The distribution pattern of critically short telomeres in human osteoarthritic knees
© Harbo et al.; licensee BioMed Central Ltd. 2012
Received: 17 July 2011
Accepted: 18 January 2012
Published: 18 January 2012
Telomere shortening is associated with a number of common age-related diseases. A role of telomere shortening in osteoarthritis (OA) has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultra-short single telomeres (below 1,500 base pairs), that is, the telomere subpopulation believed to promote cellular senescence.
Samples were obtained from human OA knees at two distances from the central lesion site. Each sample was split into three: one was used for quantification of ultra-short single telomeres through the Universal single telomere length assay (STELA), one for histological Mankin grading of OA, and one for mean telomere length measurement through quantitative fluorescence in situ hybridization (Q-FISH) as well as for assessment of senescence through quantification of senescence-associated heterochromatin foci (SAHF).
The load of ultra-short telomeres as well as mean telomere length was significantly associated with proximity to lesions, OA severity, and senescence level. The degree of significance was higher when assessed through load of ultra-short telomeres per cell compared with mean telomere length.
These in vivo data, especially the quantification of ultra-short telomeres, stress a role of telomere shortening in human OA.
The factors contributing to osteoarthritis (OA) have been classified into hereditary, mechanical, and age-related factors, where the latter represent the most prominent risk factor [1, 2]. Aging in OA does not simply consist in wearing of the cartilage matrix, but also involves aging of the chondrocyte, the cell responsible for cartilage maintenance. Aged chondrocytes respond differently to cytokines and growth factors, exhibit discoordinated gene expression, and are dysfunctional, performing anarchic proteolysis of matrix without appropriate repair [1, 2]. Such behavior is typical of cell-cycle-arrested senescent cells, a phenotype where telomere shortening is believed to be one of the critical players [3, 4]. Telomeres consist of non-coding DNA protecting the ends of mammalian chromosomes. If they shorten down to a critical level, they lose their protective capability and trigger a DNA-damage response leading to cell cycle arrest and cellular senescence . Interestingly, chondrocytes close to OA lesions are positive for the senescence marker senescence-associated β-galactosidase (SA β-gal), in contrast to those further away , and ex vivo expanded chondrocytes from OA cartilage show a decreased mean telomere length, which is compatible with a role of telomere shortening in OA .
However, a definitive conclusion about a role of telomere shortening in OA requires further validation. First, telomere lengths should be assessed in vivo and zone-specifically, rather than after expanding chondrocytes in a culture, which is likely to affect telomere length by itself. Second, there should be awareness that telomere shortening may occur by two superimposed processes : (i) gradual linear shortening reflecting the number of cell divisions (that is, 'replicative' shortening); (ii) a more stochastic process, causing sudden extensive shortening of a single telomere, and induced by various stimuli, including oxidative damage (that is, 'stress-induced' shortening). The latter process deserves special attention with respect to cell senescence, because there are indications that it is the critical shortening of a few or even a single telomere, rather than a decrease in the mean length that leads to dysfunction and induces senescence [8–10]. In chondrocytes, these two processes are likely to occur to a different extent. Replicative shortening is probably less common because articular cartilage is usually considered as a post-mitotic tissue, where cell renewal is virtually absent . However, a modest contribution from cell replication may be present, since the presence of putative chondrocyte progenitor cells has recently been demonstrated in human articular cartilage [11, 12]. In contrast, stress-induced shortening is likely to be a common process, because oxidative stress is believed to be enhanced by cyclic compressions such as those occurring at the loading zone , and such oxidative stress is thought to contribute to telomere shortening also in chondrocytes . Thus, it can be imagined that telomere shortening plays a role in initiation of OA even before lesions actually appear. If single critically short telomeres are the prevailing products of stress-induced telomere shortening and if they are responsible for induction of senescence, it appears essential to assess these fragments and not merely mean telomere length in chondrocytes.
In the present study we therefore characterized the distribution of these critically short telomeres in the cartilage of human OA knee with special emphasis on whether the load of ultra-short telomeres increases closer to the central lesion site. We used a newly developed Universal single telomere length assay (STELA) , a PCR-based method that measures the telomere length on single human chromosome arms. In addition, we measured mean telomere length by quantitative fluorescence in situ hybridization (Q-FISH) [14, 15] for comparison, assessed senescence through counting of cells showing senescence-associated heterochromatin foci (SAHF) [16, 17], and performed histopathological scoring of OA.
Materials and methods
Histopathology for OA grading
Biopsies for histopathology were fixed in 3.7% neutral-buffered formalin overnight and decalcified in Idranal III solution (7% EDTA) (Sigma-Aldrich, Broendby, Denmark). Following dehydration in ethanol, they were embedded in paraffin by standard pathological procedures and serial sections of 5 μm were cut. Sections were stained with hematoxylin and Safranin O/Fast Green FCF according to standard protocols. The severity of cartilage damage was graded by two independent observers through blinded observations, by using the Histologic/Histochemical Grading System of Mankin .
