Ph.D Aleksandar Backovic
email: aleksandar.backovic@i-med.ac.at

Dolores Wolfram

Silicone mammary implants -can we turn back the time?

Mankind remains infatuated with finding the ″fountain of youth″, and plastic surgery has become a very important component in the search for eternal youth. Invasive procedures such as face-lifts, body contouring, and implantation of silicone mammary implants (SMI) as well as less invasive procedures such as wrinkle decreasing protocols using filler substances or botulinum toxin, effectively reshape and rejuvenate the aging face or body. However, despite the improved cosmetic appearance of the individual, these treatments disrupt normal aging processes on cellular and molecular level. For example, silicone degradation products promote protein denaturation and activate cells of both the innate and adaptive immune systems, thus perpetuating chronic proinflammatory response of the local tissue. In this review, we concentrate on SMI and summarize the current clinical approaches, and the immunological and biochemical effects of those interventions.

Introduction

The desirability of youth and beauty have been a constant in the continually evolving history of mankind [START_REF] Pitanguy | Evaluation of body contouring surgery today: a 30-year perspective[END_REF][START_REF] Zimbler | Lost beauty and The Fountain of Youth[END_REF]. Current fashion trends and beauty stereotypes are promulgated via print and electronic medias and highlight the importance of fitness, youthfulness and beauty in our lives. Plastic surgery, unsurprisingly, has increased in popularity as a way to look younger despite chronological age [START_REF] Rohrich | Plastic versus cosmetic surgery: what' s the difference?[END_REF]. Medical advances in this century have enabled safe and efficient surgical corrections of contour deformities resulting from the aging process, and the use of less invasive "anti-aging" procedures, such as dermal filler substances or botulinum toxin is a rapidly growing sub-field in plastic surgery [START_REF] Coleman | Combination therapy with BOTOX and fillers: the new rejuvnation paradigm[END_REF][START_REF] Wise | Injectable treatments for the aging face[END_REF][START_REF] Wolfram | Surgery for foreign body reactions due to injectable fillers[END_REF].

A C C E P T E D

M A N U S C R I P T 3 Defining aging as an illness makes the benefits of surgical interventions self-evident.

If volume loss of the aging face or body is considered a pathological rather than a physiological process, dissatisfaction with appearance is inevitable and youthpromoting procedures are indispensable for a personal perception of good health. The phrases like "treatment of the aging face" are often used in literature in the same ways as treatment of cancer or hypertension. By using the aging face or body as an object of treatment, aging is falsely defined as a disease, and this concept of aging is reinforced by stereotypes of beauty in contemporary consumerist societies. However, there is a theoretical problem with defining aging as illness, because it would implicitly define life as an illness as well [START_REF] Ringel | The morality of cosmetic surgery for aging[END_REF], since it is a process with the inevitable result being death.

From a surgical point of view, aging is a complex process involving two important factors: volume loss, and repetitive muscle contractions causing wrinkles and folds [START_REF] Glashofer | Fat transplantation for treatment of the senescent face[END_REF][START_REF] Zimbler | Anatomy and pathophysiology of facial aging[END_REF]. The aging process thus leads to alterations of facial or general body contours that, in a youth-oriented culture, usually result in a decline in body image that provokes a strong psychological motivation for surgical correction [START_REF] De Maio | The minimal approach: an innovation in facial cosmetic procedures[END_REF]. According to the American Association of Plastic Surgeons, more than 128,000 face lifts were performed in 2003, representing an increase of 9% over the year before, and Botulinum toxin injections increased by more than 150% during the same period (Christie ,2004). In France, breast implant sales rose by 383% (61 800 implants) from 1995 to 2004 [START_REF] Petit | Breast implants in France. New regulations, and qualitative and quantitative study of breast implants sold between 1995 and 2004[END_REF].

A C C E P T E D M A N U S C R I P T 4
Although the mentioned surgical interventions enhance the appearance of the individual, they often have deleterious effects on the molecular and cellular levels and can actually enhance the normal aging processes, for example by disrupting normal inflammatory processes [START_REF] Bailey | The quantification of cellular viability and inflammatory response to stainless steel alloys[END_REF][START_REF] Kossovsky | A pathophysiological examination of the biophysics and bioreactivity of silicone breast implants[END_REF][START_REF] Wolf | Human immune response to polydimethylsiloxane (silicone): screening studies in a breast implant population[END_REF], causing posttranslational modifications or protein denaturation [START_REF] Hamilton | Nondisposable materials, chronic inflammation, and adjuvant action[END_REF][START_REF] Somasundaram | Collagens serve as an extracellular store of bioactive interleukin 2[END_REF][START_REF] Sun | Protein denaturation induced by cyclic silicone[END_REF], and development of extensive local fibrotic reactions [START_REF] Hu | Molecular basis of biomaterial-mediated foreign body reactions[END_REF][START_REF] Shanklin | Dynamics of wound healing after silicone device implantation[END_REF][START_REF] Wong | A critical assessment of the relationship between silicone breast implants and connective tissue diseases[END_REF].

This review concentrates on the use of silicone mammary implants (SMI) for body contour improvement in cosmetic surgery as an increasingly utilized intervention in the aging population, and discusses their adverse effects from medical, immunological and biochemical viewpoints in the context of normal aging processes.

A C C E P T E D M A N U S C R I P T 5

Silicone mammary implants

Indications for silicone breast implants

Breast augmentation with mammary implants continues to be one of the most commonly performed procedures in plastic surgery. Despite the controversy about the safety and efficacy of silicone gel implants [START_REF] Bar-Meir | Silicone gel breast implants and connective tissue disease--a comprehensive review[END_REF][START_REF] Janowsky | Meta-analyses of the relation between silicone breast implants and the risk of connective-tissue diseases[END_REF][START_REF] Teuber | Severe migratory granulomatous reactions to silicone gel in 3 patients[END_REF], the desire for enhanced self-image and perfectly-formed breasts has led to a boom in cosmetic breast augmentation. The medical indication for breast augmentation is glandular hypoplasia, which may be developmental or involutional ("age-related"). Patients undergoing breast augmentation range from stable, well-adapted, and socially outgoing persons to depressed women with low self-confidence and self-esteem [START_REF] Birtchnell | Motivational factors in women requesting augmentation and reduction mammaplasty[END_REF][START_REF] Meyer | Augmentation mammaplasty--psychiatric and psychosocial characteristics and outcome in a group of Swedish women[END_REF].

Implant types, operation techniques and complications of SMI

Currently, the two implant types in widespread use are silicone gel-filled or salinefilled implants, both of which have a highly polymerized silicone shell. Both implant types may be smooth or microtextured, spherical or anatomic. Both types of implants can be placed in a subglandular or submuscular location using three common incisions well-described in literature, namely the transaxillary, periareolar and inframammary, depending on surgeon's preference [START_REF] Mofid | Nipple-areola complex sensitivity after primary breast augmentation: a comparison of periareolar and inframammary incision approaches[END_REF][START_REF] Tebbetts | Axillary endoscopic breast augmentation: processes derived from a 28-year experience to optimize outcomes[END_REF]. Although serious complications are rare after cosmetic breast augmentation, two categories of complications, early and late, can occur. Early complications are considered to be those arising from the operative procedure (e.g. hematoma, seroma, infection, asymmetry), while later complications are those based on the body's response to the implant (e.g. capsular contracture, deflation or rupture, systemic complications) [START_REF] Henriksen | Incidence and severity of short-term complications after breast augmentation: results from a nationwide breast implant registry[END_REF][START_REF] Henriksen | Surgical intervention and capsular contracture after breast augmentation: a prospective study of risk factors[END_REF]. Whether silicone breast

A C C E P T E D M A N U S C R I P T

6 implant exposure results in systemic manifestations (e.g. connective tissue diseases, fibromyalgia-like syndrome) in some recipients remains controversial in the literature [START_REF] Gabriel | Risk of connective-tissue diseases and other disorders after breast implantation[END_REF][START_REF] Tugwell | Do silicone breast implants cause rheumatologic disorders? A systematic review for a court-appointed national science panel[END_REF][START_REF] Vasey | Where there' s smoke there' s fire: the silicone breast implant controversy continues to flicker: a new disease that needs to be defined[END_REF]. Large epidemiologic studies and meta-analyses have been performed by the FDA and other institutions to determine whether there was a relationship between breast implants and connective tissue diseases, [START_REF] Janowsky | Meta-analyses of the relation between silicone breast implants and the risk of connective-tissue diseases[END_REF] and a clear-cut causal connection between SMI and the appearance of systemic side effects could not be verified. However, these studies have been challenged and contradict single case reports describing autoimmune phenomena in women with breast implants [START_REF] Asherson | An unusually complicated case of primary Sjogren' s syndrome: development of transient "lupustype" autoantibodies following silicone implant rejection[END_REF][START_REF] Gurvits | Silicone pneumonitis after a cosmetic augmentation procedure[END_REF][START_REF] Varga | Systemic sclerosis after augmentation mammoplasty with silicone implants[END_REF][START_REF] Williams | Breast implants in patients with differentiated and undifferentiated connective tissue disease[END_REF].

