5. Discussion
5.5. Apoptosis in the development of OA and the role of NO in this process …
Adult articular cartilage is considered as a postmitotic tissue with resident cells enclosed in their lacunae, however several studies have shown that there is a low proliferative activity in OA chondrocytes (Meachim and Collins, 1962; Rothwell and Bentley, 1973;
Mankin et al., 1971) leading to chondrocyte clustering, a characteristic feature of OA cartilage (Sandell and Aigner, 2001). On the other hand lacunar emptying has been shown in OA cartilage (Mitrovic et al., 1983; Bullough, 1997) and it was suggested that chondrocyte death is a main reason of OA cartilage degeneration (Bullough, 1992;
Vignon et al., 1976; Meachim et al., 1965) and that chondrocytes die by apoptosis (Blanco et al., 1998; Hashimoto et al., 1998; Kim et al., 2000; Kouri et al., 2000).
Authors suggest that cell death in cartilage samples range from 22 to 51% of cells in OA and from 5 to 11% in normal cartilage. However as remarked by Aigner these numbers are overestimated, because if they were correct even normal cartilage would loose soon the capacity to undergo biosynthesis and other biochemical parameters measured in cartilage would be impossible to assess. Indeed, Aigner observed apoptotic cell death in OA cartilage but at a very low rate with approximately 0,1% of the total cell population (Sandell and Aigner, 2001; (Aigner and Kim, 2002). A significant but lesser then in OA increase in the empty lacunae was also observed with age in normal cartilage (Aigner et al., 2001).
We made similar observations as TUNEL assays performed on paraffin sections of OA cartilage revealed almost no apoptotic cells in this tissue.
It has been also shown that cultured chondrocytes are highly resistant to apoptosis in comparison to other cells (Ogawa et al., 2003) probably because of the powerful free radical scavenging system (Toda et al., 2002).
On the other hand it has been suggested that NO triggers chondrocyte apoptosis in OA (Blanco et al., 1995; Kim et al., 2003; Relic et al., 2002; Yoon et al., 2003).
Therefore we investigated the effect of NO donors and endogenously generated NO after IL-1 and TNF stimulation on cell death in these cells.
In our experimental settings treatment with IL-1β or TNFα did not increase the rate of chondrocyte apoptosis. NO-donors: DETA NONOate or Spermine NONOate induced apoptosis but only at very high concentrations (from 0,5mM). The peroxynitrite-generating SIN-1 was a more potent inducer of apoptosis. Already 250µM SIN-1 decreased cellular viability to only 30 % of control. This result was not due to the amount of NO generated by particular donors, as diazeniumdiolates are more potent NO generators than SIN-1.
In contrast to several publications these results indicate that endogenous or exogenous NO is not cytotoxic to chondrocytes, however peroxynitrite at high doses can induce chondrocyte apoptosis.
The mechanism of chondrocyte apoptosis mediated by peroxynitrite is not very well elucidated. However mitochondrial dysfunction and energy depletion through ONOO -was suggested in cell death of chondrocyte-like ATDC5 cells (Yasuhara et al., 2005).
Furthermore Whiteman et al. could show in human articular chondrocytes that peroxynitrite mediates a calcium-dependent mitochondrial dysfunction that leads to caspase-independent apoptosis mediated by calpains (Whiteman et al., 2004). Results obtained by Mistry et al. in murine OA model confirm that caspases are unlikely to be involved in apoptosis pathway in chondrocytes (Mistry et al., 2004).
In many previous studies iNOS expression or NO production was correlated with the level of apoptosis in the tissue. If NO would be an initial signal for apoptosis than we should also observe apoptosis in all cells that were iNOS positive after IL-1 stimulation.
However we have not observed increased levels of apoptosis after IL-1β or TNFα stimulation. This is consistent with recently published data (Kuhn et al., 2000; Kim and Song, 2002).
There is also a possibility that a lack of chondrocyte apoptosis after IL-1 or TNF stimulation, despite NO production, is due to an anti-apoptotic mechanism induced by
these cytokines. Treatment with IL-1 was previously suggested to protect chondrocytes from apoptosis by a mechanism that involves tyrosine phosphorylation events and NFκB-dependent gene activation (Kuhn et al., 2000).
