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Inflammation
Inflammation is a protective biological response initiated by the immune system that involves complex communication between immune cells to respond to harmful stimuli such as pathogens, damaged cells, or irritants. Acute inflammation consists of leukocyte recruitment from the circulation, with initial recruitment of polymorphonuclear granulocytes followed by monocytes and macrophages. However, chronic inflammation, as found in cardiovascular disease, can lead to destruction of tissues. The inflammatory response involves the regulation of pro- and anti-inflammatory mediators in resident tissue cells and recruited leukocytes through the coordination of various signaling pathways.
Cytokines are mediators of the inflammatory response and are classified into several classes: interleukins; tumor necrosis factors (TNF); interferons (IFN), colony stimulating factors (CSF), transforming growth factors (TGF), and chemokines. Helper-T (Th) cells are principal players in adaptive immunity and are classified according to the cytokines they secrete. A cell-mediated immune response (Th1) is associated with the secretion of IL-2 and IFN- , while a humoral immune response (Th2) is associated with the secretion of IL-4, IL-5, IL-10, and IL-13. Cytokines are also characterized based on the pathway in which they mediate their effects. Most ILs, CSFs, and IFNs belong to the JAK-STAT pathway. IL-1 (including IL-1 , IL-1 , IL1ra, and Il-18) and TNF activate NF- B and MAP kinase signaling pathways, while TGF- superfamily members activate signaling proteins of Smad family (See Figure 4). Cytokines are classified according to either pro- (IL1/12/18, IFN- , TNF) or anti- (IL4/10/13, TGF-B) inflammatory activities.
Cytokine Signaling Pathways
NF-せB. NF- B is one of the principle pathways activated in response to pro-inflammatory cytokines such as TNF-a, IL1, and IL-18. In its inactive form, NF- B is sequestered in the cytoplasm by inhibitor proteins, I Bs. Various stimuli that activate NF- B cause the subsequent degradation of I B. The activated NF- B complex can then translocate into the nucleus and induce gene expression. This negative-feedback loop gives rise to oscillations in NF- B translocation. Its activation regulates genes encoding pro-inflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes such as COX2 and iNOS.
NF- B is a redox-sensitive transcription factor and is highly influenced by the intracellular redox status of the cell (11). Antioxidants, such as aspirin, NAC, and flavonoids, can inhibit activation of NF- B. HO-1 is an example of an anti-inflammatory pathway induced in response to TNF and IL-1-induced inflammation (12, 13).
JNK/AP-1. JNK phosphorylation is mediated by two MAPK kinases: MAP2K4 and MAP2K7. JNK pathway regulates many pro-inflammatory genes, including those encoding for TNF- , IL-2, IL-6, E-selectin, ICAM-1, VCAM-1, MCP-1, COX2, and MMPs (-1, -9, – 12, -13).
JAK/STAT. JAK and/or STAT proteins can be activated by IFNs, as well as several cytokines (especially IL-6), growth factors, and hormonal factors. IL-4 activates STAT6 and promotes differentiation of Th2 cells (14), while IL-12 activates STAT4 and promotes differentiation of IFNg-producing Th1 cells (15).
SMADS. TGF-B-triggered signals are transduced by Smad family proteins. Smad3 has antagonistic properties and plays a major role in TGF- -dependent repression of vascular inflammation by inhibiting AP-1 activity (16). IFNg-induced expression of Smad7 acts as a negative regulator of the TGF-B/Smad pathway (17).
Link between Oxidative Stress & Inflammation
ROS such as O2·-, H2O2, and OH are produced in response to activation by cytokines, including TNF and IL-1. The production of ROS activates redox-sensitive signaling pathways that induce inflammatory gene expression. In adequate quantities, ROS are considered to be second messengers. ROS have been considered to be general messengers for the induction of NF-B activation (19), although possibly not through direct mechanisms (20). Recent evidence supports the notion the ROS may oxidize KF-B subunits, thereby impairing DNA binding and transcriptional activities of NF-B (21). In addition, ROS can strongly activate JNK though the oxidative inactivation of the endogenous JNK inhibitors (21).
iNOS is produced as a result of endotoxin and cytokine activation. It produces large, toxic amounts of NO in a sustained manner to help kill or inhibit the further growth of invading microorganisms. Cytokines released from infected host cells, such as TNF-a and IL-1b, can activate NO production. The NFkB pathway and IFN-g, via the activation of JAK and STAT pathways, can trigger iNOS transcription (22, 23). While iNOS is protective against certain infectious diseases chronic inflammatory conditions can cause DNA damage or tumorigenesis (24). Anti-inflammatory cytokines such as TGF- , IL-4, or IL-10 (25), and PPAR (26) can inhibit these pathways, reducing iNOS production.
