• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of taccaLink to Publisher's site
Trans Am Clin Climatol Assoc. 2007; 118: 209–214.
PMCID: PMC1863591

Novel Approaches to Treat Oxidative Stress and Cardiovascular Diseases


Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS) such as hydrogen peroxide and superoxide have been identified as important chemical processes that regulate signal transduction. The findings of increased ROS in association with endothelial dysfunction has given rise to the “antioxidant hypothesis”: since ROS are increased in hypertension, atherosclerosis and vascular injury, then inhibiting oxidative stress with antioxidants should decrease cardiovascular events. Preliminary efforts with antioxidant vitamins like beta-carotene, vitamin C and vitamin E have shown no clinical benefits. Here we discuss a specific “redox signaling hypothesis.” We propose that physiologic stimuli such as steady laminar flow regulate the redox state of cells and tissues thereby modulating signaling molecules that are redox sensitive. Here we show that steady laminar flow inhibits tumor necrosis factor (TNF) signaling and inflammation in endothelial cells. We have identified a specific redox molecule—thioredoxin interacting protein (TXNIP)-as a key redox regulator of inflammation in blood vessels. We suggest that modifying the redox state of the vasculature is an attractive therapeutic approach if we target specific redox dependent pathways such as TXNIP.


Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS) have been identified as important chemical processes that regulate signal transduction. In this paper ROS will refer to H2O2, O2 and OH. Because increased ROS may be a risk factor for cardiovascular events such as unstable angina, myocardial infarction and sudden death, understanding the biological processes that generate ROS and the intracellular signals elicited by ROS will be important to gain insight into the pathogenesis of these diseases. An increase in the number of ROS present in a tissue is defined as oxidative stress. The two causes for oxidative stress would be increased generation and impaired degradation. By far the dominant situation is increased generation due to alterations in mitochondrial metabolism and metabolism of fatty acids and carbohydrates. There are some unusual situations in which impaired degradation contributes to oxidative stress, such as in amyotrophic lateral sclerosis (ALS), where there are presumed decreases in superoxide dismutase.

Oxidative stress is an unavoidable consequence of living in air. About 1–5% of all inhaled oxygen becomes a ROS. This is 400,000,000,000,000,000,000,000 molecules per person per day or 25 billion molecules per cell per day. ROS are produced in cells by oxidases. There are several types of oxidases, including NADPH oxidases, cyclo-oxygenases, P450 cytochrome oxygenases, and lipoxygenase (1,2). These enzymes generate superoxide. Superoxide is normally metabolized by superoxide dismutase to hydrogen peroxide, and then catalase metabolizes hydrogen peroxide to oxygen and water. This is particularly important in the vasculature, because superoxide (O2) reacts extremely rapidly with nitric oxide (NO) to form peroxynitrite (ONOO).

Formation of peroxynitrite is a pathophysiologic process, because nitric oxide is an essential endogenous vasodilator. Nitric oxide is generated by endothelial nitric oxide synthase and is a gas that bubbles from the endothelial cell to the smooth muscle cell. Nitric oxide has many beneficial effects, including inhibiting thrombosis, inhibiting inflammation, promoting survival of endothelial cells, and inhibiting recruitment of macrophages to the vessel wall. In the context of increases in oxidative stress, excess superoxide combines with nitric oxide to form peroxynitrite, which is not a vasodilator. The decrease in bioavailable nitric oxide reduces vasodilatory capacity and “sensitizes” the vasculature to hypertension.

Within the blood vessel it is important to realize that there are three major biomechanical forces (3). These are pressure, which is perpendicular to the vessel wall; stretch, the longitudinal and circumferential distension of the wall; and shear stress. Shear stress is the dragging frictional force exerted by the blood, similar to the way a river carves out a canyon. Shear stress is particularly important, because the distribution of atherosclerosis in vessels is non-random and related to shear stress. In regions where shear stress is at physiologic levels (greater than 15 dynes per cm2) and laminar (no turbulence), the vasculature is relatively protected from atherosclerosis. In contrast where the shear stress is low and the flow is disturbed, the region is predisposed to atherosclerosis. A good example would be the carotid bifurcation. The common carotid rarely develops atherosclerosis where the carotid bulb is a site for early atherosclerosis. These findings have given rise to the concept that flow may have an important atheroprotective role. We now know that a key mechanism by which flow is atheroprotective is by the release of nitric oxide.

It has become a paradigm of modern cardiovascular medicine that increases in oxidative stress, by removing the protective effect of nitric oxide, lead to endothelial dysfunction (4). Endothelial dysfunction is manifest by impaired vasorelaxation to endothelium-dependent dilators, such as acetylcholine. All the common risk factors that we associate with coronary artery disease, including elevations in LDL cholesterol, cigarette smoking, diabetes, hypertension, postmenopausal state and hyperhomocysteinemia, are associated with increases in superoxide levels in the endothelium and the vessel wall. In addition to loss of vasodilation, endothelial dysfunction is associated with endothelial cell apoptosis, increased binding of leukocytes and monocytes, enhanced accumulation of lipid and a predisposition to thrombosis. These events lead to a state of vascular inflammation.

