Antioxidant and Atherosclerosis

Oxidation of LDL and of lipids by free radicals is one of the most important factors for the initiation of atherosclerosis (Parthasarathy et al, 1992).

ƒSignificantly less progression of atherosclerosis in men but not in women in the Antioxidant Supplementation in Atherosclerosis Prevention study (Salonen et al 2003). ƒInverse correlation between coronary events and vitamin C intake in the Nurse’s Health study (Stampfer et al., 1993). ƒBeta carotene was associated with reduced coronary risk, vitamin E only in the smokers and no benefit of vitamin C in Health Professionals Follow-up Study (Ascherio et al., 1999). ƒNo cardiovascular benefits of vitamin E and beta-carotene in Finnish Alpha-Tocopherol-Beta-Carotene prevention study (Virtamo et al., 1998) and in the Physicians Health study (Muntwyler et al., 1996). Antioxidants in Mouse Models of Atherosclerosis

Inhibitory effect of vitamin E (Cyrus et al, 2003, Pratico et al., 1998), Coenzyme Q(10) (Thomas et al., 2001), Tocotrienols (Black et al., 2000), N,N’-diphenyl-1,4-phenylenediamine (Tangirala et al., 1995), or a combination of vitamin C, E and beta carotene (Crawford et al., 1998). No effects of vitamin E (Munday et al., 1998) or its combination with beta- carotene (Shaish et al., 1999). Probucol has detrimental effects and increased plaque sizes (Zhang et al., 1997, Moghadasian et al., 1999). Chronic vitamin C deficiency does not influence either the initiation or progression of atherosclerosis but significantly limits collagen deposition in vitamin C-dependent apoE-/- mice (Nakata et al, 2002).

Like other treatments, protective effect of antioxidants can be seen only when the treatments are started early. Accelerated Atherosclerosis in Diabetes

Excess generation of mitochondrial ROS due to hyperglycemia initiates a vicious circle of oxidative stress through increased polyol, AGE, PKC and hexosamine pathways (Brownlee, 2004).

Oxidation of LDL and of lipids by free radicals is one of the most important factors for the initiation of atherosclerosis (Parthasarathy et al, 1992).

Despite overwhelming evidence on the damaging consequences of oxidative stress, large scale clinical trials with classic antioxidants failed to demonstrate any benefit for diabetic patients. Antioxidants and Atherosclerosis in Diabetic Mice

Protective Role of Lipoic Acid

Xianwen Yi

Treatment with alpha-lipoic acid (600 mg/day i.v.) over 3 weeks is safe and significantly improves both positive neuropathic symptoms and deficits to a clinically meaningful degree in diabetic patients with symptomatic polyneuropathy: a meta-analysis (Zieglar et al., 2005). R-lipoic acid O

* OH

S S

• LA is a water- and fat-soluble antioxidant that can easily cross cell membranes.

• Removes catalysts by metal-chelating e.g. Fenton reaction 2+ - 3+ H2O2 + Fe OH* + OH + Fe

• A strong reducing agent and recycles other antioxidants like reduced glutathione (GSH) LA is an essential cofactor for several key enzyme complexes in metabolism

glucose glucose-6-phosphate fructose-6-phosphase

fructose-1,6-bisphosphase

glyceraldehyde 3-phosphate

Gluconeogenesis 1,3-bisphophoglycerate Glycolysis

Cytosol 3-phosphoglycerate 2-phosphoglycerate

oxaloacetate phosphoenolpyruvate

lactate pyruvate

PDH malate oxaloacetate acetyl CoA Mitochondrion citrate Effects of LA on STZ Treated ApoE-/- Mice

Diet STZ Lipoic acid I. Early Diabetic LA group Weeks 82201612 248 32

500 Diabetic

400 HF

300 **

(mg/dl) ** ** 200 HF+LA ** Plasma glucose 100 12 16 20 24 28 32 Weeks Diet STZ Lipoic acid II. Late Diabetic Weeks LA group 82201612 248 32

Diet 500 HF 400

HF+LA 300 ** ** (mg/dl) 200 Plasma glucose 100 12 16 20 24 28 32 Weeks Oxidative stress is increased in diabetic mice

