Several factors are involved in the complex process of human skin ageing. Chronological ageing, which is genetically programmed and unavoidable, and extrinsic ageing, which is induced by combined environmental factors and results in a more or less rapid and severe phenotype.
Skin ageing in humans is clinically correlated with loss of elasticity and architectural regularity, i.e. dry, unblemished, lax, finely wrinkled, atrophic skin with a transparent quality.1,2 Mechanisms responsible for ageing have been thoroughly investigated. Loss of cellular homeostasis seems to contribute to the ageing phenomenon initiation. Studies on chronically aged skin models have shown that senescence–associated changes include DNA repair and stability, cell cycle and apoptosis, mitochondrial function, ubiquitin-induced proteolysis.1,3 A depressed metabolism, associated with a mitochondrial function decline, is an hallmark of aged cells.4,5 Alteration of the dermal compartment plays a key role in skin’s biomechanical properties modification and can also affect epidermis homeostasis. Aged dermis is characterised by extracellular matrix general disorganisation, atrophy, decreased cellularity (especially of fibroblasts), in connection with decreased cell proliferation.6,7 In addition, an imbalance between anabolism and catabolism of extracellular matrix (ECM) components has been reported for aged dermis. Accordingly, as a consequence of the natural ageing process, fibroblasts synthesise less collagen and more matrix metalloproteinase 1 (MMP1).7 Interestingly, resulting tissue disorganisation and fragmentation also impacts cell metabolism,8 thus dermal ageing likely results from a vicious circle set up where ECM constituents production decline causes tissue disorganisation that further perturbs fibroblast metabolism. To model the link between fibroblast metabolism and tissue organisation, we have designed an original 3D in vitro system. In this model fibroblasts embedded in collagen gels can bind to and reorganise surrounding collagen fibrils, both mechanisms resulting in a progressive gel contraction. This contraction process results from traction forces, which are a consequence of cell migration and morphology modifications as interactions between fibroblasts and matrix are taking place.9 An optimal cellular metabolism is needed in order to synthesise proteins involved in these mechanisms. To mimic age-related metabolic decline, lattices were serum-deprived. Alistin active molecule, Decarboxycarnosine (DC), an antioxidant and antiglycation peptidomimetic naturally present in several mammalian tissues including skin were tested in this model.
Experimental model
Dermal equivalents, also called fibroblastspopulated collagen lattices (FPCL), were prepared according to Bell’s model, adding low population doubling human dermal fibroblasts (HDF) to a neutralised solution of bovine collagen type I, and incubating Petri dishes at 37°C/5% CO2. HDF interaction with collagen fibres and matrix remodelling can be visualised by a progressive contraction (Fig. 1b). Lattices culture medium contained different concentrations of fetal calf serum (FCS): 10%; 5%; 1%. Gel contraction (measurement of lattices diameter), was monitored over a 160 hour period. FCSdeprivation resulted in a delayed and less efficient contraction (Fig. 1a). This model provides clear evidence that fibroblasts metabolic slowdown induced by serum deprivation impairs collagen fibrils organisation into a dense, well-packed gel arrangement. Further investigations were carried out in order to better define parameters driving contraction in this model. In line with previous reports,10 we did not find any relationship between lattice contraction and cell proliferation. Analysis of the proliferation marker Ki67 revealed that for all lattices, at any FCS concentration, most of the fibroblasts were quiescent. As a consequence, FCS deprivation had no measurable effect on cell number within the lattices. Addition of Epidermal Growth Factor in lattice medium did not modulate contraction (data not shown). Incidence of collagen degradation on lattices contraction was estimated by monitoring Matrix Metalloproteinases (MMPs) expression levels. MMPs are zinccontaining proteinases that specifically degrade ECM constituents, and MMP-1 production is increased in aged and photoaged skin.7,11 We found no correlation between FCS concentration and MMP-1 amounts released in lattices media. Deregulated catabolic process does not seem to account for serum deprivationdriven contraction decrease. Actually, addition of the broad spectrum MMPs inhibitor, Marimastat, did not significantly modify lattice contraction (data not shown). Taken together, these results support a limited involvement of MMPs in the contraction process for this model. Finally, since ROS-mediated dermal equivalent contraction has been reported,12 the effect of serum deprivation on intracellular ROS formation was examined by flow cytometry (CMDCF-DA fluorescent probe). We found no significant increase of intracellular ROS in HDF cultivated in 1% FCS medium compared to 10% FCS medium (Fig. 2).
