Association of accelerated dynamics of telomere sequence loss in peripheral blood leukocytes with incident knee osteoarthritis in Osteoarthritis Initiative cohort.

Osteoarthritis (OA) is a chronic degenerative joint disease, being the main cause of laboral inability. Decreased telomere size in peripheral blood leukocytes (PBL) has been correlated with age-related pathologies, like knee OA. In a dynamic approach, telomere-qPCR was performed to evaluate the relative percentage of PBL telomere loss after a 6-year follow-up, in 281 subjects from the prospective osteoarthritis initiative (OAI) cohort. A radiological Kellgren-Lawrence (KL) grade ≥ 2 was indicative of knee OA. Individuals with knee OA at recruitment (n = 144) showed a higher PBL telomere loss after 6 years than those without knee OA at baseline (n = 137; p = 0.018). Moreover, individuals that developed knee OA during the follow-up (n = 39) exhibited a higher telomere loss compared to those that remained without OA (n = 98; p < 0.001). Logistic regression analysis showed that PBLs telomere loss was not significantly associated with knee OA at recruitment, but behaves as an independent risk factor associated with incidence after follow-up (OR: 1.043; p = 0.041), together with maximum KL grade (OR: 3.627; p = 0.011), body mass index-BMI (OR: 1.252; p < 0.001) and WOMAC-index (OR: 1.247; p = 0.021), at recruitment. The telomere decay in PBLs is faster in individuals with incident knee OA, possibly reflecting a systemic-global accelerated aging that enhances the cartilage degeneration.

Design of a digital-PCR assay to quantify fragmented human mitochondrial DNA.

Digital PCR (dPCR) has been adapted to quantify the proportion of mitochondrial DNA (mtDNA) molecules without and with double-strand DNA breaks (DSBs). This is based on a break-apart approach of two differentially labeled target sequences distantly located in the circular DNA molecule. When the two targets amplify in separated reaction partitions, the original mtDNA molecule should be fragmented by two DSBs at least, each in a different segment between targets. When both targets amplify in the same partition, it must correspond to a circular or linear mtDNA molecule. These two possibilities may be distinguished through a restriction endonuclease (RE) induced unique DSB within a DNA segment between the targets. After RE-digestion, separation of both target signals in different partitions must indicate the presence of a previous linear mtDNA molecule. Otherwise, joint amplification in the same partition would correspond to an initial circular mtDNA that has been linearized by the endonuclease. The procedure was validated by assaying different proportions of mtDNA fragmented by in vitro digestion with REs, evidencing a perfect accordance between the expected theoretical values and dPCR quantification. Samples from peripheral blood cells, cellular and extracellular DNA from the U2OS cell line, as well as cells incubated with ethidium bromide to induce mtDNA depletion, were evaluated. The technique may be of interest to complement the studies of mtDNA in relation to aging and human disease, as well as to assess possible adverse effects of certain drugs that could be related to affectation of mtDNA.

Leukocyte Telomere Length in Patients with Radiographic Knee Osteoarthritis.

Relative mean telomere sequence amount was determined by quantitative PCR (qPCR) of peripheral blood leukocyte (PBL) samples obtained at recruitment (n = 310) from individuals from the Osteoarthritis (OA) Initiative consortium. Knees were radiologically evaluated according to the Kellgren-Lawrence (KL) score, ranging from 0 to 4, considering a KL grade ≥ 2 as radiographic evidence of OA (n = 124). Telomere size decreased as baseline KL score increased, being significantly shorter in subjects with KL ≥2 (Mann-Whitney U-test, P < 0.0001). PBL telomere size was also associated with age, hypertension, body mass index (BMI) and waist circumference. Nevertheless, logistic regression analysis showed that PBL telomere size was a consistent risk factor for concurrent knee OA, independent of these health parameters. Shorter PBL telomeres may indicate a premature aging status which enhances chondrocyte senescence and degenerative joint disease. Environ. Mol. Mutagen. 60:298-301, 2019. © 2018 Wiley Periodicals, Inc.

Individual telomere length decay in patients with spondyloarthritis.

Telomere length was sequentially determined in peripheral blood leukocytes (PBL) from patients with ankylosing spondylitis (AS; n = 44) and psoriatic arthritis (PsA; n = 42) followed through 2.93 ± 0.99 years, using a quantitative PCR (qPCR) assay. The initial telomere size from PsA patients was higher than those with cutaneous psoriasis only (n = 53), possibly due to the inflammatory condition. The qPCR assay was sensitive enough to evidence a significant telomere length shortening in PBL from practically all subjects and PsA patients showed a higher rate of loss of telomere sequence than patients with AS during the follow-up time.

Genome-wide DNA methylation analysis of articular chondrocytes reveals a cluster of osteoarthritic patients.

