Kim PM, Kim JA, Harper CM, et al. Lipoxygenase products
increase monocyte adhesion to human aortic endothelial
cells. Arteriosclerosis Thromb Vasc Biol 1999;19:2615–2622.
86. West IC. Radicals and oxidative stress in diabetes. Diabet
Med 2000;17:171–180.
87. Nakao-Hayashi J, Ito H, Kawashima S. An oxidative
mechanism is involved in high glucose-induced serum protein
modification causing inhibition of endothelial cell
proliferation. Atherosclerosis1992;97:89.
88. Curcio F, Ceriello A. Decreased cultured endothelial cell
proliferation in high glucose medium is reversed by
antioxidants: new insights on the pathophysiological
mechanisms of diabetic vascular complications. In Vitro Cell Dev
Biol 1992;28A:787.
89. Kashiwagi A, Asahina T, Nishio Y, et al. Glycation, oxidative
stress, and scavenger activity: glucose metabolism and radical
scavenger dysfunction in endothelial
cells. Diabetes 1996;45(suppl):84.
90. Sharpe PC, Yue KKM, Catherwood MA, et al. The effects of
glucose-induced oxidative stress on growth and extracellular
matrix gene expression of vascular smooth muscle
cells. Diabetologia1998;41:1210.
91. Ceriello A. The possible role of postprandial hyperglycaemia
in the pathogenesis of diabetic
complications. Diebetologia 2003;46(suppl 1):M9–M16.
92. Diwadkar VA, Anderson JW, Bridges SR, et al. Postprandial
low density lipoproteins in type 2 diabetes are oxidized more
extensively than fasting diabetes and control samples. Proc Soc
Exp Biol Med1999;222:178–184.
93. Ahmed MU, Thorpe SR, Baynes JW. Identification of N-
carboxymethyl-lysine as a degradation product of fructoselysine
in glycated protein. J Biol Chem 1986;261:4889.
94. Brownlee M, Cerami A, Vlassara H. Advanced glycosylation
end products in tissue and the biochemical basis of diabetic
complications. N Engl J Med 1988;318:1315.
95. Mullarley CJ, Edelstein D, Brownlee M. Free radical
generation by early glycation products: a mechanism for
accelerated atherogenesis in diabetes. Biochem Biophys Res
Commun 1990;173:932.
96. Sakurai T, Sugioka K, Nakano M. O -generation and lipid
2

peroxidation during the oxidation of a glycated polypeptide,

glycated polylysine, in the presence of iron-ADP. Biochim
Biophys Acta 1990; 1043:27.
97. Hunt JV, Smith CCT, Wolff SP. Autoxidative glycosylation
and possible involvement of peroxides and free radicals in LDL
modification by glucose. Diabetes 1990;39:1420.
98. Baynes JW. Role of oxidative stress in development of
complications in diabetes. Diabetes 1991;40:405.
99. Bierhaus A, Chevion S, Chevion M, et al. Advanced glycation
end product-induced activation of NF-κB is suppressed by α-
lipoic acid in cultured endothelial cells. Diabetes 1997;46:1481–
1490.
100. Arai K, Maguchi S, Fujii S, et al. Glycation and inactivation
of human Cu-Zn-superoxide dismutase: identification of the in
vitro glycated sites. J Biol Chem 1987;262:16969.
101. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic
complications. Diabetes 1999;48:1.
102. Wolff SP, Dean RT. Glucose autoxidation and protein
modification: the potential role of autoxidative glycosylation in
diabetes. Biochem J 1987;245:243.
103. Ceriello A, Giugliano D, Quatraro A, et al. Vitamin E
reduction of protein glycosylation in diabetes: new prospect for
prevention of diabetic complications? Diabetes
Care 1991;14:68.
104. Horie K, Miyata T, Maeda K, et al. Immunohistochemical
colocalization of glycoxidation products and lipid peroxidation
products in diabetic renal glomerular lesions. J Clin
Invest 1997;100:2995.
105. Giardino I, Edelstein D, Brownlee M. Nonenzymatic
glycosylation in vitro and in bovine endothelial cells alters basic
fibroblast growth factor activity: a model for intracellular
glycosylation in diabetes. J Clin Invest 1994;94:110–117.
106. Bucala R, Tracey KJ, Cerami A. Advanced glycosylation
products quench nitric oxide and mediate defective
endothelium-dependent vasodilation in experimental diabetes. J
Clin Invest1991;87:432–438.