Effects of Exercise on Oxidative Stress & Inflammation
Physical inactivity accentuates the negative components of diseases such as atherosclerosis, diabetes, obesity, and metabolic syndrome (27). On the other hand, exercise training is able to reduce and prevent these diseases (28, 29). It is now well documented that acute exercise increases ROS and inflammation. However, the body adapts to a continual practice of exercise by increasing anti-oxidants and anti-inflammatory agents to compensate for the overproduction of ROS and inflammation. In this section, I will review the role of acute and chronic exercise on oxidative stress and inflammation.
a Acute Exercise.
Aerobic exercise can cause an increase of up to 15-fold in the rate of oxygen consumption throughout the body, and as much as 100-fold in the oxygen flux in active muscles (30). As oxygen consumption and flow increases, so does the possibility of ROS production. There are several theories as to how ROS is produced during exercise, including i) leakage in mitochondrial electron transport chain, ii) ischemia/reperfusion, and iii) neutrophils.
It is commonly understood that inadequate coupling of electron transfers between complexes in the mitochondrial electron transport chain (ETC) causes the leak of superoxide radicals (31, 32). However, other studies have suggested that a decrease in mitochondrial pO2 is the cause of exercise-induced increase in ROS production rather than an ETC leakage (32, 33). This is supported by studies that show that isometric exercises do not induce an increase in VO2 but still causes an increase in oxidative stress possibly due to reduce mitochondrial pO2 (34). Secondly, another hypothesis suggests that mechanisms similar to ischemia-reperfusion injury could be responsible for ROS increase. During exercise blood flow is redistributed to active tissues leading to transient tissue hypoxia. The ischemic condition could trigger the production of XO. The reoxygenation period that follows could produce superoxide from XO. Studies have found an increase in superoxide and XO levels following exercise (35, 36), which can be ameliorated using allopurinol, an XO inhibitor (36–38).
Finally, cytokines released during an acute bout of exercise can facilitate an influx of leukocytes at the site of inflammation. Neutrophils can infiltrate affected areas, leading to a respiratory burst which produces ROS such as superoxide and hydrogen peroxide. Within neutrophils is an iron-containing enzyme called myeloperoxidase (MPO) that can convert hydrogen peroxide into hypochlorous acid, a highly potent oxidant. It has been demonstrated that exercise leads to an increase in neutrophil and MPO levels (40–43).
The consensus of most human and animal studies has found that acute aerobic (44–46) and anaerobic (46, 47) exercise increases oxidative stress. In 1978, Dillard et al (49) found that exercise at 75% VO2max increased levels of lipid peroxidation compared to resting subjects. It was then shown that exhaustive exercise increased liver and muscle free radical concentration two- to three-fold (44). Long duration exercise also increases free radical concentration in skeletal muscle and myocardium (50). However, other studies show no change in acute exercise compared to control. The discrepancies between results can be due to intensity (48, 49) and duration (50) of the exercise protocol, training status of subjects (51–53), and age (54).
Antioxidants. In response to acute physical activity, the body’s antioxidant defense system may be temporarily reduced as it combats the increase in ROS. Therefore, the measurement of antioxidants can be used as a marker of oxidative stress. During exercise and immediately after the exercise bout, antioxidants may be reduced (55, 56) indicating an increase in ROS. Further into the recovery period, antioxidants may increase above basal levels (34, 55–57) indicating a return to more normal levels of ROS. It is possible that those studies which found no significant changes in anti-oxidant capacity may not have taken enough samples throughout the protocol (44, 58).