Greisberg performed double staining of the same OA cartilage sections for apoptosis (TUNEL) and nitrotyrosine and there was no difference in the nitrotyrosine staining between in apoptotic and not apoptotic cells. He concluded also that it is improbable that all the cells, which were nitrotyrosine positive, were about to undergo apoptosis (Greisberg et al., 2002).
Recently nitrite was even found to exert a protective effect upon hypochlorous acid-induced chondrocyte toxicity (Whiteman et al., 2003).
The discrepancy between results from different studies demonstrating chondrocyte apoptosis after incubation with NO donors may be attributed to the use of chemical NO donors, which generate not only NO but additional toxic agents e.g. SNP (sodium nitroprusside). Primary byproducts of the decomposition of SNP, such as cyanide anion or pentacyanoferrate complex, might contribute to its cytotoxicity (Kim et al., 2005).
In summary our results indicate that NO is not cytotoxic to chondrocytes even in high concentrations. Therefore a revision of the opinion on the role NO plays in chondrocyte apoptosis is necessary.
We think that the effect of apoptosis on the pathology of OA is limited as rapid loss of the cells (in rates indicated by many authors) would lead to a complete degradation of the whole cartilage in some months or even weeks. It has been also shown that age predisposes articular cartilage to apoptosis and its possible that such changes are a prelude to the age-related development of OA (Todd Allen et al., 2004).
5.6. NO, O2-
andONOO-
The reactivity of NO per se has been greatly overestimated in vitro. In fact NO in cellular environment is relatively stable and persist in solutions for several minutes in micromolar concentrations. The rapid diffusion of NO between cells allows to locally
integrate the responses of tissues e.g. blood vessels or neuronal networks (Beckman and Koppenol, 1996).
NO reacts with oxygen to form much stronger oxidants like nitrogen dioxide. ●NO reacts also with ●O2- in a very fast, only diffusion limited reaction. Astoundingly both molecules
●O2- and ●NO are not very reactive towards biological macromolecules, however ONOO -is a very potent oxidant. Therefore the availability of O2 and its reduced state regulate the actions of NO. In contrast to NO and ONOO-, ●O2- cannot easily pass biological membranes therefore compartment-specific actions of this molecule can be supposed.
Concentrations of NO and O2 are decisive for the final product of the reaction of both molecules. Equal rates of ●NO and ●O2- generation result in the formation of ONOO-, however excess of ●NO can lower the ONOO- levels because than production of NO2 is favored. NO2 can then react further to produce the nitrosating agent N2O3.
Finally the acidic form of peroxynitrite ONOOH can decompose to the highly reactive oxidants ●NO2 and ●OH.
The oxidative stress induced alterations in physiological responses are discussed as important factors in the aging processes and in the development of several diseases.
Production of oxygen radicals have been shown in articular chondrocytes and ROS were implicated in the regulation of redox sensitive pathways in chondrocytes (Rathakrishnan and Tiku, 1993; Hiran et al., 1998; Rathakrishnan et al., 1992; Tiku et al., 1990). Furthermore ROS production has been found to increase in OA (Henrotin et al., 2003).
There are several sources of ●O2- in the cell, as NADPH oxidases, xanthine oxidase and uncoupled NOSes but also mitochondria release this radical during the course of aerobic respiration.
The presence of NADPH oxidases was demonstrated in chondrocytes. Moulton et al.
detected various components of the NADPH oxidase complex in an immortalized human chondrocyte line: p22-phox, p40-phox, p47-phox and p67-phox were present at mRNA level. Western blot analysis showed the presence of p47-phox and p67-phox polypeptide components. However no significant superoxide generation was seen using
cytochrome c assay if the cells were stimulated with IL-1β, IL-4, TNFα. Stimulation with ionomycin or PMA enhanced the rate of superoxide generation by only 24 or 31%
respectively (Moulton et al., 1997).
Hiran et.al. detected p67-phox in porcine chondrocytes (Hiran et al., 1997). Further expression of gp91-phox mRNAin the immortalized chondrogenic cell line C-20/A4, as well as in chondrocytes derived from a patient undergoing joint-replacement therapy was shown (Moulton et al., 1998).
