oxidative stress, inflammation, and diabetic complications

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Diabetes Mellitus: A Fundamental and ClinicalText3rd Edition

© 2004 Lippincott Williams & Wilkins

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101Oxidative Stress, Inflammation, andDiabetic ComplicationsMilagros 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

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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 hydrogenperoxide. 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 cel ls 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 byinducing 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 ). Theleukocyte-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 hyperglycemicconditions (14). Table 101.1 summarizes several potential

























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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-lipoxygenasepathway in cardiovascular disorders

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

pathway)2. Inhibition of prostacyclin synthesis3. Direct vasoconstriction of certain vascular beds4. 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

cells6. May be involved in oxidative modification of low-density

lipoprotein7. Increased adhesion of monocytes to endothelial cells

8. Activation of protein kinase C and key growth- and stress-related mitogen-activated protein kinases and transcriptionfactors

9. Regulation of macrophage cytokine production includinginterleukin 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) F2 a,

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-PGF2 a 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 theultimate 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 inturn 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 tomaintain 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 vasodilator2. Inhibition of platelet aggregation and adhesion3. Inhibition of leukocyte adhesion to activated endothelium

4. Inhibition of vascular smooth muscle cell migration andproliferation

5. Reduction of macrophage-dependent oxidation of low-densitylipoprotein

6. Inhibition of expression endothelin and platelet-derived growthfactor by the vascular endothelium

A wide variety of studies, therefore, have demonstrated the

beneficial actions of NO in the prevention of cardiovasculardisease. 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 diabeticmilieu 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 canlead to free radicals and lipid peroxidation










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1. Direct autoxidation of glucose2. Induction and activation of various lipoxygenase enzymes3. Activation of glycation pathways and receptor for advanced

glycation and products (RAGE)4. Stimulation of protein kinase C activity5. Promotion of the interaction of nitric oxide with superoxide

anions to produce peroxynitrite and hydroxyl radicals6. Reduction of the activity of the antioxidant defense

mechanisms7. 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 cellsmaintained in normal glucose concentrations (73). We have















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found that elevated glucose concentrations increase the rate of

porcine VSMC growth (74), and this accelerated growth waspartially 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 andtumor necrosis factor-α (TNF -α)–induced superoxide generation byporcine vascular smooth muscle cells (VSMCs). VSMCs growing in anormal glucose (NG) medium were placed in medium containing 12.5mM glucose or high glucose (HG; 25 mM ) and 10% fetal calf serum (FCS)for 1 week. Confluent cells were made quiescent for 24 hours inDulbecco’s modified Eagle medium (NG, 12.5 or 25 mM glucose) + 0.2%bovine serum albumin (BSA) + 0.4% FCS. Washed cells were then placedin fresh medium containing 0.2% BSA only. Cells were then incubatedwith 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 < 0.01vs. 5.5 mM basal; b, p < 0.001 vs. 5.5 mM basal; c, p < 0.02 vs. 5.5mM 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 24hours 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) andglyceraldehyde-3-phosphate dehydrogenase probes (B). (ReproducedfromNatarajan R, Gu JL, Rossi J, et al. Elevated glucose and angiotensin IIincrease 12-lipoxygenase activity and expression in porcine aortic smoothmuscle 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-induceddiabetic 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 l eads 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 aloneor with 5 ng/mL of tumor necrosis factor (TNF )-α. Nuclear proteins were

prepared and subjected to electromobility shift assays (EMSAs) todetermine activation of NF-MB using an NF-MB consensus vascular celladhesion molecule (VCAM) oligonucleotide. For the competition studiesshown 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 DNAbinding reactions with the labeled VCAM oligonucleotide and EMSA.Results demonstrate specificity of the binding. For the supershiftexperiments with p65 and p50 antibodies (fifth and sixth lanes), nuclear