A C C E P T E D M A N U S C R I P T 7

The stability of silicone polymer in vivo

Medical grade silicone (polydimethylsiloxane) is used both as the gel filling of a silicone breast implant and in construction of the outer shell. Although the chemical structure of silicones suggests great stability, they nevertheless undergo degradation with aging, and although the role of their breakdown derivates in the tissue around the implants is unclear, immunogenic properties have been shown for at least some low molecular weight silicone compounds [START_REF] Hamilton | Nondisposable materials, chronic inflammation, and adjuvant action[END_REF][START_REF] Naim | Adjuvancy effect of different types of silicone gel[END_REF]). In the connective, fat and muscle tissue around the SMIs, siloxane levels in the range of 1-1400 ng/g, and silicone from 9-85 µg/g were found. The same study found that siloxane concentrations in the fatty tissue were strongly associated with "gel bleeding" of an individual SMIs [START_REF] Flassbeck | Determination of siloxanes, silicon, and platinum in tissues of women with silicone gel-filled implants[END_REF]. In addition to inducing protein denaturation [START_REF] Sun | Protein denaturation induced by cyclic silicone[END_REF]), these silicone degradation products constitute a protracted stimulus to cells such as macrophages [START_REF] Rhie | Efficient in vitro model for immunotoxicologic assessment of mammary silicone implants[END_REF][START_REF] Tavazzani | In vitro interaction between silicone gel and human monocyte-macrophages[END_REF]) that increase their cytotoxic properties, but the exact nature of such signals remains to be elucidated.

The interaction of silicone and its degradation products with various cell types has been described in several studies reporting various outcomes [START_REF] Ciapetti | Assessment of viability and proliferation of in vivo silicone-primed lymphocytes after in vitro re-exposure to silicone[END_REF][START_REF] Ojo-Amaize | Silicone-specific blood lymphocyte response in women with silicone breast implants[END_REF][START_REF] Tavazzani | In vitro interaction between silicone gel and human monocyte-macrophages[END_REF][START_REF] Zhang | Proteins and cells on PEG immobilized silicon surfaces[END_REF]. They agree that the cell response to silicone resembles a silent perpetuated foreign body reaction, but the exact mechanism of cell activation remains unclear. Elevated secretion of TNF-has been found in fibroblasts and macrophages after silicone uptake, as well as the recruitment of T-cells to the surrounding tissue [START_REF] Smalley | Lymphocyte response to silica among offspring of silicone breast implant recipients[END_REF][START_REF] Wick | Immunohistochemical Analysis of the Connective Tissue Capsule Formation and Constriction Arround Mammary Silicone Prostheses[END_REF]. We have also noticed large silicone particles in fibroblasts and macrophages isolated directly from the fibrotic capsule surrounding the SMIs, as shown in Figure 1.

A C C E P T E D M A N U S C R I P T 8
Platinum complexes are used in the production of SMIs as catalysts for cross-linking of the silicone chains. In addition to being a potent allergen itself, depending on the amount of ionized Pt that is liberated from the SMIs by the degradation process, proteins interacting with those ions may become vulnerable to denaturation [START_REF] Brook | Platinum in silicone breast implants[END_REF]. Denaturation most likely occurs through binding of ionized Pt by the sulfhydryl bridges that control the folding of the proteins [START_REF] Lykissa | Total platinum concentration and platinum oxidation states in body fluids, tissue, and explants from women exposed to silicone and saline breast implants by IC-ICPMS[END_REF]. Furthermore, significantly elevated Pt concentrations have been found in blood (470-688pmol/L), urine (0.4-2.95µg/L) and local binding tissue (0.003-0.272µg/g) as well as in hair, nails, sweat and breast milk [START_REF] Flassbeck | Determination of siloxanes, silicon, and platinum in tissues of women with silicone gel-filled implants[END_REF][START_REF] Lykissa | Total platinum concentration and platinum oxidation states in body fluids, tissue, and explants from women exposed to silicone and saline breast implants by IC-ICPMS[END_REF].

Although they are highly irritant, hydrophobically modified amorphous silicates are used to reinforce the shell of SMIs, improving strength and tear resistance [START_REF] Brook | Platinum in silicone breast implants[END_REF][START_REF] Leyva | Pathological mineralizations: calcifications and Si-bearing particles in soft tissues and their eventual relationship to different prostheses[END_REF]Shen and Ojo-Amaize ,1996). Silicates have been detected

in the fibrotic capsule surrounding the SMIs [START_REF] Jensen | Silicate antibodies in Danish women with silicone breast implants[END_REF]Shen et al. 1996), and we have found silicate crystals in the liquid inside the autoinflated SMIs.

Interestingly, several groups have reported the presence of silicate antibodies in carriers of SMIs [START_REF] Brandon | Invited discussion[END_REF][START_REF] Brody | On the safety of breast implants[END_REF][START_REF] Marotta | Silicone gel breast implant failure: Evaluation of properties of shells and gels for explanted prostheses and meta-analysis of literature rupture data[END_REF][START_REF] Vermeulen | Rupture of silicone gel breast implants and symptoms of pain and fatigue[END_REF], indicating their active biological role in those individuals.

Data from our and other groups [START_REF] Backovic | Identification and Dynamics of Proteins Adhering To the Surface of Medical Silicones in Vivo and in Vitro[END_REF][START_REF] Jansson | In vitro preparation and ellipsometric characterization of thin blood plasma clot films on silicon[END_REF][START_REF] Wettero | On the binding of complement to solid artificial surfaces in vitro[END_REF][START_REF] Yoshida | Immunotoxicity of silicone: implications of oxidant balance towards adjuvant activity[END_REF][START_REF] Zhang | Proteins and cells on PEG immobilized silicon surfaces[END_REF]) yielded a noteworthy amount of evidence that silicone does not stay intact in vivo, as was suggested according to the chemical properties of silicone polymer. Although silicone itself has proteomic strategies can be employed as well [START_REF] Dierick | Proteomics in experimental gerontology[END_REF][START_REF] Westergren-Thorsson | Proteomicsthe protein expression technology to study connective tissue biology[END_REF][START_REF] Zerkowski | Proteomics strategies in cardiovascular research[END_REF]). Proteins on the surface of SMIs play a decisive role in the subsequent reaction of local tissue, since a decrease in protein adsorption leads to a concomitant decrease in cellular attachment. Moreover, upon binding to hydrophobic surfaces, many proteins tend to unfold, and in a short period undergo surface denaturation. Early interactions between silicone implants and inflammatory cells are thus mediated by a layer of host proteins on the material surface [START_REF] Chittur | FTIR/ATR for protein adsorption to biomaterial surfaces[END_REF][START_REF] Ortega-Vinuesa | Stagnant versus dynamic conditions: a comparative adsorption study of blood proteins[END_REF].

In an equilibrium state spontaneous protein adsorption is mainly controlled by the adsorption probability or sticking coefficient of each of the proteins. Sticking coefficients of proteins are generally found to be in the range 10 -5 -10 -8 [START_REF] Tang | Molecular determinants of acute inflammatory responses to biomaterials[END_REF], and they are depending on the protein structure, temperature, adhering surface, and the ionic strength of the surrounding milieu [START_REF] Weaver | Sticking coefficients of adsorbing proteins[END_REF].

Although albumin, fibronectin and IgG predominate on many types of biomaterials [START_REF] Elwing | Protein absorption and ellipsometry in biomaterial research[END_REF], the adsorption of proteins is a very dynamic process. To understand these dynamics, flow parameters, conformational changes, displacement effects, early events in blood clotting and complement activation all must be considered in addition

A C C E P T E D M A N U S C R I P T

11 to sticking coefficient. Although the adhesion of each protein from serum is somewhat related to its concentration in the surrounding liquid, over a time frame of ~100 min, proteins present in higher amounts in solution (e.g. blood) are replaced by those present in much smaller concentrations, but with higher surface affinities [START_REF] Ortega-Vinuesa | Stagnant versus dynamic conditions: a comparative adsorption study of blood proteins[END_REF]). This phenomenon is known as the "Vroman effect", which is defined as the time-and concentration-dependant exchange of adsorbed fibrinogen with kininogen when blood plasma is incubated with silicone surfaces.

After the state of equilibrium has been reached, around 4-16 mg of protein adhere to cm 2 of silicone surface [START_REF] Zhang | Proteins and cells on PEG immobilized silicon surfaces[END_REF].