We found NOX2 mRNA expression in cartilage tissue samples (both normal and OA) and in chondrocytes in alginate beads, however no expression of NOX2 was detected in hMSC pellets (Affymetrix, data not shown). NOX2 is a phagocytic NADPH-oxidase, a membrane-bound enzyme complex that generates large quantities of superoxide and microbicidal oxidants upon activation.
We found also mRNA expression of some gp91-phox homologs in human chondrocytes. NOX4 (renal NADPH oxidase), which has been shown to generate ●O2
-constitutively (Maturana et al., 2002); expression was found in cartilage tissue samples (normal and OA), in all hMSC pellets and in chondrocytes cultivated in the half of investigated alginate beads samples.
NOX5, which produces ●O2- in a Ca2+-dependent manner (Banfi et al., 2001) was expressed mainly in chondrocytes in culture (beads and hMSC pellets) and was seldom detected in the tissue samples.
Another source of ●O2- in chondrocytes, which was discussed in the literature, is the mitochondrium. However chondrocytes are mostly anaerobic working cells.
Articular cartilage is an avascular and low oxygen environment therefore chondrocytes are highly glycolytic, as also confirmed by the prominence of lactate dehydrogenase (LDH) in chondrocytes (Tushan et al., 1969). Interestingly the basal respiratory rate of chondrocytes in culture is very low e.g. less than 10% of that in cultured fibroblasts or hepatocytes (Stefanovic-Racic et al., 1995; Johnson et al., 2000). Mitochondrial respiration accounts for only up to 25% of total in situ ATP production in articular chondrocytes and possibly more under conditions of increased energy demands
associated with tissue stress (Terkeltaub et al., 2002). Therefore it has been suggested that although in healthy cartilage mitochondria probably do not play an important role in energy generation, in conditions of biomechanical or inflammatory stress increased supply of ATP is required for an adaptation and e.g. matrix synthesis. In such conditions mitochondrial impairment and insufficient energy production would contribute to pathogenesis of cartilage. Indeed alterations in mitochondrial function were implicated in the development of OA (Terkeltaub et al., 2002).
At the beginning of OA an increase in a number and size of mitochondria was observed what would confirm the role of aerobic respiration in matrix synthesis as at the beginning of OA increased anabolic activity of chondrocytes is observed. In addition at the end stages of OA the number of mitochondria in chondrocytes decreases (Weiss and Mirow, 1972; Weiss, 1973). Mitochondrial dysfunction would be a parallel between OA and other age-related diseases. However it is still not clear if oxidative damage is a primary or secondary event in pathogenesis of age-related diseases. Due to the glycolytic activity of chondrocytes in normal cartilage the secondary role of superoxide production due to mitochondrial dysfunction in the development of OA is more probable.
NOS enzymes were shown to generate ●O2- under particular conditions.
In case of reduced availability of L-arginine or under oxidative conditions accompanied by decreased levels of the NOS cofactor BH4 uncoupling can take place and the enzyme instead of NO releases ●O2- (Stuehr et al., 2001). Kuzkaya reported that BH4 is a target for oxidation by peroxynitrite, and this oxidation is 6-10 times faster than reaction of ONOO- with ascorbate or thiols (Kuzkaya et al., 2003). Especially under these conditions inhibition of uncoupled iNOS would be a rationale to prevent the generation of ROS. However to our knowledge till now there are no reports on the uncoupling of iNOS in chondrocytes.
We could not assess the source of ●O2- in chondrocytes as in our experiments levels of
●O2- generated in induced chondrocytes were very low and direct measurements were almost impossible. Measurement of ●O2- in the cell generating NO is very difficult because the reaction of ●O2- with NO is much faster than with e.g. cytochrome c. The rate constants for the reaction of O2- with NO and cytochrome c are 6.7x109 M-1s-1 and
1.1x106 M-1s-1 respectively. Additionally reduced cytochrome c can be reoxidized by peroxynitrite, further diminishing its effectiveness in measuring superoxide (Thomson et al., 1995) This could be an explanation for our results where we measured slightly higher levels of superoxide in control cells probably due to trapping of ●O2- by NO in stimulated cells. However levels of ●O2- measured in cells stimulated in the presence of iNOS inhibitors were also not higher (data not shown). This can be explained by the fact that even very small concentrations of NO are sufficient to quench superoxide, and iNOS inhibition was not 100%. Another explanation could be also a higher anti-oxidative potential of stimulated cells as we detected expression of several genes of proteins involved in radical scavenging as SOD and metallothioneins after IL-1 stimulation (Affymetrix analysis). Interestingly MnSOD was one of the highest up-regulated genes after IL-1 stimulation in chondrogenic pellets. MnSOD upregulation after cytokine stimulation was already reported in OA chondrocytes (Mazzetti et al., 2001).