2e. Hypoxia/Reperfusion Injury
A common repercussion of sickle cell disease due to adhesion and vaso-occlusion is the occurrence of hypoxia. The cessation of blood flow to tissues causes an ischemic or hypoxic environment. During this state, the limited concentration of oxygen available to tissues results in an inadequate amount of nutrients delivered to support the metabolic needs. Reactive oxygen species can be generated at various points during this undesirable environment: during hypoxia in both the mitochondria and in tissues, as well as during the reperfusion phase that follows. This phase can cause reperfusion injury, referring to the damage caused to the vessel and tissue when oxygen is reintroduced to the tissues, leading to an increase in the concentration of radical species (43). Under hypoxic conditions, as seen in SCD, adenosine triphosphate (ATP) is consumed to adenosine diphosphate and adenosine monophosphate. If oxygen supply continues to decrease to certain levels, adenosine monophosphate is catabolized, leading to the accumulation of hypoxanthine and xanthine in the tissue (42, 43). The produced xanthine oxidase (XO) can then lead to deleterious effects due to the restitution of blood flow and therefore oxygen to the cells. It is when oxygen is reintroduced to these tissues (“reoxygenation”) and reacts with the hypoxia-formed XO, that the damaging effects of oxidative stress are seen. This reaction results in the conversion of hypoxanthine and xanthine by XO into superoxide (38, 42, 43) (See Figure 10). Hypoxanthine + O2 uric acid + NADPH + O2·.
2g Anti-oxidant therapeutic strategies
Several treatment studies have been shown to be effective to reduce pathological consequences of the disease (See Wood et al (10); Nur et al (53) for review). Susceptibility to peroxidation, due to an increase in ROS or during conditions of reduced oxygen, can increase 3-fold in sickle cell patients. In vitro, pre-incubation of sickle erythrocytes with vitamin E decreased the susceptibility to peroxidation. This finding is maintained in vivo, where those with a vitamin E deficiency increased levels of peroxidation compared to control subjects (54). A study evaluating the effects of SCD by Natta et al, found that after only 10 weeks of vitamin E supplementation, the percentage of irreversibly sickled red cells decreased from 25% to 11% (55). A decrease in sickled RBC was also observed during a supplementation period with vitamin E possibly due to the almost 2-fold decrease of ROS and the 1.2-fold increase in the concentration of GSH (13).
The additions of ascorbic acid and dehydroascorbic acid supplements were able to inhibit dense RBC formation and lipid peroxidation levels in sickle cell patients (56). A another study by Amer et al found that supplementation of vitamin C helped to decrease ROS production almost 4-fold while increasing the concentration of GSH almost 2-fold (13). It may be through this mechanism that ascorbic acid supplements were shows to prevent H2O2-
induced hemolysis (13). Finally, in vitro supplementation of alpha-lipoic acid, known to have potent antioxidant properties, can inhibit RBC sickling by 50% (56), decrease oxidation (57), protect peroxyl radical-induced hemolysis, and increase GSH synthesis (58).
Taken together these promising results should encourage the development of such antioxidant therapy. However the conclusions of clinical trials using antioxidant therapeutic treatment in SCD are not so enthusiastic (23). Wood et al (23) suggest that the SCD-induced ROS generation may greatly overwhelm the capacity of the exogenous antioxidants, leading to the conclusion that a cocktail of multiple antioxidants may be more efficient (56).
Therapeutic strategies that focus on decreasing ROS production, instead of increasing their neutralization, were also recently studied. Hydroxyurea (HU) can reduce the occurrence of vaso-occlusive crises and pulmonary events by inducing fetal hemoglobin. However, in addition to fetal hemoglobin, HU can limit ROS production and NO scavenging via a decrease in hemolysis. To determine mechanisms, in vitro studies have found a decrease in adhesion between HbS RBC and the endothelium, as well as a decrease in adhesion molecules on sickle cell reticulocytes (59). Some studies have suggested that HU induces a NO response (60), as NO is well documented in the regulation of adhesion. Because of the importance in maintaining proper NO levels, exogenous NO treatment is often beneficial. Inhaled NO was shown to increase plasma NOx in some case studies, and decrease adhesion and ischemia/reperfusion injury in animal studies (10). Arginine therapy is shown to have a positive effect on vasodilation, possibly through the reduction of oxidative stress and hemolysis. Kaul et al (26) suggested that arginine therapy in SCD mice prevented the oxidative stress-induced hemolysis of RBC. An increase in the antioxidant GSH and enhanced NO production was also observed in these mice, possibly due to a reversal of eNOS uncoupling (26).