107. Nishikawa T, Edelstein D, Brownlee M. The missing link: a
single unifying mechanism for diabetic complications. Kidney
Int 2000;58 (suppl):26–30.
108. Culotta VC. Superoxide dismutase, oxidative stress, and
cell metabolism. Curr Top Cell Regul 2000;36:117–132.
109. Opara EC, Abdel-Rahman E, Soliman S, et al. Depletion of
total antioxidant capacity in type 2
diabetes. Metabolism 1999;48:1414–1417.
110. Maxwell SRJ, Thomason H, Sandler D, et al. Antioxidant
status in patients with uncomplicated insulin-dependent and
non–insulin-dependent diabetes mellitus. Eur J Clin
Invest 1997;27:484–490.
111. Uchimura K, Nagasaka A, Hayashi R, et al. Changes in
superoxide dismutase activities and concentrations and
myeloperoxidase activities in leukocytes from patients with
diabetes mellitus. J Diabetes Complications 1999;13:264–270.
112. Ceriello A, Giugliano D, Quatraro A, et al. Metabolic control
may influence the increased superoxide generation in diabetic
serum. Diabet Med 1991;8:541.
113. Tsai EC, Hirsch IB, Brunzell JD, et al. Reduced plasma
peroxyl radical trapping capacity and increased susceptibility of
LDL to oxidation in poorly controlled
IDDM. Diabetes 1994;43:1010.
114. Altomare E, Vendemiale G, Chicco D, et al. Increased lipid
peroxidation in type 2 poorly controlled diabetic
patients. Diabetes Metab 1992;18:264.
P.1499

115. Santini SA, Marra G, Giardina B, et al. Defective plasma

antioxidant defenses and enhanced susceptibility to lipid
peroxidation in uncomplicated IDDM. Diabetes 1997;46:1853–
1858.
116. Jain SK, McVie R, Duett J, et al. Erythrocyte membrane
lipid peroxidation and glycosylated hemoglobin in
diabetes. Diabetes 1989;38: 1539.
117. Rajeswari P, Natarajan R, Nadler JL, et al. Glucose induces
lipid peroxidation and inactivation of membrane-associated ion-
transport enzymes in human erythrocytes in vivo and in vitro. J
Cell Physiol 1991;149:100.
118. Antonipillai I, Nadler J, Jost-Vu E, et al. A 12-lipoxygenase
product, 12-hydroxyeicosatetraenoic acid, is increased in
diabetics with incipient and early renal disease. J Clin
Endocrinol Metab1996;81:1940.
119. Gerrity RG, Nadler JL, Natarajan R. Oxidant stress in a new
swine model of diabetes-induced accelerated atherosclerosis
[Abstract]. Circulation 1997;96:I-175.
120. Gerrity RG, Natarajan R, Nadler JL, et al. Diabetes-induced
accelerated atherosclerosis in swine. Diabetes 2001;50:1654–
1665.
121. Hofmann MA, Schiekofer S, Kanitz M, et al. Insufficient
glycemic control increases nuclear factor-MB binding activity in
peripheral blood mononuclear cells isolated from patients with
type 1 diabetes. Diabetes Care 1998;21:1310.
122. Hofmann MA, Schiekofer S, Isermann B, et al. Peripheral
blood mononuclear cells isolated from patients with diabetic
nephropathy show increased activation of the oxidative-stress
sensitive transcription factor NF-κB. Diabetologia 1999;42:222–
232.
123. Jain SK, McVie R, Jackson R, et al. Effect of
hyperketonemia on plasma lipid peroxidation levels in diabetic
patients. Diabetes Care 1999;22:1171–1175.
124. Valabhji J, McColl AJ, Richmond W, et al. Total antioxidant
status and coronary artery calcification in type 1
diabetes. Diabetes Care 2001;24:1608–1613.
125. Vucic M, Gavella M, Bozikov V, et al. Superoxide dismutase
activity in lymphocytes and polymorphonuclear cells of diabetic
patients. Eur J Clin Chem Clin Biochem 1997;35:517–521.
126. Yoshida K, Hirokawa J, Tagami S, et al. Weakened cellular
scavenging activity against oxidative stress in diabetes mellitus:
regulation of glutathione synthesis and
efflux. Diabetologia1995;38:201–210.
