"To flee or not to flee": Responses to persecution and the issue of relocation

Christof Sauer
"To flee or not to flee": Responses to persecution and the issue of relocation
C68103F45853322BA6D61F70B347EDA4RelocationpersecutionBelievers of Muslim Background
Religious persecution of believers contributes to migration and relocation.Three alternative responses to persecution or the threat of persecution can be discerned in Scripture: Avoiding, resisting, and enduring for those affected, and mandatory solidarity for the rest of the body of Christ.Advocacy and mission agencies are faced with the question whether relocation of persecuted believers is a viable option and provides a long-term holistic solution.The results of the discussion of a working group on the issues faced by Christian believers of Muslim background in the Middle East on the pros and cons of relocation within a country, within a region, outside a region are presented.

Introduction

Whether "to flee or not to flee" was a hotly debated topic in the Early Church.It pertained to the question whether bishops or elders were permitted to flee persecution as Jesus had warned of 'hirelings' who abandoned their flock to the wolves instead of laying down their lives for them .The church finally adopted the position that a bishop may not flee out of fear or cowardice, but could do so if it served the interests of his church.Such a decision could only be made under the Holy Spirit's leading in any specific situation (cf. Schirrmacher 2008:59f;Pettersen 1984).

In a broader sense the issue of "fleeing" or relocation is also relevant for Christians today.Two thirds of humanity live in countries with severely restricted religious freedom.This affects a large number of Christians (Pew 2011).Many are questioning if they should emigrate from the societies into which they were born -either because the circumstances deteriorated gradually or changed suddenly.This is but one among a multitude of reasons for migration.Some people are fleeing from armed conflict, or migrating because of political restructuring, natural disaster, or famine.Some are seeking a better lifestyle, employment, and improved economic prospects.Others are trying to escape poverty or overpopulated living-conditions.This equally applies to Christians, including those facing discrimination and persecution.There are varied reasons for migration.This article focuses on Christians who are confronted with acute religious persecution and have to consider whether relocation is a solution to their problem.It concentrates on new Christians who have left Islam, particularly in the Middle East and Asia, often referred to as "Believers of Muslim Background", abbreviated "BMB" (Brother Andrew & Janssen 2007).

They form a particularly vulnerable group among Christians in their societies, but they are not necessarily the only Christians facing social hostilities and repressions at the hands of the state.In most Muslim majority countries, even adherents of ancient Christian churches are suffering from their status as "dhimmi", being relegated to second-class citizens.There has been a constant exodus of Christians from the Middle East, e. g. from Egypt which is home to the largest number of Christians of all countries in the Middle East.The largest recent exodus of Christians has probably occurred from Irak.Another group are the churches that emanated from Christian missionary work in the 19th century after these countries had been Islamized, mainly in the 19th century.Often times, a newly established Christian tradition within a country has a weaker legal status and suffers a higher degree of oppression than older Christian traditions.

A third group are expatriate Christians.In extreme cases, like in Saudi Arabia, expatriate Christians found to gather for worship in private homes (for lack of churches) or in possession of a Bible are imprisoned and deported.Those who are caught sharing their faith with Muslims, which is their human right, and inviting them to share their experience might simply be deported -if they are fortunate enough to hail from a powerful country.However they may face the death penalty and actually be executed if they are among the mass of migrant workers from less powerful countries like the Philippines.Expatriate Christians are usually targeted only when found to be active in missionary work.

However, national converts from Islam are viewed as endangering the unity of the Islamic Ummah and bringing shame to the honour of their families.They are viewed by civil society as causing an offence, and thus viewed by state authorities as threatening the harmony of the community, possibly even national security (Schirrmacher 2012, Schirrmacher 2010, CSW 2008).

The following three case studies of believers of Muslim background, taken from Andrews (2011:36-38) are based on true incidents and illustrate the varying experiences with relocating.The identities of the persons involved or the places cannot be revealed for security reasons.

Single man evades assaults by studying abroad

N., a citizen of an Arab country, was assaulted by his neighbours.Even after relocating to another suburb he was traced and questioned by the security police.With the assistance of a Western colleague from work he moved to a Far Eastern country where, together with those supporting him he decided to apply for theological courses.While he failed to get a student visa for a European country, it was possible for him to study at a college in a second Far Eastern country.N. plans to return to his home country approximately nine months later.2

Family with two children forced to suddenly relocate

Farouk and his wife Ruth were compelled to leave their Arabic home country after his father sought custody of their two children in an Islamic court, which was likely to be granted.His father would undoubtedly have been granted immunity from prosecution in case of physically harming Farouk or Ruth.Prior to leaving, Farouk had already been assaulted by relatives of an in-law.The couple was well known to Christians in their home country, as well as to Western and fellow Arabic Christians in a Western country who arranged the initial entry-visa for them.Upon arrival in a Western country the family needed various facets of practical help as well as post-traumatic counselling due to the abrupt, total change of location and life style.Farouk was able to continue working as a translator with a foreign organization, which enabled him to be partly self-supporting.They decided to apply for asylum in order to be granted permanent residence.Using the asylum system has advantages and decided disadvantages.The Islamic court decision in their country to formally annul their marriage strengthened their case for acceptability as permanent residents in that Western country.Despite this very strong evidence, their application still remains pending after two and a half years.

Single man fleeing to the West

In the 1990s, due to intense pressure, a BMB decided to flee from an Arab country.A church in a European country offered to sponsor him, arranged a temporary visa and paid for his flight.Upon arrival they welcomed him, helped him apply for asylum and provided for all his physical needs.After six months he walked into the immigration office and said, "Everything in my asylum application is true.However, life is worse here than it was back home.What do I need to do to return?"He was on a plane within three days.What happened?The cultural adjustment was too great and the church was unable to provide effective long-term support.

Challenges for believers of Muslim background

The issues believers of Muslim background are facing have only briefly been alluded to in the above case studies.Their challenges emanate from various sources.BMBs are often cut off by their parents and extended families, which amounts to great shame in their cultures.Some converts are imprisoned by their families in order to pressurize them to revert to Islam or to conceal the shame brought on the family.Many are disinherited.Single girls face the fear of rape and forced marriage.The relatives of those with children may seek custody of the children on the grounds that the children are legally Muslims but not raised as such.

Society is frequently discriminating against believers of Muslim background and their children in the fields of education and employment.Converts are commonly regarded as Western spies, agents of Israel or Christian Zionists.

The legal systems of almost all predominantly Muslim countries (USCIRF 2012; Marshall 2011) do not permit converts from Islam to change their religious affiliation in their legal documents, forcing them to lead a double life.This leads to discrimination in several countries when such converts are applying for passports and other official documents.Annulment of marriage and the loss of custody of children to relatives are a very real possibility.Perpetrators of assault, abuse and discrimination against converts are often granted impunity.

