Transition metals redox: reviving an old plot for diabetic vascular disease

Vincent M. Monnier

Case Western Reserve University, Institute of Pathology, Cleveland, Ohio 44106, USA.

Phone: (216) 368-6613; Fax: (216) 368-0495; E-mail: vmm3@po.cwru.edu.

Considerable effort over the past 25 years has focused on the role
of oxidant stress in aging and in the pathogenesis of age-related
diseases — diabetes, Alzheimer’s disease, end-stage renal disease, and
atherosclerosis, among others. Research on redox signaling and the
chemistry of the aging process has led to major insights, including the
identification of oxidant stress–responsive transcription factors, such
as NF-{kappa}B,
which regulate tissue remodeling and therefore control the progression
of pathological lesions; the role of mitochondria in generating
reactive oxygen species and activating apoptotic pathways; the role of
sulfhydryl homeostasis in redox signaling; and the development of mass
spectrometry methods to identify and quantify protein damage in aging
or stressed tissues.

Because of the prevalence and the dire consequences of the diseases
involved, the stakes in this field are high. However, despite the great
interest in developing drugs that might block oxidant or carbonyl
stress, clinical studies involving antioxidant or carbonyl-trapping
agents have had mixed success, suggesting a greater degree of
complexity than anticipated. Thus, in the diabetic rat, treatment with
various antioxidants or carbonyl-trapping agents has had impressive
effects in delaying, if not altogether preventing, complications of
diabetes such as cataracts, retinopathy, nephropathy, vascular
abnormalities, nerve conduction velocity, plasma lipid oxidation, and
fetal malformations. In the diabetic human, conversely, while
intra-arterial infusion of vitamin C improved endothelium-dependent
vasodilation (1) and oral intake of vitamin E improved retinal blood flow and creatinine clearance (2), chronic treatment with vitamin E did not reduce cardiovascular risk (3). Similarly, the antioxidant {alpha}-lipoic
acid decreased plasma hydroperoxides in diabetic subjects but had
equivocal efficacy in polyneuropathy and cardiac autonomic neuropathy (4, 5). A similar “antioxidant paradox” has also been observed in other diseases associated with oxidant stress (6).

Protein oxidation in diabetes

In diabetes, the controversy has focused on the origin, the type,
the magnitude, and the localization of oxidant stress in relation to
hyperglycemia, and how this combination promotes the progression of
micro- and macrovascular disease. For example, high glucose levels were
associated early on with an altered cellular redox state, aldose
reductase activation, and impaired glutathione homeostasis in selected
tissues. In experimental diabetes, antioxidants or transition metal
chelators can ameliorate retinopathy and neuropathy, suggesting that
oxidant stress contributes to this condition (7, 8). On the other hand, Williamson et al., noting an increased cellular NADH/NAD ratio (9),
have proposed that diabetes is a state of “reductive” stress and
“pseudohypoxia,” raising the question of how oxidative damage might
arise in a reducing environment.

As the glycation theory of diabetic complications unfolded,
metal-catalyzed glucose autooxidation and oxidation of glycated
residues emerged as potent sources of free radicals and were proposed
as the primary culprits in tissue damage (10, 11).
In vitro, exposing proteins to high levels of glucose causes oxidative
protein fragmentation and damage to amino acid residues, with the
accumulation of methionine sulfoxide, o-tyrosine, m-tyrosine,
and other modifications. Many of the glucose-associated oxidative
modifications have been attributed to Fenton chemistry carried out by
transition metals like copper and iron, which are normally present in
phosphate buffer (11).
Wolff et al. and Baynes therefore proposed a key role for oxidation and
glycoxidation chemistry in the pathogenesis of diabetic complications (10, 11).
More recently, however, the model of generalized oxidant stress lost
support because of a lack of evidence for increased levels of oxidized
skin collagen in diabetic individuals (reviewed in ref. 12).
Instead, Baynes and Thorpe propose a greater role for overload of
metabolic pathways as the primary culprit in oxidant and carbonyl
stress in diabetes: “Treatment of diabetes with antioxidant therapy,”
they write, “is like applying water to a burning house, certainly
helpful in limiting the conflagration, but also a little bit late in
the process” (12).

Oxidative damage in atherosclerosis research has also focused
investigators on the effects of transition metals. Cu 2+-catalyzed LDL
oxidation has become a useful and widely studied, albeit controversial,
model for oxidative events in the arterial wall. Coincubation of LDL
with glucose or glycated proteins significantly increases lipoprotein
oxidation by adventitious transition metals, thus offering a potential
explanation for the acceleration of atherosclerosis in diabetes (13). In this context, the useful terms “glycoxidation” and “lipoxidation” have found their way in the literature (12).

However, another potentially relevant mechanism of oxidation emerged
with the exciting discovery that myeloperoxidase, derived from
macrophages of the arterial wall, potently oxidizes LDL to generate the
same oxidative modifications found in LDL isolated from atheromatous
plaques. Both Cu 2+-mediated and myeloperoxidase-mediated oxidation
lead to an increase in o-tyrosine and m-tyrosine, but only the latter selectively generates dityrosine from tyrosine radical (reviewed in ref. 14).
The finding that dityrosine was selectively increased in fatty streaks
and intermediate atheromatous lesions, whereas hydroxyl radical damage
was elevated only in more advanced lesions, led Semenkovich and
Heinecke (15)
to propose “a new plot” for oxidative events in diabetes and
atherosclerosis, in which myeloperoxidase, rather than transition
metals and hydroxyl radicals, initiates the oxidant cascade.