127. Obrosova IG, Fathallah L, Liu E, et al. Early oxidative
stress in the diabetic kidney: effect of DL-α-lipoic acid. Free
Radic Biol Med 2003;34:186–195.
128. Kakkar R, Mantha SV, Radhi J, et al. Antioxidant defense
system in diabetic kidney: a time course study. Life
Sci 1997;60:667–679.
129. Low PA, Nickander KK, Tritschler HJ. The roles of oxidative
stress and antioxidant treatment in experimental diabetic
neuropathy. Diabetes 1997;46(suppl):38–42.
130. Li W, Yanoff M, Jian B, et al. Altered mRNA levels of
antioxidant enzymes in pre-apoptotic pericytes from human
diabetic retinas. Cell Mol Biol 1999;45:59–66.
131. Altomare E, Grattagliano I, Vendemaile G, et al. Oxidative
protein damage in human diabetic eye: evidence of a retinal
participation. Eur J Clin Invest 1997;99:457–468.
132. Gurler B, Vural H, Yilmar N, et al. The role of oxidative
stress in diabetic retinopathy. Eye 2000;14:730–735.
133. Tesfamariam B. Free radicals in diabetic endothelial cell
dysfunction. Free Radic Biol Med 1994;16:383.
134. Pieper GM, Mei DA, Langenstroer P, et al. Bioassay of
endothelium-derived relaxing factor in diabetic rat aorta. Am J
Physiol 1992;263: H676.
135. Tesfamariam B, Brown JL, Cohen RA. Elevated glucose
impairs endothelium-dependent relaxation by activating protein
kinase C. J Clin Invest 1991;86:1643.
136. Craven PA, Studer RK, DeRubertis FR. Impaired nitric
oxide-dependent cyclic guanosine monophosphate generation in
glomeruli from diabetic rats: evidence for protein kinase C-
mediated suppression of the cholinergic response. J Clin
Invest 1994;93:311.
137. Hink U, Huige L, Hanke M, et al. Mechanisms underlying
endothelial dysfunction in diabetes mellitus. Circ
Res 2001;88:e14–e22.
138. Gupta S, Sussman I, McArthur CS, et al. Endothelium-
dependent inhibition of Na -K ATPase activity in rabbit aorta by
+ +
hyperglycemia: possible role of endothelium-derived nitric
oxide. J Clin Invest 1992;90:727.
139. Gupta S, Tieken K, Ruderman N. Inhibition of nitric oxide
synthase activity by hyperglycemia in endothelial cells
[Abstract]. Diabetes 1994;43(suppl 1):100A.
140. Graier WF, Wascher TC, Lackner L, et al. Exposure to
elevated d-glucose concentrations modulates vascular
endothelial cell vasodilatatory
response. Diabetes 1993;42:1497.
141. Langenstroer P, Pieper GM. Regulation of spontaneous
EDRF release in diabetic rat aorta by oxygen free radicals. Am J
Physiol 1992; 263:H257.
142. Koo JR, Vaziri ND. Effects of diabetes, insulin and
antioxidants on NO synthase abundance and NO interaction with
reactive oxygen species. Kidney Int 2003;63:195–201.
143. Coffer MJ, Coles B, O’Donnell VB. Interactions of nitric
oxide-derived reactive nitrogen species with peroxidases and
lipoxygenase; Free Radic Res 2002;35:447–464.
144. Johnstone MT, Creager SJ, Scales KM, et al. Impaired
endothelium-dependent vasodilation in patients with insulin-
dependent diabetes mellitus. Circulation 1993;88:2510.
145. Calver A, Collier J, Vallance P. Inhibition and stimulation of
nitric oxide synthesis in the human forearm arterial bed of
patients with insulin-dependent diabetes. J Clin
Invest 1992;90:2548.
146. Hogan M, Cerami A, Bucala R. Advanced glycosylation end
products block the antiproliferative effect of nitric oxide: role in
the vascular and renal complications of diabetes mellitus. J Clin
Invest1992;90:1110.
147. Tilton RG, Chang K, Hasan KS, et al. Prevention of diabetic
vascular dysfunction by guanidines: inhibition of nitric oxide
synthase versus advanced glycation end-product
formation. Diabetes 1993; 42:221.
148. Scherrer U, Randin D, Vollenweider P, et al. Nitric oxide
release accounts for insulin’s vascular effects in humans. J Clin
Invest 1994; 94:2511.