When believers of Muslim background want to get married they face significant difficulties either legally or from family and society.However, the life style as a single is unusual in their societies and leads to social ostracism.

Psychologically, converts face a significant challenge in forming a sense of identity.The ostracism they almost inevitably face, may bring a sense of fear and uncertainty.They face a painful dilemma: They want to love and honour their family, but are considered as disloyal and traitors and made to feel that way.

The relocation of large numbers of refugees from distant cultures to Western countries, such as Irakis to Germany, or the almost automatic extraction of Somali BMBs to the USA has been questioned and criticized by various authors.In the first instance, it was claimed that the remaining church would be weakened, if leaving the country was an attractive option.But this argument failed to consider that Iraqis accepted in a resettlement programme by the EU and Germany, were refugees with no prospect of returning to their home country nor to remain in their country of refuge (Schirrmacher 2009).In the second instance, a reasonable argument was made, that no local Somali church would ever grow if new believers were extracted for fear of their martyrdom (Gupta 2005;Ripken 2008).

Taking the above into consideration, we need to evaluate the responses to persecution as reported in Scripture.Thereafter we will discuss relocation options and current experiences.

Christian responses to persecution

An international theological symposium recently dealt with Christian responses to imminent or acute persecution.The symposium statement of monographic length distinguishes three legitimate reactions to persecution by those persecuted, namely avoiding, enduring and resisting persecution (Bad Urach Statement 2010:81-86).For third parties a mandatory response, namely solidarity, is seen as the prerogative of Scripture. 3his assessment is based on the view that an essential part of the training Jesus provided his disciples with, was preparing them for persecution and their response to it (e.g.Matt 10:16-42).This preparation should enable them to face the natural human responses to persecution namely worry and fear, which lead to the dangers of denying Christ, loving one's family or one's own life more than Christ, and refusing to grant refuge to those who are being sought by the authorities because of their witness (Matt 10:33-42).

Jesus' teaching to be as harmless as doves, as shrewd as serpents, being sent out "as sheep among wolves" is considered to be a reality until today (Matt 10:16-18).In this view, therefore, the key question to be considered is not how persecution can be avoided at all cost, but "how the mission of proclaiming the Gospel which Christ gave his church can be faithfully accomplished."

Within this framework several mandatory responses to persecution are seen as commanded by Scripture, namely staying faithful to Christ when enduring persecution, showing solidarity with the persecuted and praying for the persecutors.Two additional responses are considered permissible or called for under certain circumstances, namely avoiding or fleeing persecution and resisting persecution.

The main focus of the discussion concentrates on the tension between avoiding persecution and enduring persecution, and how resisting persecution may relate to them.Grounds and examples for all these actions are sought and found in Scripture and the challenge is seen in discerning which response or which combination of responses is appropriate in each individual instance according to the leading of the Holy Spirit.

Avoiding persecution

In situations where the church is exposed to a stronger degree of persecution, it is considered legitimate to wisely ask whether and how persecution could be avoided without compromising the witness to Christ.Following the way of the cross is not considered as "seeking persecution", nor the call to endure persecution as a weak apathetic or passive acceptance of the event.Occasions are recognized in which God directed the believers to avoid or flee.The motive for fleeing is considered critical.Fleeing in order to avoid suffering is not considered a sufficient motive.The mission of the kingdom of God above all else should be the main priority.Therefore, if the mission were threatened by persecution, withdrawal can be taken into consideration, based on scriptural precedents.

God himself instructed the prophet Elijah to avoid the persecution by King Ahab by hiding (1.Kgs 19:1-18).He equally instructed Joseph to flee to Egypt with the new-born Jesus (Matt 2:13-18).Jesus himself at times went into hiding (Matt 4:12, John 8:59; Matt 12:14-15; John 7:1) "because his time had not yet come" (John 7:30; 8:20,59; 10:39).His escape from suffering and death was, however, only a postponement.Nevertheless, Jesus did not pull away from confrontation with the religious leaders of his day.His ministry was not characterized by 'tactical moves', compromise, a 'watering down' of his message or avoidance of suffering.

When Jesus sent out his disciples on a training mission, and his instructions expanded to events in the future, he told them when they were persecuted in one town to withdraw to the next in order that the gospel may continue to spread (Matt 10:23).The Jerusalem church in fact later implemented such withdrawal (Acts 8:1).This is also a pattern in the missionary work of Paul (Acts 8:1, 9:25, 11:19, 14:5-6).The flight was not, therefore, a flight from suffering, but a flight in order to fulfill the mission of Christ.While God's word can go out forcefully through the testimony of martyrdom, it is sometimes better that people remain alive in order to proclaim it (Acts 14:5-6).

In a few exceptional situations, believers did go to meet certain death.Jesus and Paul both returned to Jerusalem to be arrested (Acts 10:19-25).These were, however, key situations and key people in salvation history and they were commanded directly by God to do so (e.g.Acts 20:22-23)."(Bad Urach Statement 2010:82f).This is considered to suggest that at times the church is meant to implement certain strategies to avoid persecution.Through wise, temporary 'retreat' churches in some contexts may be able to resume their missionary witness at a later and quieter time.Equally, Christian converts are advised not to seek confrontation in their families, but to witness to their new found faith in patience and love.However, they should beware of the dangers of avoiding persecution to the degree of adapting to the ideology of a hostile system or of denying the Christian faith.

There are also patterns of behaviour recognized in the history of persecution that have led to emigration merely out of fear and for the purpose of finding a more peaceful and prosperous environment.What these emigrants did not realize in the opinion of the authors, however, is that even if they were able to leave their homeland successfully they would never fully escape persecution and hardship.Even more, the churches they left behind were left with an even greater burden of existing and maintaining a voice as an ever-increasing minority.Equally the phenomenon of 'inner emigration', where individuals isolated themselves from the surrounding society, and often develop a ghetto mentality and a lifestyle that is marked by a high degree of legalism and insulation, is considered as preventing them from having a positive influence on their society or a meaningful witness for Christ among their contemporaries (cf.Kuzmic 1996:65f andTieszen 2008:71).

In summary, flight or avoidance of persecution is advised and considered permitted under certain circumstances, but considered forbidden where obedience to God's commandments and Christ's commission and love for others would be jeopardized.The avoidance of distress and pain is not accepted as the supreme good, while obedience is, regardless of the cost: "When persecution arises, careful consideration must be given to determine whether or not remaining in a situation of suffering is necessary in order to accomplish the will of God." (Bad Urach Statement 2010:84).

Enduring persecution

It is realized that avoiding persecution or fleeing may at times be impossible, impractical or inappropriate.

"In these cases God's people are called to stand firm where they are and remain faithful, even unto death.Enduring persecution as an expected event for the greater purposes of God is in fact the most broadly attested biblical directive regarding a response to persecution."(Bad Urach Statement 2010:84).