Yet a third potential mechanism of oxidation arose from observations
that endothelium-derived nitric oxide together with superoxide might
lead to increased levels of the highly oxidizing and atherogenic
molecule peroxynitrite (reviewed in ref. 14). Whereas peroxynitrite has hydroxyl radical–like properties and can generate o- and m-tyrosine
from phenylalanine, it also generates the highly specific
3-nitrotyrosine, which is increased 80-fold in atherosclerotic lesions
compared with plasma LDL, and can serve as a marker for the reactive
nitrogen pathway (14).

Hydroxyl radicals revisited

Many of the data implicating myeloperoxidase in early
atherosclerotic lesions were limited to nondiabetic tissue. In this
issue of the JCI, Pennathur et al. (16) have now examined the oxidative chemistry occurring in early atherosclerosis in Cynomologus
monkeys after 6 months of streptozotocin-induced diabetes and feeding
of a Western-type diet. Working with a protein-rich extract from
thoracic aorta, these authors quantified o- and m-tyrosine, o,o’-dityrosine,
and 3-nitrotyrosine as markers of damage from hydroxyl radical,
myeloperoxidase activity, and peroxynitrite, respectively. They report
that o-, m-, and dityrosine, but not
3-nitrotyrosine, are significantly elevated in diabetic aortae,
indicating that peroxynitrite is an unlikely source of damage. To test
the correlation of the data from these chemical analyses with the
extent of hyperglycemia, the authors also quantified glycated
hemoglobin. They report that two of the oxidative products, o- and m-tyrosine,
are tightly correlated with this physiological parameter, but that
dityrosine levels are not, consistent with hydroxyl radical–mediated
damage but not with a principle role for myeloperoxidase. They further
show that glucose autoxidation does not explain their data. Thus, in
all likelihood, redox-active transition metals are involved in this
form of atherosclerosis.

Figure 1
summarizes one possible sequence of events that may explain how
diabetes initiates atherosclerotic lesions without involving
inflammatory cells. First, diabetes-associated hyper- and dyslipidemia
are expected to accelerate LDL deposition in the arterial wall, while
hyperglycemia promotes the formation of the highly reducing Amadori
products in both LDL and collagen. Hyperglycemia also leads to the
conversion of methylglyoxal to carboxyethyl-lysine (CEL) (reviewed in
refs. 11, 17).
All these processes occur nonoxidatively. Oxidation of polyunsaturated
fatty acids in LDL, mediated by high glucose-driven superoxide
formation by mitochondria and NADH oxidase (18, 19), will yield glyoxal, a potent precursor of N-carboxymethyl-lysine (CML) (17). Indeed, CML has been detected immunochemically in early atheromatous lesions (20).
Evidence for the presence of CEL in such lesions is still pending, but
it is expected based on findings of elevated CEL in diabetic tissues (17).

Figure 1. Proposed sequence of events leading to hydroxyl radical–mediated
protein damage in early atherosclerosis in diabetes. The data from
Pennathur et al. (16)
show a strong relationship between hydroxyl radical damage and
hemoglobin glycation. Because these authors found no evidence for
increased nitration-mediated damage, it appears that formation of the
initial lesion does not involve inflammatory cells. A likely scenario
involves increased glycation and the formation of the redox-active
center due to the formation of carboxymethyl-lysine (CML) and
carboxyethyl-lysine (CEL), which can bind redox-active copper and
perhaps iron. Amadori products and ceruloplasmin (not shown) are also
expected to be potent precursors of oxidative damage.
Hyperglycemia-catalyzed superoxide formation from mitochondrial and
cytoplasmic sources is expected to initiate the lipoxidation cascade
and release of glyoxal, a potent CML precursor. PUFA, polyunsaturated
fatty acid.

The strong relationship observed between glycated
hemoglobin and hydroxyl radical damage suggests a concomitant process
in which CML originates from Amadori products through hydroxyl
radical–mediated oxidation (21). Proteins rich in CML (22)
and methylglyoxal-treated proteins (R. Subramaniam and V.M. Monnier,
unpublished results) have been found to bind redox-active Cu 2+,
providing a possible mechanism for the protein damage reported by
Pennathur et al. (16). Of major interest in this context is the recent suggestion of Saxena et al. (23)
that ascorbic acid, which is also found in atheromatous plaques, can
generate CML and become a pro-oxidant in the presence of transition
metals.

Still unclear is the exact source of the transition metals.
Possibilities include the transfer of loosely bound metals to
CML/CEL-rich proteins, which could result from glycation of superoxide
dismutase, ceruloplasmin, or ferritin (24), and the possible binding of redox-active iron by Amadori products (25). However, intact ceruloplasmin can also oxidize lipoproteins (26), and its levels are increased in selected patients with diabetes.

The apparent absence of myeloperoxidase- and nitration-mediated
oxidation suggests that inflammatory cells are not involved at the very
early stage of atherogenesis in diabetes. This scenario may be specific
for diabetes, since previous data from apparently nondiabetic
individuals suggest the contrary (14).
However, once they become oxidized and accumulate CML and other
advanced glycation products, vessel-associated LDL and other proteins
can act as signals and chemotactic factors for activation of
inflammatory cells by binding to RAGE, CD36, or other receptors (27-29). Once that barrier has been crossed, it is not surprising that many forms of protein damage ensue.

If the mechanisms put forward in Figure 1
apply to the early phase of atherosclerosis in diabetes, then
therapeutic antioxidants will be needed much earlier in the process
than previously appreciated. Transition metal–chelating agents and
hydroxyl radical scavengers may prove useful as adjuvants to other
forms of therapy.

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