Additionally it has been also demonstrated in other cell types that cytokines besides inducing radical production can in parallel modulate the expression and activity of radical scavengers (Flanders et al., 1997; Niwa et al., 1996). In particular IL-1 and TNFα are able to induce SOD expression (Sugino et al., 1998; Tannahill et al., 1997).
Except for SOD human chondrocytes constitutively express catalase, glutathione peroxidase (GPX) and peroxiredoxins (Henrotin et al., 2005; Chae et al., 1999; Knoops et al., 1999).
Different cell types differ in their antioxidative capacity and sensitivity to oxidative and nitrosative stress. We think that chondrocytes are very robust cells that have a high antioxidative potential, similar to e.g. smooth muscle cells as shown by Schildknecht (Schildknecht et al., 2005). This antioxidant potential could be due to the presence of several radical scavengers, which expression was very prominent in chondrocytes and also in differentiating stem cells and to high activities of GSH-reductase, thioredoxin or glutoredoxin.
5.7. Protein tyrosine nitration
Nitrotyrosine formation has been shown in numerous tissues under pathological conditions. However the opinion about tyrosine nitration changes during the last years.
Recently the group of Stuehr postulated that this protein modification is observed under normal conditions in all tissues and is a reversible process (Koeck et al., 2004; Aulak et al., 2004). It could represent a novel mechanism of regulation of tyrosine kinase signaling. Initially it was suggested that nitration of tyrosine residues in tyrosine kinase substrates may prevent phosphorylation and therefore inhibit tyrosine kinase signaling (Kong et al., 1996). Recently it has been reported that peroxynitrite promotes the nitration and/or phosphorylation of regulatory sites at tyrosine kinase receptors coupled to well-known antiapoptotic pathways, such as those involving phosphoinositide 3-kinase/Akt or mitogen-activated protein kinases (Bolanos et al., 2004a), what would implicate a regulatory role of tyrosine nitration.
Still the mechanisms, regulation and role protein tyrosine nitration plays in biological systems are controversial.
Nitrotyrosine was present in human OA cartilage samples as demonstrated by immunohistochemical staining and western blot analysis. A detailed analysis revealed that a number of proteins were nitrated in chondrocytes. Interestingly although experiments were performed several times we obtained very reproducible results.
As nitrated following proteins were found in human chondrocytes:
annexin A2, actin, vimentin, MnSOD, and enzymes of the glycolytic pathway: alpha enolase, pyruvate kinase M1/M2, fructose-bisphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase.
Reproducibility of results indicates that tyrosine nitration could be a strikingly controlled and selective process.
Some proteins are more susceptible to tyrosine nitration because of:
• their structure and nature as e.g. formation of tyrosyl radical during the course of catalysis (as PCS and ribonucleotide reductase)
• distance to the source of nitrating agent.
It is also possible that with present methods detection and identification of only very abundant nitrated proteins becomes possible and with more sensitive tools also other proteins would be detected. From the nitrations observed so far one might speculate on metabolic changes occurring upon chondrocyte differentiation.
Annexin II is a component of plasma membrane vesicles and is involved in regulation of membrane trafficking events (Liemann and Lewit-Bentley, 1995). In chondrocytes annexin II has been shown to play a role in the mineralization process of cartilage (Kirsch et al., 2000b) and is therefore considered as a marker of terminally differentiated chondrocytes. It has been shown that OA chondrocytes express annexin II and undergo terminal differentiation leading to cartilage mineralization and destruction (Kirsch et al., 2000a). However this is the first report showing nitration of annexin II in OA chondrocytes. Previously annexin II nitration was shown in A549 cells, a lung epithelial cell line, treated with ONOO- (Rowan et al., 2002). Results of this study suggest that liposome aggregation was inhibited by nitration of annexin II in these cells. This could indicate that nitration of annexin II in human chondrocytes could have positive effect on cartilage structure due to inhibition of mineralization.
