Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Autocrine regulation of proliferation and extracellular matrix homeostasis in human fibroblasts.

In the late stages of the tissue repair process, as well as during normal tissue turnover, tissue homeostasis may rely mostly on autocrine mechanisms. Accordingly, we have cultured normal human fibroblasts on plastic surfaces and within three-dimensional collagen gels in order to study, in this environment, the action of autologous medium conditioned by the same cells. We have observed that inside collagen gels the autologous medium strongly restrains cell proliferation, due to fibroblast-secreted growth factors, whose inhibitory effect can be annulled by suramin. Furthermore, concerning extracellular matrix formation, conditioned medium has no effect on novel collagen synthesis, while it up-regulates collagenase MMP-1 only in cultures on plastic. On the other hand, it strongly inhibits the secretion of the collagenase inhibitor TIMP-1, irrespective of the substratum. This effect is completely blocked by SB 203580, an inhibitor of the p38 MAP kinase. The above suggest the presence of an autoregulatory mechanism involved in tissue homeostasis.

Fibroblast responses to exogenous and autocrine growth factors relevant to tissue repair. The effect of aging.

The aging process is often associated with impaired wound healing, but the cellular and molecular mechanisms implicated are not completely understood. Accordingly, we have investigated the response of human fibroblasts from donors of various ages to platelet-derived and autocrine growth factors, in terms of mitogenicity as well as extracellular matrix synthesis and degradation. Our data indicate that fibroblast responses persist during aging, suggesting the involvement of systemic factors in the regulation of the healing process. In this context, we have found that neutral endopeptidase-24.11, a metalloproteinase controlling the action of neuroendocrine peptides and also of immunocyte chemotaxis, is overexpressed during aging. Finally, the connection between these data and those from in vitro aging studies is discussed.

Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing.

Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1-24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity.

The highly reducing sugar 2-deoxy-D-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption.

2-deoxy-D-Ribose (dRib), the most reducing sugar, induces apoptosis in normal human fibroblasts, as judged by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation and mitochondrial depolarization. This effect is independent from culture conditions, such as cell density and the presence or absence of serum in the culture milieu, suggesting that dRib-induced apoptosis is cell cycle-independent. dRib was found also to provoke disruption of the actin filament network and detachment from the substratum, while at the same time, interestingly, it increases the expression of several integrins and cell adhesion molecules. Furthermore, dRib was found to reduce the intracellular levels of reduced glutathione (GSH). The apoptotic process was not affected by the macromolecular-synthesis inhibitors cycloheximide and actinomycin D. On the contrary, the antioxidant N-acetyl-L-cysteine (NAC) fully blocks the dRib-induced apoptosis by preventing GSH depletion, while it also inhibits actin-filament-network disruption and mitochondrial depolarization. The above indicate that dRib induces apoptosis in human fibroblasts by a mechanism involving glutathione metabolism and oxidative stress, as well as disturbance of cytoskeletal integrity and cell adhesion.

Autocrine growth regulation in fetal and adult human fibroblasts.

Medium conditioned (CM) by human fetal fibroblasts stimulates proliferation in sparse cultures. This effect is inhibited by suramin and staurosporine, indicating the presence of autocrine growth factors in CM. On the contrary, CM inhibits DNA synthesis in confluent cultures, suggesting a regulatory role for the secreted factors. The growth regulatory profile of CM persists during in vitro ageing. However, it changes dramatically during the fetal-to-adult transition, as adult human fibroblasts are stimulated by CM, regardless of the culture density. These effects are similar to those that TGF-beta is known to have on fetal and adult human fibroblasts. Indeed TGF-beta is present in media conditioned by human fibroblasts, but CM-activity cannot be ascribed solely to this factor. Fibroblasts originating from different tissues exhibit the same autocrine regulatory features, suggesting the general character of this mechanism.

The growth-inhibitory block of TGF-beta is located close to the G1/S border in the cell cycle.

Transforming growth factor-beta (TGF-beta) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF). The inhibition of the PDGF-BB action by TGF-beta was independent of the induction of mRNAs for the PDGF-A chain and PDGF-beta receptor, the predominant types of PDGF receptor in human fibroblasts. The TGF-beta-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (GO) state to the S phase of the cell cycle. Indeed, TGF-beta upregulated the "early" genes c-myc, c-fos, and junB and downregulated the growth arrest-specific (gas) genes. These results suggest that the inhibition of DNA synthesis by TGF-beta in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-beta block comparatively late in the GO to S transition. In cultures of senescent human fibroblasts TGF-beta stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.

Restoration of down-regulated PDGF receptors by TGF-beta in human embryonic fibroblasts. Enhanced response during cellular in vitro aging.

The study of [125I]PDGF-BB binding to normal human embryonic lung fibroblasts, quiescent when cultured at sparsity in the presence of minute concentrations of homologous PDS, reveals approximately 2 x 10(5) binding sites for PDGF per cell; this number significantly increases during prolonged quiescence of the culture. As late as 48 h after down-regulation of PDGF receptors, the cells restore only partially their capacity to bind PDGF, with aged cells (above CPD 45) responding more rapidly and efficiently than younger ones. TGF-beta significantly enhances restoration of PDGF receptors and, in aged cells in particular, its presence results in total receptor recovery within 24 h, suggesting a concerted action of PDGF and TGF-beta regulating the proliferation of human fibroblasts in tissue regeneration.

Stimulation of human embryonic lung fibroblasts by TGF-beta and PDGF acting in synergism. The role of cell density.

The concerted action of TGF-beta and PDGF on a diploid human embryonic lung fibroblast cell strain (Flow 2002) grown in an homologous environment is investigated here. In sparse cultures, TGF-beta stimulates DNA synthesis over a broad concentration range (0.1-10 ng/ml). Furthermore, it acts in synergism with PDGF, a phenomenon which persists also during in vitro aging of the cells. Preincubation of TGF-beta with the fibroblasts up to 12 hours reduces the subsequent PDGF binding to the cells, while prolonged preincubation restores PDGF binding to control levels. Finally, in cultures of higher cell densities, TGF-beta ceases to stimulate DNA synthesis, whereas PDGF continues even at cell confluency, retains its stimulatory activity suggesting different roles for the two growth factors during the wound healing process.

Quiescence and proliferative response of normal human embryonic fibroblasts in homologous environment. Effect of aging.

A cell culture system for the study of human serum growth factors is described. It is based on a diploid human embryonic lung fibroblast cell strain (Flow 2002) with a finite life span (60 +/- 3 CPDs). When maintained in homologous (human) plasma-derived serum (PDS) at concentrations as low as 0.05%, the cells attain quiescence. Under these conditions, they remain viable for at least 14 days and they readily respond when stimulated to proliferate by human serum or human PDS, as well as by growth factors, such as PDGF, EGF, FGF. During in vitro aging, the cells retain their responsiveness to these growth stimuli, although the net proliferative effect decreases as they approach senescence.

Multiple hemoglobins in Triturus cristatus: their study by analytical isoelectrofocusing.

Electrophoretic separation of the hemoglobin of healthy adult Triturus cristatus reveals four components. Isoelectric focusing of the same hemolysates in various commercial ampholytes of different chemical composition and pH range results in the separation of eight individual hemoglobin bands. The bands obtained by electrophoresis are not homogeneous as revealed by individual gel electrofocusing. They finally separate into the same eight components, as in the whole hemolysate. From the above findings it is concluded that this species has not four but eight individual hemoglobin molecular forms. Our results demonstrate lack of hemoglobin polymorphism in this species.

Factors controlling the DNA-synthesis in 3T3 and EAT cells.

Beta globulins, (Cohn Fr. III), are a major source of molecules affecting the DNA-synthesis of 3T3 and EAT cells. Growth inhibitors for both cell types, chromatograph at the same position, corresponding to a mol. wt of about 50,000. A very basic, (pI 10.1), factor is isolated by gel electrofocusing, which stimulates the DNA-synthesis of 3T3 and EAT cells. Because of its extremely high cationic charge and its adsorption on gels, the estimation of the exact molecular weight and its preparative isolation, becomes very difficult. Some of the above mentioned molecules are heat-stable and express their action even after boiling for 10 min at pH 3.

Plasma lipoprotein pattern in relation to liver histology after toxic hepatitis and experimental biliary obstruction in rabbits.

Plasma lipoproteins were studied in relation to liver histology in rabbits in the course of toxic hepatitis and compared to those after experimental biliary obstruction. The lipoprotein electrophoretic pattern became deeply abnormal during the acute phase of toxic hepatitis and correlated with the degree of liver injury, improving during recovery. Liver damage was more severe after carbon tetrachloride than after alcohol and milder after allylo-isopropyl-acetamide, a porphyrinogenic substance. Lipoprotein abnormalities were not followed by significantly reduced levels of cholesterol esters in the plasma. In comparison, animals with biliary obstruction developed milder liver damage presented gross abnormalities of plasma lipids and lipoproteins, followed by relative deficiency of cholesterol esterification. It is concluded that lipoprotein changes in acute liver injury, although non-specific, are a sensitive index of liver damage and recovery. Serious acute liver injury can exist without significant fall in cholesterol esters.

Human beta-globulin factors affecting growth of human lymphocytes and mouse fibroblasts.

Evidence is presented to show the existence in human beta-globulin (Cohn fraction III) of 2 growth-promoting factors that stimulate DNA synthesis and cell division in human lymphocytes and 3T3 B mouse fibroblasts. After extraction of human beta-globulins at pH 3.0 in 0.1 M NaCl followed by sieve chromatography on Sephadex, 2 distinct fractions were obtained containing the biological activity; one of polypeptide nature and molecular weight of approx. 10 000 Daltons and the other consisting of a homogeneous ribonucleic acid, as revealed by polyacrylamide gel electrophoresis. The above-mentioned factors appear to lack both cell and species specificity, since they are mitogenic agents for cells as diverse as mouse fibroblasts and human lymphocytes.

Abnormal lipoproteins in multiple myelomatosis.

In a study of the lipoprotein pattern in multiple myelomatosis electrophoresis on agarose gel showed abnormal lipoproteins, named paralipoproteins (p-Lp), in 24 out of 30 normolipidaemic patients. These paralipoproteins were grouped according to their mobility into one or another of the following types: (1) p-Lp1 with a mobility identical with that of gamma-globulin, (2) p-Lp2 with a mobility between that of beta- and gamma-globulin, (3) p-Lp3 with a mobility identical with that of beta-globulin. On ultracentrifugation the abnormal lipoproteins were found to have a density above 1-063 g/ml.

A new approach to the diagnosis of beta-thalassaemia.

By use of isoelectric focusing in polyacrylamide gel rods we were able to detect traces of HbA (approx. 1%) as a sharp and discrete band. By overloading the gel considerable amounts of HbA (slightly contaminated with HbF) could be detected and isolated. The focused HbA was retrieved from the gels, separated from the carrier-ampholytes and concentrated by a one-step electrophoresis technique. With 3H-leuci ne-labelled haemolysates, after globin chain separation on CM-cellulose, an increase of the beta-chain counts relative to gamma-chain counts was obtained. The study of two cases of high HbF homozygous beta-thalassaemia has demonstrated that this technique may be a valuable tool in detecting minute amounts of HbA mainly in high HbF beta-thalassaemias.

Isolation of a cationic polypeptide from human serum that stimulates proliferation of 3T3 cells.

A basic polypeptide that stimulates DNA synthesis and cell division in confluent populations of mouse Balb/c-3T3 cells has been isolated from whole human serum, and has been separated from the heterogenous group of molecules with insulin-like activity. This highly purified basic polypeptide has a molecular weight of 1.3 x 10(4) and an isoelectric point of 9.7. Approximately 10(7) polypeptide molecules in the growth medium allow the replication of one density-inhibited cell.