2-1.1 Introduction of Reactive Oxygen Species
Reactive oxygen species (ROS) are a group of oxygen-containing and highly reactive species which are partially reduced from molecular oxygen, including molecules such as hydrogen peroxide (H2O2), radicals such as hydroxyl radical (•OH), ions such as hypochlorite ion (OCl⎯), and both an ion and a radical such as superoxide anion (O2•⎯) [46, 87]. Under normal physiological conditions, the formation of ROS is a natural consequence of cellular metabolism, where about 1% to 3% of oxygen uptook by the body is reduced into ROS [88, 89]. The major source of ROS exists in the mitochondrial aerobic respiratory electron transport chain (ETC). Among four multi-protein complexes of ETC, Complex I, II, and III were believed to produced superoxide anion byproduct by leaking electrons to oxygen (Figure 13) [90, 91].
Figure 13 Generation of superoxide anion in the mitochondrial respiratory electron transport chain (ETC). (Modified from reference [91])
After reducing oxygen by one electron, the produced superoxide anion (O2•⎯) can serve as the precursor of most other ROS. For example, superoxide anion can undergo dismutation to become hydrogen peroxide (H2O2), which can be further partially reduced to one of the strongest oxidants, hydroxyl radical (•OH) [92].
Furthermore, superoxide anion may also react with other radicals to produce new oxidants, such as peroxynitrite, an oxidant produced by the reaction of superoxide anion and nitric oxide (•NO) [93]. In addition to generating ROS by mitochondrial respiratory system, microsomal cytochrome P450 enzymes, flavoprotein oxidases, and peroxisomal enzymes involved in fatty acid metabolism also serve as the intracellular sources of ROS [88, 94].
According to current research, the biological functions of ROS constitute a paradox. They are not only involved in signal transduction and homeostasis of tissue turnover but also involved in cell death and cellular degradation (Figure 14) [67, 95].
Under normal physiological conditions, the toxic effects of ROS are prevented by various cellular anti-oxidant systems, which mean that the production of ROS and the scavenging of ROS by antioxidants are in balance. Therefore, the resulting low concentration of ROS within cells act as intracellular second messenger molecules which regulate the expression of a number of genes, such as ECM components, MMPs, and cytokines [96, 97]. However, in several pathological circumstances, the ROS are somehow over-produced and the antioxidants defenses become insufficient, which lead to destroy the balance between intracellular and extracellular redox state [67, 98]. The resulting so-called “oxidative stress” is an abnormal catabolic state which can induce structural or functional changes in cells and tissues by oxidizing polyunsaturated fatty acids to increase membrane fluidity and permeability, oxidizing guanine to 8-hydroxyguanine to cause DNA damage, and oxidizing amino acids such
Figure 14 The biological functions of ROS constitute a paradox.
2-1.2 Reactive Oxygen Species Formation in Cartilage
The often produced ROS in chondrocytes are superoxide anion (O2•⎯) and nitric oxide (•NO), which can further generate derivative radicals such as ONOO⎯ and H2O2
(Figure 15) [102, 103]. Although O2•⎯ in vivo can be enzymatically or nonenzymatically produced, in cartilage, O2•⎯ is produced by the nicotinamide dinucleotide phosphate (NADPH) oxidase, which is a complex enzyme system consisting two membrane bound peptides: one is a two-peptide formed flavocytochrome, the other is a regulatory peptide called (Rap1A) [104].
Similarly, •NO in cartilage is also produced by an enzyme system, called NO synthase (NOS) [105]. Among three isoforms of NOS, chondrocytes express endothelial NOS (eNOS) and inducible NOS (iNOS) to generate nitric oxide [106]. On the other hand, in an in vitro environment, it was reported that O2•⎯ may be generated by tumor necrosis factor-α (TNF-α) stimulation or by cyclic stretch [107], and •NO generation may be stimulated by IL-1β, TNF-α, shear stress, or mechanical compression.
Figure 15 Schematic representation of the often produced ROS in chondrocytes.
(Modified from references [46] and [67] )
2-1.3 Antioxidant Systems in Cartilage
In order to prevent the toxic effects of ROS, chondrocytes possess various anti-oxidant defensive systems to scavenge the over-produced ROS, which include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxins (PRDX) (Figure 16) [108-111]. SODs are metalloproteins which possess metals in their reactive centers [112]. In chondrocytes, cytosolic Cu/Zn SOD and mitochondrial Mn SOD are constitutively expressed to catalyze two superoxide anions into one hydrogen peroxide and one molecular oxygen [113]. Because O2•⎯ is the most frequent and abundant ROS produced by normal cellular metabolism, the breakdown of O2•⎯ becomes the first defense mechanism against ROS [112]. However, the resulting hydrogen peroxide is also another kind of ROS and its accumulation is regarded as an oxidative stress as well. Therefore, catalase and glutathione peroxidase coexist and are functioned as scavengers of H2O2 [113, 114]. Besides this, peroxiredoxins, a newly found peroxidase family which possesses six isoforms in mammals, expresses type V isoform and eliminates H2O2 in cartilage [111].