Actin and vimentin are members of the cytoskeletal system of filaments in nonmuscle cells. These microfilaments are involved in cell motility, organelle transport and cytokinesis. Although nitration of vimentin has never been detected previously there are some reports on actin nitration. In sickle cell disease nitration of actin tyrosine residues at positions that significantly modify actin assembly led to disorganisation of the actin fibers that altered cytoskeleton and caused cell death (Aslan et al., 2003). Peroxynitrite dependent increase in permeability of pulmonary microvessel endothelial monolayers was also associated with generation of nitrated actin and disorganisation of cell cytoskeleton (Neumann et al., 2005). Interestingly actin was markedly (>50%) carbonylated and nitrated in inflamed tissues of active IBD (Inflammatory Bowel Disease), and less in normal appearing tissues suggesting that oxidant induced cytoskeletal disruption is a part of the tissue injury (Keshavarzian et al., 2003).
MnSOD was found previously nitrated in human renal allografts and nitration probably inhibited MnSOD activity (MacMillan-Crow et al., 1996). Consistent with this hypothesis MnSOD is inactivated by peroxynitrite treatment in vitro (Yamakura et al., 1998).
Nitration of MnSOD responsible for scavenging of superoxide in the mitochondria, may cause mitochondrial dysfunction (Davies et al., 2001).
On the other hand MnSOD is a very abundant protein. Very high levels of SOD are necessary to provide a protection against superoxide since the formation of peroxynitrite in the reaction of superoxide with NO is much quicker than reaction of dismutation and therefore only much higher concentrations of SOD than NO can prevent formation of this reactive molecule. Aerobic cells generally contain enormous concentrations of SOD; it is the major fraction of cellular protein. The concentration of Cu,Zn SOD is a billion times greater than the concentration of superoxide itself (Beckman, 1999).
Interestingly there are several reports showing the prominent regulatory role of NO and O2- preferably in mitochondria. Peroxynitrite was reported to inhibit mitochondrial complexes I, II, IV and V, inhibit aconitases that catalyse the isomerization of citrate and creatinine kinase. Peroxynitrite was also shown to induce mitochondrial swelling, depolarization, calcium release, membrane damage and permeability transition (Yamakura et al., 1998; Terkeltaub et al., 2002). Additionally peroxynitrite can contribute to DNA damage and inappropriate transcription of mitochondrial proteins.
Therefore inhibition of MnSOD resulting in increased ·O2- generation in mitochondria, which can react with NO, easily passing mitochondrial membrane, leading to the formation of peroxynitrite can be critical forming a cycle contributing to further damage of mitochondria resulting in dysfunction and decrease in energy production.
In addition we found several enzymes of glycolytic pathway nitrated in human chondrocytes: alpha enolase, pyruvate kinase M1/M2, fructose-bisphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase. Nitration of enzymes belonging to glycolytic pathway could be due to their abundance in chondrocytes. On the other hand nitration could be also a mechanism of the regulation of energy metabolism in chondrocytes. Till now there were data indicating an effect of peroxynitrite on the
respiratory rate of mitochondria in chondrocytes. Our results indicate that also glycolytic pathway can be regulated by this oxidant.
A number of studies revealed nitration of enzymes involved in energy metabolism in other tissues. Aldolase A and glyceraldehyde-3-phosphate dehydrogenase (interestingly also annexin II) were nitrated in rat retina and this nitration was modulated by light (Miyagi et al., 2002). Glyceraldehyde-3-phosphate dehydrogenase was found nitrated in vivo in livers of LPS-treated rats (Aulak et al., 2001). Pyruvate kinase, aldolase A and glyceraldehyde-3-phosphate dehydrogenase were nitrated in aging skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well
A number of studies revealed nitration of enzymes involved in energy metabolism in other tissues. Aldolase A and glyceraldehyde-3-phosphate dehydrogenase (interestingly also annexin II) were nitrated in rat retina and this nitration was modulated by light (Miyagi et al., 2002). Glyceraldehyde-3-phosphate dehydrogenase was found nitrated in vivo in livers of LPS-treated rats (Aulak et al., 2001). Pyruvate kinase, aldolase A and glyceraldehyde-3-phosphate dehydrogenase were nitrated in aging skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well