OBJECTIVE: Alterations in DNA methylation patterns have been found to correlate with several diseases including osteoarthritis (OA). The aim of this study was to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA cartilage and healthy control cartilage samples. METHODS: DNA methylation profiling was performed using Illumina Infinium HumanMethylation27 in 25 patients with OA and 20 healthy controls. Subsequent validation was performed by genome-wide expression analysis using the Affymetrix Human Gene 1.1 ST array in an independent cohort of 24 patients with OA. Finally, the most consistent genes in both assays were amplified by quantitative reverse transcriptase PCR in a validation cohort of 48 patients using microfluidic real-time quantitative PCR. Appropriate bioinformatics analyses were carried out using R bioconductor software packages and qBase plus software from Biogazelle. RESULTS: We found 91 differentially methylated (DM) probes, which permitted us to separate patients with OA from healthy controls. Among the patients with OA, we detected 1357 DM probes that identified a tight cluster of seven patients who were different from the rest. This cluster was also identified by genome-wide expression in which 450 genes were differentially expressed. Further validation of the most consistent genes in an independent cohort of patients with OA permitted us to identify this cluster, which was characterised by increased inflammatory processes. CONCLUSIONS: We were able to identify a tight subgroup of patients with OA, characterised by an increased inflammatory response that could be regulated by epigenetics. The identification and isolation of this subgroup may be critical for the development of effective treatment and disease prevention.

mtDNA haplogroup J modulates telomere length and nitric oxide production.

BACKGROUND: Oxidative stress due to the overproduction of nitric oxide (NO) and other oxygen reactive species (ROS), play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. Therefore, the goal of this work is to describe the difference in telomere length of peripheral blood leukocytes (PBLs) and Nitric Oxide (NO) production between mitochondrial DNA (mtDNA) haplogroup J and non-J carriers, as indirect approaches of oxidative stress. METHODS: The telomere length of PBL was analyzed in DNA samples from 166 healthy controls (114 J and 52 non-J) and 79 OA patients (41 J and 38 non-J) by means of a validated qPCR method. The NO production was assessed in 7 carriers of the haplogroup J and 27 non-J carriers, by means of the colorimetric reaction of the Griess reagent in supernatants of cultured chondrocytes. Inducible nitric oxide synthase (iNOS) mRNA from these samples was analyzed by qPCR. Appropiated statistical analyses were performed RESULTS: Carriers of the haplogroup J showed a significantly longer telomere length of PBLs than non-J carriers, regardless of age, gender and diagnosis (p = 0.025). Cultured chondrocytes carrying the mtDNA haplogroup J also showed a lower NO production than non-J carriers (p = 0.043). No significant correlations between age and telomore length of PBLs were detected neither for carriers of the haplogroup J nor for non-J carriers. A strong positive correlation between NO production and iNOS expression was also observed (correlation coefficient = 0.791, p < 0.001). CONCLUSION: The protective effect of the mtDNA haplogroup J in the OA disease arise from a lower oxidative stress in carriers of this haplogroup, since this haplogroup is related to lower NO production and hence longer telomere length of PBLs too.

Horizontal transfer of the OXA-24 carbapenemase gene via outer membrane vesicles: a new mechanism of dissemination of carbapenem resistance genes in Acinetobacter baumannii.

The resistance of Acinetobacter baumannii strains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of ß-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes among A. baumannii strains are not fully understood. In this study we used two carbapenem-resistant clinical strains of A. baumannii (AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borne bla(OXA-24) gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate that A. baumannii releases outer membrane vesicles (OMVs) during in vitro growth. The use of hybridization studies enabled us to show that these OMVs harbored the bla(OXA-24) gene. The incubation of these OMVs with the carbapenem-susceptible A. baumannii ATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring the bla(OXA-24) gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboring bla(OXA-24) were released by A. baumannii ATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates of A. baumannii may release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surrounding A. baumannii bacterial isolates.

Decreased length of telomeric DNA sequences and increased numerical chromosome aberrations in human osteoarthritic chondrocytes.

Length of telomeric DNA sequences and numerical chromosome aberrations from uncultured human osteoarthritic (OA) articular chondrocytes were compared with those from peripheral blood leukocytes (PBL) from the same individual and from chondrocytes and PBL from control subjects. Cells were both obtained from 39 OA patients (age range: 43-80 years) and from 20 control subjects (age range: 39-94 years). Mean length of telomeric DNA sequences was determined using a quantitative real-time polymerase chain reaction (qPCR) assay and numerical chromosome aberrations were identified in interphase nuclei by Fluorescence In Situ Hybridization (FISH) using cocktails of specific DNA probes for chromosomes 7, 8 and for 18, X and Y. Chondrocytes revealed higher telomere size than PBL, both in control subjects and in OA patients, being 2 and 1.6 times higher respectively, thus revealing cell type specific differences. However, chondrocytes from OA patients showed significantly shorter telomere size than chondrocytes from control subjects (T/S ratio 1.64±0.41 vs. 1.99±0.54; mean±sd; p=0.008). Regarding the percentage of numerical chromosome aberrations, OA chondrocytes showed 1.7 times higher than chondrocytes from control subjects (19.80±3.31 vs.11.48±4.11; p<0.01) and 1.5 times average higher than that from PBL from the own OA patient (13.06±1.45; p<0.001). Moreover, PBL from OA patients also showed 1.4 times more anomalies than PBL from controls (13.06±1.45 vs. 9.54±1.61; p<0.001). No significant differences were found between chondrocytes and PBL in control subjects. Chromosome loss was the more frequent aneuploidy, mainly monosomy 18. The decreased telomere size and increased chromosome instability in chondrocytes from OA affected joints may imply a local advanced senescence that could contribute to the pathogenesis or progression of the degenerative articular disease. Moreover, the increased chromosomal abnormalities in PBL from OA patients suggest a more general accelerated senescence phenotype that could promote the age-related degenerative joint pathology.