149. Stampfer MJ, Hennekens CH, Manson JE, et al. Vitamin E
consumption and the risk of coronary disease in women. N Engl
J Med 1993;328:1444.
150. Steinberg D. Antioxidant vitamins and coronary heart
disease. N Engl J Med 1993;328:1487.
151. Jain SK. Should high-dose vitamin E supplementation be
recommended to diabetic patients? Diabetes
Care 1999;22:1242–1243.
152. Bursell SE, Clermont AC, Aiello LP, et al. High-dose vitamin
E supplementation normalizes retinal blood flow and creatinine
clearance in patients with type 1 diabetes. Diabetes
Care 1999;22:1245–1251.
153. Kunisaki M, Bursell S-E, Umeda F, et al. Normalization of
diacylglycerol-protein kinase C activation by vitamin E in aorta
of diabetic rats and cultured rat smooth muscle cells exposed to
elevated glucose levels. Diabetes 1994;43:1372.
154. Gazis A, White DJ, Page SR, et al. Effect of oral vitamin E
(alpha-tocopherol) supplementation on vascular endothelial
function in type 2 diabetes mellitus. Diabet Med 1999;16:304–
311.
155. The Heart Outcomes Prevention Evaluation Study (HOPE)
Investigators. Effects of ramipril on cardiovascular and
microvascular complications outcomes in people with diabetes
mellitus: results of the HOPE Study and MICRO-HOPE
Study. Lancet 2000;355:253–259. [Demonstrated that ramipril
treatment in patients with diabetes is associated with a
reduction in cardiovascular events and mortality. This finding
was only partially related to blood pressure reduction
suggesting a direct vascular protective effect of ramipril.]
156. Bowry VW, Ingold KU, Stocker R. Vitamin E in human low-
density lipoprotein: when and how this antioxidant became a
pro-oxidant. Biochem J 1992;288:341.
157. Hunt JV, Bottoms MA, Mitchinson MJ. Ascorbic acid
oxidation: a potential cause of the elevated severity of
atherosclerosis in diabetes mellitus? FEBS Lett 1992;311:161.
158. Young IS, Tate S, Lightbody JH, et al. The effects of
desferrioxamine and ascorbate on oxidative stress in the
streptozotocin diabetic rat. Free Rad Biol Med 1995;18:833–
840.
159. Mooradian AD. The effect of ascorbate and
dehydroascorbate on tissue uptake of
glucose. Diabetes 1987;36:1001.
P.1500

160. Mayer-Davis EJ, Bell RA, Reboussin BA, et al. Antioxidant

nutrient intake and diabetic retinopathy: the San Luis Valley
Diabetes Study. Ophthalmology 1998;105:2264–2270.
161. Mooradian AD, Failla M, Hoogwerf B, et al. Selected
vitamins and minerals in diabetes. Diabetes Care 1994;17:464.
162. Zhang WJ, Frei B. α-Lipoic acid inhibits TNF-α-induced NF-
κB activation and adhesion molecule expression in human aortic
endothelial cells. FASEB J 2001;13:1845–1854.
163. Borcea V, Nourooz-Zadeh J, Wolf SP, et al. Alpha-lipoic
acid decreases oxidative stress even in diabetic patients with
poor glycemic control and albuminuria. Free Rad Biol
Med 1999;26:1495–1500.
164. Ziegler D, Feljanovic M, Mehnert H, et al. α-Lipoic acid in
the treatment of diabetic polyneuropathy in Germany: current
evidence from clinical trials. Exp Clin Endocrinol
Diabetes 1999;107:421–430.
165. Ziegler D, Schatz H, Conrad F, et al. Effects of treatment
with the antioxidant alpha-lipoic acid on cardiac autonomic
neuropathy in NIDDM patients: a 4-month randomized
controlled multicenter trial (DEKAN study). Diabetes
Care 1997;20:369–373.
166. Morcos M, Borcea V, Isermann B, et al. Effect of α-lipoic
acid on the progression of endothelial cell damage and
albuminuria in patients with diabetes mellitus: an exploratory
study. Diabetes Res Clin Pract 2001;52:175–183.
167. Watts GF, Playford DA, Croft KD, et al. Coenzyme
Q 10 improves endothelial function of the brachial artery in type
II diabetes mellitus. Diabetologia 2002;45:420–426.