SAHF measurement of senescence
Tissue sections for fluorescent detection of SAHF were fixed in 3.7% neutral-buffered formalin overnight, dehydrated in ethanol and embedded in paraffin by standard pathological procedures. Paraffin-embedded tissue sections of 5 μm were cut and deparaffinized in xylene, dehydrated in ethanol and pre-treated by incubation with Proteinase K solution (DakoCytomation, Glostrup, Denmark) overnight at room temperature. Slides were washed, dehydrated in ethanol, air-dried and mounted with ProLong Gold anti-fade reagent with DAPI nucleic acid stain from Molecular Probes (Invitrogen, Taastrup, Denmark). Fluorescent images were obtained at 100 × magnification with a standard DAPI filter and were analyzed for SAHF formation after normalization of the DAPI intensity with a Laplacian filter as described by Lawless et al. . A total of 15 to 25 cells per zone were analyzed and those showing condensation of chromatin in the nuclei were scored as positive for SAHF and were expressed as percentage of total number of cells.
Universal STELA measurement of ultra-short single telomeres
Purification of DNA for molecular telomere measurement was performed with Blood & Tissue Genomic DNA Extraction Miniprep System from Viogene (Kem-En-Tec, Taastrup, Denmark) according to the manufacturer's manual. Briefly, the cartilage was homogenized and lysed by incubation at 60°C overnight in Viogene lysis buffer containing 4 μg/μl Proteinase K (Fisher Scientific, Slangerup, Denmark). Following incubation the enzyme was deactivated at 70°C and extraction buffer from the kit was added in a 1:1 ratio to the lysis buffer. The DNA was precipitated with 99% ethanol, washed on a silica-gel membrane (from the kit) and eluted with sterile water. Universal STELA was performed as described by Bendix et al. . In short, purified DNA was digested by a 1:1 mixture of restriction enzyme MseI and NdeI (Medinova, Glostrup, Denmark). After digestion, annealing with a double-stranded synthetic oligonucleotide having a sticky end corresponding to MseI and NdeI digests was followed by ligation of this double-stranded oligonucleotide to the proximal end of the telomeric fragments. Next, a single-stranded oligonucleotide, with part of the sequence complementary to the telomeric overhang, was ligated to the distal end of the telomeric fragment. PCR was then performed using the two ligated oligonucleotides as targets for the PCR primers. PCR reactions were performed in a 12 μl volume containing 40 to 80 pg of ligated DNA, 1× Failsafe PCR PreMix H from Epicentre (VWR, Herlev, Denmark), 0.1 μM primers (that is, teltail and adapter) and 1.25 U Failsafe Enzyme from Epicentre (VWR, Denmark). The reactions were carried out under the following conditions: 1 cycle of 68°C for 5 minutes (fill-in step), 1 cycle of 95°C for 2 minutes, 26 cycles of 95°C for 15 seconds, 58°C for 30 seconds and 72°C for 12 minute, 1 cycle of 72°C for 15 minutes. Subsequent detection of the telomeric products by Southern blotting was carried out with TeloTAGGG Telomere Length Assay from Roche Applied Science (Hvidovre, Denmark) according to the manufacturer's manual with few adaptions. The size of the PCR products was calculated on the basis of a DIG-labeled molecular weight marker using the software VisionWorksLS Acquisition and Analysis Software from UVP (AH Diagnostics, Aarhus, Denmark). The number of bands at a length below 1,500 bp were counted, calibrated in regard to PCR template concentration and presented as the number of telomeres below a length of 1,500 bp per genome equivalent of template DNA.
Q-FISH measurement of mean telomere length
Biopsies for Q-FISH were prepared and pre-treated as described for SAHF. Q-FISH was performed as described by Graakjaer et al.  using a fluorescent telomere-specific peptide nucleic acid probe, which binds almost stoichiometrically to the telomeres. A total of 100 ng of the probe  was mixed with 100 μl working solution (70 μl 70% formamide, 5 μl MgCl2, 10 μl MEN blocking solution (Roche Applied Science, Denmark) and 180 μl H2O). This mixture was added to the pre-treated slides and covered with a cover slip. Slides were then heated to 80°C for 5 minutes followed by hybridization for 60 minutes at room temperature. Subsequently, cover slips were removed followed by removal of non-specific staining and background by a short immersion of the slides in Rinse buffer (DakoCytomation, Denmark) followed by 5 minutes incubation at 62°C in Wash buffer (DakoCytomation, Denmark). Slides were dehydrated in ethanol, air-dried and mounted with ProLong Gold anti-fade reagent with DAPI from Molecular Probes (Invitrogen, Denmark). Fluorescent images of the nuclei were obtained at 100 × magnification using a standard DAPI filter and telomere images were recorded using a standard FITC filter. The intensity of telomere spots was determined by a dedicated image analysis software originally developed by DakoCytomation (Telomere Quantifier v. 1.0), followed by calculation of mean spot intensity per nucleus. A total of 20 to 30 nuclei per zone were analyzed and the mean nuclear spot intensity was calculated and expressed as arbitrary fluorescence units reflecting mean telomere length. Slide to slide and day to day normalizations proved not to be necessary probably due to the almost stoichiometric binding of the probe.