In this sense, it is important to determine the degree to which the hydrophobic silicone surface promotes exposure of cryptic antigens and/or "altered self", and how these events correspond to the local inflammatory and fibrotic processes, and perhaps also systemic immune effects. These findings will provide the knowledge needed to modify silicone to avoid immune reactions and fibrosis are minimize the risks for implant patients.

Aged proteins and silicone

Accumulation of altered proteins and their modification with age have been recently

widely discussed in this journal [START_REF] Hipkiss | Accumulation of altered proteins and ageing: causes and effects[END_REF][START_REF] Schoneich | Protein modification in aging: an update[END_REF], thus we focused solely on the effects of aged proteins in the reaction to silicone. Protein aging is usually defined as a progressive, age-dependent, covalent and nonenzymatic modification of a protein backbone and/or side group/s. This aging process is important in the context of reactions with silicone because the post-translational modifications that proteins undergo in the circulation and in situ may lead to the loss

A C C E P T E D M A N U S C R I P T
12 of immune tolerance [START_REF] Berlett | Protein oxidation in aging, disease, and oxidative stress[END_REF][START_REF] Dierick | Proteomics in experimental gerontology[END_REF][START_REF] Glomb | Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction[END_REF][START_REF] Grune | Protein oxidation and degradation during postmitotic senescence[END_REF][START_REF] Hudson | Soluble levels of receptor for advanced glycation endproducts (sRAGE) and coronary artery disease: the next Creactive protein?[END_REF][START_REF] Newkirk | Advanced glycation end-product (AGE)damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis[END_REF].

Conversely, posttranslational modifications of proteins can alter their biological activity and function.

Although aged proteins are detected and removed via several mechanisms, some aged proteins consistently escape this process. They are usually seen as foreign by the immune system, but because they share structural motives of native proteins, tolerance is often broken and autoimmune reactions towards their native conformations subsequently also occur. Furthermore, accumulation of aged proteins occurs in numerous diseases such as atherosclerosis, synovitis, cataract, rheumatoid We have obtained such a capsule from a patient undergoing SMI reimplantation after diagnosed "gel-bleeding" from a damaged implant. The fibrotic capsule was digested with collagenase type IV (Sigma, Vienna, Austria) and we attained a single cell suspension that was cultivated in plastic 6-well plates. In most of the cells accumulations of silicone particles were noticed, as depicted here.

  , its degradation products induce a strong host response, and can be associated with numerous side effects. film forms on the surface of silicone almost instantaneously after the implantation[START_REF] Tang | Fibrin(ogen) mediates acute inflammatory responses to biomaterials[END_REF]. Laterally Resolved Ellipsometry and Fourier Transform Infrared Attenuated Total Internal Reflection are methods routinely used to study conformational changes of proteins on such surfaces as implanted devices and materials[START_REF] Ortega-Vinuesa | Stagnant versus dynamic conditions: a comparative adsorption study of blood proteins[END_REF]), but numerous other

  arthritis and diabetes. Silicone surfaces promote deposition and formation of nonenzymatically-modified proteins, comprising a plausible link with autoimmune-like syndromes that occur in some SMI patients. Modified proteins on the surface of silicone can trigger an immune response, and are responsible for at least part of adverse reaction development.5. ConclusionYouth has been the foundation of human concepts of beauty and well-being from the dawn of civilization, and the technological advances of the last decades have only reinforced this ideal. The natural processes of senescence are scientifically understood as a disease, and both cosmetic surgeons and the pharmaceutical industry continually develop elaborate armentarium to fight the new "War on Aging". Conversely, once the initial excitement over new surgical procedures and presumably biocompatible materials such as silicone in tissue remodelling has faded, inevitably the scientificA C C E P T E D M A N U S C R I P T13 community is inundated with case reports of various adverse effects these techniques and materials may cause. Herein, we have focused on the side effects of cosmetic procedures that influence known mechanisms of aging on molecular and cellular levels. Although such procedures indeed yield the promised outcome of a more youthful appearance, they also interfere with the normal physiological processes of tissue remodelling. Silicone degradation products activate chronic pro-inflammatory responses in the surrounding tissues, and the surface of the silicone implant itself presents numerous modified autologous proteins to the host defence system.Therefore, we believe the public should be constantly reminded that the promises of "anti-aging" treatments are often only skin-deep, and that the attendant risk of detrimental side effects are clearly presented to anyone seeking cosmetic interventions. It is likely that the future will see improvement in both the results and diminished risk of cosmetic procedures thanks to findings from diverse scientific and clinical areas.

  Figure 1. Caption Cells isolated from the fibrotic capsule formed around the silicone mammary implant (SMI) Fibrotic capsule forms around the SMIs in a period from several months to several

Table 1

 1 Silicone degradation products in vivo. In comparison to normal population, carriers of SMIs have elevated levels of silicone degradation products in various tissues.

	Tissue	Silicone	Concentration	Reference
		component
	Local binding	siloxane	1-1400 ng/g	Flassbeck et al. 2003
	tissue
	Local binding	Low molecular	9-85 µg/g	Flassbeck et al. 2003
	tissue	weight silicone
	Blood	Platinum	470-688pmol/L	Lykissa and Maharaj,
				2006
	Urine	Platinum	0.4-2.95µg/L	Lykissa and Maharaj,
				2006
	Local binding	Platinum	0.003-0.272µg/g	Lykissa and Maharaj,
	tissue			2006
	Local binding	Silicates	Concentration not	Jensen et al. 2003
	tissue		determined*
	Blood	Silicates	Concentration not	Shen et al. 1996
			determined*

*These studies compared relative silicate levels in SMI carriers.

Acknowledgments

The authors would like to thank Georg Wick and Hildegunde Piza for their unselfish mentorship and constant support, Barbara Del Frari and Tanja Schuetz for their generous help with the clicinal specimens, and M. Kat Occhipinti-Bender for editorial assistance. The silicone project was financed through grants from Kompetenzzentrum Medizin Tirol and Udo and Lore Saldow foundation.


Krzysztof Ksią

Marek Winckiewicz

Ryszard Staniszewski

Andrzej Bręborowicz

Janusz Witowski

Krzysztof Ksi Ek

Andrzej Br Borowicz

Prof Janusz Witowski

Correlation between the donor age and the proliferative lifespan of human peritoneal mesothelial cells in vitro: is TGF-β1 a link?

Keywords: ageing, mesothelial cells, peritoneum, replicative senescence, TGF-

In the present study we have examined the relationship between calendar age of the donor and the proliferative lifespan and TGF-1 production by human peritoneal mesothelial cells (HPMC) in culture. The experiments were performed on primary omentum-derived HPMC isolated from patients undergoing elective abdominal surgery. There was an inverse relationship between the calendar age of the tissue donor and the replicative lifespan of HPMC in vitro (n=49, r= -0.3991, p<0.005). There was also a positive correlation between the donor's age and the magnitude of TGF-1 production by first-passage HPMC (n=28, r=0.5400, p<0.004). In turn, the TGF-1 levels correlated inversely with the proliferative lifespan of HPMC in vitro (n=28, r= -0.4671, p<0.02). These findings indicate that the reduced proliferative capacity of HPMC isolated from older donors may be associated with increased TGF-1 release, which may, in turn, result from the age-related accumulation of senescent HPMC in the peritoneum.

Introduction

Transforming growth factor-1 (TGF-1) is a multifunctional mediator that plays a central role in many biological processes, including cell growth and differentiation, inflammation, angiogenesis, and extracellular matrix accumulation. Because of this pleiotropic activity, the overproduction of TGF-1 has been implicated in numerous pathologies, such as atherosclerosis, tumorigenesis and tissue fibrosis (see [START_REF] Blobe | Role of transforming growth factor beta in human disease[END_REF] for review).

Interestingly, certain polymorphisms in TGF-1 gene have been found to associate with human longevity [START_REF] Carrieri | The G/C915 polymorphism of transforming growth factor beta1 is associated with human longevity: a study in Italian centenarians[END_REF]. Moreover, increased TGF-1 levels have been found both in plasma of elderly people [START_REF] Forsey | Plasma cytokine profiles in elderly humans[END_REF] and in fibroblasts undergoing either replicative [START_REF] Kim | Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway[END_REF] or stress-induced senescence in vitro [START_REF] Frippiat | Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts[END_REF]. It remains unclear, however, whether the changes in TGF-1 levels observed during organismal and cellular ageing are in any way related, or if they should rather be considered as two independent phenomena. Human peritoneal mesothelial cells (HPMC) cover the peritoneal membrane with a fine monolayer that serves many functions. Its anti-wear, lubricating and fibrinolytic properties protect the peritoneum against injury and adhesions. Mesothelial cell-derived mediators control the course of peritoneal inflammation and wound healing. In addition, the mesothelium contributes to the transperitoneal movement of fluid and particulate matter (see [START_REF] Mutsaers | The mesothelial cell[END_REF][START_REF] Yung | Peritoneal mesothelial cell culture and biology[END_REF] for recent reviews). Vast knowledge of HPMC pathobiology has been largely derived from studies with cultured cells. However, the issue of HPMC senescence was addressed only sporadically [START_REF] Thomas | Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells[END_REF]. We have recently analyzed HPMC senescence in a more systematic manner and found a borderline negative correlation between the proliferative lifespan of HPMC in vitro and the calendar age of individuals from whom the cells were isolated [START_REF] Ksiazek | Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16 INK4a -mediated premature senescence[END_REF]. We have also demonstrated that senescence of HPMC in vitro is associated with increased TGF-1 release [START_REF] Ksiazek | Accelerated senescence of human peritoneal mesothelial cells exposed to high glucose: the role of TGF-beta1[END_REF]. In the present study, we have increased the number of tissue donors analyzed to establish more firmly whether there exists a correlation between (i) the calendar age of the donor and the replicative lifespan of HPMC in vitro, (ii) the donors' age and the ability of their HPMC to release TGF-1 in vitro and (iii) the production of TGF-1 and the replicative lifespan of HPMC in vitro.