168. McCarty MF. Coenzyme Q versus hypertension: does CoQ
decrease endothelial superoxide generation? Med
Hypotheses 1999;53:300–304.
169. Salvemini D, Wang ZQ, Zweier JL et al. A nonpeptidyl
mimic of superoxide dismutase with therapeutic activity in
rats. Science 1999;286:304–306.
170. Staprans I, Rapp JH, Pan X-M, et al. The effect of oxidized
lipids in the diet on serum lipoprotein peroxides in control and
diabetic rats. J Clin Invest 1993;92:638.
171. Nadler JL, Malayan S, Luong E, et al. Intracellular free
magnesium plays a key role in increased platelet reactivity in
type II diabetes. Diabetes Care 1992;15:835.
172. Resnick LM, Gupta RK, Bhargava KK, et al. Cellular ions in
hypertension, diabetes and obesity. Hypertension 1991;17:951.
173. Weglicki WB, Phillips TM. Pathobiology of magnesium
deficiency: a cytokine/neurogenic inflammation hypothesis. Am
J Physiol 1992; 263:734.
174. Nadler JL, Buchanan T, Natarajan R, et al. Magnesium
deficiency produces insulin resistance and increased
thromboxane synthesis. Hypertension 1993;21:1024.
175. Schmidt LE, Arfken CL, Heins JM. Evaluation of nutrient
intake in subjects with non-insulin-dependent diabetes
mellitus. J Am Diet Assoc 1994;94:773.
176. Shechter M, Sharir M, Paul-Labrador M, et al. Oral
magnesium therapy improves endothelial function in patients
with coronary artery disease. Circulation 2000;102:2353–2358.
177. Shechter M, Bairey Merz CN, Stuehlinger HG, et al. Effects
of oral magnesium therapy on exercise tolerance, exercise-
induced chest pain, and quality of life in patients with coronary
artery disease. Am J Cardiol 2003;91:517–521.
178. Libby P, Ridker PM. Novel inflammatory markers of
coronary risk: theory versus
practice. Circulation 1999;100:1148–1150.
179. Devaraj S, Jialal I. Low-density lipoprotein postsecretory
modification, monocyte function, and circulating adhesion
molecules in type 2 diabetic patients with and without
macrovascular complications: the effect of α-tocopherol
supplementation. Circulation 2000;102 (2):191–196.
180. Srinivasan S, Yeh M, Danziger EC, et al. Glucose regulates
monocyte adhesion through endothelial production of
interleukin-8. Circ Res 2003;92:371–377.
181. Renier G, Mamputu JC, Desfaits AC, et al. Monocyte
adhesion in diabetic angiopathy: effects of free-radical
scavenging. J Diabetes Complications 2003;17:20–29.
182. Yui S, Sasaki T, Araki N, et al. Induction of macrophage
growth by advanced glycation end products of the Maillard
reaction. J Immunol 1994;152:1943–1949.
183. Huo Y, Ley K. Adhesion molecules and atherogenesis. Acta
Physiol Scand 2001;173:35–43.
184. Hwang SJ, Ballantyne CM, Sharrett AR, et al. Circulating
adhesion molecules VCAM-1, ICAM-1 and E-selectin in carotid
atherosclerosis and incident coronary heart disease cases: the
Atherosclerosis Risk in Communities (ARIC)
Study. Circulation 1997;96(12): 4219–4225.
185. Stehouwer C, Gall MA, Twisk JWR, et al. Increased urinary
albumin excretion, endothelial dysfunction, and chronic low-
grade inflammation in type 2 diabetes: progressive, interrelated
and independently associated with risk of
death. Diabetes 2002;51:1157–1165.
186. Devaraj S, Jialal I. Low-density lipoprotein postsecretory
modification, monocyte function, and circulating adhesion
molecules in type 2 diabetic patients with and without
macrovascular complications: the effect of α-tocopherol
supplementation. Circulation 2000;102: 191–196.
187. Bagg W, Ferri C, Desideri G, et al. The influences of
obesity and glycemic control on endothelial activation in
patients with type 2 diabetes. J Clin Endocrinol
Metab 2001;86:5491–5497.
188. Rasmussen LM, Schmitz O, Ledet T. Increased expression
of vascular cell adhesion molecule-1 (VCAM-1) in cultured
endothelial cells exposed to serum from type 1 diabetic
patients: no effects of high glucose concentrations. Scand J Clin
Lab Invest 2002;62:485–494.
