N-acetyl-L-hydroxyproline - A potent skin anti-ageing active preventing advanced glycation end-product formation in vitro and ex vivo.

OBJECTIVE: Advanced glycation end-products (AGEs) represent a large group of compounds generated by a non-enzymatic reaction between reducing sugars and amino groups. The formation and accumulation of AGEs in the skin lead to protein crosslinking, dermal stiffening and yellowing, which ultimately contribute to cutaneous ageing. Amino acids have been described to exhibit anti-glycation effects. The objective of this study was to understand the inhibitory role of the amino acid derivative N-acetyl-L-hydroxyproline (NAHP) as an anti-glycation active for human skin. METHODS: A cell-free assay investigating the inhibition of glycation of serum albumin by NAHP was used to determine the capability of NAHP to decrease AGE formation. Also, by assessing the amount of the AGE N-(carboxymethyl)lysine (CML) the anti-glycation abilities of NAHP were investigated utilizing dot blot analysis. The improvement of cell-matrix interaction by NAHP was determined in vitro using a glycated fibroblast-populated collagen lattice (FPCL) dermis model. In skin biopsies, AGE autofluorescence was determined after treatment with NAHP and/or glucose ex vivo. RESULTS: NAHP significantly and dose-dependently inhibited levels of AGEs, which were induced by the glycation of a protein solution. This decrease could be visualized by showing that the brownish appearance as well as the AGE-specific fluorescence of glucose-treated samples were reduced after the application of increasing amounts of NAHP. Also, CML formation was dose-dependently inhibited by NAHP. In FPCLs, the contractile capacity of fibroblasts was significantly disturbed after glycation. This could be prevented by the addition of NAHP. Compared to glyoxal-treated samples, the co-application of NAHP significantly decreased the diameter as well as the weight of glycated FPCLs. Ex vivo application of glucose to skin explants showed a higher AGE fluorescence signal compared to control explants. Co-treatment with NAHP and glucose decreased the level of AGE fluorescence in comparison to glucose-treated explants. CONCLUSION: These data provide clear evidence that under glycation stress conditions treatment with NAHP inhibited AGE formation in vitro and ex vivo and prevented the loss of cellular contractile forces in a glycated dermis model. Thus, NAHP obviously provides a beneficial treatment option to counteract AGE-related changes in human skin such as dermal stiffening and yellowish skin appearance.

OBJECTIF: Les produits finis de glycation avancée (AGE) représentent un grand groupe de composés générés par une réaction non enzymatique entre des sucres réduits et des groupes amino. La formation et l'accumulation d'AGE dans la peau entraînent une réticulation protéique, un raidissement de la peau et un jaunissement, qui finissent par contribuer au vieillissement cutané. Les acides aminés ont été décrits comme ayant des effets d'anti­glycation. L'objectif de cette étude était de comprendre le rôle inhibiteur du dérivé d'acide aminé N­acétyl­L­hydroxyproline (NAHP) en tant qu'actif anti­glycation pour la peau humaine. MÉTHODES: Un test acellulaire étudiant l'inhibition de la glycation de l'albumine sérique par la NAHP a été utilisé pour déterminer la capacité de la NAHP à diminuer la formation d'AGE. En évaluant la quantité de l'AGE N­(carboxyméthyl)lysine (CML), les capacités d'anti­glycation de la NAHP ont également été étudiées à l'aide d'une analyse par dot blot. L'amélioration de l'interaction cellule­matrice par la NAHP a été déterminée in vitro à l'aide d'un modèle de derme de lattices de collagène composées de fibroblastes glyqués. Dans des biopsies cutanées, l'autofluorescence des AGE a été déterminée après un traitement par NAHP et/ou glucose ex vivo. RÉSULTATS: La NAHP a inhibé de manière significative et dose­dépendante les taux d'AGE induits par la glycation d'une solution protéique. Cette diminution a pu être visualisée en montrant que l'aspect brunâtre ainsi que la fluorescence spécifique aux AGE des échantillons traités par glucose ont été réduits après l'application de quantités croissantes de NAHP. En outre, la formation de CML était inhibée de manière dose­dépendante par la NAHP. Dans des lattices de collagène composées de fibroblastes, la capacité contractile des fibroblastes était significativement perturbée après la glycation. Cela a pu être évité par l'ajout de NAHP. Par rapport aux échantillons traités au glyoxal, la co­application de NAHP a significativement réduit le diamètre ainsi que le poids des lattices de collagène composées de fibroblastes glyquées. L'application ex vivo de glucose sur les explants de peau a montré un signal de fluorescence des AGE plus élevé que les explants témoins. Le traitement concomitant par NAHP et glucose a réduit le niveau de fluorescence des AGE par rapport aux explants traités par glucose. CONCLUSION: Ces données fournissent des preuves évidentes que, dans des conditions de stress par glycation, le traitement par NAHP a inhibé la formation d'AGE in vitro et ex vivo, et a prévenu la perte des forces contractiles cellulaires dans un modèle de derme glyqué. Ainsi, la NAHP constitue manifestement une option de traitement bénéfique pour contrer les changements liés aux AGE dans la peau humaine, tels que le raidissement du derme et l'aspect jaunâtre de la peau.

Ageing is associated with a reduction in markers of mitochondrial energy metabolism in the human epidermis.

The decline of mitochondrial function throughout the lifespan is directly linked to the development of ageing phenotypes of the skin. Here, we assessed alterations in markers of epidermal mitochondrial energy metabolism as a function of skin age. Human skin samples from distinct anatomical regions were obtained during routine dermatological surgery from 21 young (27.6 ± 1.71 year) and 22 old (76.2 ± 1.73 year) donors. Sections of skin samples were analysed by immunohistochemistry for mitochondrial subunits of each electron transport chain complex (I-V)/oxidative phosphorylation (OXPHOS), as well as proteins serving as a marker of mitochondrial mass (VDAC1) and the regulation of DNA transcription (TFAM). Staining intensities of ATP5F1A (comprising complex V) and TFAM in the epidermis of older subjects were significantly decreased compared with younger donors. Moreover, these effects were independent of UV exposure of the stained skin section. Overall, we demonstrate that ageing is associated with reduced protein levels of complex V of the mitochondrial respiratory chain and TFAM. These alterations may impair essential mitochondrial functions, exacerbating the cutaneous ageing process.

Assessing Cellular Uptake of Exogenous Coenzyme Q10 into Human Skin Cells by X-ray Fluorescence Imaging.

X-ray fluorescence (XRF) imaging is a highly sensitive non-invasive imaging method for detection of small element quantities in objects, from human-sized scales down to single-cell organelles, using various X-ray beam sizes. Our aim was to investigate the cellular uptake and distribution of Q10, a highly conserved coenzyme with antioxidant and bioenergetic properties. Q10 was labeled with iodine (I2-Q10) and individual primary human skin cells were scanned with nano-focused beams. Distribution of I2-Q10 molecules taken up inside the screened individual skin cells was measured, with a clear correlation between individual Q10 uptake and cell size. Experiments revealed that labeling Q10 with iodine causes no artificial side effects as a result of the labeling procedure itself, and thus is a perfect means of investigating bioavailability and distribution of Q10 in cells. In summary, individual cellular Q10 uptake was demonstrated by XRF, opening the path towards Q10 multi-scale tracking for biodistribution studies.

Inter-layer and inter-subject variability of diurnal gene expression in human skin.

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.

Anti-ageing effects of ubiquinone and ubiquinol in a senescence model of human dermal fibroblasts.

Coenzyme Q10 (CoQ10) is an endogenous lipophilic quinone found in equilibrium between its oxidised (ubiquinone) and reduced (ubiquinol) form, ubiquitous in biological membranes and endowed with antioxidant and bioenergetic properties, both crucial to the ageing process. CoQ10 biosynthesis decreases with age in different tissues including skin and its biosynthesis can be modulated by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors such as statins. Statin-induced CoQ10 deprivation has previously been shown to be associated with the development of a senescence phenotype in cultured human dermal fibroblasts (HDF), hence this model was used to further investigate the role of CoQ10 in skin ageing. The present study aimed to compare the bioavailability of exogenously added CoQ10, in the form of ubiquinone or ubiquinol, to CoQ10-deprived HDF, and to determine their efficacy in rescuing the senescent phenotype induced by CoQ10 deprivation. First, additional senescence markers were implemented to further support the pro-ageing effect of statin-induced CoQ10 deprivation in HDF. Indeed, numerous senescence-associated secretory phenotype (SASP) markers such as p21, IL-8, CXCL1, and MMP-1 were upregulated, whereas components of the extracellular matrix were downregulated (elastin, collagen type 1). Next, we showed that CoQ10 supplementation to statin-treated HDF was able to counteract CoQ10 deprivation and rescued the development of selected senescence/ageing markers in HDF. Ubiquinol resulted more bioavailable than ubiquinone at the same concentration (15 µg/mL) and it significantly improved the cellular oxidative status even within isolated mitochondria highlighting an effective subcellular delivery. Ubiquinol was also more efficient compared to ubiquinone in reverting the expression of the senescent phenotype, quantified in terms of ß-galactosidase positivity, p21, collagen type 1, and elastin at the gene and protein expression levels. In conclusion, our results highlight the pivotal role of CoQ10 for skin vitality and strongly support the use of both forms as a beneficial and effective anti-ageing skin care treatment.

Modulation of Coenzyme Q10 content and oxidative status in human dermal fibroblasts using HMG-CoA reductase inhibitor over a broad range of concentrations. From mitohormesis to mitochondrial dysfunction and accelerated aging.

Coenzyme Q10 (CoQ10) is an endogenous lipophilic quinone, ubiquitous in biological membranes and endowed with antioxidant and bioenergetic properties, both crucial to the aging process. In fact, coenzyme Q10 synthesis is known to decrease with age in different tissues including skin. Moreover, synthesis can be inhibited by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors such as statins, that are widely used hypocholesterolemic drugs. They target a key enzymatic step along the mevalonate pathway, involved in the synthesis of both cholesterol and isoprenylated compounds including CoQ10.In the present study, we show that pharmacological CoQ10 deprivation at concentrations of statins > 10000 nM triggers intracellular oxidative stress, mitochondrial dysfunction and generates cell death in human dermal fibroblasts (HDF). On the contrary, at lower statin concentrations, cells and mainly mitochondria, are able to partially adapt and prevent oxidative imbalance and overt mitochondrial toxicity. Importantly, our data demonstrate that CoQ10 decrease promotes mitochondrial permeability transition and bioenergetic dysfunction leading to premature aging of human dermal fibroblasts in vitro.

Topical treatment with coenzyme Q10-containing formulas improves skin's Q10 level and provides antioxidative effects.

Ubiquinone (coenzyme Q10, Q10) represents an endogenously synthesized lipid-soluble antioxidant which is crucial for cellular energy production but is diminished with age and under the influence of external stress factors in human skin. Here, it is shown that topical Q10 treatment is beneficial with regard to effective Q10 replenishment, augmentation of cellular energy metabolism, and antioxidant effects. Application of Q10-containing formulas significantly increased the levels of this quinone on the skin surface. In the deeper layers of the epidermis the ubiquinone level was significantly augmented indicating effective supplementation. Concurrent elevation of ubiquinol levels suggested metabolic transformation of ubiquinone resulting from increased energy metabolism. Incubation of cultured human keratinocytes with Q10 concentrations equivalent to treated skin showed a significant augmentation of energy metabolism. Moreover, the results demonstrated that stressed skin benefits from the topical Q10 treatment by reduction of free radicals and an increase in antioxidant capacity.

Age and skin site related differences in steroid metabolism in male skin point to a key role of sebocytes in cutaneous hormone metabolism.

Hormone concentrations decline with aging. Up to now it was not clear, whether the decrease of hormone concentrations in blood samples are also present in cutaneous suction blister fluids, and whether skin from different anatomical sites shows different hormone concentrations.Analysis of suction blister fluids and paired blood samples from young (mean 27.8 y) and old (mean 62.6 y) male subjects by UPLC-MS/MS showed that DHEA concentration in blood samples was age-dependently significantly reduced, but increased in suction blister fluids, while androstenedione behaved in an opposite manner to DHEA. Testosterone decreased age-dependently in blood samples and in suction blister fluids. Regarding skin sites, DHEA was lower in samples from upper back compared with samples from the forearm. In contrast, the concentrations of androstenedione and testosterone were higher in samples from upper back.In vitro analyses showed that SZ95 sebocytes, but neither primary fibroblasts nor keratinocytes, were able to use DHEA as precursor for testosterone biosynthesis, which was confirmed by expression analysis of 3ß-hydroxysteroiddehydrogenase in skin biopsies.In conclusion, we show an inverse pattern of DHEA and androstenedione concentrations in blood vs. suction blister fluids, highlighting age-dependent changes of dermal testosterone biosynthesis, and a stronger metabolism in young skin. Furthermore, sebocytes play a central role in cutaneous androgen metabolism.

Krüppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes.

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Dermal penetration of creatine from a face-care formulation containing creatine, guarana and glycerol is linked to effective antiwrinkle and antisagging efficacy in male subjects.