Materials and methods.

Cell culture

Human peritoneal mesothelial cells (HPMC) were isolated from the pieces of omentum as detailed by Stylianou et al. [START_REF] Stylianou | Isolation, culture and characterization of human peritoneal mesothelial cells[END_REF]. Specimens of omentum were obtained from 49 patients undergoing elective abdominal surgery. Age of the donors ranged from 17 to 77 years. They were not diabetic and had no evidence of peritoneal inflammation and malignancy. The study was approved by the institutional ethics committee and all patients gave their informed consent. Cells were propagated in Earl's-buffered M199 medium (Sigma, St. Louis, MO, USA) supplemented with 10% v/v fetal calf serum (Invitrogen, Karlsruhe, Germany). HPMC were serially passaged until their growth ceased and cells failed to divide within four weeks. The number of cumulative population doublings (CPD) was calculated as described previously [START_REF] Ksiazek | Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16 INK4a -mediated premature senescence[END_REF].

Measurement of transforming growth factor

Statistics

Statistical analysis was performed using GraphPad Prism™ 4.00 (GraphPad Software, San Diego, USA). Correlations were analyzed using the Spearman test. The comparison of TGF-1 release by HPMC from donors below and above 30 years of age was performed with the Mann-Whitney test for unpaired data, and the results were expressed as medians and ranges.

Statistical significance was acknowledged when the P-value was less than 0.05.

Results

In a group of 49 tissue donors, there was a negative correlation between their calendar age and the replicative lifespan of HPMC in culture (Fig 1A). From this cohort, a subgroup of 28 donors aged 17 to 63 was selected for further analysis. The selection was based solely on the availability of cell culture supernatants in quantities allowing reliable measurement of TGF-1. In the subgroup selected, the relationship between the donors' age and the lifespan of HPMC in culture was equally evident (Fig. 1B). Cells from younger individuals (<30 years) reached as many as 7.2 (range 5.1 to 9.6) CPD, compared to 5.6 (range 2.4 to 8.9) CPD reached on average by HPMC from donors above 30 years of age (p=0.0597). Furthermore, we have observed a strong positive correlation between the calendar age of HPMC donors and the production of TGF-1 by these cells at their first passage (Fig. 2). Median values for TGF-1 release for donors below and above 30 years were 25 pg/10 5 cells (range 14 to 99) and 60 pg/10 5 cells (range 20 to 116), respectively (p<0.03). In addition, we have observed that the magnitude of TGF-1 release by first-passage HPMC correlated with the subsequent number of population doublings achieved by HPMC before entering senescence (Fig. 3).

Discussion.

It is not entirely clear how the number of cell divisions in vitro relates to the calendar age of the donor. Examining fibroblasts, Hayflick was the first to propose that these parameters were inversely correlated [START_REF] Hayflick | The limited in vitro lifetime of human diploid cell strains[END_REF]. Since then several studies have shown such a relationship in many cell types, including keratinocytes [START_REF] Rheinwald | Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells[END_REF], endothelial cells [START_REF] Hoshi | Isolation, growth requirements, cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium[END_REF]) and adrenocortical cells [START_REF] Yang | Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells[END_REF].

On the other hand, however, the concept has been met with some criticism when the study, in which health status of the donors and biopsy conditions were carefully controlled, failed to demonstrate the correlation between replicative lifespan of skin fibroblasts and the donors' age [START_REF] Cristofalo | Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation[END_REF]. We have previously characterized replicative senescence of HPMC in vitro and on this occasion we have observed a borderline negative correlation between HPMC replicative lifespan and the donor's age [START_REF] Ksiazek | Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16 INK4a -mediated premature senescence[END_REF]. Since the effect was not entirely certain, we have analyzed more cell preparations to establish more firmly whether or not this relationship exists. Indeed, by increasing the number of subjects examined we have been able to demonstrate clearly the inverse correlation between the lifespan of HPMC and the donors' age. The relationship observed was relatively strong given that biopsy conditions could be controlled only partially. The impact of systemic disease such as diabetes and uremia was excluded, as well as of overt peritonitis and peritoneal carcinomatosis. Obviously, however, the tissue was sampled from patients with abdominal diseases that frequently -like in intraperitoneal adhesions -involved the omentum.

2 (

 2 in cell culture supernatants were measured with a specific enzyme-linked immunosorbent assay (ELISA), which employed an extracellular domain of human TGF-receptor type II fused to the Fc region of human IgG (R&D Systems, Abingdon, UK) as a capture. The multi-well modules (Nunc, Roskilde, Denmark) were coated with this recombinant chimeric protein (100 ng/ml in PBS overnight) and then blocked with 10% Roti®-Block (Carl Roth, Karlsruhe, Germany). The samples and standards of human recombinant TGF-1 (ranging from 31.3 to 2000 pg/ml; R&D Systems) were added in duplicates and incubated at 4°C overnight. Captured molecules were detected after the sequential incubation with biotinylated chicken anti-TGF-1 (50 ng/ml for 2 hours) and streptavidin-horseradish peroxidase solution (both from R&D Systems). The color was developed following the exposure to the substrate solution of tetramethylbenzidine and H 2 O ICN Biomedicals, Irvine, CA, USA). The reaction was stopped with 2 N H 2 SO 4 and the optical density was measured at 450 nm with a reference wavelength of 540 nm. Sensitivity of the assay was calculated to be 15 pg/ml. For the assay the samples were activated by a 15minute incubation with 1 N HCl and subsequently neutralized with 1.2 NaOH / 0.5 M HEPES.

Furthermore, the mesothelial

  cell monolayer is an extremely fragile structure, the integrity of which may easily be jeopardized by routine surgical procedures. Prolonged exposure to air, swabbing, pressure from surgical instruments and sutures, lavages with saline -are all the factors with potentially detrimental impact on HPMC[START_REF] Ryan | Mesothelial injury and recovery[END_REF][START_REF] Von Ruhland | Can artifact mimic the pathology of the peritoneal mesothelium?[END_REF][START_REF] Polubinska | Time to reconsider saline as the ideal rinsing solution during abdominal surgery[END_REF].We have recently shown that the senescent phenotype in HPMC can be induced by TGF-1[START_REF] Ksiazek | Accelerated senescence of human peritoneal mesothelial cells exposed to high glucose: the role of TGF-beta1[END_REF]. We have therefore analyzed TGF-1 release by HPMC in relation to the age of HPMC donor. We found a strong positive correlation in this regard, with earlypassage HPMC from older donors secreting more TGF-1 than cells from younger individuals. Moreover, these TGF-1 levels correlated inversely with the number of HPMC population doublings. One may envisage a scenario in which the percentage of senescent HPMC in the omentum increases with age. Once isolated, these cells produce more TGF-1, which in turn inhibits HPMC growth.The consequences of peritoneal accumulation of aged cells secreting more TGF-1 remain hypothetical. While it is now clear that TGF-1 plays a pivotal role in wound healing, excessive or prolonged TGF-1 expression may lead to increased matrix accumulation and intraperitoneal adhesions and/or fibrosis. In this respect, increased TGF-1 expression was detected in post-operative peritoneal adhesions[START_REF] Holmdahl | Overproduction of transforming growth factor-beta1 (TGF-beta1) is associated with adhesion formation and peritoneal fibrinolytic impairment[END_REF]. Furthermore, by inhibiting HPMC proliferation, TGF-1 may contribute to impaired wound healing seen in the aged.

Fig. 1 .

 1 Fig. 1. Correlation between the calendar age of the omentum donors and the cumulative number of population doublings achieved by HPMC in vitro. (A): results from 49 patients, (B): results from a subgroup of 28 patients, HPMC from whom were further analyzed for TGF-1 release (see text for details).