Perseverance is expounded as a crucial virtue.Multiple biblical characters are portrayed as examples to follow, such as Shadrach, Meshach, and Abdnego who refused to bow before King Nebuchadnezzar's golden image, fully aware of the consequences (Dan 3:8-30).The Apostle Paul is seen to have several times demonstrated his readiness to endure the consequences of his ministry, even unto death (Phil 1:20-26; 2:17), and to have been steadfast in going to Jerusalem even if it meant persecution (Acts 21:10-14).

Christian spirituality, in the view of the authors is not defined by the fact in itself that Christians experience persecution, but rather in the way in which they endure it and choose to respond to it.Their credibility is seen in their solidarity towards each other and their ethics that demonstrate love that includes neighbour and enemy and which foregoes all violence.This is considered a stark contrast to the antagonistic context in which they are living.

Resisting persecution

Bringing together the call for all Christians to endure persecution and the option of avoiding it under certain circumstances, a third response to persecution is seen as an option, which may be a part of these responses.This is the possibility that God may call Christians to resist persecution as well.

"There are times when it is appropriate to fight for one's legal rights.Jesus defended himself at one point during his trial (John 18:23), not to protest his suffering but as a testimony to his innocence, while remaining silent throughout the rest of his trial.Paul demonstrated resistance through his appellation to Roman law (22)(23)(24)(25)(26)(27)(28)(29).With his knowledge of the law and his own rights as a citizen of the Roman Empire, Paul was able to avoid persecution by resisting it.However, it is important to note that both Jesus and Paul exercised this choice under specific premises and for specific purposes.Like fleeing, resisting is permissible unless it hinders the furtherance of the kingdom of God." (Bad Urach Statement 2010:85).

Christians are considered to have a right to be angry about situations of persecution with a holy indignation and a righteous outrage which leads to attempts to transform such conditions.Such transformation could occur through legislative change, which is regarded as potentially being expedited through civil disobedience, publications, political lobbying, or public demonstrations.However, a standard is raised to which resistance must submit: "Christian resistance to religious persecution must not distort, diminish or contradict the gospel, God's purposes in persecution, Christ's mission in the world or the Holy Spirit's leading to respond to persecution in another manner."

The authors conclude: "We are not called at all times to be subjected to ungodly treatment, nor are we at all times to shirk such treatment.Ultimately, God himself will defend his church, but in the same way, there are times when God will lead his people to rise up in holy indignation and resist efforts to squelch his people."(Bad Urach Statement 2010:85).

Solidarity with the persecuted

While the other three responses to persecution are seen as necessitating the seeking of God's direction as to the appropriate response to persecution, solidarity is considered an obvious response:

"Christians can only ask God how they should help their persecuted brothers and sisters, not if.Even Christians whose persecution is intensely hostile can show solidarity with the experience of others through prayer.However, it remains a primary responsibility of those whose experience of persecution is presently mild to stand for and with those whose experience is intensely hostile."(Bad Urach Statement 2010:85f).

Praying for the persecutors and the authorities is regarded as an additional element that should unite all responses discussed above.

Relocation options and current experiences

Current reflection of practitioners of advocacy for persecuted Christians distinguishes different types of relocation according to its durationtemporary or permanent -and according to its distance from the original location, both in terms of physical distance and cultural distance.I am employing the parameter of distance to structure the discussion on relocation, namely relocation within a country, within a region and outside that region.4

Relocation within a country

If relocation within one's own country is to be viable, there needs to be sufficient distance between the original location and the destination to ensure appropriate anonymity.There should also be a community of believers which the person forced to relocate can join as his or her "new family".A typical scenario might involve moving from a rural village setting to a large town or city.In some countries Christians provide safe houses as a place of refuge for others.However in small countries such as some Gulf States relocation within-country is rarely a viable option for lack of distance and anonymity.Generally speaking, relocation within the same country is rarely an option for single female believers of Muslim background due to the strong cultural expectation that girls remain at home until they marry.Being away from home brings suspicion and the presumption of shame on the individual and her family.

In cases where state authorities are pursuing a Believer of Muslim Background, legal exit from the country is rarely an option as the Christian would face apprehension at the point of departure.In-country relocation is then the only legal option.However, in the majority of cases the main perpetrators of persecution are family members and the state's role is omission of due protection to the convert, condoning the actions of family members and the maintenance of discriminatory legal and administrative systems.

Relocation within the region

Relocation outside of the home country but staying within the region is frequently possible.This may be relatively easy to achieve, at least in the short term, as visas may be readily available or not required.There can be clear advantages regarding language and culture, those relocating can often blend in easily, and in some countries in the region there are churches speaking the same language.Even where Arab governments oppose their own nationals attending churches, some tolerate Arabs of other nationalities, such as migrant workers and students attending Arabic churches.The same applies for Farsi language groups in several Gulf States.

Relocation outside the region

When it comes to relocation outside the Middle East and other Muslim majority countries, it seems appropriate to distinguish between relocation to the Western world and other areas of the world.

Relocation to the West

Relocation to the West is getting harder due to tighter visa regulations motivated by several factors, including the heightened political sensitivity of immigration and asylum (cf. Burkhardt & Schirrmacher 2009).

Those seeking long-term relocation need to secure long-term residency.The ideal means of securing long-term residency is by obtaining a work permit or student visa.However, if a convert has been denied qualification certificates or they have been withheld or if there has been adverse discrimination in education and employment, such a person can then face difficulties in proving suitability for jobs or study courses.

Bible schools are sometimes willing to sponsor believers of Muslim background to engage in theological study.This arrangement can be expected to provide valuable insights for fellow students, especially any preparing to work in Muslim majority areas.

However the majority of converts seeking relocation to the West actually lack the skills and qualifications necessary to obtain work or study visas.One route for them is to obtain a short-term visa and to then apply for asylum upon arrival in the Western country.This can be problematic as sponsorship is required for short-term visa applicants, and sponsorship can be sensitive.Many Christian ministries will not sponsor those they know are planning to apply for asylum, since the authorities are likely to refuse subsequent applications by these ministries to sponsor visas for others.

Many from the Middle East find it difficult to settle in the West.Language and cultural differences are often compounded by a marked gap between expectation and reality -the streets of the West are not paved with gold.Many will find Western morality shocking, leading to disillusionment or even to succumbing to new temptations.Some Western church cultures can also be very different from expectations, and many Christian communities can struggle to truly understand and welcome believers of Muslim background.Some Arabic language churches in the West, for example, struggle to accept converts from Islam.

Relocation to the Non-West

The option of relocation to a non-Western country outside the home region of a believer of Muslim background has several attractions.Many Western Christians perceive that the difficulties for such persons would be less pronounced in non-Western countries than in the Western world.For example, practical difficulties in securing visas may be less prohibitive, and financial support costs may be lower.The scope for, and merits of, non-Western relocation needs further exploration.Some believers of Muslim background have also come to South Africa, but not all have stayed.