BACKGROUND: The dermal extracellular matrix provides stability and structure to the skin. With increasing age, however, its major component collagen is subject to degeneration, resulting in a gradual decline in skin elasticity and progression of wrinkle formation. Previous studies suggest that the reduction in cellular energy contributes to the diminished synthesis of cutaneous collagen during aging. AIMS: To investigate the potential of topically applied creatine to improve the clinical signs of skin aging by stimulating dermal collagen synthesis in vitro and in vivo. PATIENTS/METHODS: Penetration experiments were performed with a pig skin ex vivo model. Effects of creatine on dermal collagen gene expression and procollagen synthesis were studied in vitro using cultured fibroblast-populated collagen gels. In a single-center, controlled study, 43 male Caucasians applied a face-care formulation containing creatine, guarana extract, and glycerol to determine its influence on facial topometric features. RESULTS: Cultured human dermal fibroblasts supplemented with creatine displayed a stimulation of collagen synthesis relative to untreated control cells both on the gene expression and at the protein level. In skin penetration experiments, topically applied creatine rapidly reached the dermis. In addition, topical in vivo application of a creatine-containing formulation for 6 weeks significantly reduced the sagging cheek intensity in the jowl area as compared to baseline. This result was confirmed by clinical live scoring, which also demonstrated a significant reduction in crow's feet wrinkles and wrinkles under the eyes. CONCLUSIONS: In summary, creatine represents a beneficial active ingredient for topical use in the prevention and treatment of human skin aging.

Biochemical rationale and experimental data on the antiaging properties of CoQ(10) at skin level.

Coenzyme Q(10) (CoQ(10) ) is a key component of the mitochondrial respiratory chain and, therefore, is essential for the bioenergetics of oxidative phosphorylation. It is also endowed with antioxidant properties, and recent studies pointed out its capability of affecting the expression of different genes. In this review, we analyze the data on the mechanisms by which CoQ(10) interacts with skin aging processes. The effect of CoQ(10) in preserving mitochondrial function cooperates in maintaining a proper energy level, which serves to prevent the aging skin from switching to anaerobic energy production mechanisms. Furthermore, the antioxidant capacity of CoQ(10) contributes to a positive effect against UV-mediated oxidative stress. Some of these effects have been assessed also in vivo, by the sensitive technique of ultraweak photoemission. Finally, CoQ(10) has been shown to influence, through a gene induction mechanism, the synthesis of some key proteins of the skin and to decrease the expression of some metalloproteinase such as collagenase. These mechanisms may also contribute to preserve collagen content of the skin.

Folic acid and creatine improve the firmness of human skin in vivo.

BACKGROUND: The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. AIMS: Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. PATIENTS/METHODS: Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. RESULTS: The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. CONCLUSIONS: Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.

A circadian clock in HaCaT keratinocytes.

To anticipate daily environmental changes, most organisms developed endogenous timing systems, the so-called circadian (∼24 hours) clocks. Circadian clocks exist in most peripheral tissues and govern a huge variety of cellular, metabolic, and physiological processes. Recent studies have suggested daytime-dependent variations in epidermal functions such as barrier recovery and pH homeostasis. However, a local circadian clock in epidermal keratinocytes has not been reported yet, and as such the molecular link between the circadian system and epidermal physiology remains elusive. In this study we describe a functional cell autonomous circadian clock in human adult low calcium temperature (HaCaT) keratinocytes. Using live-cell bioluminescence imaging and mRNA expression time series, we show robust circadian transcription of canonical clock genes in synchronized HaCaT keratinocytes. Genetic and pharmacological perturbation experiments as well as the phase relations between clock gene rhythms confirm that the molecular makeup of the HaCaT keratinocyte clock is very similar to that of other peripheral clocks. Furthermore, temperature was identified to be a potent time cue (Zeitgeber) for the epidermal oscillator. Temperature cycles entrain HaCaT keratinocytes, leading to the identification of rhythmic expression of several genes involved in epidermal physiology such as cholesterol homeostasis and differentiation. Thus, we present HaCaT keratinocytes as an excellent model to study the regulation of keratinocyte physiology by the circadian clock in a simple yet robust in vitro system.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.

Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.

Modification of vimentin: a general mechanism of nonenzymatic glycation in human skin.

In a recent study, we were able to show that the intermediate filament protein vimentin aggregates in human dermal fibroblasts because of modification by the advanced glycation endproduct carboxymethyllysine (CML). In this work, we investigated the formation of intracellular CML in relation to the concentration of glucose in the culture medium. The natural degradation product of glucose, methylglyoxal, was able to induce the aggregation of vimentin. This dicarbonyl leads to the formation of the modifications MG-H1 and carboxyethyllysine (CEL) as a result of the reaction with arginine and lysine residues of proteins. Furthermore, we found that the protein vimentin was modified, not only by CML and CEL, but also by pentosidine and pyrraline. These findings underline the special position of vimentin as a preferential target of the Maillard reaction in human skin.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology.

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging.

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.