Fig. 2 .

 2 Fig. 2. Correlation between the calendar age of the omentum donors and the production of TGF-1 by early-passage HPMC.

Fig. 3 .

 3 Fig. 3. Correlation between the basal production of TGF-1 by HPMC at passage early and the cumulative number of population doublings.


Mónica Santos-González

Consuelo Gómez Díaz

Plácido Navas

PhD José Manuel Villalba

MODIFICATIONS OF PLASMA PROTEOME IN LONG-LIVED RATS FED ON A COENZYME Q 10 -SUPPLEMENTED DIET

Keywords:

Dietary coenzyme Q 10 prolongs lifespan of rats fed on a PUFAn-6-enriched diet.

Our aim was to analyze changes in the levels of plasma proteins of rats fed on a PUFAn-6 plus coenzyme Q 10 -based diet. This approach could give novel insights into the mechanisms of lifespan extension by dietary coenzyme Q 10 in the rat. Serum albumin, which decreases with aging in the rat, was significantly increased by coenzyme Q 10 supplementation both at 6 and 24 months. After depletion of the most abundant proteins by affinity chromatography, levels of less abundant plasma proteins were also studied by using 2D-electrophoresis and MALDI-TOF mass fingerprinting analysis. Our results have shown that lifelong dietary supplementation with coenzyme Q 10 induced significant decreases of plasma hemopexin, apolipoprotein H and interalpha-inhibitor H4P heavy chain (at both 6 and 24 months), preprohaptoglobin, fibrinogen γ-chain precursor, and fetuin-like protein (at 6 months), and alpha-1antitrypsin precursor and type II peroxiredoxin (at 24 months). On the other hand, coenzyme Q 10 -supplementation resulted in significant increases of serine protease inhibitor 3, vitamin D-binding protein (at 6 months), and Apo A-I (at 24 months). Our results support a beneficial role of dietary coenzyme Q 10 decreasing oxidative stress and cardiovascular risk, and modulating inflammation during aging.

INTRODUCTION

Aging is an endogenous process characterized by a progressive loss of function with decreased fertility, increased mortality and augmented susceptibility to the onset of age-related diseases [START_REF] Kirkwood | Why do we age?[END_REF]. Among the different theories that have been elaborated over the past 35 years to explain the causes of aging, the oxidative damage theory of aging is one of the most accepted today. This theory postulates that aging is the result of the accumulation of cellular and tissue damage caused by the continuous and endogenous generation of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals, mainly of a mitochondrial and plasma membrane origins [START_REF] Wallace | Mitochondrial genetics: a paradigm for aging and degenerative diseases?[END_REF][START_REF] Barja | Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals[END_REF][START_REF] Sohal | Oxidative stress hypothesis of aging[END_REF][START_REF] Sohal | Mechanisms of aging: an appraisal of the oxidative stress hypothesis[END_REF][START_REF] Harman | The free radical theory of aging[END_REF][START_REF] Hekimi | Genetics and the specificity of the aging process[END_REF][START_REF] Barja | Free radicals and aging[END_REF][START_REF] Schriner | Extension of murine lifespan by overexpression of catalase targeted to mitochondria[END_REF]. This theory has gained a significant support with the recent demonstration that overexpression of mitochondria-targeted catalase prevents ROS production and increases life span in transgenic mice [START_REF] Schriner | Extension of murine lifespan by overexpression of catalase targeted to mitochondria[END_REF], and an increase of mitochondrial biogenesis with higher bioenergetic efficiency in supporting calorie restriction effect on lifespan extension [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF].

Currently there is a great interest in the development of anti-aging therapies.

Since oxidative stress in involved in aging, it is considered that any strategy to reduce oxidative damage would have significant anti-aging effects [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]. From a practical and therapeutic point of view, dietary modifications may be a feasible approach to counteract the deleterious effects of prooxidants during aging. The impact of diet and dietary constituents on aging and age-associated diseases is well documented, mostly regarding calorie restriction and antioxidant nutrient supplementation [START_REF] Finkel | Oxidants, oxidative stress and the biology of aging[END_REF][START_REF] Sohal | Mechanisms of aging: an appraisal of the oxidative stress hypothesis[END_REF][START_REF] Finkel | A toast to long life[END_REF][START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF].

Coenzyme Q (CoQ, ubiquinone) (2,3-dimethoxy-5-methyl-6-polyprenyl-1,4benzoquinone) is a prenylated benzoquinone that is present in all cellular membranes and in high and low density lipoproteins as well. CoQ participates as an electron carrier in both mitochondrial and extramitochondrial membranes and, in its hydroquinone form (ubiquinol), it is a powerful antioxidant [START_REF] Turunen | Metabolism and function of coenzyme Q[END_REF]. It has been recently demonstrated that lifelong supplementation with CoQ 10 produced significant increases of mean and maximum life span in rats fed a polyunsaturated fatty acids (PUFA)enriched diet [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]. Compared to rats fed lifelong on a PUFA-alone diet, animals that were fed on a PUFAn-6 plus CoQ 10 diet showed significantly higher levels of CoQ 10 in liver mitochondria and plasma membrane, less DNA double-strand breaks in blood lymphocytes, lower oxidative stress and improved mitochondrial function in the heart, and enhanced glutathione-dependent antioxidant capacity in liver [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]Ochoa et al., 2005;[START_REF] Bello | Enhanced anti-oxidant protection of liver membranas in long-lived rats fed on a coenzyme Q 10 -supplemented diet[END_REF].

These previous studies were focused to the biochemical analyses of classical antioxidant proteins that have established roles in oxidative stress resistance. However, it is clear that changes in many other proteins, still to be identified, could be also related to the observed increase of longevity in CoQ 10 -supplemented rats. The proteomic approach offers an unbiased way to study changes in protein levels induced by different experimental conditions. This approach has revealed successful for the identification of novel mechanisms of cell responses against oxidative stress [START_REF] Keightley | Proteomic analysis of oxidative stressresistant cells: a specific role for aldose reductase overexpression in cytoprotection[END_REF]. A major challenge of proteome research is detecting clinically useful biomarkers of disease, treatment response and aging [START_REF] Anderson | Candidate-based proteomics in the search for biomarkers of cardiovascular disease[END_REF]. Serum and plasma offer particularly promising resources for biomarker discovery because collection of these samples is minimally invasive and the blood is thought to contain the majority of protein constituents found in the body [START_REF] Echan | Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma[END_REF].

To better understand the possible mechanisms of lifespan extension by CoQ 10 in rats fed a PUFA-enriched diet, we have initiated a proteomic study to identify plasma proteins whole levels are altered due to CoQ 10 -supplementation during aging. Our results support a potential beneficial role of CoQ 10 decreasing oxidative stress and cardiovascular risk, and modulating inflammation during aging

MATERIALS AND METHODS

Animals, diets and blood samples

Animals and diets used in this work were the same as those utilized in previous works carried out by the group of Prof. José Mataix at the Universidad de Granada, Spain [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]Ochoa et al., 2005) and by ours at the University of Córdoba, Spain [START_REF] Gómez-Díaz | Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues[END_REF][START_REF] Bello | Enhanced anti-oxidant protection of liver membranas in long-lived rats fed on a coenzyme Q 10 -supplemented diet[END_REF]. Briefly, groups of 10 male Wistar rats (Rattus norvegicus) initially weighing 80-90 g were housed under standard conditions and maintained on a 12 h light/12 h darkness cycle and at a temperature of 22º C ± 1º C. Animals were provided ad libitum access to water and a fresh semisynthetic and isoenergetic diet with 8% fat (containing 61% of total fatty acids as PUFA) over a 24 months period. Animals were randomly assigned into two groups which differed in the dietary supplementation or not with CoQ 10 (intake of 0.7 mg/kg/day). Eight rats per group were sacrificed by cervical dislocation and further decapitation at 6 months (young) or 24 months (old) from the start of the experiment.

Blood was collected by decantation onto glass tubes coated with lithium heparin and plasma was then recovered by centrifugation. No evidence of haemolysis was observed.

Samples were immediately frozen in liquid nitrogen and then stored at -80ºC for later analysis. Animals were handled at the facilities of the University of Granada (Spain) according with guidelines of Spanish Society for Laboratory animals and the experiment had the approval of the Ethical Committee of the University of Granada.

Coenzyme Q determinations

Plasma samples (about 50 µl) were mixed with an equal volume of 2% SDS.

Afterwards, four volumes of 95% ethanol-5% isopropanol were added and the mixture was vigorously vortexed. CoQ was recovered from SDS-alcoholic solution by extraction with ten volumes of hexane. After hexane evaporation under vacuum, the lipid extract was dissolved in ethanol and used for quantification of CoQ 9 and CoQ 10 by reversed-phase HPLC. Separation was accomplished at 1 ml/min with a C18 column (5µm particle, 5 x 0.45 cm) and a mobile phase composed of a mixture of methanol and npropanol (1:1) containing lithium perchlorate (2.12 g/L). Monitoring was carried out with a Coulochem II electrochemical detector (ESA, Chelmsford, MA) fitted with a Model 5010 analytical cell with the electrodes set at potentials of -500 mV and +300 mV. Quinones were detected from the signal obtained at the second electrode.

