Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.     

Dicarbonyl-induced accelerated aging in vitro in human skin fibroblasts

Dicarbonyls glyoxal (GO) and methylglyoxal (MGO) produced during the autoxidation of reducing sugars are a source of macromolecular damage in cells. Since an accumulation of damaged macromolecules is a universal characteristic of aging, we have tested whether GO and MGO which cause oxidative damage to proteins and other macromolecules can bring about accelerated aging in normal human skin fibroblasts in vitro. A treatment of cells with 1.0 mM GO or 400 μM MGO leads to the appearance of senescent phenotype within 3 days, as judged by the following criteria: morphological phenotype, irreversible growth arrest and G₂ arrest, increased senescence-associated β-galactosidase (SABG) activity, increased H₂O₂ level, increased N^ξ-(carboxymethyl)-lysine (CML) protein level, and altered activities of superoxide dismutase and catalase antioxidant enzymes. This experimental model of accelerated cellular aging in vitro can be useful for studies on testing the effects of various physical, chemical and biological conditions, including natural and synthetic molecules, for the modulation of aging.     

Autofluorescence of human skin fibroblasts during growth inhibition and in vitro ageing

The increase in autofluorescence (AF) of human skin fibroblasts during their in vitro ageing and growth inhibition was investigated by means of flow cytophotometry. The cellular AF of in vitro ageing cultures increased while the relative number of (3H)-thymidine incorporating cells decreased. Therefore, the rate of accumulation of cellular AF during in vitro ageing of the cultures is inversely related to the proliferation rate of the culture. The rates of increase of AF varied widely among the cell strains, being the highest in cells from patients with Werner's syndrome. Upon growth inhibition in a confluent culture the net rates of increase of cellular AF were found to vary widely among the cell strains. The respective net rates of increase of AF of the cells from patients with Werner's syndrome and the Spielmeyer-Vogt syndrome were within the range covered by the normal cell strains. The ultrastructure of the bright AF cells from patients with Werner's syndrome and the Spielmeyer-Vogt syndrome differed from the ultrastructure of AF cells from control persons with regard to the morphology of their residual bodies, those from the patients contained more multilamellar and multivesicular structures. In sorted non-AF cells vitamin E was found to completely inhibit the accumulation of AF without affecting the formation of 'residual bodies'. We infer that cellular AF is caused by lipid peroxidative reactions and that the accumulation of AF is due to a decrease in cellular proliferation rate.     

Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro

Our studies have shown that zeatin, (6-[4-hydroxy-3-methyl-but-2-enylamino]adenine), a cytokinin plant growth factor, has gerontomodulatory, youth preserving and anti-aging effects on serially passaged human adult skin fibroblasts undergoing aging in vitro. There were no immediate negative or toxic effects in terms of cell attachment, cell proliferation, cell survival, cytoskeletal organization, and cellular growth by treatment with zeatin concentrations between 1 and 200 microM. During long-term treatment, cells could be maintained throughout their replicative lifespan in the presence of 40, 80, and 200 microM zeatin, but the optimal concentration of zeatin's anti-aging and youth preserving effects was found to be 80 microM. Life-long serial passaging of human skin fibroblasts in the presence of zeatin resulted in the prevention of cell enlargement, reduction of intracellular debris, prevention of actin polymerization, and enhancement of cellular ability to decompose hydrogen peroxide and to cope with ethanol and oxidative stresses. Most importantly, anti-aging and beneficial effects of zeatin were observed without any induction of additional cell proliferation or an increase in the maximum proliferative capacity, thus ruling out any potentially harmful and carcinogenic effects.     

Effect of donor age on clonal differentiation of human skin fibroblasts in vitro

Human skin fibroblasts of about the same population doubling level, derived from donors of different age, were cloned. After 14 days of growth, the clones were morphologically classified, and cell counts were performed. Significant differences between the three age groups (infant, young adult, old adult) examined concerned the percentual clone type distribution. The size of a single clone depended on the clone type referring to the differentiation level of the cells in all age groups.     

Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts.

Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antisense oligonucleotide inhibited the irradiation-dependent induction of both the heme oxygenase I enzyme and ferritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in ferritin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanism, involving ferritin, in human skin fibroblasts.     