When it comes to theological studies, some believers of Muslim background actively recommend their fellow believers undertake their initial theological study in non-Western countries rather than in the West because there they will find a context closer to their own culture.I would also assume that the prospects of returning to one's home country are better if the alienation from one's inherited culture is less intense.

Bringing theology and praxis together

From a theological perspective it has become clear that there is a range of legitimate responses to persecution.Fleeing or avoiding persecution is one of them as long as the mission to which Christ has called his church is not abandoned and the Gospel is not betrayed.However, the response of avoiding persecution is not the only one.There may be situations where resistance to persecution or enduring persecution are called for under the guidance of the Holy Spirit.This assessment, primarily in view of those affected by persecution, should also be kept in mind by those who follow the imperative of assisting the persecuted in Christian solidarity.

When moving from foundational theological and ethical guidelines to current reality of believers from Muslim background in the specific context of the Middle East, practitioner experience leads to further differentiation as to the best practice of Christian advocacy agencies and receiving churches.It appears that, whenever possible, assistance to persecuted believers of Muslim background should be provided within their respective situations.In addition, it should be a given that in all actions to support persecuted believers, their informed choices regarding their own response to persecution should be respected.This includes seeking and respecting the advice of those close to the victims of persecution and their situations.

The following guidelines and insights emerge from the collective experience and wisdom of those assisting the persecuted:5 Relocation outside of one's own country is rarely easy and has long-term consequences and involves potential struggles.Relocation should be considered only when the alternatives of standing up for one's rights and making a conscious decision to accept persecution are either not available or clearly not desirable.There are circumstances when relocation is the only viable option.In some cases, those forced to relocate are able to return after a short period away.The ideal mechanisms for relocation are to find work or enrol in a formal study course or vocational training program.Refugee and asylum systems should be regarded as options of last resort.

So ultimately, practical reality and theological options are much more complex than the question "to flee or not to flee".For persecuted Christians and those assisting them the guiding criterion is to fulfill the mission of Christ.
 Dr Christof Sauer (*1963) is Associate Professor Extraordinary at the Department of Practical Theology and Missiology, Theological Faculty, Stellenbosch University. He is also Co-Director of the International Institute for Religious Freedom (Bonn, Cape Town, Colombo) <www.iirf.eu>. Contact: PO Box 535, Edgemead 7407, South Africa, firstname.lastname@example.com.
In another similar case, the relocation to a foreign country was no longer necessary because a return to the original home became possible because neighbours became tolerant of the conversion.
This is following Tieszen's terminology(Tieszen 2008:67-78). Also cf. Glenn Penner's earlier triad of flight, fortitude and fight(Penner 2004:132f).
The material presented in this section emanates from a working group of the "Religious Liberty Partnership" that studied the issue of relocation of Believers of Muslim Background.
See the reference above to the working group of the RLP. For an extensive treatment of the practical aspects cf. Andrews 2009.

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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

María C De Andrés
Emilia Maneiro
Miguel A Martín
Joaquín Arenas
Francisco J Blanco
Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism
R E S E A R C H A R T I C L E Open Access
Introduction: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro.There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. Methods: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[ 3 H]glucose (2-[ 3 H]DG). Statistical analysis was performed using the Mann-Whitney U test. Results: NOC-12 liberates approximately ten times more NO 2 than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[ 3 H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[ 3 H]DG than healthy cells. Conclusions: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.

Introduction

Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source and as a precursor for glycosaminoglycan synthesis and a regulator of gene expression. Degradation of articular cartilage is a hallmark of osteoarthritis (OA) [1] and is associated with aberrant glucose metabolism [2,3]. On the other hand, the pathogenesis of OA is characterized by the production of high amounts of nitric oxide (NO), consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines, such as IL-1β and TNFα, and other factors [4][5][6][7].

Although it has been reported that NO causes chondrocyte apoptosis [8][9][10], production of high levels of endogenous NO by over-expression of the iNOS gene in transfected chondrocytes has not been found to cause cell death [11]. Other reports have proposed NO to be a physiologic regulator of mitochondrial respiration in chondrocytes [10,12,13]. A variety of NO donors have been demonstrated to suppress energy production by mitochondrial respiration in different cell types [14,15], an effect enhanced at low oxygen tensions [16], and firstly reported in chondrocytes by Johnston and collaborators [12].

Chondrocytes are highly glycolytic resident cells of articular cartilage that metabolize glucose as a primary substrate for ATP production [17]. However, oxygen does diffuse into articular cartilage and articular chondrocytes possess mitochondria and respire in vivo [18]. The superficial and middle zones of articular cartilage are not anoxic [19], and in this context, mitochondrial oxidative phosphorylation (OXPHOS) is 18 times as efficient in ATP generation as is glycolysis [14,20]. Furthermore, OXPHOS may account for up to one fourth of total steady-state ATP production within articular cartilage, and possibly more under conditions of increased energy demands associated with cartilage stress [21]. Besides this, mitochondria are important in regulating both caspase-dependent and caspase-independent apoptotic pathways [22][23][24][25][26].

It is generally accepted that the quantities of available oxygen and glucose can fluctuate considerably in connective tissues such as articular cartilage, growth plates and the intervertebral disc [27][28][29]. Articular chondrocytes consume less oxygen in comparison with most other cell types [30]. Consequently, anaerobic glycolysis forms the principal source of cellular ATP in cartilage.

The direct investigation of the function of exogenous NO production on articular chondrocytes has been hampered by the lack of uniformity between the different types of NO donor compounds [31]. Since the past decade, the diazeniumdiolates began to replace traditional donors, such as SIN-1 (3-morpholinosydnonimine), SNP (sodium nitroprusside), SNAP (S-nitroso-N-acetylpenicillamine) and S-nitrosogluthathione, as sources of exogenous NO production [32], because have been shown to be reliable sources of NO under a variety of culture conditions [33,34]. The main advantages of these compounds are: known rates of NO generation, NO generation rates covering a wide range, spontaneity of NO generation and tenable generation of NO redox forms [34].

For all these reasons, besides the classical donor SNP, we used a diazeniumdiolate, NOC-12 (N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine), with a half-life of 327 minutes (determined at 22°C, pH 7.4) [33], for exogenous NO production, to further investigate the conditions in which NO is cytotoxic to chondrocytes, and compare the different effects induced by the two different types of NO donors. We wished to determine whether NO modulates the pathogenesis of OA, inducing apoptosis by means of the inhibition of mitochondrial function

Materials and methods

Cartilage acquisition and cell isolation

Normal human cartilage from femoral heads (joint replacement surgery) and knees (joint replacement surgery and autopsies) was obtained from 11 adult donors without history of joint disease and who had macroscopically normal cartilage (mean age ± SD 46.5 ± 10.5 years); human OA cartilage was obtained from the femoral heads of 12 patients (age 70 ± 12.6 years). All patients and healthy donors have signed the informed consent and the project was approved by the Regional Ethical Committee from Galicia (Spain). Small cartilage fragments were digested as previously described [35].

