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Laboratory of Cell Proliferation & Ageing

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Research Staff

Dimitris Kletsas, Research Director

Harris Pratsinis, Associate Researcher

Eleni Mavrogonatou, Post-doctoral Fellow

Adamantia Papadopoulou, Post-doctoral Fellow

Sotirios-Spyridon Vamvakas, Post-doctoral Fellow

Eleni Liakou, Graduate Student (PhD)

Maria Angelopoulou, Graduate Student (PhD)

Anastasios Kouroumalis, Graduate Student (MSc)

 

 

Research interests

The Laboratory is focusing on tissue repair during development and ageing with an emphasis on the role of growth factors, and especially that of TGF-β. The action of growth factors on cell proliferation and extracellular matrix production, as well as the responsible signaling pathways are investigated. Alternative mechanisms of cell proliferation and differentiation, such as autocrine regulation, cell-matrix interactions, exogenous stresses and the effect of mechanical forces are also studied.

Main goal of the Laboratory is the investigation of the mechanisms of ageing and longevity. The structural and functional characteristics of the senescent cell - as a result of successive duplications or of exogenous stresses - in comparison to that of the young or the cancer cell are investigated. Especially, we are interested on the role of the senescent – somatic and stem - cell in the process of ageing and the development of age-related diseases, including cancer. In this direction, we study the interaction between the senescent stromal fibroblasts and adjacent cancer cells. Emphasis is given in tissues, such as the intervertebral disc, the degeneration of which provokes severe dysfunctions during ageing.

Aim of our studies is the elucidation of the mechanisms underlying the regulation of tissue homeostasis, especially during ageing, and furthermore the contribution, through research networks, in the development of cell replacement therapies. Finally, we study natural products and new synthetic compounds with putative anti-cancer, anti-ageing/anti-oxidant and wound healing action, as well as their mode of action.



Current Research Projects

The role of growth factors in tissue homeostasis and repair

TGF-β is a multifunctional growth factor. Especially concerning the regulation of cell proliferation it is well known that it can inhibit epithelial and endothelial cells, while it can stimulate cells of mesenchymal origin. Having in mind the crucial role of TGF-β in wound healing and the different strategies in skin repair followed between fetuses and adults, we studied the effect of this growth factor on the proliferation of fibroblasts from these two developmental stages. We found that TGF-β exerts a differential effect on the proliferation of these cell types: while it inhibits the proliferation of fetal fibroblasts (via the activation of PKA and subsequently of the up-regulation of cell cycle inhibitors p21 and p15), it stimulates cells from adult individuals (mediated by the release of FGF-2 and the consequent activation of the MEK-ERK pathway) (Figure 1). Currently, we are investigating the role of the extracellular environment in this phenomenon, and especially of the presence of components of the extracellular matrix, such as collagen and hyaluronate.In parallel, other growth factors, such as PDGF, bFGF, IGF-I (exogenously administered or produced in an autocrine/paracrine manner) are being studied in the Laboratory, along with the signaling pathways they activate, for the control of cell proliferation or other cellular functions in normal and pathologic conditions.

Relevant publications:

Pratsinis et al. (2004) Wound Repair Regen. 12: 374

Giannouli and Kletsas (2006) Cell. Signal. 18: 1417

Liontos et al. (2007) Cancer Res. 67: 10899

Pratsinis and Kletsas (2007) Eur. Spine J. 16: 1858

Gioni et al. (2008) Mol. Cancer Res. 6: 706

Chrissouli et al. (2010) Wound Rep. Regen. 18: 643

Mavrogonatou and Kletsas (2010) J. Orthop. Res. 28: 1276

Pratsinis et al. (2012) J. Orthop. Res. 30: 958

 

Figure 1.Molecular mechanisms of the differential response of fetal and adult skin fibroblasts to TGF-β.



Cellular senescence: Molecular mechanisms and role in tissue homeostasis

Cellular senescence is considered nowadays a major parameter of tissue homeostasis. It represents a potent anticancer mechanism and is involved in several age-related pathologies.

  1. A novel biomarker of cellular senescence

The identification of senescent cells in vitro and in vivo is a major task for understanding their role in tissue homeostasis. Recently, a novel such biomarker has been developed, based on a specific lipofuscin staining, with two major advantages over existing markers: the recognition of senescent cells is independent of culture conditions and it can be used in cryo-preserved archival materials (Figure 2).

Figure 2. Identification of senescent cells by the Sudan Black B staining


 

  1. Molecular mechanisms of cellular senescence

Various types of stress can lead to a senescent phenotype. Oncogene-induced senescence (a major anticancer mechanism) is linked with an activation of a DNA Damage Response (DDR) pathway [Bartkova et al. (2006) Nature 444: 633] (Figure 3). On the other hand, anticancer treatments, such as ionizing radiation or genotoxic anti-cancer drugs also provoke premature senescence and activate a DDR.

Figure 3. Oncogene overexpression provokes premature senescence in normal fibroblasts by activating a DNA damage response.