Relation between hemodynamic and ventilatory responses in determining exercise capacity in severe congestive heart failure

The cause of exercise intolerance in congestive heart failure is unclear. Hemodynamic and ventilatory responses were measured during symptomatic maximal upright bicycle exercise in 28 patients with chronic severe left ventricular failure who achieved a maximal oxygen uptake of only 12 +/- 4 ml/min/kg (+/- standard deviation). All patients reached anaerobic metabolism as the respiratory exchange ratio rose and arterial pH fell significantly. Pulmonary capillary wedge pressure increased from 20 +/- 10 mm Hg at rest to 38 +/- 9 mm Hg at peak exercise and cardiac index increased from 2.51 +/- 0.73 to 4.54 +/- 1.65 liters/min/m2 (both p less than 0.001). Systemic vascular resistance decreased, but pulmonary vascular resistance did not change during exercise. Despite the marked pulmonary venous hypertension at peak exercise, blood gases were unchanged (PaO2, 96 +/- 15 mm Hg; PaCO2, 35 +/- 7 mm Hg). Systemic arterial oxygen content increased from 16 +/- 2 to 17 +/- 2 vol% (p less than 0.01). Changes in pulmonary capillary wedge pressure did not correlate with changes in arterial oxygen content. Results were similar whether patients were limited by dyspnea or fatigue. Thus, exercise intolerance in patients with severe left ventricular failure is associated with marked elevation of pulmonary capillary wedge pressure and anaerobic metabolism without hypoxemia or altered carbon dioxide tension. These findings suggest that exercise ability in congestive heart failure is more dependent on cardiac output than on ventilatory consequences of pulmonary congestion.     

Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro

The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.     

Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology

The aging process is known to be regulated by specific genes in various organisms, including yeast, the nematode C. elegans, fruitflies and mice. To explore the novel genes involved in aging process, we applied cDNA microarray technology to a replicative senescence model of human dermal fibroblasts (HDF). Eighty-four genes, including inflammatory genes, cell cycle regulatory genes, cytoskeletal genes, and metabolic genes were found to show more than two fold expressional differences in young and old fibroblasts. Furthermore, 31 genes were confirmed to be up- or down-regulated during replicative senescence by semi-quantitative RT-PCR. The overexpressions of several genes including CD36, putative lymphocyte G0/G1 switch gene (G0S2), tumor protein D52-like 1 (TPD52L1), chemokine (C-X-C motif) ligand 6, myxovirus resistant gene 1 (MX1), and the down-regulation of the immunoglobulin superfamily containing leucine-rich repeat (ISLR), neurotrimin, insulin-like growth factor 2 associated protein (IGF2A), and apoptosis-related RNA binding protein (NAPOR3) were newly identified. These results suggest that fibroblasts show the deregulation of various cellular processes, such as inflammatory response, mitosis, cell adhesion, transport, signal transduction, and metabolism during replicative senescence.     

Association between tumor DNA aneuploidy and in vitro tetraploidy of skin fibroblasts in patients with colorectal neoplasms

In vitro tetraploidy (IVT) in cultures of skin fibroblasts was compared with tumor DNA ploidy, as determined by flow cytometry on paraffin-embedded material, in 99 patients with colorectal neoplasm. In 63 patients with non-heritable carcinoma we found a significant correlation between the number of aneuploid stemlines in the tumor and IVT in the fibroblast culture. Furthermore, tumor aneuploidy was significantly correlated to the size of the tetraploid subpopulation in the fibroblasts. There was no correlation between aneuploidy and Dukes's stage or the degree of differentiation. In 36 patients with adenoma no correlation between tumor aneuploidy and fibroblast IVT was demonstrated, whereas the number of tumor stemlines was significantly correlated to histopathologic stage and grade of dysplasia. IVT in cultured skin fibroblasts, which has been reported to reflect a genetic predisposition to colorectal cancer in heritable colon cancer syndromes, thus seems to be relevant also for the understanding of tumor formation and progression in the 'non-heritable' type of colorectal cancer.     