Decarboxycarnosine effect on fibroblasts tensile properties
Decarboxycarnosine (DC), is a small antioxidant and anti-glycation molecule naturally present at low concentration in skin and other mammalian tissues. DC is a peptidomimetic that shares high structural homology with the ubiquitous dipeptide L-carnosine (Fig. 3). Being a peptidomimetic, DC is much less sensitive to cutaneous proteinase and thus more bioavailable than L-carnosine.13,14 Addition of DC to serum-deprived dermal equivalents enhanced lattice contraction (Fig. 4). This effect was only noticeable when cells were metabolically depressed, and DC had no effect on contraction when added to dermal equivalents maintained in a medium with optimum serum concentration (10% FCS). Since our model system is responsive to TGF?1 (data not shown), a growth factor known to enhance fibroblasts transdifferentiation into myofibrolasts endowed with increased tensile properties,15 we investigated DC ability to induce HDF trans-differentiation. Immunolabelling of ?-smooth muscle-actin (?-sm actin), a myofibroblast-specific intracellular fibril, showed that ?-sm actin expression is almost undetectable in serum deprived (1% FCS) HDF (Fig. 5), and that DC has no effect on the number of ?-sm actin-positive cells. In agreement with published results, under the same conditions TGF?1 induces a strong increase of ?-sm actin positive fibroblasts. The same result was observed in dermal equivalents. We can thus exclude the participation of myofibroblasts in contraction improvement observed in DC-treated lattices. Taken together, these data suggest that DC improvement of serum-deprived dermal fibroblasts tensile capacity results from cellular metabolism stimulation.
Decarboxycarnosine effect on human fibroblasts metabolism
To further investigate DC effect on cellular metabolism, a MTT assay (reflecting enzymatic mitochondrial activity) was performed on HDF cultivated during 24 hours in 1% FCS-medium supplemented with DC 20 mM-50 mM. Cells were counted in parallel. DC dose-dependently increased metabolic transformation of MTT, while no change in cell number was noticeable (Fig. 6). These results account for “metabolic activation”, and not for cytostimulation. It was verified that DC-dependent increased MTT metabolisation was not related to an augmented number of mitochondria. Indeed mitochondrial biogenesis is an early response to stress enabling cells to meet augmented energy needs for repair and proteins neosynthesis.16 Mitochondrial mass, which is indicative of total mitochondria number, was monitored by flow cytometry, using the fluorescent probe MitoGreen (specific labelling of the mitochondria). In our experimental conditions, no mitochondrial mass increase was observed when cells were incubated with increasing concentrations of DC (Fig. 7). Sirtuins are NAD+-dependent deacetylases that have emerged as key players in ageing, stress resistance and metabolic regulation.17 The mitochondrial sirtuine 3 (SIRT3), is a metabolic sensor responsible for basal ATP level maintenance and mitochondrial electron transport regulation.18 Energy production in serum-deprived HDF treated with DC was assessed by immunolabelling SIRT3. In accordance with our previous results, we found that SIRT3 expression is dosedependently overexpressed upon treatment with DC (Fig. 8). We finally examined collagen I production which is critical for dermis organisation maintenance that account for firmness. Ageing is associated with collagen I reduced expression and a loss of intact collagen fibres.6 Monitoring of pro-collagen I secretion by metabolically depressed HDF incubated with DC, confirmed that metabolic stimulation is paralleled with increased collagen production (Fig. 9). On the other hand, we did not detect any measurable modification of the MMP1/TIMP1 ratio in lattice medium. These results support a mitochondrial function improvement relevant for an increased metabolic activity.