Primary culture of chondrocytes

Chondrocytes were recovered and plated at high density (4 × 10 6 per 162 cm 2 flask; Costar, Cambridge, MA, USA) in DMEM (Life Technologies, Paisley, UK) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 1% glutamine, and 10% FCS (Life Technologies). Chondrocytes were incubated at 37°C in a humidified gas mixture containing 5% CO 2 balanced with air. Chondrocytes were used at weeks 2 to 3 at confluence in primary culture. Cell viability was assessed by trypan blue dye exclusion, and stained cells were discarded to carry out experiments.

General procedure and NO donor compounds employed NO donor compounds were added from 60 mM stock solution dissolved in 0.1 M NaOH (NOC-12) and 10 mM stock solution dissolved in medium (SNP). This NO donor compound was freshly prepared before each experiment but NOC-12 was stored as 60 mM stock solutions in 0.1 mM NaOH at -20°C. Chondrocytes were first seeded in DMEM with 5% FCS inactivated for 24 hours, and then the NO donor compound was added directly to the culture medium and allowed to incubate for an additional 5-, 12-, 24-and 48-hour period, depending on each experiment. Experiments without glucose were carried out in DMEM glucose-free medium (Life Technologies) supplemented in the same way as the standard.

Quantification of nitrites

The NO production of chondrocyte cells was measured by estimating nitrite accumulation using the Griess reagent (1% sulphanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% H 3 PO 4 ) (Sigma, St Louis, MO) as previously described [36]. Chondrocytes were cultured in 96-well plates (5 × 10 4 ) and stimulated with different NO donors for 5, 24 and 48 hours.

DNA labelling technique with propidium iodide for flow cytometry analysis

Chondrocytes (500,000 cells/well in a 6-well plate) were incubated with different NO donors (0.5, 1.0 and 2.0 mM SNP and NOC-12) for 12, 24 and 48 hours. Then cells were fixed in 70% ethanol at 4°C for 60 minutes, washed and incubated with RNAse (50 μg/ml) and propidium iodide (PI, 100 μg/ml) for 15 minutes at room temperature in the dark and kept at 4°C. PI fluorescence of nuclei was measured by flow cytometry on a FACScan (Becton and Dickinson, Mountain View, CA, USA) using a 560-nm dichromatic mirror and a 600-nm band pass filter. Data are expressed as percent apoptotic (hypodiploid) nuclei.

Morphological evidence of apoptosis

For morphological studies, chondrocytes were cultured in 8-well slides (Costar) and treated with 1 mM of different NO donors for 24 hours. The cells were then washed with cold PBS, fixed in acetone/ethanol 70% (1:1) for 10 minutes at 4°C, stained with 49,6-dianidino-2phenylindole dihydrochloride (DAPI, 2 mg/ml) (Sigma) for 10 minutes in the dark, mounted in glycergel (DAKO, Hamburg, Germany), and observed by fluorescence microscopy (Olympus, BX-UCB, Center Valley, PA).

Measurement of the MRC complex activities in digitoninpermeabilized chondrocytes

Untreated, SNP-treated (5 hours) and NOC-12-treated (24 hours) chondrocytes (at least 10 × 10 6 ) were collected by trypsinization, washed with PBS, and sedimented at 150 g for 5 minutes at 4°C. Digitonin-permeabilized chondrocyte homogenates (10 to 50 μl per ml of test volume) were used to measure the activities of the respiratory chain enzymes and citrate synthase (CS) in a DU-650 spectrophotometer (Beckman Instruments, Palo Alto, CA, USA) as previously described [35].

Determination of mitochondrial membrane potential (ΔΨm)

To measure the ΔΨm of chondrocytes, the fluorescent probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide) (Molecular Probes, Inc., Eugene, OR, USA) was used. JC-1 exists as a monomer at low values of ΔΨm (green fluorescence), whereas it forms aggregates at high ΔΨm (red fluorescence). Briefly, chondrocytes were cultured in 6-well plates (5 × 10 5 ) and stimulated with different NO donors for 5, 12 and 24 hours; after that they were prepared as previously described [35].

Assay of intracellular ATP

To assay intracellular ATP, we used a commercial biolumiscence kit (ATPLite, PerkinElmer Inc., Boston, MA, USA). Chondrocytes were cultured in 96-well plates (5 × 10 4 ) and stimulated with different NO donors for 24 hours (100 μl); after this, 50 μl of lysis solution were added and mixed for 5 minutes; subsequently the enzymatic substrate (luciferase/luciferine) was added. The kit supplies a standard that gives reference values. Readers were carried out in a microbeta counter (PerkinElmer Inc.).

2-Deoxy-[ 3 H] glucose uptake

To determinate the glucose uptake levels, normal chondrocytes were cultured in 24-well plates (Visiplate, PerkinElmer Inc.) at 2 × 10 5 cells per well in DMEM without glucose and 5% inactivated calf serum for 24 h at 37°C. Later, cells were stimulated with 10 μM of different donors for 24 hours at 37°C in DMEM without glucose and subsequently 10 μCi 2-deoxy-( 3 H)glucose (2-( 3 H)DG) was added to cells in DMEM without glucose for 0 minutes, 15 minutes and 1 hour at 37°C. Cells were washed with cold PBS pH 7.4; later, 50 μl of solvable (PerkinElmer Inc.) was added to lysate cells and mixed vigorously for 5 minutes. Lastly, 500 μl of scintillation liquid was added (Ultima Gold, PerkinElmer, Inc.) and mixed for 2 minutes; glucose uptake was estimated by means of a microbeta counter (MicroBeta TriLux (PerkinElmer, Inc.)).

Quantification of lactic acid

Enzymatic determination of lactic acid in chondrocyte culture supernatants was performed using Lactate Reagent (Spinreact, Girona, Spain). Chondrocytes were cultured in 96-well plates (5 × 10 4 ) and stimulated with different NO donors for 24 hours; 10 μl of supernatant were mixed with 10 μl of lactate reagent and incubated for 5 minutes at room temperature. The absorption was estimated by an automated plate reader (Ultrospec 1000, Amersham Pharmacia Biotech, Uppsala, Sweden) at 505 nm; this method is linear towards lactate values of 150 mg/dl.

Data analysis

Data analysis was performed with SPSS software, version 12.05 (SPSS, Chicago, IL, USA). Results are expressed as the mean ± SD. Individual donors were studied in duplicate; cells from different donors were not pooled in any experiment. Comparisons between groups were carried out using the Mann-Whitney two-tailed U-test. P-values ≤ 0.05 were considered significant.

