Diabetes Mellitus: A Fundamental and Clinical Text
3rd Edition

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

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) F2a, 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-PGF2a 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,

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

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),

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 H2O2 (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

“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

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

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 86Rb-uptake by L-arginine and sodium nitroprusside (SNP) in 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 86Rb-uptake. The asterisk denotes values that are 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

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 B2, 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 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 Q10, 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 Q10 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

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

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

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.
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.
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.
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