Figure 16 Schema of the antioxidant systems. (Modified from reference [91, 115])
2-1.4 Reactive Oxygen Species on Cartilage Matrix Degradation
In pathological conditions, such as OA, reactive oxygen species have been described as an important factor on cartilage matrix degradation because they can cause cellular or organic changes by either directly oxidizing the amino acids of collagen and proteoglycan molecules or by modifying the expression of anabolic and catabolic related enzymes [46, 99-101, 116]. From several in vitro experiments, it have been reported that ROS directly attacked ECM components. For example, when treating the incubated type I collagen with O2•⎯, collagen was degraded and the ability of fibrils formation was lost [117, 118]. Moreover, in the •OH and oxygen coexisting environment, collagen was degraded into small peptides where the cleavages often happened on proline or 4-hydroxyproline residues, and the increase of glutamic acid and aspartic acid in the peptides were also observed [119]. Furthermore, HOCl was discovered to possess the ability to cleave hyaluronic acid and hence decrease the viscosity of synovial fluid [120]. On the other hand, some data also showed that ROS can change the response of chondrocytes to ECM-related anabolic enzymes or upregulate the expression of ECM-related catabolic enzymes. For instance, the concurrent generation of O2•⎯ and •NO could decrease the sensitivity of chondrocytes to IGF-1 and hence inhibit the synthesis of proteoglycan [121, 122]. Moreover, •NO could inhibit the sulfation of newly synthesized proteoglycan under the induction of IL-1 [123], and •NO also played an important role in activation of metalloprotease enzymes in articular cartilage [124]. Besides this, HOCl could directly activate the proenzymes of metalloprotease and inactivate the tissue inhibitors of metalloprotease (TIMPs) [125, 126].
2-1.5 Reactive Oxygen Species on Cartilage Senescence
Oxidative stress is an important factor of the aging process in cartilage. Because adult articular cartilage is considered as a post-mitotic tissue with the characteristic of limited ability to achieve tissue turnover [127], its aging process is not attributed to intrinsic replicative senescence accompanying with telomere shortening but is related to oxidative stress-induced extrinsic senescence [66, 128]. Several reports showed that the ability of chondrocytes to detoxify ROS and turnover the damaged macromolecules is dramatically inefficient over time [112]. Therefore, oxidative damage products such as peroxidated lipids [129], nitrotyrosine [130], and mutagenic 8-oxoguanine [131] gradually accumulate in chondrocytes and disturb normal cellular functions until the chondrocytes can not endure these structural changes. Notably, the production of mutagenic 8-oxoguanine has been reported to result from O2•⎯ attack which directly cause telomere erosion by injuring the guanine repeats in telomere DNA [132]. Moreover, it also has been reported that cell aging process correlates with the degeneration of mitochondria which may lead further leakage of electron transport chain and hence increase the production of ROS [133]. The accumulated ROS in mitochondria then attack mitochondrial 16-kilobase circular DNA, and the resulting mtDNA mutations contribute to the feature of accelerated aging [134].
2-1.6 Reactive Oxygen Species on Chondrocyte Death
In adult tissues, cell death plays an important role in maintenance of the balance of cell proliferation and the constancy of cell numbers [135]. Apoptosis is an active process of programmed cell death which is characterized by a series of morphological changes including DNA fragmentation, chromatin condensation, nucleus break-up, and cell fragmentation, while necrosis is the accidental death of cells that resulting from a pathologic injury [136].
It has been noted that aging [110], mechanical damage [137], and pathology such as osteoarthritis [138] often result in increased chondrocyte death in cartilage, and because of adult cartilage has limited ability for self-repair, the damaged cells may further accelerate the progression of the lesion [139]. From Blanco et al.’s research, it has been revealed that •NO is a primary inducer of chondrocyte apoptosis and is down-regulated by caspase-3 and tyrosine kinase activation [140]. However, from recent research, it has revealed that the toxicity of •NO needs to be further modulated by other ROS. For example, Del Carlo et al.’s research showed that the chondrocyte cell death mediated by •NO requires the generation of O2•⎯, which suggests that ONOO⎯ plays an important role in the process [141]. Moreover, in Kurz et al.’s finding, they found that both O2•⎯ and H2O2 play important roles in mechanically induced cell death [142]. From these examples, it might be suggested that O2•⎯ also plays an important role as •NO in chondrocyte cell death [46].
Several methods have been developed to detect cell death. However, it is often not sufficient to distinguish between apoptosis and necrosis by only applying a single assay. Therefore, combination of two to three methods is necessary to measure the morphological changes as well as the intracellular cell death parameters. Table 1 summarizes some methods for the characterization of cell death [136].
Table 2 Some methods for the characterization of cell death [136].
Cell Death Parameter Method Specific
Detection of Apoptosis Loss of cell attachment Staining of cells with crystal violet or
fluorescent DNA-binding dyes
－
Cellular ultrastructure Electron Microscopy ＋ Externalization of
phosphatidyl serine
Annexin V binding －
Release of cytosolic compounds
51Cr release, 3H-labeled proteins, enzymatic activities in culture supernatants
－
Uptake of dyes Vital dyes (counting of cells) or fluorescent dyes for FACS
－
DNA laddering Agarose gel electrophoresis ＋
In situ DNA cleavage TUNEL －
Nuclear condensation and fragmentation
DAPI ＋
DNA degradation DNA content in sub G1 cells (FACS), agarose gel electrophoresis, DNA fragmentation ELISA
－
Internucleosomal DNA fragmentation
DNA fragmentation ELISA, agarose gel electrophoresis
Aggregation, uptake, sequestration of fluorescent dyes
－
Caspase activity Conversion of fluorogenic substrates ＋ Caspase processing Western blot, immunhistochemistry ＋ Cleavage of caspase
substrates
Western blot, immunhistochemistry ＋