Results

NO release by different NO donors

We observed that kinetic liberation of NO changes between different NO donors. The diazeniumdiolate NOC-12 liberated approximately ten times more NO 2 than SNP. NO 2 accumulation in supernatants of normal chondrocytes treated with different NO donors depending on time and concentration ( Figure 1).

Effect of NO on cell death of normal human articular chondrocytes

NO donors damage the nuclear DNA of human articular chondrocytes in very different ways. By means of flow cytometry with PI, we could observe that the percentage of death cells (expressed as hypodiplod DNA percentage) was much higher with SNP than NOC-12 (Additional file 1); with 1 mM SNP at 24 hours: 25.9 ± 23.3 versus 0.8 ± 0.5 (P ≤ 0.05) compared with 1 mM NOC-12 at 24 hours: 4.3 ± 1.9 versus 0.8 ± 0.5 (P ≤ 0.05) ( Figure 2A). However, with higher concentrations of NOC-12 (2 mM at 48 hours), the percentage of apoptosis reached 21.6 ± 8.5% (Additional file 1). Besides this, when the cells were stained with DAPI we could see that the only NO donor able to induce the fragmentation of the nucleus and the formation of apoptotic bodies was SNP ( Figure 2B). The only effect of NOC-12 on the nucleus of chondrocytes was the acquisition of a globule-like aspect ( Figure 2B).

NOC-12 alters the activity of the complexes of the mitochondrial respiratory chain (MRC) in articular chondrocytes

Previous results obtained in our laboratory showed that the activity of the complex IV is significantly lower in normal chondrocytes stimulated with 1 mM SNP at 5 hours than in control cells [10] (Table 1). In relation with the enzymatic activity of the MRC of normal chondrocytes treated with the diazeniumdiolate compound NOC-12, the activities of all complexes were significantly lower than in control cells, except complex II (Table 1). Enzyme activities were referred to the specific activity of CS to correct for mitochondrial volume.

NO causes depolarization of the mitochondria in normal chondrocytes

The relative ratio of red/green fluorescence (ratio of normal mitochondrial polarization to mitochondrial depolarization) intensity values showed that in normal human chondrocyte cultures 1 mM SNP at 24 hours decreased the ratio of red/green fluorescence in comparison with untreated cells (1.91 ± 1.9 versus 3.5 ± 2.9; P ≤ 0.01) ( Figure 3B). In addition, 1 mM SNP caused an increase in the cell population with mitochondrial depolarization (12.2 ± 6.6 versus 33.6 ± 13.1%; P ≤ 0.05) ( Figure 3A). On the other hand, NOC-12 induced mitochondrial depolarization, as the percentage of cells with normal polarization diminished, with 1 mM NOC-12 at 24 hours: 27.7 ± 17.9 versus 14.1 ± 3.6; P ≤ 0.05; (Figure 3A), and the percentage of cells with depolarization increased: 12.2 ± 6.6 versus 18.8 ± 11.3; P ≤ 0.05 ( Figure 3A). This finding also can be observed with decreasing ratio of red/ green fluorescence in comparison with untreated cells (2.2 ± 1.5 versus 3.5 ± 2.9; P ≤ 0.05) ( Figure 3B). However, the NO donor that induced the strongest changes in the mitochondrial membrane potential was SNP.

NO abolishes ATP generation by chondrocytes in culture

NO has a detrimental effect on the generation of ATP by normal chondrocytes (Additional file 2). With NOC-12, the intracellular ATP levels were significantly lower than in control cells, with 1mM NOC-12 at 24 hours: 0.40 ± 0.16 versus 0.57 ± 0.19 (P ≤ 0.05). Again, the NO donor that induced the most dramatic changes was SNP, as it reduced the intracellular ATP levels practically to zero ( Figure 4).

Chondrocytic lactate production is strongly influenced by NO donor type NO donors have very different effects on lactate release by normal articular chondrocytes. The only donor that     Table 2).

SNP reduces glucose uptake by normal articular chondrocytes

Uptake of 2-( 3 H)DG by normal chondrocytes cultured under μM SNP concentrations (10 μM) for either 15 minutes or 1 hour was approximately 20% lower than that found in their respective controls ( Figure 5). On the contrary, 2-( 3 H)DG uptake in normal chondrocytes stimulated with NOC-12 did not change relative to the control. In this set of experiments, μM NO donors concentrations were employed; the reason is because in most of these cases, when mM concentrations were used, this caused the cells to rise (above all, SNP) and the quantifications showed false positives.

Detrimental effect of NO on chondrocyte viability depends on glucose levels

To assess the impact of glucose levels on cell viability after NO treatment, we carried out experiments with both NO donors (SNP and NOC-12) using only one constant concentration (1 mM) and increasing glucose concentrations (from 0.75 to 5 mM). The percentage of death cells decreased as glucose concentration increased, only when SNP was employed as NO donor ( Figure 6). No significant results were found in this issue when NOC-12 was used.

Glucose uptake by OA chondrocytes in basal conditions is more efficient than by normal chondrocytes Chondrocytes were maintained for 15 minutes or 1 hour in culture media without glucose and a non-metabolizable analogue of glucose: 2-( 3 H)DG. Basal 2-( 3 H)DG uptake was identical in normal and OA chondrocytes incubated for 15 minutes; however, the basal 2-( 3 H)DG uptake in OA chondrocytes was significantly higher than in normal chondrocytes (23889.9 ± 9941.6 versus 14669.5 ± 2776.1; P ≤ 0.05) (Figure 7), when cells were maintained for 1 hour in culture.

Discussion

Traditionally, the increase of endogenous NO production by human articular cartilage has been associated with joint degeneration. NO donors have been used so far to mimic the OA process in vitro, and they represent a powerful tool of study. However, in vitro models with different NO donors have not resolved what the role of NO is in cartilage degradation due to the lack of uniformity that exists between the different types of NO compounds [31]. The differential effects of NO are partly due to the type of NO donors and cell used [37]. The   (7) 1 mM NOC-12 2.90 ± 0.66 (7) 2 mM NOC-12 2.86 ± 0.77 (7) Values are the mean ± SD (number). NO, nitric oxide; SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine.  biochemistry of NO is complex because of the reactions of NO itself, the interactions of secondary products of NO and the overall chemical environment under which NO is produced [38]. In our study, we employed two NO donor types: the traditional compound SNP, that is used in the majority of studies, and one diazeniumdiolate: NOC-12. It has been reported that the traditional donor SNP does not spontaneously release NO in the absence of redox activation [34]. Diazeniumdiolates, also denominated as NONOate, or NOC, have begun to replace traditional donors as sources of exogenous NO production [32] and have been shown to be reliable sources of NO under a variety of culture conditions [33,34]. For these reasons, the diazeniumdiolate compound NOC-12, with a half-life of 327 minutes (determined at 22°C, pH 7.4) [33] was used for exogenous NO production to further investigate the conditions in which NO is cytotoxic to chondrocytes. A primary basis for the use of diazeniumdiolates is that many of them decompose spontaneously in aqueous media to release the critical bioregulatory species [34]. The main advantages of these compounds are known rates of NO generation, NO generation rates covering a wide range, spontaneity of NO generation and tenable generation of NO redox forms.