Concentrations were calculated by integration of peak areas and comparison with external standards [START_REF] Bello | Enhanced anti-oxidant protection of liver membranas in long-lived rats fed on a coenzyme Q 10 -supplemented diet[END_REF]. Since CoQ 9 is the predominant CoQ homolog in the rat, CoQ 10 supplementation was assessed by a decrease in the CoQ 9 /CoQ 10 ratio.

One-dimensional SDS-electrophoresis

For protein analysis, plasma samples were pooled according to supplementation group and duration of dietary intervention (n=8). Protein content was measured by the dye binding method with bovine γ-globulin as standard. Levels of albumin in plasma were assessed by quantifying the intensity of the 66 kDa band in gels obtained after separation of 3 µg plasma protein by SDS-discontinuous electrophoresis (10% polyacrylamide). Electrophoresis was developed at a constant voltage of 200 V using a MINI-PROTEAN 3 Cell (BioRad) and gels were stained with colloidal Coomassie stain (BioSafe, Bio-Rad). Gel images were obtained using a GS-800 Calibrated Densitometer (Bio-Rad) and quantification of the albumin band was carried out with Quantity One software (Bio-Rad). Results were expressed as optical density (OD) x mm 2 .

Proteomic analysis

Aliquots of pooled samples (60 µl) were depleted from albumin and immunoglobulins using commercial disposable affinity columns (Proteoextract Albumin/IgG removal kit, Calbiochem) according to manufacturer´s recommendations.

Afterwards, lipids and salts were removed from the samples to improve resolution of 2D gels. Delipidation and salt depletion were accomplished by mixing one volume of plasma sample (that had been depleted from albumin and IgG) with four volumes of methanol. After thoroughly mixing with the vortex, one volume of chloroform was added, the mixture was vortexed again and then, three volumes of water (HPLC grade) were incorporated. After centrifugation at 12,500 RPM for 5 min at 4ºC, the upper phase was discarded. Three volumes of methanol were then added and the resultant mixture was vortexed again and centrifuged. The supernatant was discarded and the pellet containing precipitated plasma protein was air-dried for 5-10 min. About 0.7 mg of delipidated and salt-depleted plasma proteins were dissolved in 125 µl of sample solubilization solution (8.0 M urea, 50 mM DTT, 4% CHAPS, 0.2% carrier ampholytes and 0.0002% bromophenol blue). IPG strips (7cm long, pH range 4-7, BioRad, Hercules, CA, USA) were then passively rehydrated for 13h with the protein lysate.

Isoelectrofocusing was carried out at 20 ºC, using a PROTEAN IEF system (Bio-Rad).

Focusing was started with a conditioning step of 250 V for 20 min, followed by a voltage ramping step to 4,000 V for 2 hours, and a final focusing step of 10,000 Vh.

Thereafter, the strips were soaked in equilibration solution (50 mM Tris-HCl, pH 8.8, 6 M urea, 20% glycerol and 2% SDS) containing 20 mg/ml DTT for 10 min and then in the same solution containing 25 mg/ml iodoacetamide for an additional period of 10 min.

Second dimension was carried out in 12.5% polyacrylamide gels at a constant voltage of 200 V using a MINI-PROTEAN 3 Cell (BioRad). Proteins in 2-D gels were fixed with 40% methanol-10% acetic acid for 1 h at room temperature. Gels used for peptide identification and analysis were stained with colloidal Coomassie stain (BioSafe, Bio-Rad). Gel images were obtained using a GS-800 Calibrated Densitometer (Bio-Rad) and analyzed with the PDQuest 7.0 2-D analysis software (BioRad). To accurately compare spot quantities between gels, image spot quantities were normalized dividing the raw quantity of each spot in a gel by the total quantity of all the valid spots in that gel. Fluctuations in the protein expression levels among the control maps were monitored in densitometric analysis, and a coefficient of variation (CV) was generated for the mean value of each spot in order to better define changes in protein expression due to CoQ supplementation. Only those changes that were statistically significant and that involved at least two-fold increases or half decreases of protein levels were considered in our study.

Protein spots of interest were manually excised from preparative gels, transferred to eppendorf tubes and automatically digested with trypsin according to standard protocols in a ProGest station (Genomic Solutions). Spots were destained twice for 30 min at 37ºC with 200 mM ammonium bicarbonate/ 40% acetonitrile, and dehydrated for 5 minutes with pure acetonitrile. Then, spots were rehydrated with 25mM ammonium bicarbonate for 5 min and dehydrated again for 10 min with pure acetonitrile. After that, gel plugs were dried out for 4 h at room temperature. Then, 10 µL of 12.5 ng/µL trypsin solution in 25 mM ammonium bicarbonate were added to each sample. Gels were allowed to rehydrate for 10 min at room temperature with the enzyme. The temperature was then adjusted to 37ºC and digestion was carried out for

RESULTS

Animals supplemented on CoQ 10 reached a significantly higher mean (11.7%) and maximum (24%) lifespan [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]. Levels of CoQ 9 and CoQ 10 were quantified by HPLC in plasma samples obtained from rats fed for 6 or 24 months either with a PUFAn-6-alone or a PUFAn-6 plus CoQ 10 (0,7 mg/kg/ day) diet. As depicted in Figure 1, CoQ 9 /CoQ 10 ratio was 1.8 ± 0.48 in plasma of 6 months old rats fed on the PUFAn-6-alone diet, and this value increased to 3.4 ± 0.54 at 24 months. Feeding animals with a CoQ 10 -supplemented diet produced a significant decrease of the plasma CoQ 9 /CoQ 10 ratio both at 6 and 24 months.

Once we demonstrated that our lifelong supplementation schedule based on a low CoQ 10 concentration led to a good adaptation pattern, we studied how the CoQ 10enriched diet affected the levels of plasma proteins. Aging is associated with changes in the concentration of various serum proteins, and one of the most consistent findings is an age-dependent decrease in the level of albumin and increase in the content of some globulins [START_REF] Chlebovská | Favourable effect of K, Mg aspartate on serum proteins in aging rats[END_REF][START_REF] Kim | Age-related changes in redox status of rat serum[END_REF]. Thus, we first studied putative changes of plasma albumin levels as a function of aging and/or dietary CoQ 10supplementation. As depicted in Figure 2A andB, in our study we also observed a decrease of albumin levels in aged (24 months) compared to young (6 months) animals, which is consistent with previous observations. Although plasma albumin decreased with aging both in the control and in CoQ 10 -supplemented group, dietary administration of CoQ 10 increased significantly albumin at both ages in such a way that albumin levels at 24 months in the CoQ 10 -group were equivalent to those found at 6 months in the control group.

To obtain information about putative changes in less abundant plasma proteins that could be elicited by dietary CoQ 10 -supplementation, we carried out a proteomic study of rat plasma after depletion of albumin and IgG. At least four replicates were performed for all samples, similar protein spot patterns being obtained within each sample type. Total numbers of protein spots identified in Coomassie blue-stained gels were: 244 (young rats, PUFAn-6-alone diet), 218 (young rats, PUFAn-6 plus CoQ 10 diet), 262 (old rats, PUFAn-6-alone diet) and 232 (old rats, PUFAn-6 plus CoQ 10 diet).

Near 99% of all spots were matched on duplicate gels (the gels of the sample were produced, assayed, and processed in parallel), and the intensity of the same spot from different gels showed no significant change. A representative gel showing those proteins whose levels in plasma were altered in CoQ 10 -supplemented animals is depicted in Figure 3. Only proteins that increased or decreased by a factor of at least two were considered in this study. This gel is annotated to show the location of spots excised and identified by peptide mass fingerprinting and the proteins identified are listed in Table I. The predicted molecular weight and pI of protein spots were used to corroborate protein identifications. Details of 2D gels showing those proteins whose levels in plasma from CoQ 10 -supplemented animals were significantly (p<0.05) altered compared with their corresponding controls are depicted in Figure 4.