Differing patterns of peripheral blood leukocyte telomere length in rheumatologic diseases.

Telomeres progressively shorten with repeated somatic tissue cell division, their length being an indicator of cellular ageing. Telomeric dysfunction may be implicated in a variety of diseases. We measured mean telomere length in peripheral blood leukocytes (PBL) from patients with various rheumatologic diseases. Mean PBL telomere length was measured using real-time quantitative polymerase chain reaction (Q-PCR) assay in a control population (n=130; age range: 3-94 years) and in subjects diagnosed with rheumatoid arthritis (RA; n=86; age range: 31-82 years), psoriatic arthritis (PA; n=56; age range: 26-79 years) and ankylosing spondylitis (AS; n=59; age range: 21-75 years). These diseases are associated with chronic systemic inflammatory activity. Telomere length was also quantified in subjects with osteoarthritis (OA; n=34; age range: 43-82 years) and osteoporosis (OP; n=35; age range: 59-95 years), diseases without a chronic systemic inflammatory component. Telomere length in OA showed no differences from age-matched controls (p=0.234), but was significantly shorter in OP (p=0.001). Telomere length was significantly longer than controls in RA (p=0.015), PA (p<0.001) and AS (p<0.001). Different patterns in telomere length from PBL are evidenced in rheumatologic pathologies, possibly dependent on the presence or absence of chronic systemic inflammation.

Interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to nitric oxide damage, and DNA-PKcs has a specific local role in its repair.

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was used to analyze DNA single-strand breaks (SSBs) and alkali-labile sites induced by exposure to the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnomine hydrochloride (SIN-1) in the whole genome and in long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cells. The relative density of DNA damage generated in the ITRS by X-rays was similar to that induced in the genome overall, whereas it was 1.7 times higher when the alkylating agent MNNG was assayed. Nevertheless, after SNP or SIN-1 treatment, ITRSs proved to be 2.8 and 2.7 times relatively more damaged, respectively, than the whole genome. When the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was not active, as in XR-C1 mutant cells, the repair kinetics in the whole genome did not differ from that in the parental cell line with X-ray or SNP exposure. However, whereas the SSBs and alkali-labile sites induced in the ITRS by X-rays exhibited rejoining kinetics similar to that of the parental cell line, the damage induced by SNP was more slowly rejoined. This implies a role for DNA-PKcs in the repair of DNA damage induced by NO, especially in ITRSs. The results demonstrated intragenomic heterogeneity of NO-induced DNA damage and repair; there was a higher density of DNA damage in the ITRS blocks, possibly because of their guanine richness. This suggests that a parallel process may occur in the terminal telomeres, which has implications for premature aging and neoplastic development by chronic NO exposure in vivo.

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells.

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.

Angiotensin II type 1 receptor blocker irbesartan ameliorates vascular function in spontaneously hypertensive rats regardless of oestrogen status.

BACKGROUND: Angiotensin II type 1 (AT1) receptor overexpression may play a decisive role in endothelial dysfunction during oestrogen deficiency in spontaneously hypertensive rats (SHRs). Similarly, exaggerated production of angiotensin II and enhanced expression of AT1 receptor have been reported in vessels of SHRs compared with normotensive rats. OBJECTIVE: To test the hypothesis that antihypertensive treatment with the AT1 receptor antagonist, irbesartan, could not only decrease blood pressure but also ameliorate endothelial dysfunction associated with both hypertension and oestrogen deficiency. METHODS: Ovariectomized and sham-ovariectomized SHRs were treated with 50 mg/kg irbesartan per day, administered with chow for 30 weeks. Sham-ovariectomized and ovariectomized rats receiving no treatment were used as control groups. At the end of the treatment period, the vascular reactivity of aortic rings was studied. RESULTS: In the irbesartan-treated groups, vasoconstriction induced by Nomega-nitro-l-arginine methyl ester (l-NAME) was increased and the response to phenylephrine exhibited greater potentiation in the presence of l-NAME, demonstrating a greater availability of basal nitric oxide in these groups. In addition, chronic treatment with irbesartan similarly enhanced the responsiveness of aortic rings from ovariectomized or sham-ovariectomized rats to acetylcholine and sodium nitroprusside. Incubation with indomethacin did not significantly alter acetylcholine- and sodium nitroprusside-induced relaxations in the irbesartan-treated rats. However, relaxations induced by acetylcholine and sodium nitroprusside in aortic rings from non-treated rats were significantly greater in the presence of indomethacin. CONCLUSION: Our data suggest that irbesartan enhances basal nitric oxide availability and ameliorates vascular relaxations in SHRs, by decreasing the production of cyclooxygenase-dependent contracting factors in smooth muscle cells, regardless of oestrogen status.