Immortalization of Fibroblasts Using the Catalytic Subunit of Human Telomerase (TERT)

The limit on cell proliferation in primary cells represents an obstacle to the study of cultivated patient fibroblasts, especially since many rounds of cell division are used to produce large quantities of cells required for bio-chemical analysis. It has been shown that the forced expression of exogenous human telomerase (TERT) is sufficient to produce telomerase activity in Tangier and Progeria fibroblasts and prevent the erosion of telomeres and circumvent the induction of senescence and crisis (Ouellette et al, 2000; Walter et al, 2004). The expression of TERT efficiently extends the lifespan of human fibroblasts without changing the key characteristics (Bodnar et al, 1998; Morales et al, 1999). Thus ectopic expression of telomerase is a way to circumvent the lack of critical experimental material.

Moreover telomerase immortalized cells of affected patients can be used to distinguish effects intrinsic to the genetic defect from those due to cell senescence. For example, a preliminary biochemical characterization of immortalized Tangier fibroblasts indicated the existence of a novel cholesterol removal pathway that may help to prevent early atherosclerosis in Tangier disease. This ATP binding cassette transporter 1 (ABCA1)-independent cholesterol secretion pathway is sensitive to aging phenomena ex vivo and possibly in vivo and therefore hard to detect in primary cells (Walter et al, 2004). Further studies in Tangier and Progeria patients may help to identify new candidate genes involved in (replicative) aging, HDL metabolism and atherogenesis. The role of these genes can later be examined in appropriate animal models, selected patients and appropriate study collectives such as young survivors of myocardial infarction or centenarians.



Characterization of the Cellular Phenotype of Tangier and Progeria Fibroblasts

A low HDL cholesterol plasma level is one of the most predictive coronary risk factors. Reverse cholesterol transport, the transport of cholesterol from peripheral tissues to the liver, is an important atheroprotective function of HDL. However, the mechanism by which HDL mobilizes cellular cholesterol is still obscure. The discovery of ATP-binding cassette transporter A1 (ABCA1) as the defective protein in the autosomal co-dominant genetic HDL deficiency syndrome Tangier disease (Rust et al., 1998; Rust et al., 1999; Bodzioch et al, 1999; Brook-Wilson et al., 1999) and the finding that cholesterol efflux is impaired in Tangier cells (Walter et al., 1994; Francis et al, 1995, Rogler et al., 1995) initiated a tremendous number of new studies in this field. These studies indicated that ABCA1 is essential in the initial steps of HDL formation and that impaired function of ABCA1 in Tangier disease is associated with higher coronary risk. However, the exact physiological role of ABCA1 is still unclear. Moreover, it is not understood why the overall coronary risk in Tangier patients is increased (Serfaty-Lacrosniere et al., 1994), whereas atherosclerosis in many Tangier patients occurs at a relatively advanced age and is apparently completely lacking in some patients (Walter et al., 1994). This may at least partly be explained by age-sensitive cholesterol secretion pathways and other compensatory mechanisms (Kannenberg et al., 2013).

By contrast, all patients suffering from the premature aging syndrome Hutchinson-Gilford progeria develop severe atherosclerosis at young age and die before age 20, in most cases from atherosclerotic complications. The disease is caused by  mutations in lamin A. This protein is an essential scaffolding (supporting) component of the nuclear envelope, which surrounds the nucleus in cells. Mutations that cause Hutchinson-Gilford progeria syndrome disrupt the normal production of the lamin A protein. The altered protein makes the nuclear envelope unstable and progressively damages the structure and function of the nucleus. It is however not clear how these changes lead to the characteristic disease features, including severe premature atherosclerosis.

Preliminary data from our lab suggest that impaired HDL-inducible cholesterol secretion is at least partly responsible for the severe athero-sclerosis in these patients. We currently characterize the consequences of ABCA1 and lamin A loss of function on the regulation of cellular cholesterol homeostasis. Aim of this project is (i) to expand our knowledge of the anti-atherogenic mode of action of HDL in reverse cholesterol transport, and (ii) to obtain new insights into the molecular mechansims enabling target-specificity and regulation of intracellular cholesterol trafficking in normal and aged cells.


Characterization of interrelationships between atherogenesis and replicative aging.

The mechanism by which shortened telomeres (caused either by many cell divisions or much cellular stress) cause growth arrest is not well understood. One of the major functions of telomeres is to hide the end of chromosomes from being recognized as double-strand breaks, needing repair. Thus, telomere dysfunction provokes chromosomal fusions, leading to induction of a p53/Rb DNA damage signal and consecutively to senescence and/or apoptosis (Shay et al, 1991). In addition, transcriptional silencing of genes adjacent to telomeres, a mechanism called telomere position effect, may contribute to telomere-mediated senescence (Baur et al, 2001). The effects associated with telomere shortening may act in concert with other known damaging signals such as free radical formation, protein crosslinking or mitochondrial dysfunction to trigger growth arrest (Wright & Shay, 2002; Shay & Wright, 2004).

We have characterized the HDL-inducible cell signalling pathways making use of various cell and disease models. It is of interest that many of the involved signalling molecules are likely influenced by aging and stress phenomena. These include activation of phosphatidylcholine-specific phospholipases C and D (Walter et al, 1995; Walter et al, 1996), activation of small GTP binding proteins of the Rho/Rac/cdc42 family (Utech et al 2001), activation of JNK-1 (Nofer et al 2003) and serine-/threonine-specific phosphatases (Höbbel et al, 1998). Some of these pathways are involved in HDL-inducible lipid secretion, others may influence cell cycle regulation (Nofer et al. 2000; Nofer et al 2001) or may mediate other potentially anti-atherogenic effects of HDL on nitric oxide synthesis or apoptosis. Moreover HDL is not only target but may also modulate the aging process. For example, increased HDL levels were shown to protect against the detrimental effect associated with defects in the survival protein KLOTHO (Arking et al, 2003), and most HDL-inducible signalling pathways overlap with cellular survival pathways.

The aim of this project is (i) to identify the HDL-inducible signaling pathways that are influenced by aging and stress, (ii) to distinguish effects due to a telomere position effect from secondary changes, and (iii) to characterize possible protective effects of HDL on replicative aging, defined as the ability to produce a greatly extended lifespan by preventing telomere shortening.


Establishment of new atherothrombotic risk factors.

We will continue the establishment and validation of new risk factors for atherothrombosis. A traditional focus (a collaboration of the Institute of Clinical Chemistry and Laboratory Medicine, the Leibniz Institute of Arteriosclerosis Research, the Department of Pediatric Haematology/ Oncology and the Department of Otolaryngology, Head and Neck Surgery) is the examination of thrombophilic factors that influence both venous and arterial thrombosis and atherothrombosis.

Recently, we could demonstrate a partial overlap between classical coronary risk factors and risk factors for sudden hearing loss (SHL). Hypercholesterolemia and hypoalphalipoproteinemia (low HDL cho-lesterol levels) are apparently no major risk factors for SHL, whereas the Glycoprotein Ia C807T polymorphism, elevated fibrinogen levels, and smoking are associated with an increased risk for SHL. Remarkably, significantly more SHL patients were current smokers (56% vs. 19% in the control group). Moreover, we identified the first genetic (and a relatively common) SHL risk factor: GPIa C807T. Altogether these findings suggest a vascular involvement in the pathogenesis of SHL and may have implications for the development of therapeutic and preventive strategies (Rudack et al 2006). The functional background of the observed clinical and epidemiological relationships is currently investigated on a cellular level.