For the paired comparison of the central and the peripheral biopsy within the same orthogonal direction we used the sign test, assuming as the null hypothesis that measurement obtained from the central biopsy of each pair is as likely to be higher as to be lower than the measurement from the peripheral biopsy. For the correlations of Universal STELA and Q-FISH measurements with Mankin scores or percentage of SAHF positive cells, we used Pearson's correlation coefficient.
The Mankin scores showed a highly consistent distribution pattern in agreement with daily clinical experience, namely higher Mankin scores at the medial plateau compared to the lateral plateau, and higher scores close to the cartilage lesion compared to the cartilage from the peripheral part of the joint surface (Figure 2A). The histopathological findings were consistent with the clinical grading of OA. All the paired comparisons shown in Figure 3A showed higher scores towards the central area compared to the periphery (P = 0.000001), irrespective of the OA scores of the peripheral biopsies, which varied over a wide range. The percentage of SAHF-positive cells followed a distribution parallel to the Mankin scores (Figure 2B) supporting previous data, where SA β-gal was used as another senescence marker . All 21 but 1 paired comparisons between positions close and distant to the lesion showed a higher percentage of SAHF positive cells close to the lesion (P = 0.000002) (Figure 3B).
Overall, Figure 2C shows that the number of ultra-short telomeres exhibits the same general distribution pattern as the Mankin score and the percentage of SAHF-positive cells. Mean telomere length exhibits a similar pattern, but less clearly. When analyzing individual orthogonal directions it is obvious that the number of ultra-short telomeres is increasing when moving towards the central lesion. This phenomenon is further illustrated in Figure 3C, where it is seen that in all but one of the 20 eligible orthogonal directions, the number of ultra-short telomeres increased when moving towards the central region of the plateau (P = 0.000004), and the only pair of biopsies that did not show an increase showed identical values. Again, the increase was seen irrespective of the number of ultra-short telomeres in the peripheral biopsies, which varied over a wide range. When plotting the Q-FISH data in the same way (Figure 3D), the expected decline in mean telomere length when moving towards the cartilage lesion was less consistent, as it was observed in only 17 out of 21 orthogonal directions (P = 0.007).
The reason why we also find an association between OA changes and mean telomere length could be due to the fact that the two mechanisms of telomere shortening, that is, gradual replicative shortening and more sudden, stochastic telomere damage are not totally independent. It is likely that telomere damage due to, for example, persistent oxidative stress in the long run will lead also to a decrease in mean telomere length, which can explain why mean telomere length correlates with both distance to lesion and parallel Mankin score. This view is in line with recent reports on cultured chondrocytes showing that oxidative stress leads to a decrease in mean telomere length [20, 21]. It is also interesting that the response of mean telomere length, reported by Brandl et al., is not immediate but occurs gradually .
Our analysis is based on the variations of telomere length within restricted areas of the same cartilage tissue, that is, 20 comparisons between cells next to and away from the lesion, the latter being used as a 'control'. Further studies performed on more than three patients, could also address the inter-patient variability of the number of ultra-short single telomeres and their relation to the global OA grade of the patient. It would also be useful to extend the present study to other joints, such as the hip. Interestingly, shortening of telomeres was recently also associated with chondrocyte senescence in degenerate intervertebral discs [22, 23], thereby supporting the concept that telomere shortening may be a general player in chondrocyte senescence, and not restricted to OA.
Recent efforts to identify critical players in OA include elegant approaches based on gene expression profiling and gene polymorphisms [24, 25]. These approaches allow very systematic screenings but miss factors such as telomere shortening, which may very well be important to take into account in a number of age-related diseases. In the present study, we used a newly-developed assay, which allows quantifying in cartilage biopsies, the minute subpopulation of ultra-short single telomeres believed to trigger cellular senescence, and we show its association with OA.
polymerase chain reaction
quantitative fluorescence in situ hybridization
- SA β-gal:
senescence-associated heterochromatin foci
single telomere length assay.
The authors thank Birgit McDonald, Tinna Herløv Jensen and Marianne Mose Hansen for technical assistance, Jane Schwartz Leonhardt for recruiting patients and Per Wagner Kristensen for collecting the cartilage biopsies.
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