5mo post STZ treatment

50 * 4

40 * 3 30 * 2 20 * 1 10

Erythrocyte GSH (mg/ml) 0 0 Plasma TBAR (mmole/ml) n132 92019 n132 92019 LA -- LA LA -- LA Diabetic Nondiabetic Diabetic Nondiabetic

LA significantly improves antioxidant status LA in diet prevents enhanced atherosclerosis and kidney damage induced by diabetes in apoE-/- mice

(5mo post STZ) 400 125 ) 2

m 100 μ

3 300 g/day) μ 75 200 * 50 * 100 25 Aortic Lesion (10 Aortic Lesion Urine Albumin ( Urine Albumin 0 0 n118 12 610 n17 15 10 LA -- LA - LA - Diabetic Nondiabetic Diabetic Nondiabetic HF HF+LA

4 X 4 X 50 400 * 800 40 * 300 600 * 30 * 200 400 BW (g) 20

100 200 Glucose (mg/dl) Glucose 10 Cholesterol (mg/dl)

0 0 0 n132 92019 n132 92019 n132 92019 LA -- LA LA -- LA LA -- LA Diabetic Nondiabetic Diabetic Nondiabetic Diabetic Nondiabetic

Plasma cholesterol was significantly elevated in diabetic mice with a control diet at 5 mo post STZ treatment.

Increased plasma TC may be the consequence of dehydration. It may be contributing to the increased atherosclerosis. Protective effects of LA are partly due to protection from pancreatic β cell damage by STZ and their regeneration over time

Control Diabetic/HF+LA Diabetic/HF

X 10

(Immunostaining of beta cells with insulin) • LA, as a natural antioxidant, is effective against atherosclerosis & kidney damage in STZ-induced diabetic apoE-/- mice.

• LA has robust protective and therapeutic functions partly through protection of beta cells.

• Other diabetic models? Mammals can produce LA in all the cells

1.5 P< 0.01

1

0.5 Lias Expression 0 Control DM

Lipoic acid synthase (Lias) gene expression in the kidney of STZ induced DM C57BL6 mice is 50% normal Endogenous production of LA is essential Lias-/- embryos are developmentally retarded and die immediately after implantation

7.5 dpc

Dietary LA supplement at 1.65 g/kg to Lias+/- mothers failed to rescue Lias-/- embryo

Lipoic acid synthesis From Booker, 2004 LipB

octanoyl acid LipA, SAM (Lias)

In E.coli But not in yeast LplA α lipoic acid

LplA

Growth of yeast mutants lacking Lias or ACP cannot be rescued by LA in media (Sulo and Martin, 1993) Lias +/- heterozygotes have a trend increased lactate

Liver Lactate Liver PDH activity 2 ( mmol/mg protein) 2.5 (μmole/min/mg protein)

P=0.09 2 1.5

1.5 1 1

0.5 0.5

0 0 +/+ +/- +/+ +/- Lias+/- heterozygotes have a trend reduced antioxidant capacity

Plasma TBAR Erythrocyte GSH (nmole/ml) 25 40 (mg/ml) P<0.005 20 30

15 20 10

10 5

0 0 +/+ +/- +/+ +/- Conclusion

• Dietary supplement of LA, as a natural antioxidant, has athero-protective effects in STZ-induced diabetic apoE-/- mice.

• Endogenous production of LA is essential for development and cannot be replaced by exogenous LA.

• Heterozygotes with reduced Lias may be useful for studying the role of LA in disease. Vitamin C Deficiency and Atherosclerosis in Diabetic Gulo-/-Apoe-/- Mice

400 )

2 300 m μ

VitC in water Liver Plasma 3 at 0.33g/L (μg/g) (μg/ml) 200 1X 150±26 9.6±3.7 100

0.1X 30±13 3.0±0.8 lesion (10 N=7 N=6 0 0.1X 1X Vitamin C Complex plaque morphologies in diabetic apoE-/- mice (129/SvEv background, chow diet) Organized Thrombi (?)

Inflammation

Thrombi (?)