Work from our laboratory and many other laboratories has shown that hypertension is a setting in which increased oxidative stress is associated with endothelial dysfunction. For example, in the spontaneously hypertensive rat or the genetically hypertensive rat of New Zealand, there is increased generation of ROS in the vessel wall compared to normotensive control animals (5). Similarly, in animal models of vascular injury such as induced by a wire or by a balloon, there are increases in superoxide production in the injured vessel during the repair process. The findings of increased ROS in association with endothelial dysfunction has given rise to the “antioxidant hypothesis”: since ROS are increased in hypertension, atherosclerosis and vascular injury, then inhibiting oxidative stress with antioxidants should decrease cardiovascular events. Preliminary efforts in this area focused on antioxidant vitamins, like beta-carotene, vitamin C and vitamin E. However, a number of publications over the last 10 years have shown that there is no benefit of vitamin C and vitamin E to limit myocardial infarction, stroke or death from cardiovascular disease. Among the most definitive trials was the MRC/BHF Heart Protection Study (6). In this study 20,000 high risk patients were followed for five years. They were randomized to placebo or to 20 mg beta-carotene, 200 mg vitamin C and 600 mg vitamin E daily. There was no significant reduction in cardiovascular events with the antioxidants. Does the failure of beta carotene, vitamin C and vitamin E to prevent cardiovascular disease mean that the antioxidant hypothesis is wrong? Below I will argue that the answer is “Not yet.”

There are several reasons why antioxidant vitamins may have been ineffective in limiting cardiovascular disease. First, they are not very strong antioxidants, and they are not catalytic in their ability to remove ROS. Second, during the chemical reaction between vitamin E and vitamin C with superoxide, a free radical may form (e.g., vitamin E radical) that may, in fact, be deleterious itself. Third, the ability of the vitamins to penetrate tissues and to have access to the regions of cells that are susceptible to ROS is unclear. Finally, the ability to compete with superoxide to prevent nitric oxide forming peroxynitrite is likely limited. Thus, we have proposed a specific redox target hypothesis.

The specific hypothesis is that physiologic stimuli, such as steady laminar flow, regulate the redox state of cells and tissues in a manner that affects the activity of specific pathways that are themselves regulated by cell redox state. As an example, it is well known that stimuli such as steady laminar flow decreases ROS generation and prevents atherosclerosis; conversely, TNF increases ROS and activates intracellular pro-inflammatory events, like c-Jun N-terminal kinase (JNK), that leads to progression of atherosclerosis. The specific hypothesis we tested is that steady laminar should inhibit TNF signaling and inhibit JNK activation (7).

Specifically, we identified the thioltransferase molecule thioredoxin as a key mediator of the atheroprotective antioxidant effects of steady laminar flow. Thioredoxin is a thioltransferase that takes oxidized protein cysteines (S-S) and reduces them to their sulfhydryl form (S-H). It is known that thioredoxin binds to a protein termed ASK1 for Apoptosis Signal regulated Kinase. In response to TNF and ROS, thioredoxin becomes oxidized and stops binding to ASK1. Dissociation of thioredoxin promotes ASK1 activation, which contributes to endothelial cell apoptosis, dysfunction and inflammation (7,8).

In our laboratory, we used both cells in culture and intact blood vessels to study the effects of flow on activation of ASK1 and JNK by flow. We showed that flow inhibits the ability of TNF to activate JNK by more than 70% (8,9). As expected, the anti-inflammatory effect of flow was associated with an increase in thioredoxin activity. However, unexpectedly there was no change in thioredoxin expression, suggesting that the effect of flow was to change the function of a thioredoxin regulatory protein. Recently, a protein that interacts with thioredoxin was identified that is called TXNIP for ThioredoXiN Interacting Protein. This is a 46,000 dalton protein that is ubiquitously expressed in all cells. It has been shown that TXNIP inhibits thioredoxin by interacting with the catalytic site of thioredoxin. Thus, our model for flow-mediated regulation of TXNIP is that increases in flow would decrease TXNIP expression. This would increase thioredoxin activity that would now inhibit ASK1 and JNK activity, and inhibit vascular inflammation (10).

To prove this occurs physiologically, we decreased TXNIP expression using the small interference RNA (siRNA) technique. In response to the decrease in TXNIP expression, we observed that the ability of TNF to activate JNK (as measured by inflammatory molecule expression, such as vascular cell adhesion molecule-1 (VCAM-1), was inhibited by 80% (10). Finally, we obtained a mouse termed the HcB-19 mouse that has a spontaneous mutation in the TXNIP gene. This mutation results in a 100% decrease in the protein expression level of TXNIP in all tissues. Using the aorta from these mice we exposed them to TNF for six hours and then measured the expression of VCAM-1. We observed an 80% reduction in VCAM-1 expression in TXNIP deficient mouse aorta compared to aorta from the wild type mouse. These data strongly suggest that TXNIP is a key regulator of inflammation in the endothelium.