  1. Effect of cellular senescence on tumor development

Although cellular senescence is a potent anticancer mechanism, after becoming senescent, stromal cells can enhance tumor development, a mechanism that involves increased expression of matrix metalloproteases (Figure 4).

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Figure 4. Senescent cells enhance the growth of cancer cells in vitro (A) and in vivo (B).



  1. Cellular senescence impairs the differentiation of somatic and stem cells

We have found that cellular senescence affects considerably the ability of several cell types to differentiate. Human periodontal ligament fibroblasts that became senescent after replicative exhaustion of exposure to ionizing radiation have a decreased expression of the major transcription factors for osteoblastic differentiation (i.e. Runx2 and Osterix), as well as of alkaline phosphatase, leading to a decreased differentiation, due to the activation of the tumor suppressor p53 in senescent cells. Similarly, we observed that replicative senescence or premature senescence after the exposure of cells to ionizing radiation or genotoxic drugs have also a significantly decreased ability for adipogenic or chondrogenic differentiation (Figure 5).

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Figure 5. Decreased differentiation (A. osteoblastic, B. chondrogenic and C. adipogenic) of senescent cells (right in each panel) vs. early passage ones (left).



  1. Low Back Pain: a major age-related pathology. Stresses, proliferation and senescence in the intervertebral disc.

Low Back Pain (LBP) is considered one of the major chronic age-related pathologies, with a great impact in patients’ quality of life. It is linked with the degeneration of the intervertebral discs (IVD), the joints of the spine. IVD cells are characterized by a very low proliferative rate. However, during degeneration the cells proliferate more vigorously, probably as an attempt towards tissue repair. We have shown that under these conditions cells respond to exogenous growth factors and activate pivotal signaling pathways, such as MEK-ERK and PI3K-Akt (Figure 6A). In addition, we have shown the presence of an increased number of senescent cells in the aged and degenerated IVDs (Figure 6B). In order to understand the low proliferation and the increased senescence in these cells we studied one of the major stresses they are subjected to during daily activities, i.e. hyperosmolality. We have found that hyperosmolality activates stress-induced kinases (i.e. the p38 MAPK). In addition, it provokes a DNA damage response (Figure 6C) leading to the activation of the p53-p21WAF1-pRb axis. As a consequence, cells are arrested in both the G1 and the G2 phases of the cell cycle (Figure 6D). Increased osmolality also negatively affects the response of IVD cells to exogenous growth factors, while it provokes premature senescence. Finally, cDNA microarray experiments followed by bioinformatic analysis revealed a large number of differentially expressed genes in high osmolality-treated IVD cells in comparison to untreated ones. Results from transcriptomics analysis have been verified and the physiological role of selected genes is currently under investigation. These studies have been conducted within the framework of several EU-funded research networks such as EURODISC , MYJOINT and GENODISC.

Relevant publications:
Gorgoulis et al. (2005) Nature 434: 907
Gorgoulis et al. (2005) Lab. Invest. 85: 502
Bartkova et al. (2006) Nature 444: 633
Roberts et al. (2006) Eur. Spine J. 15 : S312
Pratsinis and Kletsas (2007) Eur. Spine J. 16: 1858
Mavrogonatou and Kletsas (2009) DNA Repair 8: 930
Mavrogonatou and Kletsas (2010) J. Orthop. Res. 28: 1276
Chandris et al. (2010) Biogerontology 11: 421
Papadopoulou and Kletsas (2011) Int. J. Oncol. 39: 989
Pratsinis et al. (2012) J. Orthop. Res. 30: 958
Mavrogonatou and Kletsas (2012) J. Cell. Physiol. 227: 1179
Konstantonis et al. (2013) Biogerontology 14: 741
Georgakopoulou et al. (2013) Aging 5: 37
Velimezi et al. (2013) Nature Cell Biology 15: 967
Mavrogonatou and Kletsas (2013) Spine (Phila Pa 1976). 38: 308
Neidlinger-Wilke et al. (2013) Eur Spine J. (in press)

 

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Figure 6. Activation of signaling pathways (A) and increased senescence (B) in the degenerated IVD. DNA damage (C) and activation of signaling pathways (D) as a response to hyperosmolality.



Effect of biomaterials on tissue physiology

Several compounds leaking from biomaterials used in therapeutic treatments seriously affect tissue physiology. In this vein, we have shown that the resin TEGDMA, frequently used in dental practice, inhibits the proliferation of gingival fibroblasts via the activation of the p53-p21WAF1-pRb axis, probably in order to protect the cells from its genotoxic action. In addition, we have studied several similar compounds for their putative cytotoxic, antiproliferative or estrogenic action.



Bioactive compounds with putative therapeutic action.

Extracts from natural products, isolated bioactive compounds and new chemically synthesized molecules are studied as putative leads for the development of novel antiageing/antioxidant, wound healing or anticancer treatment approaches. Currently, within the framework of the EU-funded project AGROCOS and the GSRT-funded project ENGAGE we are studying plant extracts and isolated compounds from the Greek flora and from all over the world. So far, we have identified several interesting extracts/compounds with antioxidant and UV-protection action for the protection of human skin fibroblasts.