Conclusion
FPCL is a good model to study the biology of dermis and cell-matrix interactions. To investigate the influence of fibroblasts metabolism on these interactions, we have set up a model of dermal equivalent based on serum-deprived fibroblasts embedded in a type I collagen matrix. We observed a metabolic slowdown, which occurs during ageing, and in this in vitro model resulted in fibroblast tensile properties decreasing. Treatment with decarboxycarnosine (DC) could limit this decrease, suggesting that DC improved connective tissue remodelling, which is impaired as skin ages. We also showed that DC beneficial effect on lattice contraction may result from a stimulation of fibroblast metabolism. This increased metabolic activity resulted in an augmented expression of the major dermal macromolecule, collagen I, which quantity and integrity is crucial for maintenance of a “young phenotype”. DC has already proved to possess antistress properties, counteracting oxidation and glycation. Here, we demonstrate that DC by its ability to stimulate cellular metabolism, demonstrates additional advantageous properties that may be useful to fight against skin ageing.
References
1 Makrantonaki E, Zouboulis CC. Characteristics and pathomechanisms of endogenously aged skin. Dermatology 2007; 214 (4): 352-60. 2 Callaghan TM, Wilhelm KP. A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part 2: Clinical perspectives and clinical methods in the evaluation of ageing skin. Int J Cosmet Sci 2008; 30 (5): 323-32. 3 Laimer M, Kocher T, Chiocchetti A, Trost A, Lottspeich F, Richter K, Hintner H, Bauer JW, Onder K. Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging. Exp Dermatol 2010; 19: 912–918. 4 Mays PK, McAnulty RJ, Campa JS, Laurent GJ. Age-related alterations in collagen and total protein metabolism determined in cultured rat dermal fibroblasts: age-related trends parallel PPCC those observed in Rat Skin. In Vivo. Int J Biochem Cell Biol 1995; 27: 937-945. 5 Blatt T, Lenz H, Koop U, Jaspers S, Weber T, Mummert C, Wittern KP, Stäb F, Wenck H. Stimulation of skin’s energy metabolism provides multiple benefits for mature human skin. BioFactors 2005; 25: 179-185. 6 Uitto J, Bernstein E. Molecular mechanisms of cutaneous aging: connective tissue alterations in the dermis. J Investig Dermatol Symp Proc 1998; 3: 41–44. 7 Varani J, Warner RL, Gharaee-kermani M, Phan SH, Kang S, Chung J, Wang Z, Datta SC, Fisher GJ, Voorhees JJ. Vitamin A antagonizes decreased cell growth and elevated collagendegrading MMP and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol 2000; 114 (3): 480-486. 8 Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, He T, Varani J, Kang S, Voorhees JJ. Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin. Am J Pathol 2009; 174 (1): 101-14. 9 Dallon JC and Ehrlich HP. A review of fibroblastpopulated collagen lattices. Wound Rep Reg 2008; 16: 472-479. 10 Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. PNAS 1979; 6 (3): 1274-8. 11 Fisher GJ, Wang Z, Datta SC, Varani J, Kang S, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. New England J Med 1997; 337 (20): 1419-1428. 12 Treiber N, Peters T, Sindrilaru A, Huber R, Kohn M, Menke A, Briviba K, Kreppel F, Basu A, Maity P, Koller M, Iben S, Wlaschek M, Kochanek S, Scharffetter-Kochanek K. Overexpression of manganese superoxide dismutase in human dermal fibroblasts enhances the contraction of free floating collagen lattice: implications for ageing and hyperplastic scar formation. Arch Dermatol Res 2009; 301: 273-287. 13 Pegova A, Abe H, Boldyrev A. Hydrolysis of carnosine and related compounds by mammalian carnosinases. Comp Biochem Physiol B Biochem Mol Biol 2000; 127 (4): 443-6. 14 Nicolaÿ JF, Courbebaisse Y. Percutaneous absorption and metabolization of chemically modified dipeptides. Symposium “Skin and formulation” APGI (2003). 15 Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechanoregulation of connective tissue remodelling. Nature 2002; 3: 349-363. 16 Lee HC, Wei YH. Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. Int J Biochem Cell Biol 2005; 37 (4): 822-34. 17 Yu J, Auwerx J. The role of sirtuins in the control of metabolic homeostasis. Ann NY Acad Sci 2009; 1173: E10-E19. 18 Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX, Finkel T. A role for the mitochondrial deacetylase SirT3 in regulating energy homeostasis. PNAS 2008; 105: 14447-52.