Preprohaptoglobin was significantly decreased (3-5 times) in the CoQ 10 group at 6 months and hemopexin was significantly decreased (about 2-fold) at both 6 and 24 months. Type II peroxiredoxin levels decreased (3-4 fold) at 24 months in the CoQ 10group. Plasma apolipoprotein H (β 2 -glycoprotein I, β 2 GPI) was decreased in the CoQ 10group at both 6 and 24 months. Levels of β 2 GPI were halved at 6 months in animals fed with the CoQ 10 -diet, and the protein was almost undetectable at 24 months. Levels of another apolipoprotein, Apo A-I were also altered by dietary CoQ 10 , although in this case the protein was increased in the CoQ 10 -group (two-fold at 24 months). Fibrinogen γ-chain precursor was decreased three times by dietary CoQ 10 at 6 months, although no significant effect was observed at 24 months. Inter-alpha-inhibitor H4P heavy chain was halved at 6 and 24 months in the CoQ 10 -group. Two protein spots that were identified as inter-alpha-inhibitor H4P heavy chain fragments were also consistently found in the plasma proteome of control animals, and these spots were strongly decreased (more than ten times) in the CoQ 10 -group at 6 months (see Figures 3 and4). Levels of alpha-1-antitrypsin precursor were halved by CoQ 10 -supplementation at 24 months, although no significant effects were observed at 6 months. Serine protease inhibitor 3 was increased more than ten times at 6 months in the CoQ 10 -group, although no significant alterations were observed at 24 months. Finally, we observed changes elicited by dietary CoQ 10 in vitamin D-binding protein (which increased at 6 months in the CoQ 10 group) and fetuinlike protein (which was strongly decreased by CoQ 10 -supplementation at 6 months).

DISCUSSION

Rat plasma and tissues contain both CoQ 9 and CoQ 10 as the endogenous CoQ forms [START_REF] Turunen | Metabolism and function of coenzyme Q[END_REF]. Thus, we used the decrease of CoQ 9 /CoQ 10 ratio as an estimation of supplementation with dietary CoQ 10 . This ratio may be of importance for aging studies because longevity correlates positively with CoQ 10 levels, and negatively with CoQ 9 levels in different mammal species [START_REF] Lass | Mitochondrial ubiquinone homologues, superoxide radical generation, and longevity in different mammalian species[END_REF]. Plasma CoQ 9 /CoQ 10 ratio was significantly decreased in the CoQ 10 -group indicating an effective supplementation with exogenous CoQ 10 regardless of age. This is very important because any putative benefit of dietary supplementation with CoQ 10 might be maintained through the whole life [START_REF] Quiles | Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet[END_REF]. According to our studies [START_REF] Gómez-Díaz | Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues[END_REF][START_REF] Bello | Enhanced anti-oxidant protection of liver membranas in long-lived rats fed on a coenzyme Q 10 -supplemented diet[END_REF] this work) and those from others [START_REF] Sugiyama | Preservation of mitochondrial respiratory function by coenzyme Q10 in aged rat skeletal muscle[END_REF][START_REF] Forsmark-Andrée | Lipid peroxidation and changes in the ubiquinone content and the respiratory chain enzymes of submitochondrial particles[END_REF][START_REF] Matthews | Coenzyme Q 10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects[END_REF][START_REF] Lönnrot | The effects of lifelong ubiquinone Q 10 supplementation on the Q 9 and Q 10 tissue concentrations and life span of male rats and mice[END_REF][START_REF] Kwong | Effects of coenzyme Q 10 administration on its tissue concentrations, mitochondrial oxidant generation, and oxidative stress in the rat[END_REF], plasma and liver show a better adaptation pattern to dietary CoQ 10 than organs like brain or heart. In accordance, significant decrease in CoQ 9 /CoQ 10 ratio was also found in liver samples obtained from the same animals [START_REF] Bello | Enhanced anti-oxidant protection of liver membranas in long-lived rats fed on a coenzyme Q 10 -supplemented diet[END_REF]. Calorie restriction also decreases CoQ 9 /CoQ 10 ratio in rats and mice liver [START_REF] De Cabo | Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver[END_REF][START_REF] López-Lluch | Mouse liver plasma membrane redox system activity is altered by aging and modulated by calorie restriction[END_REF].

The actual concentration of plasma/serum proteins is a result of metabolic processes, maintaining homeostasis. With advancing age a progressive weakening of these factors occurs which results in the unfavourable effect on the whole protein turnover with subsequent changes in the concentration of individual proteins [START_REF] Chlebovská | Favourable effect of K, Mg aspartate on serum proteins in aging rats[END_REF]. Lower levels of serum albumin are associated with increased risk of all-cause and cardiovascular mortality as well with coronary heart disease and stroke incidence in humans. In addition, albumin is a negative acute-phase reactant [START_REF] Shaper | Serum albumin and risk of stroke, coronary heart disease, and mortality: the role of cigarette smoking[END_REF]. It has been proposed that the aging-related decrease of serum albumin in the rat may be partially responsible for decreased serum thiol levels and a shifted redox serum balance towards oxidation during aging [START_REF] Chlebovská | Favourable effect of K, Mg aspartate on serum proteins in aging rats[END_REF]. In addition to its antioxidant role inhibiting lipid peroxidation and regenerating α-tocopherol in serum lipoproteins [START_REF] Stocker | Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does α-tocopherol[END_REF][START_REF] Mohr | Dietary supplementation with coenzyme Q 10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation[END_REF], dietary CoQ 10 supplementation might further enhance antioxidant capacity of blood plasma by increasing albumin levels.

Besides albumin, proteins which levels were altered as a consequence of dietary CoQ 10 supplementation fall into the following functional groups: i. heme metabolism: preprohaptoglobin and hemopexin; antioxidant protection: peroxiredoxin 2 (and preprohaptoglobin and hemopexin as well); ii. lipid metabolism and transport: apolipoprotein H and apolipoprotein A1; iii. blood coagulation: fibrinogen γ-chain precursor; iv. acute-phase proteins: inter-alpha-inhibitor H4P, serine protease inhibitor 3 (and preprohaptoglobin and hemopexin as well); and ostogenesis: vitamin D-binding protein and fetuin-like protein.

Preprohaptoglobin was decreased in the CoQ 10 group at 6 months, and hemopexin was decreased at both 6 and 24 months. Haptoglobin is a serum protein that binds hemoglobin and allows its metabolization in the liver. Haptoglobin also functions as an antioxidant because it prevents oxidative damage mediated by free haemoglobin [START_REF] Melamed-Frank | Structure-function analysis of the antioxidant properties of haptoglobin[END_REF]. Haptoglobin has been shown to be a potential marker of cancer [START_REF] Thadikkaran | Recent advances in blood-related proteomics[END_REF]. Hemopexin is a plasma glycoprotein with a high binding affinity to heme, which is then transported to the liver. The presence of heme in plasma is due to oxidation of haemoglobin, which is released during the enucleation of erythroblasts or in states of intramedullary or intravascular haemolysis. In humans, haemolysis is associated with several pathologic conditions such as reperfusion injury and/or ischemia. Hemopexin also inhibits some of the oxidative reactions catalyzed by free heme and may protect tissues from oxidation under conditions in which heme release occurs [START_REF] Barnard | Protective role of hemopexin on heme-dependent lung oxidative stress[END_REF][START_REF] Tolosano | Defective recovery and severe renal damage after acute hemolysis in hemopexin-deficient mice[END_REF]. Previous studies have documented that levels of these two proteins of the acute phase increase in rat serum with aging, and their levels were further increased in animals whose aging was accelerated by irradiation [START_REF] Chlebovská | Favourable effect of K, Mg aspartate on serum proteins in aging rats[END_REF]. Interestingly, these aging-related changes were attenuated by dietary CoQ 10 .

Type II peroxiredoxin was significantly decreased at 24 months in the CoQ 10group. Peroxiredoxins are a family of thiol-specific peroxidases found in all species that can reduce a wide range of peroxides, including toxic phospholipid hydroperoxides, thus playing a major role in antioxidant protection [START_REF] Wong | Peroxiredoxin-null yeast cells are hypersensitive to oxidative stress and are genomically unstable[END_REF][START_REF] Tölle | Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells[END_REF]. Although peroxiredoxin levels decrease with aging in rodents, which could be indicative of compromised antioxidant protection with aging [START_REF] Cho | Differential expression of the liver proteome in senescence accelerated mice[END_REF], this protein is induced by oxidative stress conditions provoked by alimentary vitamin E deficiency [START_REF] Tölle | Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells[END_REF]. Thus, it is plausible to interpret that decreased levels of peroxiredoxin in plasma obtained from CoQ 10 -supplemented animals (as well as decreased hemopexin and preprohaptoglobin, see above) may be indicative of a better antioxidant status compared to non supplemented animals.