Aromatase activity in cultured human genital skin fibroblasts

Aromatase activity of human genital skin fibroblasts grown in cell culture was studied using both [1,2,6,7-3H] androstenedione (A) and [1-3H]A as substrates. With the former substrate the generation of [3H]estrogens was determined, whereas with the latter substrate, the formation of [3H]H2O was measured. Our results showed that the release of [3H]H2O from [1-3H]A provides an accurate and sensitive method for determining aromatase activity in cultured human skin fibroblasts. Because genital skin fibroblasts also possess marked 5 alpha-reductase activity, we found that addition of an alternate substrate for 5 alpha-reductase was necessary to prevent shunting of A from the aromatase pathway. Hence, all aromatase assays were carried out in the presence of 5 microM progesterone. Under these experimental conditions, no correlation was found between levels of 5 alpha-reductase and aromatase activities. The Michaelis-Menten constant (Km) of the aromatase in cultured genital skin fibroblasts measured in the presence of A and added progesterone ranged between 10 and 39 nM, and the maximum velocity (Vmax) ranged between 0.14 and 1.46 pmol product/mg protein/h. These values are in good agreement with those previously described for adipose tissue stromal-vascular cells, suggesting that the aromatase complexes are similar in skin and adipose tissue. We conclude that skin may be an important site for aromatization of androgens to estrogens in men.     

Angiotensin-converting enzyme: characteristics in human skin fibroblasts

Angiotensin-converting enzyme, although most prominent in vascular endothelium, has been identified in numerous tissues. Recent studies have indicated that several hormones, including glucocorticoids and thyroid hormone, may affect the activity of this enzyme. In the present study, angiotensin-converting enzyme was examined in homogenates of cultured human skin fibroblasts. Angiotensin-converting enzyme activity was measured by a radiometric assay using [Glycine-1-14C] Hippuryl-L-histidyl-L-leucine (1.1 mmol/L) as substrate, and was expressed as nmol hippuric acid formed per minute/mg protein. Angiotensin-converting enzyme was identified in all five cell strains tested, and the activity observed was 0.97 +/- 0.18 nmol/min/mg protein (mean +/- SE). The optimum pH was between 6.9 and 7.6, and optimum temperature was 37 degrees C, with loss of activity of 55 degrees C and higher. Buffer strength was optimized at Tris 0.025 mol/L, and 1.0 mol/L NaCl. Activity increased linearly with protein concentration and with time, and the Km = 1.14 mmol/L. The most potent inhibitor of fibroblast ACE was captopril (SQ 14,225) with an IC50 = 10(-10) mol/L; other inhibitors included SQ 20,881, EDTA, and phenanthroline. Competitive substrates included angiotensin-I, substance P, and bradykinin. Four hormones, T3 (10(-9)-10(-7) mol/L), 1,25 (OH)2D3 (10(-8)-10(-7) mol/L), dexamethasone (10(-7)-10(-6) mol/L), and a synthetic androgen, R1881 (10(-8)-10(-7) mol/L) were incubated with cells for 72 hours. In all incubations, there was no significant effect on cellular ACE activity induced by any agent. Angiotensin-converting enzyme activity in serum free media was less than 1% of cell activity and was unaltered by hormone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, N-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at approximately equal to 3 J/m2 UV, 0.5 mM MNU, or 1.0 microM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.     

Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts.

Clinical evidence suggests that skin is responsive to GH status in vivo. We sought to demonstrate the presence of GH receptors in human skin and in cultured skin fibroblasts using the techniques of immunohistochemistry and northern blotting. Human foreskin was obtained at surgery for preparation of sections and primary fibroblast cultures. Skin sections and fibroblast monolayers were immunostained using a monoclonal antibody which recognizes the hGH receptor (MAb 263). Positive immunoperoxidase staining was seen in all epidermal layers except the stratum corneum, in dermal sweat and sebaceous glands, and in dermal fibroblasts. In cultured fibroblasts capping of surface GH receptor was observed after aqueous formaldehyde fixation, whereas fixation in Carnoy's solution resulted in granular cytoplasmic staining. Fibroblast poly A+ RNA was prepared from cultured skin fibroblasts, separated by denaturing agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized to a 32P-labeled, 847 base pair (bp) hGH receptor complementary DNA (cDNA) clone. Human liver and non-pregnant rabbit liver total RNA were used as controls. Fibroblast poly A+ RNA contained a single hybridizing species of approximately 5.2 kilobase. Human liver total RNA also contained a single hybridizing species of 4.9 kilobase. We have demonstrated the presence of GH receptor protein in human skin and growth hormone receptor mRNA and protein in cultured human skin fibroblasts. These observations suggest that GH may indeed have a direct role in modulating keratinocyte and fibroblast function.