Hermann Unterluggauer

Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan

Mesenchymal stem cells (MSC) are capable of differentiating into bone, fat, cartilage, tendon and other organ progenitor cells. Despite the abundance of MSC within the organism, little is known about their in vivo properties or about their corresponding in vivo niches. We therefore isolated MSC from spongy (cancellous) bone biopsies of healthy adults. When compared with the surrounding marrow, a fourfold higher number of colony‐forming units was found within the tight meshwork of trabecular bone surface. At these sites, oxygen concentrations range from 1% to 7%. In MSC cultured at oxygen as low as 3%, rates for cell death and hypoxia‐induced gene transcription remained unchanged, while in vitro proliferative lifespan was significantly increased, with about 10 additional population doublings before reaching terminal growth arrest. However, differentiation capacity into adipogenic progeny was diminished and no osteogenic differentiation was detectable at 3% oxygen. In turn, MSC that had previously been cultured at 3% oxygen could subsequently be stimulated to successfully differentiate at 20% oxygen. These data support our preliminary finding that primary MSC are enriched at the surface of spongy bone. Low oxygen levels in this location provide a milieu that extends cellular lifespan and furthermore is instructive for the stemness of MSC allowing proliferation upon stimulation while suppressing differentiation.

Senescence-associated cell death of human endothelial cells: the role of oxidative stress.

Replicative senescence of human endothelial cells was analyzed, using primary endothelial cells from the human umbilical vein endothelial cells (HUVEC) as an experimental model system. We had shown before that senescent HUVEC arrest in the G1 phase of the cell cycle and that a subpopulation of the senescent cells undergoes cell death. We now demonstrate that cell death occurs by apoptosis, characterized by activation of caspase 3. Using the redox-sensitive dye dihydrorhodamine 123, a significant accumulation of reactive oxygen species is detected in senescent but not young endothelial cells. To determine if increased oxidative stress may contribute to the senescent phenotype, cells were treated with tert-butyl hydroperoxide (tBHP), which is known to increase oxidative stress by decreasing the intracellular glutathione levels. We show here that mild tBHP stress induces a phenotype of premature senescence in a subpopulation of the treated cells, which closely resembles the phenotype of naturally senescent HUVEC, including growth arrest, senescence-associated beta-gal activity, and apoptotic cell death. These results establish a model of premature senescence for human endothelial cells, which will be suitable to analyze mechanisms of age-associated cell death.

High-resolution respirometry--a modern tool in aging research.

Alterations in mitochondrial function are believed to play a major role in aging processes in many species, including fungi and animals, and increased oxidative stress is considered a major consequence of altered mitochondrial function. In support of this theory, a lot of correlative evidence has been collected, suggesting that changes in mitochondrial DNA accumulate with age in certain tissues. Furthermore, genetic experiments from lower eukaryotic model organisms, indicate a strong correlative link between increased resistance to oxidative stress and an extended lifespan; in addition, limited experimental evidence suggests that the inhibition of mitochondrial function by selected pharmacologically active compounds can extend lifespan in certain species. However, changes in mitochondrial function may affect aging in a different way in various tissues, and a clear statement about the role of mitochondrial deterioration during physiological aging is missing for most if not all species. At this point, respirometric analyses of mitochondrial function provide a tool to study age-associated changes in mitochondrial respiratory chain function and mitochondrial ATP production within living cells and isolated mitochondria. In the recent years, new instruments have been developed, which allow for an unprecedented high-resolution respirometry, which enables us to determine many parameters of mitochondrial function in routine assays using small samples of biological material. It is conceivable that this technology will become an important tool for all those, who are interested in experimentally addressing the mitochondrial theory of aging. In this article, we provide a synopsis of traditional respirometry and the advances of modern high-resolution respirometry, and discuss how future applications of this technology to recently established experimental models in aging research may provide exciting new insights into the role of mitochondria in the aging process.

THE NADPH OXIDASE NOX4 RESTRICTS THE REPLICATIVE LIFESPAN OF HUMAN ENDOTHELIAL CELLS

The free radical theory of aging proposes that ROS (reactive oxygen species) are major driving forces of aging, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described that might contribute to cellular senescence. Noxs (NADPH oxidases) are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in recent years non-phagocytic homologues of Nox2 have been identified that are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAPKs (mitogen-activated protein kinases) and stress-activated kinases, but could also contribute to cellular senescence, which is known to involve oxygen radicals. In HUVECs (human umbilical vein endothelial cells), Nox4 is predominantly expressed, but its role in replicative senescence of HUVECs remains to be elucidated. Using shRNA (small-hairpin RNA)-mediated knockdown of Nox4, implicating lentiviral vectors, we addressed the question of whether lifelong depletion of Nox4 in HUVECs would influence the senescent phenotype. We found a significant extension of the replicative lifespan of HUVECs upon knockdown of Nox4. Surprisingly, mean telomere length was significantly reduced in Nox4-depleted cells. Nox4 depletion had no discernable influence on the activity of MAPKs and stress-activated kinases, but reduced the degree of oxidative DNA damage. These results suggest that Nox4 activity increases oxidative damage in HUVECs, leading to loss of replicative potential, which is at least partly independent of telomere attrition.