β 2 GPI was decreased in the CoQ 10 -group at both 6 and 24 months. β 2 GPI is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome, and has been shown to regulate the activation of plasminogen. Although the exact physiological role of β 2 GPI is not well understood [START_REF] Lopez-Lira | The role of β 2 -glycoprotein I (β 2 GPI) in the activation of plasminogen[END_REF], it is known that β 2 GPI forms complexes with oxidized LDL, which represent a common metabolic product relevant to atherogenesis and a risk factor or an indirect but significant contributor for atherothrombotic complications in autoimmune patients [START_REF] Matsuura | Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis[END_REF]. Levels of another apolipoprotein, Apo A-I were also altered by dietary CoQ 10 , although in this case the protein was increased in the CoQ 10 -group (twofold at 24 months). It is important to note that high concentrations of Apo A-I are associated with lower mortality and myocardial infarction five years after coronary artery bypass graft surgery in humans [START_REF] Anderson | Candidate-based proteomics in the search for biomarkers of cardiovascular disease[END_REF]. Interestingly, dietary supplementation with α-tocopherol, another lipophilic antioxidant as CoQ 10 , also elevates plasma Apo A-I levels in healthy humans [START_REF] Aldred | Alpha tocopherol supplementation elevates plasma apolipoprotein A1 isoforms in normal healthy subjects[END_REF]. Levels of fibrinogen γ-chain precursor were decreased by dietary CoQ 10 at 6 months, although no significant effect was observed at 24 months. Fibrinogen is strongly, consistently, and independently related to cardiovascular risk [START_REF] Koenig | Fibrin(ogen) in cardiovascular disease: an update[END_REF].

Interestingly, many experimental studies have indicated that exogenous CoQ 10 treatment may be beneficial in cardiovascular pathologies [START_REF] Thomas | Dietary cosupplementation with vitamin E and coenzyme Q 10 inhibits atherosclerosis in apolipoprotein E gene knockout mice[END_REF], and a relation exists between low plasma CoQ 10 concentration and coronary artery disease in humans [START_REF] Yalcin | Coenzyme Q 10 concentrations in coronary artery disease[END_REF]. Changes in plasma proteins elicited by dietary CoQ 10 could explain, at least partially, these beneficial effects on the cardiovascular system.

Besides hemopexin and preprohaptoglobin (see above), other acute phase proteins were also decreased by dietary CoQ 10 -supplementation. Inter-alpha-inhibitor H4P belongs to a family of plasma protease inhibitors of hepatic origin, collectively termed as the inter-alpha-inhibitor family. Levels of inter-alpha-inhibitor H4P increase significantly under acute inflammatory conditions [START_REF] Daveau | Hepatic and extra-hepatic transcription of inter-alpha-inhibitor family genes under normal or acute inflammatory conditions in rat[END_REF]. Alpha-1antitrypsin precursor is also considerably increased during the acute-phase response [START_REF] Sandford | Alpha1-antitrypsin genotypes and the acute-phase response to open heart surgery[END_REF]. Although inter-alpha-inhibitor H4P heavy chain and alpha-1antitrypsin precursor were decreased significantly by CoQ 10 -supplementation, serine protease inhibitor 3, another acute-phase protein, was increased more than ten times at 6 months in the CoQ 10 -group, although no significant alterations were observed at 24 months. Changes in the levels of acute-phase proteins due to CoQ 10 -supplementation indicate that dietary CoQ 10 may play an important role in the regulation of inflammation. In accordance, a previous study has documented that co-supplementation with vitamin E and CoQ 10 reduces circulating markers of inflammation in baboons [START_REF] Wang | Cosupplementation with vitamin E and coenzyme Q 10 reduces circulating markers of inflammation in baboons[END_REF]. Finally, we observed changes elicited by dietary CoQ 10 in two plasma proteins involved in bone metabolism, vitamin D-binding protein and fetuin. Vitamin D-binding protein is an abundant serum glycoprotein which transports vitamin D sterols and binds to monomeric actin [START_REF] Cooke | Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA[END_REF]. On the other hand, fetuin is a serum protein which inhibits osteogenesis and may promote artery calcification [START_REF] Binkert | Regulation of osteogenesis by fetuin[END_REF][START_REF] Price | Serum levels of the fetuin-mineral complex correlate with artery calcification in the rat[END_REF]. In addition, fetuin concentrations are strongly associated with metabolic syndrome and an atherogenic lipid profile in humans [START_REF] Ix | Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study[END_REF]. The fact that dietary CoQ 10 increased vitamin D-binding protein and decreased fetuin-like protein might indicate that CoQ 10 status might also influence osteogenesis. It is noteworthy that rats fed lifelong on the PUFAn-6 plus CoQ 10 diet showed increased bone mineral density compared to their control counterparts (Ochoa et al., 2005). This effect could be also related to the role of serum CoQ 10 as antioxidant because it has been reported that oxidative stress levels are negatively associated with bone mineral density, and antioxidant levels are lower in osteoporosis patients [START_REF] Basu | Association between oxidative stress and bone mineral density[END_REF][START_REF] Maggio | Marked decrease in plasma antioxidants in aged osteoporotic women: results of a cross-sectional study[END_REF].

Overall, our proteomic study strongly supports that increase of lifespan provided by a CoQ 10 -enriched diet in the rat is related to decreased oxidative stress, and less cardiovascular risk and inflammation during aging.

LEGENDS FOR FIGURES

Figure 1. CoQ 9 /CoQ 10 ratio in plasma. Rats were fed for 6 or 24 months with a basal semisynthetic PUFAn-6-rich diet, either supplemented or not with CoQ 10 (intake of 0.7 mg/kg/day). Lipids were extracted from plasma and CoQ 10 and CoQ 9 quantified by HPLC. Values are means with standard errors (n=8). Significant differences between diets for a given feeding period are indicated with asterisks (*, P < 0.05; ***, P < 0.001). Statistically significant differences between feeding periods for a given diet are also indicated (a, statistically significant differences with 6 months, P < 0.05; b, statistically significant differences with 6 months, P < 0.001). We demonstrate that our dietary protocol increases CoQ 10 levels (measured as a decrease of CoQ 9 /CoQ 10 ratio in plasma) both at 6 and 24 months. ). Values in (B) are means with standard errors (n=8). Significant differences between diets for a given feeding period are denoted with asterisks (**, P < 0.01). Statistically significant differences between feeding periods for a given diet are also indicated (a, statistically significant differences with 6 months, P < 0.05; b, statistically significant differences with 6 months, P < 0.05). Our results demonstrate that albumin levels decrease with aging, whereas dietary CoQ 10 supplement increases albumin both at 6 and 24 months.

  12 h. Finally, digestion volume was recovered and peptides were extracted with 10 µL of 0,5% trifluoroacetic acid for 15 min. Peptides of each sample were analyzed in a 4700 Proteomics Station (Applied Biosystems, USA) in automatic mode. After drying, samples were analyzed in the m/z range 800 to 4000 Da with an accelerating voltage of 20 kV. Spectra were internally calibrated with peptides from trypsin autolysis. Proteins were identified by PMF and confirmed by MALDI-TOF/MS (APPLIED BIOSYSTEMS mod 4700). MASCOT searching engine (Matrixscience, UK) was used for protein identification over the database (MSDB). Only those proteins with MOWSE score above 88 (p, 0.00005) were accepted as valid identifications.

Figure 2 .

 2 Figure 2. Effect of dietary CoQ 10 supplement on albumin levels. Rats were fed for 6 or 24 months as described in the legend of figure 1 and equal amounts of plasma proteins (3 µg) were separated by SDS-PAGE (10% acrylamide). Gels were stained with colloidal Coomassie stain and albumin was then quantified by densitometric analysis of the 66 kDa band. A) Two representative samples from each condition are depicted. B) Albumin quantification by densitometry (OD x mm 2 ). Values in (B) are means with standard errors (n=8). Significant differences

Figure 3 .

 3 Figure 3. A representative 2D gel electrophoresis image showing the most abundant proteins of albumin and IgG-depleted rat plasma. The scanned image of the coomassie-stained gel was used to detect and compare protein spots. Those

Figure 4 .Function

 4 Figure 4. Close-up sections of protein spots of proteins whose levels are significantly altered in plasma of rats fed on a CoQ 10 -enriched diet. The animal age when changes were observed is denoted at the right. A) This panel depicts those proteins whose levels were decreased in the CoQ 10 -group. a) hemopexin precursor, b) apolipoprotein H precursor, c) preprohaptoglobin precursor, d) interalpha-inhibitor H4P heavy chain, e) inter-alpha-inhibitor H4P heavy chain (fragments), f-I) fetuin-like protein, f-II) fibrinogen γ-chain precursor, g) alpha-1antitrypsin precursor, h) type II peroxiredoxin. B) This panel depicts those proteins whose levels were increased in the CoQ 10 -group. i) vitamin D-binding protein precursor, j) serine protease inhibitor 3, k) Apo A-I.

ACKNOWLEDGEMENTS

Supported by Grant No. BFU2005-00137/BMC (Spanish Ministerio de Educación y Ciencia), and CVI-276 (Junta de Andalucía). M.S. and C. G.-D. were supported by CVI-276.

Antioxidant protection

Peroxirredoxin 2 (Thioredoxin peroxidase 1) 12,a)