In summary, our study shows that there are multiple specific pathways responsible for generating and handling oxidative stress. We have identified TXNIP as a key regulator of inflammation in blood vessels that works by modified thioredoxin function. Our belief is that modifying the redox state of the vasculature remains an attractive therapeutic approach if we target specific redox-dependent pathways such as TXNIP and thioredoxin.


Alexander: Atlanta: Brad, that's very creative and stimulating as usual. I wanted to ask you, you talked about the specificity of targeting redox-sensitive pathways, how would you incorporate issues of compartmentalization into that, because its becoming more and more obvious that there are multiple intracellular compartments, and you can look at cytosolic levels of glutathione, reduced glutathione and still not reflect what is going on in some of these compartments. I wonder if you can comment on that.

Berk: Rochester: TXNIP and thioredoxin in fact move between compartments. I was very interested in Dr. Fitz's discussion on exocytosis. Thioredoxin is actually secreted outside of cells. We think TXNIP is a carrier that does that. Thioredoxin moves into the nucleus. Its very important in regulating NF-kappa B-dependent transcription. So, compartmentalization is quite important. It seems TXNIP though stays in the cytoplasm, and as I said, it's highly regulated. We have identified a number of hormones that can up-regulate or down-regulate TXNIP expression. So we are looking to see in which compartments TXNIP goes up and down. There is a big advantage to trying to regulate the protein itself rather than the generation of reactive oxygen species. So we heard earlier today about CGD disease in which NADPH oxidase is modified. You could try to change the generators—that is, develop drugs that would inhibit NADPH oxidase—but you can imagine that that might not be a very good thing in terms of infection. So I think the key is to identify the specific targets, and then we will see to what extent in target cells, smooth muscle cells, endothelium would have a benefit.

Gallin: Bethesda: This was really fascinating. Thank you. There is one experiment that is in the literature that I would love to hear your thoughts on, where the CGD knockout mouse was crossed with the apoE mouse which normally or abnormally gets atherosclerosis; and when you make this hybrid knockout, it was reported that these animals did not have atherosclerosis from the aorta through the femoral arteries, though they did get lesions up in the coronary arteries.

Berk: Rochester: There has been a large amount of work done looking at the oxidases and their influence on atherosclerosis. Davis Harrision, who is at Emory, for example, has shown a significant effect on hypertension, and in atherosclerosis, knocking out various components of the vascular oxidases, which is primarily gp91phox. So I think that that does reflect, in fact, the change in the redox state, and by reducing the oxidative stress or by reducing the modification of other molecules within the developing atherosclerotic plaque, you have beneficial effects. If you over-express a superoxide dismutase to assist in the metabolism of superoxide, you also have a beneficial effect on atherosclerosis. So I think there are some specific targets. We are actually now studying atherosclerosis in our TXNIP knockout mouse. We hope that that will, in fact, be equally protective, and we have quite a bit of data already on doing the converse, that is over-expressing thioredoxin has beneficial effects to limit atherosclerosis. So I think there is a large number of these evolving targets, and I think it will just be a matter of time before we see efforts in humans. The problem, of course, is the long duration of atherosclerosis makes a surrogate necessary.


1. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part II: animal and human studies. Circulation. 2003;108:2034–40. [PubMed]
2. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part I: basic mechanisms and in vivo monitoring of ROS. Circulation. 2003;108:1912–6. [PubMed]
3. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998;18:677–85. [PubMed]
4. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000;87:840–4. [PubMed]
5. Lehoux S. Redox signalling in vascular responses to shear and stretch. Cardiovasc Res. 2006;71:269–79. [PubMed]
6. MRC/BHF. MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet. 2002;360:23–33. [PubMed]
7. Surapisitchat J, Hoefen RJ, Pi X, Yoshizumi M, Yan C, Berk BC. Fluid shear stress inhibits TNF-alpha activation of JNK but not ERK½ or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members. Proc Natl Acad Sci USA. 2001;98:6476–6481. [PMC free article] [PubMed]
8. Liu Y, Yin G, Surapisitchat J, Berk BC, Min W. Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3. J Clin Invest. 2001;107:917–23. [PMC free article] [PubMed]
9. Yamawaki H, Lehoux S, Berk BC. Chronic physiological shear stress inhibits tumor necrosis factor-induced proinflammatory responses in rabbit aorta perfused ex vivo. Circulation. 2003;108:1619–1625. [PubMed]
10. Yamawaki H, Pan S, Lee RT, Berk BC. Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells. J Clin Invest. 2005;115:733–8. [PMC free article] [PubMed]

Articles from Transactions of the American Clinical and Climatological Association are provided here courtesy of American Clinical and Climatological Association
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...