Iron chelator desferoxamine (61), a catalase mimetic (38) and an NF- B inhibitor (62) were shown to attenuate oxidative stress, adhesion and inflammation in murine models of SCD. Although not tested yet in SCD, the use of SOD mimetic (such as tempol) may potentially be beneficial to target the anion superoxide clearly identified in the pathogenesis of SCD (23).
Table of contents :
Chapter I. General Introduction.
I.1 Sickle Cell Disease vs. Atherosclerosis.
I.2 Exercise Training.
Chapter II: Oxidative Stress and Inflammation
II.1 Oxidative Stress
II.2 Inflammation
II.2a Cytokine Signaling Pathways
II.3 Link between Oxidative Stress & Inflammation
II.4 Effects of Exercise on Oxidative Stress & Inflammation
II.4a Acute Exercise.
II.4b Chronic Exercise.
II.5 Conclusion.
II.6 References:
Chapter III: Sickle Cell Disease and Sickle Cell Trait
III.1 Sickle Cell Disease
III.1a Pathogenesis
III.2 Role of Oxidative Stress (Published Review: See Annex IX.1a)
III.2a. Blood Cell Auto-oxidation
III.2b. Sickling & Hemolysis
III.2c. Fate of Nitric Oxide
III.2d RBC Adhesion & Vaso-occlusion
III.2e. Hypoxia/Reperfusion Injury
III.2f Anti-oxidants
III.2g Anti-oxidant therapeutic strategies
III.3 Role of Exercise
III.3a Exercise in SCT
III.3b Exercise in SCD
III.4 References:
Chapter IV
IV.1 Introduction to Article #1
IV.2 Exercise training blunts oxidative stress in sickle cell trait carriers
Chapter V Atherosclerosis
V.1 Plaque Development
V.1a Vascular Components: The vessel wall.
V.1b Vascular Components: The endothelium
V.2 Pathogenesis of Atherosclerosis.
V.3 Generation of oxidative stress and inflammation in Atherosclerosis.
V.3a Role of oxidative stress and inflammation in macrophages.
V.3b Role of oxidative stress and inflammation on endothelial dysfunction
V.3c Role of oxidative stress and inflammation on adhesion
V.4 Non-invasive methods for plaque vulnerability investigation
V.4a Magnetic Resonance Imaging (MRI)
V.4b Imaging in Atherosclerosis.
V.4c Animal models of advanced lesions
V.5 Effects of exercise training in atherosclerosis
V.5a LDL
V.5b Endothelial dysfunction
V.5c Macrophage/leukocyte recruitment
V.5d Adhesion
V.5e Plaque instability
V.6 References:
Chapter VI: Article #2.
VII. Conclusions and Perspectives
VII.1 Sickle Cell Disease and Trait
VII.1a Conclusion
VII.1b Perspectives
VI.2 Atherosclerosis
VII.2a Conclusion
VII. 2b Perspectives
VII.3 Final Conclusion.
VIII. Publications & Communications
VIII.1 Articles related to my published in Peer-reviewed scientific Journals
VIII.2 International Conference Proceedings
IX. Annex
IX.1 Published Papers
IX.1a Role of Oxidative Stress in the Pathogenesis of Sickle Cell Disease
IX.1b Exercise training blunts oxidative stress in sickle cell trait carriers
IX.1c Editorial: Research in Athletes with Sickle Cell Trait: Just Do It
IX.1d Effect of –thalassemia on exercise-induced oxidative stress in sickle cell trait .
IX.1e In vivo cardiac anatomical and functional effects of wheel running in mice by magnetic resonance imaging
IX.2 Supplemental Research Data (not published)
IX.2a BMDM differentiation