The precise role of NO in the induction of chondrocyte death is repeatedly debated. Treatment with classical NO donors consistently induces apoptosis in cultured chondrocytes [6,8,10,39], whereas the production of high levels of endogenous NO by the over-expression of the iNOS gene in transfected chondrocytes has not been found to cause cell death [11]. This discrepancy might be the result of using chemical NO donors, which not only generate reactive nitrogen species but also produce various secondary reactions depending on the cellular milieu with in vitro experiments [40]. Also, an anti-apoptotic role has been addressed in several review articles [41][42][43][44][45]. Specifically, del Carlo and collaborators showed that compounds that only release NO, such as the diazeniumdiolates NOC-5 and NOC-12, do not cause chondrocyte cell death and can even be protective under certain conditions of oxidative stress [39]. It is likely that persistent spontaneous release of NO is necessary for the protective effect and that peroxynitrite and cyanide contribute to the cytotoxic effect of NO donors [37]. Chondrocyte cell death from NO occurs under conditions where other reactive oxygen species (ROS) are also generated [39].  Chondrocyte death does not correlate with the amount of NO released by NO donors. Similar to other authors [46], our results showed that SNP is the least potent in terms of producing exogenous NO in chondrocyte culture, although it is the most potent inducer of chondrocyte death. The amount of NO produced by NOC-12 was 10-fold higher than the NO produced by SNP, but the level of cell death induced was not as profound as that produced by SNP. As previously shown in our laboratory [10], SNP was able to induce formation of apoptotic bodies, which are produced from cells undergoing cell death by apoptosis. However, we observed that NOC-12 increased the hypodiploid nuclei number without formation of apoptotic bodies, which is probably related to another type of programmed cell death. Recently it has been proposed that autophagy is another type of programmed cell death than happens in the human articular cartilage as well [47]. The increase in the number of hypopliod nuclei and the observation of some morphologic changes as vacuole formation seems to relate NOC-12 with autophagy (personal data).

It is believed unlikely that NO is the sole mediator of SNP-induced chondrocyte death and peroxynitrite, a reaction product of NO and superoxide anions, or the primary by-products of the decomposition of SNP, such as the cyanide aninon or pentacy-anoferrate complex, might contribute to its cytotoxicity [34,48]. It is unclear whether chondrocyte apoptosis is the major mechanism of cartilage degradation or merely a by-product of tissue degeneration [46].

Mitochondria comprise a target of NO and there is accumulating evidence that inhibition of respiration may contribute to the pro-apoptotic effect of NO by ΔΨm alteration, transition-pore opening and release of cytochrome c [45,49]. There is increasing evidence about the importance of mitochondria in OA pathology. Previously, we showed that the activity of the mitochondrial complexes II and III is lower in OA than in normal human chondrocytes; this produces a decrease in ATP levels as well as a higher ROS generation [12,35]. The relevance of the MRC inhibition in human chondrocytes is already known, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to prostaglandin E 2 (PGE2) production via mitochondrial Ca 2+ exchange, ROS production, and nuclear factor (NF)-B activation [50]. More recently, Rego and collaborators have found that the predisposition to the development of OA is related to some haplogroups of mitochondrial respiratory genes of chondrocytes [51]. Also, chondrocytes are not the only joint cells affected in OA pathology and, a recent study has shown that SNP reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle [52].

Analysis of the MRC showed that at 5 hours, SNP reduced the activity of complex IV by 30%; furthermore, SNP induced depolarisation of the mitochondrial membrane [10]. In this study we show that NOC-12 induces depolarisation of the mitochondrial membrane as well as SNP, but to a lesser extent (10%); however, it had a more radical effect on MRC activity than SNP, this donor reduces the activities of all the complexes except complex II (complex I by 80%, complex III by 33% and complex IV by 75%). These results show that the inhibition of the MRC complexes is not the main cause of cell death induction in chondrocytes by NO. On the other hand, CS activity was increased about 40% in NOC-12-treated chondrocytes, and this fact has been correlated with an increment of the mitochondrial mass [30]; Nisoli and collaborators also suggested that NO is implicated in the regulation of energy metabolism, possibly through the enhancement of mitochondria formation [53]. Similar findings were previously found in OA chondrocytes [35] but not in SNP-treated ones [10]. Therefore, an increase in mitochondrial mass could be a mechanism by which OA chondrocytes as well as NOC-12-treated cells, compensate for the electron transfer deficiency resulting from dysfunction in several complexes and the consequent low production of ATP per mitochondrion, as has already been reported by Maneiro and collaborators [35].

In relation to ATP synthesis both donors had a detrimental effect on it, but once more SNP was the compound with the most important deleterious effect. With respect to lactate production, SNP reduced the levels in a significant way compared to the control cells; on the contrary, NOC-12 increased lactate production by chondrocytes although this increment was not statistical significant. A dysfunction in complexes I, III and IV compromises the electron transfer pathway; this defect could be solved increasing the anaerobic metabolism to avoid excess production of ROS, and these findings are in agreement with the increase of lactate levels after incubation with NOC-12. These results are consistent with the findings reported by Tomita and collaborators on NOC-18-treated chondrocytes, another member of the diazeniumdiolate family [13]. Because NO inhibited the respiration of mitochondria, cellular glycolysis was enhanced significantly, the effect on cellular ATP levels was rather mild, despite the inhibition of mitochondrial respiration by NO. Thus, the enhanced glycolysis in NOC-18-treated chondrocytes could theoretically compensate for the inhibition of mitochondrial synthesis by approximately 46% [13]. On the other hand, the build-up of lactic acid will have detrimental effects on the extracellular matrix and may contribute to the pathogenesis and progression of OA [54].

Chondrocytes are highly glycolytic resident cells of articular cartilage that metabolize glucose as a primary substrate for ATP production [17]. The Pasteur effect arises in articular cartilage; in this way anaerobic glycolysis and lactate production are involved in respiratory metabolism of articular cartilage even under aerobic conditions [21,55,56]. An anaerobic metabolism could be beneficial for OA, since the products of glucose degradation (lactate and pyruvate) would act as ROS scavengers [57,58], and would assure ATP production even under conditions of mitochondrial dysfunction (defects in CRM complexes and high NO production) [35].