extracts from TNF-α–treated cells in HG were incubated with therespective antibodies for 1 hour at 4 °C and then EMSAs run as usual.Specific complexes, X and Y, are indicated. Results indicate that thebinding complex is composed of p65 and p50 subunits. B: Bar graphshowing the mean ± SEM of results from the phosphorimager quantitationof the EMSA results obtained from six experiments. * p < 0.005 vs. NGbasal; ** p < 0.01 vs. NG TNF-α, by analysis of variance using Prismsoftware (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 ), suggestingthat increased OS coupled with impaired degradation of

superoxide and hydrogen peroxide are important mechanisms

for the glucose-induced decline in endothelial cell g rowth (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 aorticendothelial cells (HAECs) on monocyte binding to HAECs. HAECs werecultured 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 ) orhigh mannitol (CH-HM ; osmolality control). Monocyte binding (with humanmonocytes) 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 ProductsNonenzymatic 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 H2O 2 (65 ), whereas Schiff bases and Amadori

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 functionof 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 studiessupport 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 inpatients 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 nondiabeticsubjects (130). Altomare et al. reported decreased g lutathione

























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 lipidperoxidation, than those without diabetic retinopathy (132).

They also described a significant correlation between markers of

lipid peroxidation and duration of DM. Valabhji et a l. 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 endof incubation, erythrocytes were washed three times in PBS, and from thealiquots, the formation of MDA and PL-MDA adduct was determined. Eachvalue represents mean ± SD (n = 25 for type 2 diabetes mellitus; n = 10for normal healthy control subjects). TBA, thiobarbituric acid. (ReproducedfromRajeswari P, Natarajan R, Nadler JL, et al. Glucose induces lipid

perioxidation and inactivation of membrane-associated ion-transportenzymes 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 animportant 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 s treptozotocin-

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 production2. Reduction of NO action by interaction with advanced

glycosylation end products3. Reaction of NO with superoxide anions to produce peroxynitrite,

which can promote oxidation of low-density lipoprotein and lead

to lipid peroxidation4. 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 86Rb-uptake by L-arginine and sodium nitroprusside (SNP) inendothelium-intact aorta. L-arginine (0.3 mM ) and SNP (10 mM ) wereadded to the incubation media during the final 30 and 10 minutes,respectively, of the 3-hour incubation; hyperglycemia failed to decreaseouabain-sensitive 86Rb-uptake. The asterisk denotes values that aresignificantly different from those in aorta incubated in 5.5 or 44mM glucose ( p < 0.05). Cont , control. (Reproduced fromGupta 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, insulinresistance, 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. Cholinergicvasodilation was less in the diabetic group than in the normal group. Thedifference between groups was significant at the 3- and 10-mg/min doses.(Reproduced fromJohnstone MT, Craeger SJ, Scales KM, et al. Impaired endotheliumdependent vasodilation in patients with insulin-dependent diabetesmellitus. 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 etal. (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’svasodilatory 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 2, and to reduce the ex vivo oxidative susceptibility of LDL

(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 renalhemodynamics (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 indiabetic 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, andperipheral 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 beneficialin 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 Q1 0, a critical intermediate of the

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 1 0 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 inendothelial 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 l ipoproteins, 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 exercisetolerance, 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 increasedmonocyte adhesion to endothelial cells to increased risk of

atherosclerotic plaque rupture (81).









Figure 101.8. The role of inflammation in atherogenesis. Vascular andintravascular proinflammatory conditions lead to release of proinflammatory cytokines, which in turn influence all stages of atherosclerosis from monocyte endothelial cell interactions to plaquerupture. Reproduced fromHuerta MG, Nadler JL. Role of inflammatory pathways in the developmentand cardiovascular complications of type 2 diabetes. Current DiabetesReports 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 toinflammatory 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 functionremains 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 inthe 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 humanaortic 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 i t 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, andpreadipocyte 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 mediatorsderived 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 theirpotential 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 solubleadhesion 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 thecumulative 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 asthe underlying mechanism leading to both development of type 2 diabetesand 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 39721and PO1 HL55798) and the Juvenile Diabetes Foundation.

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