189. Pearson TA, Mensah GA, Alexander RW, et al. Markers of
inflammation and cardiovascular disease: application to clinical
and public health practice: a statement for healthcare
professionals from the Centers for Disease Control and
Prevention and the American Heart
Association. Circulation 2003;107:499–511.
190. Ridker PM. Clinical application of C-reactive protein for
cardiovascular disease detection and
prevention. Circulation 2003;107:363–369.
191. Festa A, D’Agostino RJ, Howard G, et al. Chronic
subclinical inflammation as part of insulin resistance syndrome:
the Insulin Resistance Atherosclerosis Study
(IRAS). Circulation 2000;102:42–47.
192. Pasceri V, Wilkerson JT, Yeh ET. Direct proinflammatory
effect of C-reactive protein on human endothelial
cells. Circulation 2000; 102:2165–2168.
193. Ridker PM. High sensitivity C-reactive protein: potential
adjunct for global risk assessment in the primary prevention of
cardiovascular disease. Circulation 2001;103:1813–1818.
194. Danesh J, Whincup P, Walker M, et al. Low-grade
inflammation and coronary heart disease: prospective study and
updated meta-analyses. BMJ 2000;321:199–204.
195. Bastard JP, Jardel C, Bruckert E, et al. Elevated levels of
interleukin 6 are reduced in serum and adipose tissue of obese
women after weight loss. J Clin Endocrinol
Metab 2000;85:3338–3342.
196. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive
protein and other markers of inflammation in the prediction of
cardiovascular disease in women. N Engl J Med 2000;342:836–
843.
197. Ridker PM. Physician’s Health Study. Ann Intern
Med 1999;130: 933–937.
198. Han TS, Sattar N, Williams K, et al. Prospective study of C-
reactive protein in relation to the development of diabetes and
metabolism syndrome in the Mexico City Diabetes
Study. Diabetes Care2002; 25:2016–2021.
199. Ridker PM, Buring JE, Cook NR, et al. C-reactive protein,
the metabolic syndrome, and risk of incident cardiovascular
events: an 8-year follow-up of 14,719 initially healthy American
women.Circulation 2003;107:391–397.
200. Chinetti G, Fruchart JC, Staels B. Peroxisome-proliferator–
activated receptors (PPARs): nuclear receptors at the
crossroads between lipid metabolism and
inflammation. Inflammation Res2000;49:497–505.
201. Marx N, Kehrle B, Kohlhammer K, et al. PPAR activators as
antiinflammatory mediators in human T lymphocytes.
Implications for atherosclerosis and transplantation-associated
atherosclerosis. Circ Res 2002;90:703–710.
202. Robins SJ. PPAR-α ligands and clinical trials:
cardiovascular risk reduction with fibrates. J Cardiovasc
Risk 2001;4:203–210.
203. Ishibashi M, Egashira K, Hiasa K, et al. Antiinflammatory
and antiarteriosclerotic effects of
pioglitazone. Hypertension 2002;40:687–693.
P.1501

204. Schieffer B, Schieffer E, Hilfiker-Kleiner D, et al.

Expression of angiotensin II and interleukin 6 in human
coronary atherosclerotic plaques: potential implications for
inflammation and plaque instability. Circulation 2000;101:1372–
1378.
205. Ruiz-Ortega M, Lorenzo O, Suzuki Y, et al. Proinflammatory
actions of angiotensins. Curr Opin Nephrol
Hypertens 2001;10:321–329. [Discusses the molecular
mechanisms by which angiotensin-II contributes to the
proinflammatory pathways involved in the development of
atherosclerosis, hypertension, and renal damage.]
206. SOLVD Investigators. Effect of enalapril on mortality and
the development of heart failure in asymptomatic patients with
reduced left ventricular ejection fraction. N Engl J
Med 1992;327:568–574.
207. Pfeffer M, Braunwald E, Moye L, et al. Effect of captopril on
mortality and morbidity in patients with left ventricular
dysfunction after myocardial infarction: results of the survival
and ventricular enlargement trial. N Engl J Med 1992;327:669–
677.
208. Lindholm LH, Ibsen H, Dahlöf B, et al., for the LIFE study
group. Cardiovascular morbidity and mortality in patients with
diabetes in the Losartan Intervention for Endpoint reduction in
hypertension study (LIFE): a randomized trial against
atenolol. Lancet 2002;359: 1004–1010.

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