Only the traditional donor SNP was able to reduce glucose uptake by normal chondrocytes. Previously, we showed that the inhibition of complex IV with sodium azide modified the survival of the chondrocytes, but its effect was greater when glucose was absent. A possible explanation is that the inhibition of complex IV exclusively is not enough to induce apoptosis and other cellular events; such a reduction in the intake of glucose needs to be present to induce it [10]. The glucose dependency of chondrocytes arises with the fact that the effect of SNP on chondrocyte apoptosis correlates with glucose levels; the lower the glucose levels in the media, the highest the apoptotic levels induced.

Finally, OA chondrocytes incorporated more glucose than healthy chondrocytes under the standard experimental conditions used in this study. These findings are in consonance with a higher lactate production by OA chondrocytes than control chondrocytes (personal data). Furthermore, this up-regulation can be considered a protective mechanism that maximizes the cell's ability to capture glucose and thus to overcome stressful conditions, such as glucose scarcity or even deprivation [59,60], or just to compensate CRM defects. On the other hand, these findings can somehow explain the ROS contribution to the pathogenesis of OA [61,62]; no changes in glucose incorporation by normal chondrocytes can suggest a protective mechanism against the deleterious effects of excessive intracellular glucose, as seen in other cells [63], and the incapacity of OA chondrocytes to regulate this can trigger ROS accumulation in OA cartilage. Others authors have reported that basal glucose uptake is identical in normal and OA chondrocytes [63]; the reasons for these discrepancies are unclear but the observed differences may be related to the culture conditions used in these studies.

Conclusions

The new generation donor NOC-12 mimics the metabolic OA situation much better than the classical NO donor SNP. Taking account of all the results obtained in this study, previous findings using SNP have to be considered very cautiously, and most of the effects observed in human chondrocytes probably cannot be attributed exclusively to NO.

Additional material

Additional file 1: Table showing values

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MCdA carried out the experimental work, analysed the data and drafted the manuscript. EM participated in the study design, interpretation of data and manuscript preparation. MAM and JA measured the MRC complex activities in digitonin-permeabilized chondrocytes. FJB conceived and coordinated the project and revised the manuscript. All the authors read and approved the manuscript.

Figure 1
1Nitric oxide (NO) donors differ from the amount of NO 2 released. NO 2 accumulation in supernatants of normal chondrocytes treated with different NO donors for 24 hours in standard culture conditions. The lines show the dose-dependent increase of NO accumulation in all cases. Values are the mean ± SD; n = 5. SNP, sodium nitroprusside; NOC-12, N-ethyl-

Figure 2
2Nitric oxide (NO) effect on chondrocyte apoptosis. (A) Cell death levels in chondrocytes treated with different NO donors was determined by the iodide propidium (PI) method. Control chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 5. *P ≤ 0.05 versus untreated chondrocytes (control). (B) Cellular changes induced by NO on normal human chondrocytes were analysed by 49,6diamino-2-phenylindole dihydrochloride (DAPI staining) and fluorescence microscopy. Shown is a representative example of five experiments.

Figure 3
3Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2nitrosohydrazine (NOC-12) for 24 hours. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells and those treated with NO donors.Green fluorescence (open graph) increases, whereas red fluorescence (solid graph) decreases in the NOC-12 and sodium nitroprusside (SNP)-treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential, and therefore, a decrease in the red/green ratio. Shown is an example at 24 hours. Results are the mean ± SD; n = 5. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 4
4ATP generation by normal chondrocytes treated with different nitric oxide (NO) donors. Articular chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 2 mM N-ethyl-2 (1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Then the ATP assay was performed as described in Materials and methods. Values are the mean ± SD; n = 7. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 5
5Nitric oxide (NO) influence on 2-deoxy-( 3 H) glucose uptake by normal chondrocytes. Effect of the NO donors sodium nitroprusside (SNP) and N-ethyl-2(1-ethyl-2 hydroxy-2nitrosohydrazine (NOC-12) on the uptake of 2-deoxy-( 3 H) glucose by normal chondrocytes for 15 minutes. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 7. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 6
6The effect of nitric oxide (NO) on chondrocyte viability depends on glucose levels. Articular chondrocytes were cultured in medium with crescent glucose concentrations and treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 3.

Figure 7
7Osteoarthritis (OA) chondrocytes are efficient cells in the uptake of glucose. Differences between human articular normal and OA chondrocytes in the uptake of 2-deoxy-( 3 H)glucose for 1 hour. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 12. *P ≤ 0.05 versus normal chondrocytes.

of apoptotic cell death in normal chondrocytes treated with different nitric oxide (NO) donor compounds. Additional file 2: Table showing values of ATP production by normal chondrocytes treated with different NO donors compounds for 24 hours*. Abbreviations CS: citrate synthase; DAPI: 49,6-dianidino-2-phenylindole dihydrochloride; 2-( 3 H)DG: 2-deoxy-( 3 H)glucose; DMEM: Dulbecco's modified Eagle's medium; FCS: fetal calf serum; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; JC-1: 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide; ΔΨm: mitochondrial membrane potential; MRC: mitochondrial respiratory chain; NO: nitric oxide; NOC-12: N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine; OA: osteoarthritis; OXPHOS: mitochondrial oxidative phosphorylation; PBS: phosphate buffered saline; ROS: reactive oxygen species; SIN-1: 3-morpholinosydnonimine; SNAP: S-nitroso-Nacetylpenicillamine; SNP: sodium nitroprusside; TNFα: tumour necrosis factor α.

Table 1
1Values of mitochondrial respiratory chain complexes in cultures of normal chondrocytes treated with two NO donor compoundsNormal
chondrocytes

Cells treated with
1 mM NOC-12

Cells treated with
1 mM SNP

Table 2
2Values of lactate release in cultures of normal
chondrocytes treated with different NO donors
compounds for 24 hours

Lactate
(μM/5 × 10 4 cells)

Control normal chondrocytes
2.30 ± 0.58 (7)

0.5 mM SNP
1.83 ± 0.65 (7)

1 mM SNP
1.33 ± 0.38 (7)  †
2 mM SNP
1.01 ± 0.30 (7)  †

0.5 mM NOC-12
2.91 ± 0.58
de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115
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AcknowledgementsThe authors express appreciation to the Department of Orthopedics and the Tissue Bank of the Complejo Hospitalario Universitario de A Coruña for providing cartilage samples. This study was supported by grants from the Fondo Investigación Sanitaria, Madrid, Spain: (CIBER-CB06/01/0040; PI-12/ 00329; RETIC-RIER-RD12/0009/0018; and Proteo-Red/ISCIII); Ministerio Ciencia e Innovación, Madrid, Spain: PLE2009-0144 and FEDER (European Community).Authors' detailsSubmit your next manuscript to BioMed Central and take full advantage of:
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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism. Andrés De, Arthritis Research & Therapy. 15115Cite this article asCite this article as: de Andrés et al.: Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism. Arthritis Research & Therapy 2013 15:R115.