Increased expression of extracellular proteins as a hallmark of human endothelial cell in vitro senescence.

A convenient way to study processes of aging in distinct human tissues consists of a molecular analysis of cells from the tissue in question, that were explanted and grown in vitro until they reach senescence. Using human umbilical vein endothelial cells (HUVEC), we have established an in vitro senescence model for human endothelial cells. A major hallmark of HUVEC in vitro senescence is the increased frequency of apoptotic cell death, which occurs as a determining feature of HUVEC senescence. Senescent endothelial cells are also found in vivo in atherosclerotic lesions, suggesting that the presence of such cells may contribute to the development of vascular pathology. To elucidate mechanisms underlying endothelial cell senescence and age-associated apoptosis, gene expression analyses were carried out. In these experiments, we observed the up-regulation of genes coding for extracellular proteins in senescent HUVEC. In particular, a significant upregulation of interleukin-8, VEGI, and the IGF-binding proteins 3 and 5 was observed. Upregulation of these genes was confirmed by both RT-PCR and Western blot. In the case of interleukin-8, a roughly 50-fold upregulation of the protein was also found in cellular supernatants. The extracellular proteins encoded by these genes are well known for their ability to modulate the apoptotic response of human cells, and in the case of interleukin-8, clear links to the establishment of atherosclerotic lesions have been defined. The results described here support a new model, where changes in the secretome of human endothelial cells contribute to vascular aging and vascular pathology.

Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress.

Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.

Identification of cultivation-independent markers of human endothelial cell senescence in vitro.

Human aging processes are regulated by many divergent pathways and on many levels. Thus, to understand such a complex system and define conserved mechanisms of aging, the use of cell culture-based models is a widespread practice. An often stated advantage of in vitro aging of primary cells is the high reproducibility compared to the much more intricate aging of organisms. However, the aging process of cultured cells is, like aging of organisms, not only defined by genetic but also by environmental factors, making it difficult to distinguish between cell culture condition-induced artefacts and true aspects of aging. Therefore we investigated aging of HUVEC (human umbilical vascular endothelial cells), a well-known and widely used model system for in vitro aging, with different, already well-established cell culture protocols. Culturing conditions had indeed a strong impact on cell proliferation, the replicative lifespan and apoptosis rates. However, despite these significant differences, we found also various robust markers that define senescent HUVEC: morphological changes, increased senescence-associated β-galactosidase staining, cell cycle arrest in the G1 phase, lowered mitochondrial membrane potential and increased oxidatively modified proteins were displayed independent of cell culture protocols and could therefore be considered also as markers for in vivo aging.

Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bonafide target for AGE modification in human fibroblasts.

Insulin-like growth factor-induced signals activate mitochondrial respiration

From experiments with lower eukaryotes it is known that the metabolic rate and also the rate of aging are tightly controlled by the insulin-like growth factor (IGF)/insulin signal transduction pathway. The mitochondrial theory of aging implies that an increased metabolic rate leads to increased mitochondrial activity; increased production of reactive oxygen species due to these alterations would speed up the aging process. To address the question if mitochondrial activity is influenced by insulin/IGF signaling, we have established an experimental system to determine the influence of IGF-I-dependent signaling on mitochondrial function. We used DU145 prostate cancer cells, known for the intact IGF signal transduction pathway, to address the influence of IGF receptor activation on mitochondrial function by high-resolution respirometry. These experiments revealed that indeed mitochondrial function is regulated by IGF signaling, and up-regulation of respiration seems to require phosphoinositide 3-kinase/AKT signaling, but is independent of IGF effects on cell cycle progression. Collectively these data establish a regulatory cross-talk between insulin/IGF signal transduction and mitochondrial function, two major pathways implicated in controlling the rate of aging.

Identification of FAH domain containing protein 1 (FAHD1) as oxaloacetate decarboxylase

Background: Enzymes of the FAH superfamily catalyze a multitude of diverse chemical reactions. Results: Using molecular modeling followed by biochemical investigations, FAHD1 was identified as oxaloacetate decarboxylase. Conclusion: Our findings suggest that ODx activity can be found in eukaryotic members of the FAH superfamily. Significance: Our results identify a mammalian ODx enzyme as a so far undescribed player in mitochondrial metabolism. Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes.